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Imidazoline antihypertensive drugs: a critical review on their mechanism of action
Pharmacology & Therapeutics 93 (2002) 1 – 35
Associate editor: H. Bo¨nisch
Imidazoline antihypertensive drugs: a critical review on their mechanism of action Bela Szabo* Institut fu¨r Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universita¨t, Albertstrasse 25, D-79104 Freiburg i. Br., Germany
Abstract It was long thought that the prototypical centrally acting antihypertensive drug clonidine lowers sympathetic tone by activating a2-adrenoceptors in the brain stem. Supported by the development of two new centrally acting drugs, rilmenidine and moxonidine, the imidazoline hypothesis evolved recently. It assumes the existence of a new group of receptors, the imidazoline receptors, and attributes the sympathoinhibition to activation of I1 imidazoline receptors in the medulla oblongata. This review analyzes the mechanism of action of clonidine-like drugs, with special attention given to the imidazoline hypothesis. Two conclusions are drawn. The first is that the arguments against the imidazoline hypothesis outweigh the observations that support it and that the sympathoinhibitory effects of clonidine-like drugs are best explained by activation of a2-adrenoceptors. The second conclusion is that this class of drugs lowers sympathetic tone not only by a primary action in cardiovascular regulatory centres in the medulla oblongata. Peripheral presynaptic inhibition of transmitter release from postganglionic sympathetic neurons contributes to the overall sympathoinhibition. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Imidazoline receptor; Blood pressure; Presynaptic receptor; Rostral ventrolateral medulla; Sympathetic nervous system; a2-Adrenoceptor Abbreviations: AGN192403, 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane; CHO, Chinese hamster ovary; DOPAC, 3,4-dihydroxyphenylacetic acid; GABA, g-aminobutyric acid; HEK, human embryonic kidney; 5-HT, 5-hydroxytryptamine; IRAS, imidazoline receptor-antisera-selected; IRP, imidazoline receptor protein; NRL, nucleus reticularis lateralis (mostly used as a synonym for RVLM); NTS, nucleus tractus solitarii; RSNA, renal sympathetic nerve activity; RVLM, rostral ventrolateral medulla; S23230, (-)-(5-[2-methyl-phenoxy-methyl]-1,3-oxazolin-2-yl)amine; SHR, spontaneously hypertensive rats; SK&F86466, 6-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benzazepine HCl
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 2. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Mode of action of clonidine-like drugs: two hypotheses . . . 3.1. The a2-adrenoceptor hypothesis . . . . . . . . . . . . 3.2. The imidazoline hypothesis . . . . . . . . . . . . . . 4. Characterization of drugs used in imidazoline cardiovascular binding studies . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Agonists . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Selective a2-adrenoceptor agonists . . . . . . 4.1.2. Selective I1 imidazoline receptor agonists . . 4.2. Antagonists . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Selective a2-adrenoceptor antagonists . . . . 4.2.2. Selective I1 imidazoline receptor antagonists . 5. The central nervous site of action of clonidine-like drugs . . 5.1. Action in the rostral ventrolateral medulla. . . . . . .
* Tel.: +49-761-203-5312; fax: +49-761-203-5318. E-mail address: [email protected] (B. Szabo). 0163-7258/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 3 - 7 2 5 8 ( 0 1 ) 0 0 1 7 0 - X
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B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
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5.2. Action in the nucleus tractus solitarii . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Action in the spinal cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characterization of a2-adrenoceptors and I1 imidazoline-binding sites . . . . . . . . . . . . . . . 6.1. a2-Adrenoceptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1. Molecular identity, second messenger mechanisms . . . . . . . . . . . . . . . . . 6.1.2. Distribution in cardiovascular regulatory centres . . . . . . . . . . . . . . . . . . 6.2. I1 imidazoline receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1. Molecular identity, second messenger mechanisms . . . . . . . . . . . . . . . . . 6.2.2. Distribution in cardiovascular regulatory centres . . . . . . . . . . . . . . . . . . Action of clonidine-like drugs on cardiovascular regulatory nuclei in vitro . . . . . . . . . . . . Correlation between sympathoinhibition and affinity for I1 imidazoline-binding sites and a2-adrenoceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Cats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. Rabbits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4. Monkeys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interaction of agonists with antagonists in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. Studies supporting involvement of a2-adrenoceptors. . . . . . . . . . . . . . . . . . . . 9.2. Studies supporting involvement of I1 receptors . . . . . . . . . . . . . . . . . . . . . . Experiments in genetically modified animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . Relationship between sympathoinhibition and other a2-adrenoceptor-mediated effects. . . . . . . 11.1. Animal experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1.1. Clonidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1.2. Rilmenidine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1.3. Moxonidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2. Studies in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.1. Clonidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.2. Rilmenidine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.3. Moxonidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contribution of presynaptic inhibition of transmitter release from postganglionic sympathetic neurons to the overall reduction of sympathetic tone produced by clonidine-like drugs . . . . . . 12.1. Peripheral presynaptic inhibition in experimental models with an artificial sympathetic tone 12.2. Peripheral presynaptic inhibition under physiological conditions . . . . . . . . . . . . . . 12.3. Presynaptic imidazoline receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4. Penetration of clonidine-like drugs into the brain . . . . . . . . . . . . . . . . . . . . . . 12.5. Summary of the role of peripheral presynaptic inhibition . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Increased release of transmitter from sympathetic neurons — increased sympathetic tone — is an early link in the pathogenesis of essential hypertension in many patients (see Esler & Kaye, 1998). Two groups of the available antihypertensive drugs prevent increased activation of adrenoceptors in cardiovascular tissues, the b- and a1-adrenoceptor antagonists. Reducing transmitter release from sympathetic neurons, i.e., reducing sympathetic tone, is a logical approach, in a certain sense a causal therapy, to lower blood pressure. Clonidine-like antihypertensive drugs lower sympathetic tone. In addition to the older drugs, clonidine, a-methyldopa, guanfacine, and guanabenz, two drugs were introduced into therapy recently, rilmenidine and moxonidine (see Fig. 1 for chemical structures of the drugs). A renaissance of this class of drugs can be observed, which is also supported by new ideas regarding their mechanism of
10 11 11 11 11 11 12 12 12 13 14 14 14 15 15 15 15 18 19 20 20 20 21 23 23 23 23 23 24 24 25 26 26 27 27 28 28
action. The interest in these drugs is also increasing because they may be beneficial in cardiac failure (Manolis et al., 1995; Azevedo et al., 1999; Swedberg et al., 2000) and because they possess analgesic – anaesthetic properties (Pertovaara, 1993). While it is generally accepted that clonidine-like drugs lower sympathetic tone, several hypotheses have been developed to explain the mechanism. The aim of this review is to critically analyze these hypotheses.
2. History A short history of the pharmacology of these drugs is given at first to show the development of ideas. The discovery of imidazolines goes back to 1939. Scientists at Ciba in Basel in Switzerland wanted to obtain new drugs by combining the phenylethylamine moiety of adrenaline with the imidazole structure of histamine (Hartmann & Isler,
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
3
Fig. 1. Structure of agonists and antagonists used in imidazoline cardiovascular research. I1, affinity for I1-binding sites; a2, affinity for a2-adrenoceptors.
1939). They obtained the a-adrenoceptor antagonist tolazoline. It turned out to possess therapeutically useful vasodilating properties. Another of the discovered drugs, naphazoline, is used even today to relieve nasal congestion. In an attempt to produce a new nasal decongestant, the chemist H. Sta¨hle synthesized the compound St155 at Boehringer Ingelheim in Germany in 1962. Unexpectedly, St155 caused strong sedation, and this effect was essential for recognizing that a new class of compounds had been discovered. St155, later named clonidine, also lowered blood pressure and heart rate and inhibited saliva secretion (Hoefke & Kobinger, 1966; see also Klingspohr, 1983). Clonidine turned out to be the prototype of centrally acting antihypertensive drugs. Other centrally acting antihypertensive drugs followed: guanabenz (Baum et al., 1970), guan-
facine (Scholtysik et al., 1975), rilmenidine (Laubie et al., 1985), and moxonidine (Armah, 1987; Armah et al., 1988). The development of a-methyldopa followed a different course, which will not be outlined here. It is actually the oldest centrally acting antihypertensive drug (Oates et al., 1960). During the 20 years following the discovery of clonidine, it was shown that clonidine and its relatives decrease blood pressure by causing central sympathoinhibition and bradycardia. It was thought that activation of a2-adrenoceptors in the medulla oblongata is responsible for the central sympathoinhibition (for reviews, see Schmitt, 1977; Kobinger, 1978; Van Zwieten et al., 1984; Kobinger & Pichler, 1990). In 1984, Bousquet et al. hypothesized a new mechanism of action for clonidine-like drugs. It was suggested that a new receptor, the imidazoline receptor,
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B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
Table 1 Hypotensive and sympathoinhibitory doses of systemically administered clonidine-like drugs Species
Anaesthesia
Effect
Rat
Pentobarbitone
Decrease in blood pressure Decrease in heart rate Decrease in blood pressure and the catecholamine oxidation peak in the NRL Decrease in blood pressure and heart rate Decrease in blood pressure and heart rate
Rat
Pentobarbitone
Rat Rat
Pentobarbitone Pentobarbitone
Rat (SHR) Rat Rat
Decrease in blood pressure and heart rate Urethane Pentobarbitone
Rat (SHR) Pentobarbitone Conscious Rat (SHR) Urethane Mouse Conscious
Rabbit
Rabbit Rabbit Rabbit
Rabbit
Cat Cat Dog Humans Humans Humans
Humans
Humans Humans
Decrease in blood pressure Decrease in heart rate Decrease in blood pressure and the catecholamine oxidation peak in the NRL Decrease in blood pressure and heart rate Decrease in blood pressure and heart rate Decrease in blood pressure and heart rate Decrease in blood pressure and heart rate Decrease in blood pressure and heart rate Decrease in blood pressure and heart rate
Conscious
Decrease in blood pressure Decrease in heart rate Decrease in blood pressure Decrease in heart rate Pentobarbitone Decrease in blood pressure Pentobarbitone Decrease in blood pressure and heart rate Conscious Decrease in blood pressure, heart rate, renal sympathetic nerve activity, and plasma noradrenaline concentration Conscious Decrease in blood pressure, heart rate, renal sympathetic nerve activity, and plasma noradrenaline concentration Urethane/chloralose Decrease in the firing rate of splanchnic sympathetic nerves Urethane Decrease in the firing rate of splanchnic and renal sympathetic nerves Chloralose/urethane Decrease in blood pressure and heart rate Conscious Decrease in blood pressure and sympathetic nerve activity Conscious Decrease in blood pressure and plasma noradrenaline concentration Conscious Decrease in blood pressure, sympathetic nerve activity, plasma noradrenaline concentration and vascular resistance Conscious Decrease in blood pressure, heart rate, sympathetic nerve activity, and plasma noradrenaline concentration Conscious Decrease in blood pressure, sympathetic nerve activity, and calf vascular resistance Conscious Decrease in blood pressure and heart rate
Agonist (route of application) Clonidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.) Rilmenidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.) Rilmenidine (i.v.)
Effective doses1
Reference
ED50 2.6 mg kg ED50 1.3 mg kg 2 – 10 mg kg 1
1
100 – 1000 mg kg 300 – 1000 mg kg 3 – 30 mg kg 1 100 – 1000 mg kg 3 – 30 mg kg 1 ED50 = 250 mg kg ED50 = 350 mg kg 300 – 1500 mg kg
1
de Jonge et al., 1981a
1
Tibirica et al., 1989 van Zwieten et al., 1986 Koenig-Berard et al., 1988
1
1
1
Gomez et al., 1991
1 1
Tibiric¸a et al., 1991b
Moxonidine (i.v.) Rilmenidine (i.v.) Rilmenidine (i.v.)
3 – 30 mg kg 1 30 – 300 mg kg 1 3 – 30 mg kg 1 30 – 300 mg kg 1 40 mg kg 1 30 – 300 mg kg 1 300 – 1000 mg kg 1 100 – 1000 mg kg 1 ED50 7 mg kg 1 ED50 14 mg kg 1 ED50 180 mg kg 1 ED50 210 mg kg 1 10 – 100 mg kg 1 30 – 1000 mg kg 1 100 – 1000 mg kg 1
Moxonidine (i.v.)
3 – 100 mg kg
Guanfacine (i.v.)
ED50 = 83 mg kg
Clonidine (i.v.)
10 – 100 mg kg
Clonidine (i.v.) Clonidine (i.v.)
1 – 100 mg kg 100 – 275 mg
Clonidine (i.v.)
200 mg
Warren et al., 1991
Clonidine (p.o.)
300 mg
Muzi et al., 1992
Clonidine (i.v.) Rilmenidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.) Moxonidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.) Moxonidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.)
1
Haxhiu et al., 1994 Zhu et al., 1999
Head & Burke, 1991
Armah et al., 1988 Feldman et al., 1990 Szabo et al., 1993; Urban et al., 1994 Urban et al., 1995a
1
1
Sannajust et al., 1992b
1
Scholtysik et al., 1975 Haeusler, 1976 Hoefke & Kobinger, 1966 Wallin & Frisk-Holmberg, 1981
Moxonidine (p.o.) 400 mg
Wenzel et al., 1998
Moxonidine (p.o.) 200 – 400 mg
Greenwood et al., 2000
Rilmenidine (p.o.) 1000 – 2000 mg
Weerasuriya et al., 1984
The table gives an overview of the effective hypotensive and sympathoinhibitory doses of the drugs for comparison with doses eliciting other effects. The aim was not to give a complete list of all studies in which hypotensive and sympathoinhibitory effects are described. 1 Several of the ED50 values are approximate values read from the dose – response curves in the references.
should be made responsible for the central sympathoinhibition caused by clonidine (Bousquet et al., 1984). This suggestion was supported by observations that clonidine and its chemical relatives bind to specific imidazoline-binding sites, in addition to a2-adrenoceptors, in the medulla oblong-
ata. The two new antihypertensive drugs rilmenidine and moxonidine were shown to have high affinity and moderate selectivity for imidazoline-binding sites, and their sympathoinhibitory effects were attributed to activation of these imidazoline receptors.
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
It turned out that the imidazoline-binding sites are heterogeneous. At least three groups of binding sites were classified. The medullary binding sites mediating the sympathoinhibitory effects of clonidine-like drugs were termed I1; their molecular identity has not been clarified yet. This review deals with the role of I1 imidazoline receptors in cardiovascular regulation. A second major binding site, I2 receptor or idazoxan-preferring receptor, is characterized by high affinity for idazoxan and low affinity for clonidine. I2-binding sites are widely distributed in the CNS and in peripheral tissues. Most I2-binding sites are located on the monoamine oxidase A and B enzymes in the outer membrane of the mitochondria, and binding to these sites allosterically modulates the enzymes. The pharmacology of I2 receptors has been reviewed by Parini et al. (1996) and Eglen et al. (1998) (see also Raddatz et al., 2000). A third binding site, recently named I3, was identified in pancreatic b cells; the activation of these sites increases insulin secretion. Imidazolines can directly interact with the ionconducting pore component (Kir6.2) of the ATP-sensitive K + channel, and this may be the major mechanism of the enhanced insulin secretion (Proks & Ashcroft, 1997; Rustenbeck et al., 1997; for a review, see Morgan et al., 1999). In 1984, Atlas and Burstein (1984a, 1984b) isolated a substance from calf brain that displaced radiolabelled clonidine from its binding sites. Accordingly, the compound was named clonidine-displacing substance. A search was started to identify the chemical identity of this putative endogenous ligand of the imidazoline receptor. In 1994, agmatine was identified as an endogenous ligand of the imidazoline binding sites (Li et al., 1994). It is, however, not the only clonidine-displacing substance (see, e.g., Parker et al., 1999). The pharmacology of the endogenous ligands of imidazoline receptors has been reviewed recently (Regunathan & Reis, 1996; Eglen et al., 1998; Reis & Regunathan, 2000).
3. Mode of action of clonidine-like drugs: two hypotheses Clonidine-like drugs cause sympathoinhibition, i.e., they reduce the release of sympathetic transmitter in peripheral tissues. As a result, vascular resistance, heart rate, force of cardiac contraction, and blood pressure decrease. Enhancement of vagal tone also contributes to the cardiac effects of the drugs. Table 1 gives examples of the cardiovascular effects in different species. Regarding the mechanism of the central sympathoinhibition elicited by clonidine-like drugs, two different hypotheses have been developed: the a2-adrenoceptor hypothesis and the imidazoline hypothesis. 3.1. The 2-adrenoceptor hypothesis According to the older a2-adrenoceptor hypothesis, a2-adrenoceptors mediate all effects of clonidine. Blood pressure reduction is due to activation of sympathoinhibitory a2-adrenoceptors in the medulla oblongata. The side
5
effects — the most disturbing of which are sedation and inhibition of saliva secretion — are also elicited by activation of a2-adrenoceptors. If this hypothesis is true, then it is difficult to separate the desired and unwanted effects of clonidine-like drugs. The a2-adrenoceptor hypothesis was the original explanation of the mode of action of clonidinelike drugs (for reviews, see Schmitt, 1977; Kobinger, 1978; Kobinger & Pichler, 1990; Van Zwieten et al., 1984), and this view still enjoys considerable support (for reviews, see Guyenet et al., 1995; Guyenet, 1997; Eglen et al., 1998). 3.2. The imidazoline hypothesis According to the more recent imidazoline hypothesis, imidazoline I1 receptors in the rostral ventrolateral medulla (RVLM) are important for the sympathoinhibitory action of clonidine, and the role of I1 receptors is particularly prominent in the case of rilmenidine and moxonidine. The side effects of clonidine — sedation and dry mouth — are mediated by a2-adrenoceptors. Rilmenidine and moxonidine cause only few a2-adrenoceptor-mediated side effects because they are selective for I1 receptors. If the imidazoline hypothesis is true, then I1-selective drugs, devoid of the typical side effects of other centrally acting antihypertensive drugs, could be widely used in antihypertensive therapy. The imidazoline mode of action is supported by several recent reviews (Ernsberger et al., 1995; Ernsberger & Haxhiu, 1997; Bousquet & Feldman, 1999; Head & Burke, 2000a). The following major arguments are used to support the imidazoline hypothesis. (1) a2-Adrenoceptor agonists without affinity for I1-binding sites do not cause sympathoinhibition when they are microinjected into the RVLM. In contrast, drugs devoid of affinity for a2-adrenoceptors, but with affinity for I1 receptors, lower blood pressure. (2) Antagonists possessing affinity for I1 receptors block the cardiovascular effects of I1 imidazoline agonists more effectively than pure a2-adrenoceptor antagonists do. (3) Selective I1 imidazoline agonists lower blood pressure without simultaneously eliciting a2-adrenoceptor-mediated effects. These arguments will be discussed in detail in Sections 8– 11.
4. Characterization of drugs used in imidazoline cardiovascular research in radioligand binding studies The chemical structures of important agonists and antagonists are shown in Fig. 1. Radioligand binding experiments played an essential role in the development of the imidazoline hypothesis. The affinities of key drugs for a2-adrenoceptors and I1-binding sites are given in Tables 2 and 3. Imidazoline I1-binding sites were identified and studied in a number of tissues, e.g., in the ventral medulla oblongata of humans, cows, and rabbits; in bovine and rat corpus striatum; in rat kidney; in the bovine adrenal medulla; in PC12 cells; and in human platelets. They were labeled by [3H]clonidine, [3H]para-aminoclonidine, [3H]para-iodoclonidine, or
6
Table 2 Affinities of agonists1 that are used in cardiovascular studies for a2-adrenoceptors2 and I1 imidazoline-binding sites3 Clonidine Radioligand
Species
Tissue
a2
I1
Rilmenidine
Moxonidine
a2
a2
I1
Guanabenz a2
I1
Guanfacine I1
a2
I1
a-Methyl-noradrenaline
Noradrenaline
a2
a2
I1
I1
Reference
2105
Ernsberger et al., 1987
120,000
Ernsberger et al., 1990
> 106
Ernsberger et al., 1993 Molderings et al., 1993 Czerwiec et al., 1996 Felsen et al., 1994 Piletz & Sletten, 1993 Piletz et al., 1996
Ki or IC50 (nM)4 Cattle
[3H]pAminoclonidine
Cattle
[3H]Clonidine [3H]Clonidine [3H]Clonidine [3H]Clonidine [125I]p-Iodoclonidine [125I]p-Iodoclonidine
Cattle Cattle Cattle Dog Humans Humans
[3H]Clonidine
Humans Humans Humans Human
3
[ H]Clonidine [3H]Clonidine
Humans Humans
[3]MK-912
[3H]Clonidine [3H]Clonidine (I1) [3H]Yohimbine (a2) [3H]p-Iodoclonidine [3H]Rilmenidine [3]MK-912
Humans Humans Humans Rabbit Rabbit
Ventrolateral medulla (I1) Cortex (a2) Ventrolateral medulla (l1) Cortex (a2) Ventrolateral medulla Adrenal medulla Striatum Prostata Platelet Platelet (I1) Transfected CHO cells (a2A) (a2B) (a2C) NRL (I1) Cortex (a2) NRL Ventrolateral medulla Transfected HEK 293 cells (a2A) (a2B) (a2C) Ventral medulla Brain
255 6
PC12 cells Brain Brain cortex (a2A/D) (a2B) Kidney (a2C)
77 > 106
1 5 4
1 15 220
180
6 84 590
75
1600 55 55
9 31 9
59
36 43 13 112
33
2 78 5000 0.7 0.7 4
13 10 16
11 7
122 89,000
360 >10,000 428 240
2
35 35
2 3 1
160
2500
19
16 8 5
191
97 163 5 122
135 105 412
> 10
22 > 10 593
>20,000
5
Bricca et al., 1989b
5
Bricca et al., 1989a Bricca et al., 1994
28 5
> 10 >20,000
5600
Jasper et al., 1998; Zhu et al., 1999 62 69 135 6
1585 1738 4677 6
6
15 371
Rat Rat Guinea pig
2505
6
4266 > 10,000 > 10,000 81
22 282 447
93 1380 3890 16
>20,000
11
2427
3631 2630 2884 49
>20,000
1995 2754 1349 22
1.3
2371 6
19 147 155
713 5950 2800
Bricca et al., 1993 Hamilton et al., 1991
1065 8
0.8
>20,000
0.8 2860 1950 7700
12 460 160
17
17
Separovic et al., 1996 King et al., 1992 Uhlen et al., 1995
18 1850 834
1
The term agonist refers to the interaction with a2-adrenoceptors.
2
In most of the studies, it was not determined which subtype of the a2-adrenoceptors is involved in the binding (a2A, a2A/D, a2B, or a2C). The a2A/D denomination refers to the species ortholog of the a2A-adrenoceptor found in rats, mice, and guinea pigs.
3
Catecholamine-insensitive binding sites. Since the I1 nomenclature was developed later, the binding sites are not called I1 in several publications.
4
Only IC50 values are reported in several publications, but they are close to Ki values because the radioligands were used at concentrations close to their dissociation constants.
5
Estimation of affinity for I1-binding sites is compromised by the presence of a2-adrenoceptors.
6
Possesses affinity for a2-adrenoceptors and I1-binding sites, but exact affinities were not determined.
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
[3H]pAminoclonidine
Table 3 Affinities of antagonists1 that are used in cardiovascular studies for a2-adrenoceptors2 and I1 imidazoline-binding sites3 Idazoxan Radioligand
Species
Tissue
a2
Efaroxan I1
a2
Yohimbine I1
a2
SK&F86466 I1
a2
Phentolamine I1
a2
I1
Reference
425
Ernsberger et al., 1987 Ernsberger et al., 1990
Ki or IC50 (nM)4 [3H]p-Aminoclonidine 3
[ H]p-Aminoclonidine 3
[3H]Clonidine [3H]Clonidine [3H]Clonidine [3H]Clonidine (I1) [3H]Yohimbine (a2) [3H]p-Iodoclonidine [3H]RX821002 [3H]RX821002
[3H]Rilmenidine [3]MK-912
[3H]Yohimbine or [3H]rauwolscine
Cattle Cattle Cattle Dog Humans Humans
Humans Humans Rabbit Rabbit Rat Rat Humans Rat Rat Rat Guinea pig
Humans Rat Opossum
Ventrolateral medulla (I1) Cortex (a2) Ventrolateral medulla (I1) Cortex (a2) Ventrolateral medulla Adrenal medulla Prostata Platelet Platelet (I1) Transfected CHO cells (a2A) (a2B) (a2C) NRL Ventrolateral medulla Ventrolateral medulla Brain PC12 cells RINm5F cells (a2A/D) Platelet (a2A) Cerebral cortex (a2A/D) Lung (a2B) Brain Brain cortex (a2A/D) (a2B) Kidney (a2C) HT29 cells (a2A) Lung (a2B) OK cell (a2C)
32005 179
11
186
93,000
61
84 19
0.11
103 5 1020 1255
12 20 235
20 120
52 52
441,000 441,000
3055 1027
Bricca et al., 1989a Bricca et al., 1994 Bricca et al., 1993 Hamilton et al., 1991
1600 239
10 10 178
51 34 86
33 152 1216 1.5
11,500 568 22
> 20,000 21,810
6 180
2 10 5 2 17 10 2 145 20 2 5 0.6
11
Haxhiu et al., 1994 Molderings et al., 1993 Felsen et al., 1994 Piletz & Sletten, 1993 Piletz et al., 1996
148
5 4 1 4
2570 104 3 30 7 41 176 6 0.6 1 0.2
10 13 11 11
2 2 3
7 24 12 27 9
6 4 10
Separovic et al., 1996 Remaury & Paris, 1992 Renouard et al., 1994
King et al., 1992 Uhlen et al., 1995
Blaxall et al., 1991
1
The term antagonist refers to the interaction with a2-adreceptors.
2
In most of the studies, it was not determined which subtype of the a2-adrenoceptors is involved in the binding (a2A, a2A/D, a2B, or a2C). The a2A/D denomination refers to the species ortholog of the a2A-adrenoceptor found in rats, mice, and guinea pigs.
3
Catecholamine-insensitive-binding sites. Since the I1 nomenclature was developed later, the binding sites are not called I1 in several publications.
4
Only IC50 values are reported in several publications, but they are close to Ki values because the radioligands were used at concentrations close to their dissociation constants.
5
Estimation of affinity for I1-binding sites is compromised by the presence of a2-adrenoceptors.
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
[ H]Clonidine [3H]Clonidine [3H]Clonidine [125I]p-Iodoclonidine [125I]p-Iodoclonidine
Cattle
7
8
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
[3H]rilmenidine. The a2-adrenoceptors were labelled by the same ligands, and in some studies by [3H]yohimbine, [3H]MK-912, or [3H]RX821002 (methoxy-idazoxan). There are several contradictions between the data, and it is especially difficult to identify compounds that are selective for I1 imidazoline receptors. In a few cases, affinities for identified subtypes of a2-adrenoceptors are available. They are given for sake of completeness, but are not discussed systemically. 4.1. Agonists 4.1.1. Selective 2-adrenoceptor agonists In most studies, the catecholamines noradrenaline and a-methylnoradrenaline possess appreciable affinity for a2-adrenoceptors and very low affinity for I1-binding sites (Table 2). In fact, the low affinity of catecholamines for I1-binding sites belongs to the definition of these sites. Guanabenz and guanfacine possess high affinity for a2-adrenoceptors (except in the study of Hamilton et al., 1991). In the study of Ernsberger et al. (1993), guanabenz and guanfacine possess very low affinity for I1-binding sites, and accordingly, these guanidine compounds should be selective a2-adrenoceptor agonists. However, in several other studies, a fairly high affinity for I1-binding sites was found (Bricca et al., 1989b; Hamilton et al., 1991; Piletz et al., 1996). UK14304 (not shown in Table 2) has high affinity for a2-adrenoceptors (Ki = 1– 2 nM) (Piletz et al., 1991; Bricca et al., 1993). Its affinity for I1-binding sites was very low in the rabbit brain stem (Ki = 1531 nM) (Bricca et al., 1993), but high, comparable with its affinity for a2-adrenoceptors, in human platelets (Ki = 2 nM) (Piletz et al., 1991). It recently was shown that medetomidine (not shown in Table 2) possesses high affinity for a2-adrenoceptors (Ki = 3 nM), but low affinity for I1-binding sites (Ki = 385 –14600 nM) (Piletz & Sletten, 1993; Ernsberger et al., 1997). Thus, the catecholamines noradrenaline and a-methylnoradrenaline are undoubtedly selective for a2-adrenoceptors. Their use in vivo, however, is limited. In addition to a2-adrenoceptors, they activate, of course, other adrenoceptors. Also, their pharmacokinetic behaviour (e.g., failure to pass the blood –brain barrier; uptake by several neuronal and extraneuronal transporters) renders them unsuitable for many kinds of experiments. The radioligand binding data on UK14304, guanabenz, and guanfacine are far from being unequivocal. At the moment, medetomidine seems to be the agonist of choice if an a2-selective agonist has to be used. 4.1.2. Selective I1 imidazoline receptor agonists The key drug for developing the imidazoline hypothesis was clonidine. Its affinity for a2-adrenoceptors is in the low nanomolar range in most experiments. It also possesses affinity for I1-binding sites. In some studies, this affinity is slightly higher; in other studies, it is lower than its affinity
for a2-adrenoceptors. Altogether, clonidine is not a selective I1 agonist. Rilmenidine possesses affinity for a2-adrenoceptors (except in the study of Hamilton et al., 1991) and I1-binding sites. Only the data of Ernsberger et al. (1993) and Hamilton et al. (1991) indicate selectivity for I1-binding sites. In addition to rilmenidine, moxonidine is claimed to be selective for I1 imidazoline-binding sites. It is clear from the data in Table 2 that moxonidine possesses appreciable affinity for a2-adrenoceptors. In two studies, the affinity of moxonidine was somewhat higher for I1-binding sites than for a2-adrenoceptors (Ernsberger et al., 1993; Piletz et al., 1996). However, in the studies of Bricca et al. (1993, 1994), moxonidine had practically no affinity for I1 imidazoline-binding sites. It is remarkable that the data from different laboratories on one of the two supposedly selective I1 agonists differ so dramatically. Rilmenidine and moxonidine had rather high affinity for human a2A-, a2B-, and a2C-adrenoceptors expressed in Chinese hamster ovary (CHO) cells (Piletz et al., 1996). The affinities of these drugs for the human a2-adrenoceptor subtypes were much lower in transfected human embryonic kidney (HEK) 293 cells (Jasper et al., 1998; Zhu et al., 1999), and the reason for the discrepancy is not known. The low affinity of rilmenidine and moxonidine for the a2A/D-, a2B-, and a2C-adrenoceptors of the guinea pig (Uhlen et al., 1995) is also remarkable. King et al. (1992) studied the binding of [3H]rilmenidine to membranes prepared from rat cerebral cortex and determined two binding sites. Rilmenidine was bound with high affinity (Kd = 17 nM; Ki = 6 nM) to a2-adrenoceptors. The other, low-affinity binding site showed some similarity with the idazoxan or I2-binding sites observed in many studies. Such binding sites are not thought to participate in the cardiovascular effects of clonidine-like drugs. Association of rilmenidine with I1-binding sites was not observed. In a corresponding paper (King et al., 1995), the distribution of [3H]rilmenidine-binding sites in the brain was studied. [ 3H]Rilmenidine labelled a2-adrenoceptors throughout the brain, notably also in the locus coeruleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus, and the RVLM — areas that may be involved in the central cardiovascular effects and side effects of rilmenidine. [3H]Rilmenidine also labelled the I2-like binding sites, already seen in the cortex (see above), in many brain regions. Notably, the I2-like binding was not preferential in regions of the medulla oblongata thought to be involved in the cardiovascular effects of rilmenidine. It is altogether surprising that using one of the two ‘‘selective’’ I1 ligands, rilmenidine, no specific I1 binding was observed in the RVLM or elsewhere in the brain. Radioligand binding studies unequivocally show that rilmenidine and moxonidine possess affinity for a2-adrenoceptors. In vitro functional studies indicate that moxonidine is an agonist at a2A-adrenoceptors (notably also at the human a2A-adrenoceptor). Moxonidine inhibits electrically evoked noradrenaline release in the rabbit pulmonary artery
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
and human atrium (Molderings et al., 1991, 2000), stimulates incorporation of [35S]GTPgS in HEK 293 cells transfected with the human a2A-adrenoceptor (Zhu et al., 1999), and elicits contraction in the dog saphenous vein (Zhu et al., 1999). However, moxonidine did not activate human a2B- and a2C-adrenoceptors expressed in HEK 293 cells (Zhu et al., 1999). The behaviour of rilmenidine in functional studies is less clear. Thus, rilmenidine inhibits electrically evoked noradrenaline release in the rabbit pulmonary artery (Verbeuren et al., 1986; Molderings et al., 2000), elicits contraction in the dog saphenous vein (Verbeuren et al., 1986; Marsault et al., 1996), and stimulates incorporation of [35S]GTPgS in HEK 293 cells transfected with the mouse a2A/D-adrenoceptor, indicating agonist activity at a2A- and a2A/D-adrenoceptors in rabbits, dogs, and mice. In contrast, the a2A-adrenoceptors mediating inhibition of electrically evoked contractions of the pig tail artery and urinary bladder (Ali et al., 1998) and of electrically evoked noradrenaline release from sympathetic nerve endings of the human heart were not activated by rilmenidine (Molderings et al., 2000). In these preparations, rilmenidine behaved as an antagonist of a2A-adrenoceptors. In HEK 293 cells transfected with human a2A-, a2B-, and a2C-adrenoceptors, rilmenidine displayed affinity for the a2-binding sites without stimulating incorporation of [35S]GTPgS (Jasper et al., 1998). This pattern of effect also supports an antagonist character of rilmenidine at human a2-adrenoceptors. The pharmacological profile of rilmenidine is made even more complex by the observation that rilmenidine is an antagonist of a1-adrenoceptors in a rat vascular preparation (Cario-Toumaniantz et al., 1998). Two compounds with high selectivity for I1 imidazolinebinding sites were recently synthesized: 2-endo-amino3-exo-isopropylbicyclo[2.2.1]heptane (AGN192403) (Munk et al., 1996) and (-)-(5-[2-methyl-phenoxy-methyl]-1,3oxazolin-2-yl)amine (S23230) (Barrot et al., 2000). AGN192403 possesses high affinity for the I1-binding sites of the bovine ventrolateral medulla labelled with [3H]clonidine (Ki = 42 nM). The affinities for the human a2A-, rat a2B-, and human a2C-adrenoceptors expressed in CHO cells were much lower (Ki > 20,000 nM for all three receptor subtypes). S23230 has high affinity for the I1-binding sites in bovine adrenals labelled with [3H]clonidine (Ki = 6 nM). The affinity of this drug for a2-adrenoceptors in the bovine cerebral cortex is much lower (Ki = 7400 nM). Summarizing the data on the I1 binding profile of drugs, it must be said that no agonists exist with unequivocally proven selectivity for I1-binding sites. A selectivity ratio of about 30 was shown for rilmenidine and moxonidine in only one study (Ernsberger et al., 1993). In several other papers, only a marginal selectivity for I1-binding sites (selectivity ratio = 3 –4) (Piletz et al., 1996) or low or very low affinity for I1-binding sites was reported. Moxonidine proved to be an agonist of a2-adrenoceptors in all species studied. In contrast, rilmenidine was an agonist of a2-adrenoceptors in some species, but an antagonist in others, including humans.
9
AGN192403 behaves in binding studies as a selective ligand. It seems, however, that this compound has no cardiovascular effects (see Sections 7, 8.3, and 8.4). It is not known whether the other selective I1 ligand, S23230, elicits cardiovascular effects expected for an I1 imidazoline receptor agonist or antagonist. 4.2. Antagonists The affinities of five antagonists used in imidazoline cardiovascular research are shown in Table 3. Three (idazoxan, efaroxan, and phentolamine) possess an imidazoline ring, whereas two (yohimbine and 6-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benzazepine HCl [SK&F86466]) have different chemical structures. It is clear that all five drugs are rather potent a2-adrenoceptor antagonists. 4.2.1. Selective 2-adrenoceptor antagonists It was repeatedly found that the affinity of yohimbine and SK&F86466 for I1-binding sites is much lower than their affinity for a2-adrenoceptors. Therefore, these drugs can be considered as a2-selective antagonists. In some cardiovascular studies, the imidazoline derivative methoxy-idazoxan (also called RX821002; not shown in Table 3) was used as an a2-selective antagonist. Methoxy-idazoxan is a high-affinity a2 antagonist (Ki or Kd = 2 –10 nM) (Langin et al., 1989; Trendelenburg et al., 1996; Ernsberger et al., 1997). Since its affinity for I1-binding sites is much lower (Ki = 400 nM) (Ernsberger et al., 1997), this compound can also be considered a2-selective. 4.2.2. Selective I1 imidazoline receptor antagonists The three antagonists with an imidazoline structure — idazoxan, efaroxan, and phentolamine — possess, in addition to their affinity for a2-adrenoceptors, affinity for I1-binding sites. The affinity of idazoxan for I1-binding sites is generally lower than its affinity for a2-adrenoceptors (except in the study of Hamilton et al. [1991]). Phentolamine does not differentiate between a2-adrenoceptors and I1-binding sites. The behaviour of efaroxan is not clear. According to Haxhiu et al. (1994), it is highly selective for I1-binding sites (173-fold). This selectivity is due to the very high affinity of efaroxan for the I1-binding sites (Ki = 0.11 nM). However, much lower I1 affinity values were found by Piletz and Sletten (1993), Piletz et al. (1996), and Separovic et al. (1996). Summarizing the state of knowledge on antagonists, it can be concluded that yohimbine, SK&F86466, and methoxyidazoxan are reasonably selective for a2-adrenoceptors. In contrast, the evidence for the I1 selectivity of efaroxan is not convincing. Thus, we do not possess I1-selective antagonists. At the end of the section describing the drugs used in imidazoline cardiovascular research (Section 4), it must be critically remarked that only very few reliable drugs are available for use in in vivo experiments to determine involvement of I1 imidazoline receptors or a2-adrenocep-
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B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
tors. Thus, as pointed out above, no selective I1 imidazoline receptor agonists or antagonists have been identified. Moreover, very few binding data have been obtained on tissues of rats and rabbits, despite the fact that almost all in vivo experiments have been carried out in these species. The results of experiments carried out on rats and rabbits are regularly interpreted using the binding data of rilmenidine, moxonidine, idazoxan, and efaroxan determined on bovine tissues. Such extrapolation of data to other species is questionable because the receptors can markedly differ in various species. It is known, e.g., that the susceptibility of the a2A-adrenoceptor to agonists and antagonists differs markedly in rats, rabbits, and humans (see Trendelenburg et al., 1996; Molderings et al., 2000). It would be necessary to determine the binding and functional characteristics of the key imidazoline and a2 drugs at I1 imidazoline receptors and a2A-, a2B-, and a2C-adrenoceptors. Within one species, binding and functional studies on I1 imidazoline receptors and a2A-, a2B-, and a2C-adrenoceptors should be carried out under identical conditions. Without such comprehensive sets of data for rats, rabbits, and humans, the meaning of many in vivo observations remains unclear.
5. The central nervous site of action of clonidine-like drugs Clonidine-like drugs are often classified as ‘‘centrally acting antihypertensive drugs,’’ reflecting the view that they lower sympathetic nerve firing by a primary action in the CNS. An action in the medulla oblongata is supported, e.g., by the observations that clonidine-like drugs lower sympathetic tone after administration of low doses into the vertebral artery (e.g., Timmermans et al., 1981; van Zwieten et al., 1986) or into the cisterna cerebellomedullaris (e.g., Kobinger, 1967; Chan et al., 1996; Bock et al., 1999). In this section, the mechanism of action within the CNS will be discussed. Peripheral mechanisms can also contribute to the reduction of sympathetic tone. It will be discussed in detail in Section 12 that clonidine and its relatives can lower the amount of transmitter released per action potential from postganglionic sympathetic neurons by activating inhibitory presynaptic receptors. 5.1. Action in the rostral ventrolateral medulla The RVLM (it is alternatively called subretrofacial nucleus, ventrolateral medullary pressor area, or nucleus reticularis lateralis [NRL]1) is the final relay station of the baroreceptor reflex pathway in the brain. It contains the perikarya of sympathoexcitatory neurons projecting to sym1
The nomenclature is not uniform. Most often, NRL is used as a synonym for RVLM. In some publications, however, NRL refers to a nucleus caudal of the RVLM. In this review, we will keep the RVLM and NRL denominations, as they were used in the original references.
pathetic preganglionic neurons in the intermediolateral column of the spinal cord. Many RVLM neurons synthesize adrenaline, and these neurons constitute the C1 adrenergic cell group. The RVLM is considered as the primary site of action of clonidine-like drugs (for reviews, see Dampney, 1994; Ernsberger et al., 1995; Guyenet et al., 1995; Reis, 1996; Sun, 1996; Guyenet, 1997). The high sensitivity of the ventral surface of the medulla oblongata to locally applied clonidine was shown by Bousquet and Guertzenstein in 1973. The opinion that clonidine-like drugs lower sympathetic tone by a principal action in the RVLM is based on a set of observations made first by Punnen et al. (1987). They showed that clonidine lowers blood pressure in rats when it is microinjected into the RVLM, and this effect is prevented by microinjection of the imidazoline a2-adrenoceptor antagonist idazoxan into the RVLM. Idazoxan also prevented the hypotensive effect of systemically administered clonidine. The effects of systemically administered clonidine were identical in animals decerebrated at the level of the midbrain, indicating that structures lying rostrally, most importantly the hypothalamus, are not necessary for the hypotensive effect. Finally, Punnen et al. (1987) excluded a role of the nucleus tractus solitarii (NTS) in the sympathoinhibition produced by systemically administered clonidine by showing that inactivation of the NTS by lidocaine does not change the hypotensive action of clonidine. The observations of Punnen et al. (1987) have since been repeated by several groups. In addition, it was shown that systemically and locally microapplied clonidine inhibits the activity of sympathoexcitatory RVLM neurons (Sun & Guyenet, 1986; Haselton & Guyenet, 1989; Clement & McCall, 1991; Allen & Guyenet, 1993). A role of the RVLM in the sympathoinhibition is also supported by the observation that systemically administered clonidine and rilmenidine suppress the expression of c-fos mRNA and Fos protein in this nucleus (Li & Dampney, 1995; El-Mas & Abdel-Rahman, 2000). Importantly, it has been shown that the more recently synthesized drugs rilmenidine and moxonidine also elicit sympathoinhibition if they are microinjected into the RVLM (Feldman et al., 1990; Gomez et al., 1991; Haxhiu et al., 1994; Head & Burke, 1998, 2000b; Tolentino-Silva et al., 2000; Mayorov & Head, 2001). One group observed that clonidine caused hypotension and bradycardia, not only when it was microinjected into the RVLM, but also when it was administered into the caudal ventrolateral medulla in rats (McAuley et al., 1989). A vasodepression was also seen after injection of clonidine into the gigantocellular depressor area in the medulla of the rat (Aicher & Drake, 1999). In the cat, the medullary site of action of clonidine is lying caudal from the RVLM, in the rostral part of the NRL (Gatti et al., 1988). 5.2. Action in the nucleus tractus solitarii The NTS is the region in the medulla oblongata in which axons of baroreceptor and chemoreceptor afferents termi-
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
nate. The catecholamines noradrenaline and a-methylnoradrenaline cause hypotension and bradycardia when they are microinjected into the NTS, and the effects can be blocked by a2-adrenoceptor antagonists (e.g., Zandberg et al., 1979; Kubo & Misu, 1981; Head & Burke, 1998). The NTS was long considered to be the major site of action of clonidine-like drugs in the medulla (for a review, see Kobinger & Pichler, 1990). However, microinjection of clonidine and rilmenidine into the NTS elicited no or only a moderate hypotension (Zandberg et al., 1979; Gomez et al., 1991). This fact and the observation that lesion of the NTS does not greatly change the hypotensive effect of systemically administered clonidine led to the opinion that the NTS is not the primary medullary site of action of clonidine-like drugs (for a review, see Ernsberger et al., 1995). It was shown recently, however, that rilmenidine lowers blood pressure, heart rate, and sympathetic nerve firing rate when it is microinjected into the NTS of rabbits (Head & Burke, 1998). It is also a recent observation that neuronal activity in the NTS, measured as expression of c-fos mRNA and Fos protein, was inhibited by systemically administered clonidine and rilmenidine (Li & Dampney, 1995; El-Mas & Abdel-Rahman, 2000). 5.3. Action in the spinal cord Clonidine and several other a2-adrenoceptor agonists were also administered intrathecally at thoracal and lumbar levels. Clonidine (Connor et al., 1981; Kubo et al., 1987; Solomon et al., 1989; Eisenach & Tong, 1991; Filos et al., 1994), guanabenz (Kubo et al., 1988), B-HT920 (Kubo et al., 1987), and dexmedetomidine (Eisenach et al., 1994) lowered blood pressure and, under certain experimental conditions, also heart rate. The effects were antagonized by intrathecally injected a2-adrenoceptor antagonists (Connor et al., 1981; Kubo et al., 1987, 1988; Solomon et al., 1989; Eisenach & Tong, 1991). In several of these studies, it was verified that the effects of the a2-adrenoceptor agonists were due to a direct spinal action and not to actions elsewhere, after diffusion of the drugs into the systemic circulation. Spinal sympathoinhibition occurs also after intracerebroventricular administration of rilmenidine (Sannajust et al., 1992a). Moreover, sympathoinhibition at the level of the spinal cord was suggested to play a role after systemic administration of clonidine in spontaneously hypertensive rats (SHR) (Tibiric¸a et al., 1992). Adrenaline hyperpolarizes some of the sympathetic preganglionic neurons in isolated spinal cord slices, an effect that is sensitive to a2-adrenoceptor blockade (Miyazaki et al., 1989). Thus, activation of a2-adrenoceptors in the spinal cord causes sympathoinhibition. It is not clear, however, whether the spinal effect contributes to the overall sympathoinhibition caused by systemically administered clonidine-like drugs. In summary, clonidine-like drugs lower sympathetic tone and blood pressure when they are locally applied into the
11
RVLM, NTS, and spinal cord. The involvement of the RVLM in the sympathoinhibition produced by systemically applied drugs has been demonstrated, whereas the involvement of the NTS and spinal cord cannot be excluded. Of course, the effectiveness of clonidine-like drugs in the CNS does not exclude simultaneous peripheral actions on sympathetic neurons. 6. Characterization of A2-adrenoceptors and I1 imidazoline-binding sites 6.1. 2-Adrenoceptors 6.1.1. Molecular identity, second messenger mechanisms Three genetically defined subtypes of the a2-adrenoceptor have been identified: a2A, a2B, and a2C (Bylund et al., 1994; Docherty, 1998). Due to minor differences in the amino acid sequence, the a2A-adrenoceptors of humans, rabbits, and pigs differ pharmacologically from the a2A-adrenoceptors of rats, mice, guinea pigs, and cows. The ortholog receptor found in rats, mice, guinea pigs, and cows is denominated in this review as the a2A/D-adrenoceptor. The intracellular signal transmission mechanisms of the a2-adrenoceptors are well-characterized. These receptors typically couple to Gi- and Go-proteins, and this can lead to inhibition of adenylate cyclase and to changes in the open probability of K + channels and voltage-dependent Ca2 + channels (e.g., Summers & McMartin, 1993; Hein & Kobilka, 1995; Docherty, 1998; Goldstein, 1998). 6.1.2. Distribution in cardiovascular regulatory centres The distribution of the mRNA and the receptor protein of a2-adrenoceptor subtypes in the pons, medulla, and spinal cord is well-characterized. mRNA for the a2A/D-adrenoceptor predominated in regions relevant for cardiovascular regulation (Nicholas et al., 1993, 1996). Thus, a2A/D-mRNA was observed in the locus coeruleus, A5 pontine nucleus, RVLM, dorsal motor nucleus of the vagus, nucleus ambiguus, and intermediolateral column of the spinal cord. The distribution of the appropriate receptor protein, visualized immunohistochemically, is very similar. Labelling was seen in the locus coeruleus, A5 pontine nucleus, NTS, dorsal motor nucleus of the vagus, and the RVLM (Talley et al., 1996). The great majority of the tyrosine hydroxylase positive cells in the locus coeruleus and the C1 cell group of the RVLM contained the a2A/D-receptor protein (Rosin et al., 1993). In addition, many a2A/D-receptors were identified as presynaptic heteroreceptors located on terminals of non-adrenergic afferent axons in the vicinity of RVLM/C1 cells (Milner et al., 1999). The distribution of the a2C-adrenoceptor protein is similar to the distribution of the a2A/D-adrenoceptor, i.e., it is found in the locus coeruleus, dorsal motor nucleus of the vagus, the RVLM, and intermediolateral column of the spinal cord (Rosin et al., 1996).
12
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
In summary, the molecular structure and second messenger mechanisms of the a2-adrenoceptors are well-characterized. The a2-adrenoceptors are appropriately located in the medulla to influence the function of cardiovascular regulatory centres. 6.2. I1 imidazoline receptors 6.2.1. Molecular identity, second messenger mechanisms The molecular structure of the I1 imidazoline receptor has not been identified. Two antibodies have played an important role in the search for the receptor protein. One antibody was raised against an imidazoline receptor protein (IRP antibody) that was affinity purified on columns labelled with paraaminoclonidine or idazoxan (Wang et al., 1993). Another antibody was raised as a secondary antibody (anti-idiotypic antibody) against a primary antibody that was directed against idazoxan (Bennai et al., 1996). Solubilized proteins from extracts of different tissues were immunolabelled using one of these antibodies. Immunoreactive proteins of different sizes were detected in the rat brain (45 kDa) (Escriba´ et al., 1995), human brain (43 kDa) (Bennai et al., 1996), bovine adrenomedullary cells (31 kDa, 70 kDa, 90 kDa) (Wang et al., 1993; Ivanov et al., 1998a), human platelets (33 kDa, 95 kDa) (Ivanov et al., 1998a), and MEG-01 cells (33 kDa, 85 kDa) (Ivanov et al., 1998a). An 85-kDa protein was recognized by both antibodies in human and rat brain and PC12 cells (Ivanov et al., 1998c). Since the density of this latter protein in different brain regions correlated with the density of I1-binding sites, it was considered to be a candidate for the full-length I1 IRP. It was suggested that the smaller proteins observed in previous studies were proteolytic fragments of this 85-kDa protein. The IRP antibody and the anti-idiotypic antibody were also used to isolate a cDNA clone from a human hippocampal cDNA expression library. In the first step, a 1.8-kbp cDNA clone (5A-1B) was sequenced, corresponding to a 66-kDa protein, but this was apparently not the full-length cDNA (Ivanov et al., 1998b). Recently, the full-length cDNA was determined to be a 5.1-kbp molecule, corresponding to a 1504 amino acid protein (Piletz et al., 2000). The latter cDNA was named imidazoline receptor-antiseraselected (IRAS) cDNA and the protein product, IRAS-1 protein. Transfection of the IRAS cDNA into CHO cells increased the density of I1-binding sites in these cells, and rilmenidine and moxonidine had high affinity, Ki = 10 and 14 nM, respectively, for these binding sites (Piletz et al., 2000). The sequence analysis of the IRAS cDNA indicated that the product is not a G-protein-coupled receptor. Sequence homologies were found with chromogranins, cytokine receptors, cytochrome P450 enzymes, and the ryanodine receptor (Piletz et al. 2000). Thus, IRAS-1 protein seems to be an I1 imidazoline-binding site. It is not clear, however, whether it is a receptor capable of inducing cellular changes attributed, until now, to I1 receptors.
Only a limited number of observations are available on the intracellular transmission mechanisms of the putative I1 imidazoline receptor. The I1 imidazoline-binding sites are enriched in cellular membranes, and the binding of paraiodoclonidine is inhibited by Gpp(NH)p. These observations led to the suggestion that they represent plasma membrane-bound G-protein-coupled receptors (Ernsberger & Shen, 1997). Imidazoline agonists, like clonidine, have no effect on basal and forskolin-stimulated cyclic AMP levels, do not activate phosphatidylinositol-sensitive phospholipase C, and affect the influx of Ca2 + ions into cells only at very high concentrations (Regunathan et al., 1990, 1991; Liedtke et al., 1993). In PC12 cells, moxonidine stimulates, at the rather high concentrations of 0.1 –1 mM, phosphatidylcholine-sensitive phospholipase C, releasing diacylglyceride and choline phosphate (Separovic et al., 1996, 1997). Moreover, the hypotensive effect of moxonidine microinjected into the RVLM of rats is prevented by pretreatment with an inhibitor of phosphatidylcholine-sensitive phospholipase C (Separovic et al., 1997). The identified second messenger mechanisms do not seem to fit the properties of the IRAS-1 protein, which was identified as an I1 imidazoline-binding site (see above). 6.2.2. Distribution in cardiovascular regulatory centres The distribution of I1 imidazoline-binding sites in the medulla oblongata was studied with autoradiography using [125I]para-iodoclonidine as label (Ernsberger et al., 1995). The RVLM was strongly labelled, whereas only weak labelling was seen in the NTS. Surprisingly, a rather strong diffuse labelling was seen on the entire cross-sectional area of the rostral medulla. The binding of [125I]para-iodoclonidine in the bovine RVLM was shown to be located on (pre)synaptic membranes (Heemskerk et al., 1998). In one immunohistochemical study (Ruggiero et al., 1998), the IRP antibody (Wang et al., 1993; see Section 6.2.1.) was used to identify IRPs. The IRP antibody recognizes both I1- and I2-binding sites. Labelling was seen in the NTS, dorsal motor nucleus of the vagus, nucleus ambiguus, RVLM, and intermediolateral column of the thoracic spinal cord. Labelling of these regions was not preferential, the IRP antibody labelled many structures throughout the brain. In situ hybridization and quantitative northern blot analysis revealed an mRNA distribution pattern in the CNS that does not strongly correlate with the known distribution of I1-binding sites. High concentrations of 5A-1B mRNA (the truncated form of IRAS) were seen, e.g., in the pituitary, cerebellum, cortex, hippocampus, and extrapyramidal motor nuclei, and lower concentrations in the medulla oblongata (Ivanov et al., 1998b). In summary, the molecular identity of the I1 imidazoline receptor has not been clarified. Until now, only one protein (IRAS-1) was identified that could be an I1 imidazolinebinding site. The distribution of this protein does not fit the expected preferential localization of I1 imidazoline receptors in the medulla oblongata. Knowing the amino acid sequence
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
of IRAS-1, it is difficult to assign a function to this protein. It is also not obvious how IRAS-1 could lead to the generation of the second messengers ascribed to the putative I1 imidazoline receptor.
7. Action of clonidine-like drugs on cardiovascular regulatory nuclei in vitro The effect of clonidine-like drugs on RVLM neurons in rat brain slices has been analyzed in a few studies. In one study, clonidine lowered the firing rate of RVLM pacemaker neurons by hyperpolarizing the cells (Sun & Reis, 1995). Since the hyperpolarization was blocked by bicuculline and tetrodotoxin, it was suggested that clonidine acted indirectly, by enhancing the synaptic release of g-aminobutyric acid (GABA). Noradrenaline (in the presence of
13
prazosin) and UK14304 can also hyperpolarize and inhibit RVLM neurons. These effects are sensitive to a2-adrenoceptor blockade by yohimbine (Hayar et al., 1997). In several experiments, the effects of imidazoline and catecholamine drugs on identified spinally projecting sympathoexcitatory RVLM neurons were studied (Li et al., 1995, 1998; Hayar & Guyenet, 1999, 2000). The spinally projecting neurons were identified with the help of a retrogradely transported fluorescent label that had been injected into the spinal cord in vivo several days before killing the animals for brain slice preparation. a-Methylnoradrenaline and moxonidine inhibited the spontaneous firing of bulbospinal RVLM neurons, whereas the I1 imidazoline-binding site ligand AGN192403 was without effect (Li et al., 1995; Hayar & Guyenet, 2000; see Fig. 2). Noradrenaline and moxonidine also inhibited glutamatergic and GABAergic synaptic transmission between afferent axons and RVLM
Fig. 2. Interactions between moxonidine (Mox), AGN192403 (AGN), and SK&F86466 (SKF) in rat brain slices. Bulbospinal presympathetic neurons in the RVLM were studied with the patch – clamp technique. A: Moxonidine (10 mM) lowers the firing frequency of a neuron, and the effect is fully reversed by SK&F86466 (10 mM). B: In the presence of SK&F86466 (10 mM), moxonidine (10 mM) and AGN192403 (10 mM) do not affect the firing rate of RVLM neurons, whereas the GABAB receptor agonist baclofen (10 mM) causes strong inhibition. C: Moxonidine (10 mM) elicits an outward current in RVLM neurons, and the effect is completely prevented by SK&F86466 (10 mM). D: Moxonidine (10 mM) inhibits electrical stimulation-evoked excitatory postsynaptic currents (EPSCs) in an RVLM neuron, and the effect is antagonized by SK&F86466 (10 mM). The figure demonstrates that moxonidine inhibits sympathoexcitatory neurons in the RVLM. The antagonism by SK&F86466 indicates involvement of a2-adrenoceptors; the I1 imidazoline receptor ligand AGN192403 is without effect. Moxonidine suppressed RVLM neurons most likely by evoking an outward current (i.e., hyperpolarization) and by inhibiting the excitatory input of the neurons. Adapted from Hayar and Guyenet (2000), with kind permission of the authors.
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neurons by a presynaptic mechanism (Hayar & Guyenet, 1999, 2000; see Fig. 2). The effects of a-methylnoradrenaline, noradrenaline, and moxonidine were antagonized by the selective a2-adrenoceptor antagonists methoxy-idazoxan and SK&F86466 (see Fig. 2). In addition, noradrenaline and UK14304 inhibited high voltage-activated Ca2 + channels in RVLM neurons (Li et al., 1998). The somadendritic and presynaptic a2-adrenoceptor-mediated effects in the RVLM agree well with the localization of a2-adrenoceptors, which were observed in the soma of RVLM neurons and in the vicinity of RVLM neurons on axon terminals (see Section 6.1.2). In summary, catecholamine and imidazoline agonists inhibit the activity of bulbospinal sympathoexcitatory RVLM neurons. Direct inhibition of RVLM neurons, inhibition of their excitatory input, and enhancement of their inhibitory input may all contribute to the inhibition of activity. The involvement of a2-adrenoceptors in these effects is verified. In contrast, there was no indication for an involvement of I1 imidazoline receptors.
8. Correlation between sympathoinhibition and affinity for I1 imidazoline-binding sites and A2-adrenoceptors The observation of Bousquet et al. (1984) that drugs possessing an imidazoline structure, but devoid of affinity for a2-adrenoceptors, lower blood pressure when they are injected into the NRL (used as a synonym of RVLM) was the starting point of the imidazoline hypothesis. Since then, it has been claimed repeatedly that the sympathoinhibitory action of drugs injected into the RVLM depends on their affinity for imidazoline receptors. It was even assumed that selective a2-adrenoceptor agonists do not cause sympathoinhibition. The experimental evidence behind this important component of the imidazoline hypothesis is reviewed in Sections 8.1 –8.4. 8.1. Cats The imidazoline hypothesis emerged from the work of Bousquet et al. (1984) in cats. It was shown that three imidazolines — clonidine (an a2-adrenoceptor agonist), cirazoline (an a1-adrenoceptor agonist and an a2 antagonist), and St-587 (an a1-adrenoceptor agonist) — lowered blood pressure when they were microinjected into the NRL. In contrast, the a2-adrenoceptor agonist a-methylnoradrenaline had no effect. It was inferred that affinity for a2-adrenoceptors was not important for the sympathoinhibition. Instead, an imidazoline chemical structure was necessary to elicit hypotension. It recently was shown that cirazoline, at least in the rabbit, is a strong partial agonist at a2-adrenoceptors (EC50 = 40 nM; Emax = 80%) (Gaiser et al., 1999). If this is also true in the cat, then there is only one drug in the experiments of Bousquet et al. (1984) that lowered blood pressure without being an a2-adrenoceptor agonist, St-587. Remarkably,
St-587 did not lower blood pressure when injected into the vertebral artery of cats (De Jonge et al., 1981b), although many clonidine-like drugs lower blood pressure after such an application (e.g., van Zwieten et al., 1986). The behaviour of guanfacine, an a2-adrenoceptor agonist displaying low affinity for I1 sites in the majority of radioligand binding studies (see Table 2), fits the predictions of the imidazoline hypothesis. Thus, guanfacine did not change the blood pressure and heart rate of cats when it was topically administered to the ventral surface of the medulla in the vicinity of the RVLM. In contrast, clonidine, applied at the same site, elicited marked effects (Scholtysik et al., 1975). 8.2. Rats Several drugs with affinity for I1 receptors and a2-adrenoceptors were microinjected at one fixed and identical dose (1 nmol/injection site) into the RVLM of rats (Ernsberger et al., 1990). Most of the drugs lowered blood pressure. Drugs with high affinity for I1 receptors (clonidine, para-aminoclonidine) produced the strongest hypotensive response, whereas selective a2 -adrenoceptor agonists (noradrenaline, adrenaline, and a-methylnoradrenaline) had only weak effects. There was a correlation between the affinities of the drugs for I1-binding sites and the magnitude of the hypotensive response, whereas there was no correlation between the affinity for a2-adrenoceptors and the hypotensive response. In a similar study (Buccafusco et al., 1995), dose – response curves for the hypotensive effect of a series of intracisternally administered agonists were constructed. The selective a2-adrenoceptor agonist guanfacine had no effect on blood pressure. There was a correlation between the affinity of the drugs for I1-binding sites and their potency to produce hypotension, but no such correlation existed if the affinity of the drugs for a2-adrenoceptors was considered. The correlation between I1 affinity and hypotensive response and the missing correlation between a2 affinity and hypotension was considered as support for the imidazoline mode of action (Ernsberger et al., 1990; Buccafusco et al., 1995). Some shortcomings are apparent, however, in both studies. Only a few drugs were included that possess I1 affinity and cause substantial hypotension (clonidine, para-aminoclonidine, oxymetazoline, and cimetidine in Ernsberger et al. [1990]; moxonidine, clonidine, lofexidine, and ICI106207 in Buccafusco et al. [1995]). Importantly, the correlation between I1 affinity and the hypotensive effect is less than impressive for these respective four drugs. Another shortcoming is that affinity for a2-adrenoceptors of the catecholamines noradrenaline, adrenaline, and a-methylnoradrenaline was compared with their in vivo efficacy. Compared with their affinity in cell free binding assays, the catecholamines will appear less potent under in vivo conditions because they are subject to neuronal uptake and metabolism. In addition, their interaction with a1- and b-adrenoceptors will greatly modify their effects in vivo.
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a-Methylnoradrenaline and noradrenaline, microinjected into the RVLM, were effective in several studies. Thus, a-methylnoradrenaline lowered blood pressure and heart rate, and the maximal effect appeared after 10 nmol/injection site (Granata et al., 1986). The 10-nmol dose of a-methylnoradrenaline is similar to the doses that lowered blood pressure after injection into the NTS, a region in which the sympathoinhibitory role of a2-adrenoceptors is generally accepted (De Jong & Nijkamp, 1976; Zandberg et al., 1979; Kubo & Misu, 1981). In another study, a-methylnoradrenaline applied by microiontophoresis in the RVLM inhibited the activity of a set of bulbospinal sympathoexcitatory neurons. The same set of neurons was also inhibited by locally and systemically administered clonidine (Allen & Guyenet, 1993). Noradrenaline also caused hypotension when it was injected into the RVLM of rats (Sinha et al., 1985). Thus, catecholamines, which possess only very low affinity for I1-binding sites, consequently lower blood pressure when they are microinjected into the RVLM, probably by activating a2-adrenoceptors. 8.3. Rabbits Microinjection of a-methylnoradrenaline into the RVLM of rabbits lowered blood pressure, heart rate, and the firing rate of renal sympathetic nerves. The blood pressure – renal sympathetic nerve activity (RSNA) reflex curve was shifted to the right, and the maximum of the curve was depressed (Head & Burke, 1998, 2000b). The hypotensive doses of a-methylnoradrenaline, microinjected into the RVLM or into the NTS, were identical. Injection of AGN192403, the drug that possesses high affinity and high selectivity for I1-binding sites (see Section 4.1.2), directly into the CNS of the rabbit, elicited no cardiovascular effects (Munk et al., 1996). 8.4. Monkeys Intravenous injection of AGN192403 did not change the blood pressure of monkeys, and it also did not antagonize the hypotension produced by clonidine (Munk et al., 1996). In summary, a2-adrenoceptor agonists lower blood pressure when they are injected into the RVLM of rats and rabbits. The role of a2-adrenoceptors in the RVLM of cats is less clear; however, there are too few observations to exclude a role for a2-adrenoceptors. It is not clear why guanfacine is not active when it is administered intracisternally or to the surface of the ventral medulla; it may penetrate into the brain only poorly. It is very remarkable that the first drug, AGN192403, which in radioligand binding studies proved to be selective for I1-binding sites, has no effect on blood pressure, neither when administered alone nor when combined with clonidine. The suggestion that there is no a2-adrenoceptor-mediated sympathoinhibition in the RVLM and that the effects of clonidine-like drugs correlate with the affinity of the drugs for I1-binding sites is not sufficiently supported by experimental observations.
15
9. Interaction of agonists with antagonists in vivo Cardiovascular experiments with antagonists on anaesthetised or conscious animals are essential for determining the receptors primarily activated by clonidine-like drugs. Table 4 shows studies that support the role of a2-adrenoceptors in the sympathoinhibition produced by clonidine-like drugs. In contrast, Table 5 compiles studies in which the authors concluded that I1 receptors are involved in the sympathoinhibition. These studies are now critically evaluated. 9.1. Studies supporting involvement of 2-adrenoceptors As outlined in the Section 4.2.1 (see also Table 3), affinity for a2-adrenoceptors and lack of affinity for I1 receptors was verified for three antagonists: yohimbine, SK&F86466, and methoxy-idazoxan. Antagonism by these three a2-selective drugs of the sympathoinhibitory and hypotensive effects of clonidine, rilmenidine and moxonidine, supports involvement of a2-adrenoceptors in these effects. Yohimbine was already used in 1973 by Schmitt et al. to analyze the cardiovascular effects of clonidine (when, of course, the imidazoline hypothesis did not yet exist). Table 4 lists the studies in which yohimbine, SK&F86466, and methoxy-idazoxan were effective against clonidine, rilmenidine, and moxonidine. Involvement of a2-adrenoceptors was shown in several species in anaesthetized and also in conscious states. Several routes of drug administration were used: agonists were injected intravenously, into the vertebral artery, into the lateral cerebral ventricle, and into the cisterna cerebellomedullaris. Antagonists were frequently administered by the same way as the agonists, but, in a few cases, systemically administered agonists were combined with intracerebrally applied antagonists. Several types of interactions between agonists and antagonists were tested in order to study the role of a2-adrenoceptors. (1) In the simplest experiments, the a2-adrenoceptor antagonist counteracted the effect of clonidine-like drugs (Schmitt et al., 1973; Bolme et al., 1974; Farsang et al., 1980; Timmermans et al., 1981; Johansson et al., 1981; McConnaughey & Ingenito, 1982; van Zwieten et al., 1986; Tibirica et al., 1988; Allen & Guyenet, 1993; Szabo et al., 1993; Szabo & Urban, 1995). (2) In more complex designs, it was shown that yohimbine and SK&F86466 are as potent against rilmenidine and moxonidine as against the selective a2-adrenoceptor agonist UK14304 (Urban et al., 1994, 1995a). (3) Finally, it was shown that yohimbine and SK&F86466 were at least as effective against clonidine, rilmenidine, and moxonidine as the mixed I1/a2 antagonists idazoxan and efaroxan (Hieble & Kolpak, 1993; Vayssettes-Courchay et al., 1996; Bock et al., 1999). In our opinion, the studies of Hieble and Kolpak (1993) and Bock et al. (1999) convincingly show that a2-adrenoceptors are involved in the hypotensive actions of clonidine,
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Table 4 In vivo studies with agonists and antagonists supporting involvement of a2-adrenoceptors in the hypotension Species
Anaesthesia
Measured parameters
Rat Rat (SHR) Rat (SHR) Rat (SHR)
Chloralose Pentobarbitone Pentobarbitone a-Chloralose
Blood Blood Blood Blood
Rat
Pentobarbitone
Blood pressure, heart rate, sympathetic nerve activity
Rat
Urethane
Rat
Urethane
Firing rate of RVLM neurons Blood pressure, heart rate
Rabbit
Conscious
Rabbit
Conscious
Rabbit
Conscious
Rabbit
Conscious
Dog Cat
pressure, heart rate pressure, heart rate pressure, heart rate pressure
Agonist (route of application)
Antagonist (route of application)
Clonidine (i.v.) Clonidine (i.v.) Clonidine (i.c.v.) Clonidine (i.v.) Guanabenz (i.v.) Clonidine (i.v.)
Yohimbine (i.v.) Yohimbine (i.p.) Yohimbine (i.c.v.) Idazoxan (i.v.) SK&F86466 (i.v.) Idazoxan (i.v.) Methoxy-idazoxan (i.v.) Yohimbine (i.v.) Yohimbine (i.v.)
Bolme et al., 1974 Farsang et al., 1980 Tibirica et al., 1988 Hieble & Kolpak, 1993
Methoxy-idazoxan (gigantocellular depressor area microinjection) Yohimbine (i.v.) Methoxy-idazoxan (i.v.)
Aicher & Drake, 1999
Rilmenidine (i.v.) UK14304 (i.v.)
SK&F86466 (i.v.)
Urban et al., 1994
Clonidine (i.v.) Clonidine (gigantocellular depressor area microinjection)
Rilmenidine (i.v.)
Reference
Vayssettes-Courchay et al., 1996
Allen & Guyenet, 1993
Szabo et al., 1993; Szabo & Urban, 1995
Moxonidine (i.v.) UK14304 (i.v.)
Yohimbine (i.v.) Yohimbine (i.c.m.)
Urban et al., 1995a
Rilmenidine (i.c.m.) Moxonidine (i.c.m.) a-Methyldopa (i.c.m.) Clonidine (vertebral artery)
Cat
Chloralose + urethane a-Chloralose
Blood pressure, heart rate, sympathetic nerve activity Blood pressure
Clonidine (i.v.) Clonidine (i.c.m.) Clonidine (vertebral artery)
Cat
Pentobarbitone
Blood pressure, heart rate
Cat
Chloralose
Blood pressure
Cat
Conscious
Blood pressure, heart rate
Clonidine (vertebral and carotid artery) Rilmenidine (vertebral artery) Clonidine (i.c.v.)
Yohimbine (i.c.m.) SK&F86466 (i.c.m.) Efaroxan (i.c.m.) Yohimbine (vertebral artery) Yohimbine (i.v.) Yohimbine (i.c.m.) Yohimbine (vertebral artery) Rauwolscine (vertebral artery) Yohimbine (i.v.)
Bock et al., 1999
Pentobarbitone
Blood pressure, heart rate, sympathetic nerve activity, plasma noradrenaline concentration Blood pressure, heart rate, sympathetic nerve activity, plasma noradrenaline concentration Blood pressure, heart rate, sympathetic nerve activity, plasma noradrenaline concentration Blood pressure, heart rate, plasma noradrenaline concentration Blood pressure
Cat
Conscious
Blood pressure, heart rate
Clonidine (i.v.)
Sea gull
Pentobarbitone
Blood pressure, heart rate
Clonidine (i.v.)
Yohimbine (vertebral artery) Yohimbine (i.c.v.) Efaroxan (i.c.v.) Yohimbine (i.c.v.) Efaroxan (i.c.v.) Yohimbine (i.v.)
Schmitt et al., 1973
Timmermans et al., 1981
McConnaughey & Ingenito, 1982 van Zwieten et al., 1986 Ally, 1997 Ally, 1998 Johansson et al., 1981
i.c.m., injection into the cisterna cerebellomedullaris; i.c.v., injection into a lateral cerebral ventricle.
rilmenidine, and moxonidine. What is more, there was no indication of a role for I1 receptors in these studies. Hieble and Kolpak (1993) first determined i.v. doses of idazoxan and SK&F86466 that caused equal blockade of a2-adrenoceptors in pithed rats. The interaction of i.v.-administered antagonists, at the determined doses, with i.v.-administered clonidine was then studied in anaesthetized rats: idazoxan and SK&F86466 were equieffective at blocking the hypotensive effect of clonidine. This result suggests that the entire effect of idazoxan is attributable to blockade of
a2-adrenoceptors. An additional effect of idazoxan, due to blockade of I1 receptors, could not be seen. In the study of Bock et al. (1999), all drugs were administered intracisternally in conscious rabbits. a-Methyldopa, rilmenidine, and moxonidine lowered blood pressure, heart rate, and the plasma noradrenaline concentration. Efaroxan, yohimbine, and SK&F86466 were administered after the agonists. The hypotensive effects of all three agonists were completely antagonized by all three antagonists (see Fig. 3). The mixed I1/a2 antagonist efaroxan was the most potent of the three
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
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Table 5 In vivo studies with agonists and antagonists in which the authors conclude that imidazoline receptors are involved in the hypotension Species
Anaesthesia
Measured parameters
Rat Rat
Pentobarbitone Pentobarbitone
Rat
Urethane
Blood pressure, heart rate Blood pressure, heart rate DOPAC concentration in RVLM and LC Blood pressure
Rat
Urethane
Blood pressure
Rat
Conscious
Rat (SHR)
Urethane
Blood pressure, heart rate, sympathetic nerve activity Blood pressure, heart rate
Rat
Urethane
Blood pressure
Mouse
Urethane
Blood pressure, heart rate
Rabbit
Pentobarbitone
Blood pressure, heart rate
Rabbit
Conscious
Blood pressure, heart rate
Rabbit
Conscious
Blood pressure, heart rate
Rabbit
Conscious
Rabbit
Urethane
Blood pressure, heart rate, plasma noradrenaline concentration Blood pressure, heart rate, sympathetic nerve activity
Agonist (route of application)
Antagonist (route of application)
Clonidine (i.c.v.) Clonidine (i.v.)
Yohimbine (i.c.v.) Yohimbine (i.c.m.) Idazoxan (i.c.m.)
Tibirica et al., 1988 Tibiric¸a et al., 1991a
Clonidine (RVLM microinjection) Rilmenidine (i.v.)
Idazoxan (RVLM microinjection) SK&F86466 (RVLM microinjection) SK&F86466 (RVLM microinjection) Idazoxan (RVLM microinjection) Idazoxan (i.c.m.) Yohimbine (i.c.m.) Efaroxan (RVLM microinjection) SK&F86466 (RVLM microinjection) Yohimbine (i.v.) Idazoxan (i.v.) Efaroxan (RVLM microinjection) SK&F86466 (RVLM microinjection) Idazoxan (i.c.m.) Yohimbine (i.c.m.) Idazoxan (i.c.m.) RX821002 (i.c.m.)
Ernsberger et al., 1990
Rilmenidine (i.v.) Rilmenidine (i.c.m.) Moxonidine (RVLM microinjection; i.v.) Clonidine (i.v.) Moxonidine (RVLM microinjection; i.v.) Rilmenidine (i.c.m.) Rilmenidine (i.c.m.) a-Methyldopa (i.c.m.) Rilmenidine (i.c.m.) Moxonidine (i.c.m.) Clonidine (i.c.m.) a-Methyldopa (i.c.m.) Rilmenidine (i.c.m.) Moxonidine (i.c.m.) Guanabenz (i.c.m.) Rilmenidine (RVLM, NTS microinjection)
Reference
Gomez et al., 1991 Mayorov et al., 1993 Haxhiu et al., 1994 Mao & Abdel-Rahman, 1996 Tolentino-Silva et al., 2000 Feldman et al., 1990 Sannajust & Head, 1994
Efaroxan (i.c.m.) RX821002 (i.c.m.)
Chan et al., 1996
Yohimbine (i.c.m.) Efaroxan (i.c.m.)
Szabo & Urban, 1997
Idazoxan (RVLM, NTS microinjection) RX821002 (RVLM, NTS microinjection)
Head & Burke, 1998, 2000b
a-Methylnoradrenaline (RVLM, NTS NTS microinjection) i.c.m., injection into the cisterna cerebellomedullaris; i.c.v., injection into a lateral cerebral ventricle; LC, locus coeruleus.
antagonists against rilmenidine and moxonidine. At first glance, this would suggest a role for I1 receptors in the effects of rilmenidine and moxonidine. However, efaroxan was the most potent of the three antagonists also against a-methyldopa, the active metabolite of which, a-methylnoradrenaline, acts as an a2-adrenoceptor agonist. The data show that selective a2-adrenoceptor antagonists are capable of fully antagonizing the effects of rilmenidine and moxonidine. In addition, the antagonism by the mixed I1/a2 antagonist efaroxan could be fully attributed to blockade of a2-adrenoceptors. An additional effect of efaroxan, due to blockade of I1 receptors, was not seen. It has been argued repeatedly that a2 antagonists do not counteract the effects of agonists by blocking the same primary receptor that is activated by the agonists. Instead, they produce ‘‘functional’’ antagonism; e.g., systemically administered antagonists could inhibit transmission between sympathetic neurons and vascular smooth muscles by blocking vascular a1- and a2-adrenoceptors. In this situation, a centrally elicited sympathoinhibition by clonidine-
like drugs would not be accompanied by hypotension (see Ernsberger & Haxhiu, 1997). Such a ‘‘functional’’ antagonism by systemically administered yohimbine, SK&F86466, and methoxy-idazoxan is unlikely, since these antagonists possess relatively low affinity for a1-adrenoceptors and they did not cause major blood pressure changes when they were administered alone in the above mentioned studies. Moreover, in some studies, systemically administered yohimbine, SK&F86466, and methoxy-idazoxan antagonized the inhibition of sympathetic nerve activity, the inhibition of the firing of RVLM neurons, and the reduction in plasma noradrenaline concentration produced by clonidine, rilmenidine, and moxonidine (Schmitt et al., 1973; Allen & Guyenet, 1993; Szabo et al., 1993; Urban et al., 1994, 1995a; Szabo & Urban, 1995; Vayssettes-Courchay et al., 1996). Experiments in which yohimbine was used as an a2 antagonist were criticized because yohimbine may also interfere with 5-hydroxytryptamine (5-HT) receptors (Ernsberger & Haxhiu, 1997). Indeed, yohimbine possesses rel-
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et al., 1994). However, in cardiovascular studies, it does not elicit responses that are consistent with an effect at 5-HT1A receptors (compare, e.g., Szabo et al., 1992b with Ramage & Fozard, 1987 and Szabo et al., 1992a). It should be noted that idazoxan, the antagonist used in the majority of in vivo studies supporting a role of I1 imidazoline receptors, has an affinity for 5-HT1A receptors comparable with that of yohimbine (Ki = 62 nM) (Kawai et al., 1994). The third type of functional antagonism was suggested by Head et al. (Sannajust & Head, 1994; Chan et al., 1996; Head et al., 1998; Head & Burke, 2000a): clonidine-like drugs primarily activate I1 receptors in the medulla oblongata, but medullary a2-adrenoceptors participate in the chain of events finally leading to sympathoinhibition. a2 Antagonists would produce functional antagonism of the effects of clonidine-like drugs acting downstream from the I1 receptor. This hypothesis diminishes the discriminative power of all experiments in which selective a2 antagonists were used to determine whether a2-adrenoceptors are the primary sites of action of clonidine-like drugs. It should be noted that essential elements of this hypothesis have remained unverified. 9.2. Studies supporting involvement of I1 receptors
Fig. 3. Interactions between the agonists a-methyldopa (AMD), rilmenidine (RIL), and moxonidine (MOX) and the antagonists efaroxan (EFA), yohimbine (YOH), and SK&F86466 (SKF) in conscious rabbits. All drugs were administered into the cisterna magna (i.c.m.). At first, animals were pretreated with a-methyldopa (400 mg kg 1), rilmenidine (10 mg kg 1), or moxonidine (0.3 mg kg 1). The three agonists lowered blood pressure equally by 12 mm Hg (not shown), and the blood pressure remained at the hypotensive values in animals not receiving antagonists (not shown). Administration of efaroxan, yohimbine, and SK&F86466 dose-dependently and significantly increased blood pressure in animals pretreated with the agonists. The antagonists had no significant effects in animals that had not been pretreated with agonists (not shown). The figure demonstrates that the hypotensive effects of rilmenidine and moxonidine were antagonized by all three antagonists. Notably, the selective a2-adrenoceptor antagonists yohimbine and SK&F86466 were capable of causing full antagonism. Efaroxan, which is suggested to be a selective I1 imidazoline receptor antagonist, was 3 - to 10 - fold more potent against rilmenidine and moxonidine than yohimbine and SK&F86466. In my opinion, this is not due to the affinity of efaroxan for I1 receptors, but due to its high affinity for a2-adrenoceptors: efaroxan was the most potent of the three antagonists also against a-methyldopa, which is thought to activate a2-adrenoceptors after its conversion into a-methylnoradrenaline. Significant blood pressure increase evoked by the antagonists: * P < 0.05. Data from Bock et al. (1999).
atively high affinity for 5-HT1A-binding sites (Ki = 29 – 74 nM) and elicits behavioural effects similar to those of 5-HT1A receptor agonists (Winter & Rabin, 1992; Kawai
As pointed out in Section 4.2.2 (see also Table 3), no I1-selective antagonists are available. For lack of better drugs, idazoxan and efaroxan were used in the studies that concluded that I1 receptors are the primary sites of action of clonidine-like drugs (Table 5). Both drugs are a2-adrenoceptor antagonists, with additional affinity for I1-binding sites. Several kinds of experimental designs were used. In the study of Tibirica et al. (1988), yohimbine was ineffective against clonidine, and this was taken as an indication of the involvement of imidazoline receptors in the effects of clonidine. In other studies, the selective a2 antagonists yohimbine and SK&F86466 were compared with idazoxan and efaroxan. The effects of clonidine, rilmenidine, and moxonidine were considered to be mediated by I1 receptors, if idazoxan and efaroxan were more effective than yohimbine or SK&F86466 at antagonizing the hypotensive and sympathoinhibitory effects of clonidine, rilmenidine, and moxonidine (Ernsberger et al., 1990; Feldman et al., 1990; Gomez et al., 1991; Tibiric¸a et al., 1991a; Mayorov et al., 1993; Haxhiu et al., 1994; Mao & AbdelRahman, 1996; Tolentino-Silva et al., 2000). These studies have a major shortcoming. It was not experimentally verified that at the given doses, yohimbine and SK&F86466, on the one hand, and idazoxan and efaroxan, on the other hand, caused equal a2-adrenoceptor blockade. In other words, the greater effectivity of idazoxan and efaroxan than that of yohimbine and SK&F86466 at antagonizing the effects of clonidine, rilmenidine, and moxonidine may have been due to more complete blockade of a2-adrenoceptors and not to blockade of I1 receptors. For example, idazoxan is 10- to 20fold more potent than yohimbine at the a2A/D-adrenoceptor
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
(Table 3; Remaury & Paris, 1992; Renouard et al., 1994; Uhlen et al., 1995). a2A/D is the most abundant a2-adrenoceptor subtype in cardiovascular regulatory centres of rats. Idazoxan antagonized more effectively than yohimbine (the two antagonists were administered at equimolar doses) the effects of clonidine (Tibiric¸a et al., 1991a) and rilmenidine (Mayorov et al., 1993). These findings probably reflect the difference in affinity of the two antagonists for the a2A/D-adrenoceptor. In addition to their high affinity for a2-adrenoceptors, the imidazoline antagonists may penetrate into brain tissue more easily than the non-imidazoline antagonists, and this may enhance their effectivity in vivo. The effects of clonidine, rilmenidine, and moxonidine, administered into the cisterna cerebellomedullaris of conscious rabbits, were analyzed in three studies (Sannajust & Head, 1994; Chan et al., 1996; Szabo & Urban, 1997). These agonists were compared with guanabenz and a-methyldopa. The interaction with intracisternally administered yohimbine, methoxy-idazoxan, idazoxan, and efaroxan was also tested. A constant finding of these studies was that the a2-adrenoceptor antagonists yohimbine and methoxy-idazoxan always antagonized the effects of clonidine, rilmenidine, and moxonidine. In the study of Sannajust and Head (1994), methoxy-idazoxan was 10-fold more potent against a-methyldopa than against rilmenidine, suggesting a different mechanism of action for the two agonists. In the experiments of Chan et al. (1996) and Szabo and Urban (1997), the mixed I1/a2 antagonist efaroxan was more effective than the a2-adrenoceptor antagonists methoxyidazoxan and yohimbine (efaroxan, methoxy-idazoxan, and yohimbine were administered at doses that produced equal blockade of a2-adrenoceptors) against rilmenidine and moxonidine. Accordingly, it was concluded that I1 receptors were likely to be involved in the effects of rilmenidine and moxonidine. Interestingly, Chan et al. (1996) concluded that clonidine acts mainly through a2-adrenoceptors. It should be critically noted that the conclusions of these studies, in favour of involvement of I1 receptors, rely on 3- to 10-fold potency differences of antagonists. This value may be too low for firm conclusions. In two recent studies in anaesthetized rabbits, rilmenidine and the antagonists methoxy-idazoxan and idazoxan were microinjected into the RVLM (Head & Burke, 1998, 2000b). Rilmenidine elicited hypotension and inhibited RSNA. In addition, the blood pressure –RSNA reflex curve was shifted to the left, and the maximum of the curve was depressed. The effects of rilmenidine on blood pressure and the blood pressure –RSNA reflex curve were counteracted by methoxy-idazoxan and idazoxan. Since idazoxan had no effect against a-methylnoradrenaline, it was suggested that rilmenidine acted by activating I1 receptors. It is remarkable that in these two studies, idazoxan antagonized I1 receptor-mediated effects; yet, at the same dose, it did not counteract a2-adrenoceptor-mediated effects. From the results of binding studies (Table 3), one would expect
19
that idazoxan blocks a2-adrenoceptors more readily than I1 receptors. The findings of the in vivo agonist/antagonist interaction experiments can be summarized as follows. In the majority of experiments, pure a2-adrenoceptor antagonists were capable of preventing or abolishing the hypotensive and sympathoinhibitory effects of imidazoline antihypertensive drugs. In some experiments, only the mixed I1/a2 antagonists idazoxan and efaroxan produced full blockade of the hypotensive effects. It is doubtful, however, whether the doses of the applied selective a2-adrenoceptor antagonists were sufficient. Only three studies showed that idazoxan and efaroxan were more effective than selective a2-adrenoceptor antagonists and that this difference was not due to more complete blockade of a2-adrenoceptors by idazoxan and efaroxan.
10. Experiments in genetically modified animals Mice with genetically modified a2-adrenoceptors were also used to clarify the mechanism of action of clonidinelike drugs. In some studies, the D79N mouse strain was used (MacMillan et al., 1996; Zhu et al., 1999; TolentinoSilva et al., 2000). In these mutant mice, the coupling of the a2A/D-adrenoceptor to certain K + channels is disrupted (Surprenant et al., 1992). Other studies used mice in which the a2A/D-adrenoceptor (Altman et al., 1999), the a2B-adrenoceptor, or the a2C-adrenoceptor was deleted (Link et al., 1996). Compared with control mice, the hypotensive action of UK14304 and dexmedetomidine, injected into the carotid artery, was lost in D79N mice (MacMillan et al., 1996). The hypotensive effect of dexmedetomidine was also lost in a2A/D-adrenoceptor knockout mice (Altman et al., 1999). UK14304 and dexmedetomidine are selective a2-adrenoceptor agonists without affinity for I1 receptors (see Section 4.1.1). These results indicate, therefore, that activation of a2A/D-adrenoceptors can cause hypotension; they do not help to answer the question whether a2-adrenoceptors are involved in the action of I1 imidazoline agonists, such as clonidine, rilmenidine, and moxonidine. The hypotensive action of clonidine, rilmenidine, and moxonidine, administered i.v. in conscious animals, was lost in D79N mice (Zhu et al., 1999). In contrast to these results, the hypotensive effect of moxonidine, microinjected directly into the RVLM, was preserved in anaesthetized D79N mice (Tolentino-Silva et al., 2000). The authors of this latter study believe that the effect of moxonidine is mediated by I1 imidazoline receptors. They suggest that the effect is more pronounced in anaesthetized animals and with direct application of moxonidine into the RVLM, as compared with systemical administration in conscious animals. Still, the difference between the observations of Zhu et al. (1999) and Tolentino-Silva et al. (2000) remains unexplained.
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The hypotensive effect of dexmedetomidine was also studied in mice in which the a2B-adrenoceptor or the a2C-adrenoceptor was deleted (Link et al., 1996). Deletion of the a2C-receptor had no influence on the hypotensive effect, whereas in a2B-deficient mice, the hypotension was potentiated. The enhanced hypotension was attributed to the missing a2B-adrenoceptor-mediated vascular contraction. Fairbanks and Wilcox (1999) observed an interesting non-cardiovascular effect of moxonidine, and the results may be relevant to cardiovascular studies. Moxonidine caused spinal antinociception in mice. This effect persisted in D79N mice, and could be antagonized by the selective a2-adrenoceptor antagonist SK&F86466. The authors concluded that the effect of moxonidine was mediated by a2B- or a2C-adrenoceptors. Thus, the experiments in genetically modified animals did not really help to answer the question of the role of a2-adrenoceptors in the effects of I1 imidazoline receptor agonists. Either agonists without affinity for I1 receptors were used, or the results of the different studies are contradictory.
11. Relationship between sympathoinhibition and other A2-adrenoceptor-mediated effects Sedation and inhibition of saliva secretion are the major side effects of centrally acting antihypertensive drugs. Sedation was indeed the effect that led to the discovery of the prototype of this class of drugs, clonidine (see Section 2). The sedative effect is attributed to a2-adrenoceptor-mediated inhibition of the activity of noradrenergic neurons in the locus coeruleus (De Sarro et al., 1987; Correa-Sales et al., 1992; Scheinin & Schwinn, 1992). The a2-adrenoceptor responsible for the inhibition of locus coeruleus neurons and sedation was recently identified as the a2A/D receptor (Chiu et al., 1995; Mizobe et al., 1996; No¨renberg et al., 1997; Mateo & Meana, 1999; see also Sallinen et al., 1997). One study showed inhibition of locus coeruleus neurons mediated by a2B- or a2C-adrenoceptors (Arima et al., 1998). The imidazoline antihypertensive drugs clonidine (IC50 = 6 nM), rilmenidine (IC50 = 127 nM), and moxonidine (IC50 = 49 nM) inhibit the firing of locus coeruleus neurons in rat brain slices, and the effects are blocked by the selective a2-adrenoceptor antagonist SK&F86466 (Szabo et al., 1996). The mechanism of the inhibition of salivation by clonidine-like drugs is less clear. While a2-adrenoceptors mediate this effect, the subtype of the involved a2-adrenoceptor is not known yet. Experimental data support action at three different sites. (1) Clonidine-like drugs can inhibit salivation, with a primary action in the CNS, probably in the brain stem (Green et al., 1979; Warren et al., 1991). (2) Saliva secretion can be lowered by inhibition of acetylcholine release from postganglionic cholinergic neurons in the salivary glands (Montastruc et al., 1989; Izumi et al., 1995). (3) Finally, it was suggested that clonidine-like drugs inhibit saliva secre-
tion by direct action on the secretory cells in the salivary glands (Kaniucki et al., 1984; Lung, 1994, 1998). An important component of the imidazoline hypothesis deals with the relationship between sympathoinhibition and a2-adrenoceptor-mediated effects. It is suggested that the drugs that cause sympathoinhibition by activation of a2-adrenoceptors, i.e., guanabenz, guanfacine, and a-methyldopa, simultaneously elicit the a2-adrenoceptor-mediated side effects of sedation and dry mouth. In contrast, the imidazoline agonists rilmenidine and moxonidine, and some say even clonidine, cause sympathoinhibition, without eliciting a2-adrenoceptor-mediated effects. a2-Adrenoceptormediated effects would appear only after higher doses of these drugs. Importantly, the new drugs rilmenidine and moxonidine, due to their improved I1/a2 selectivity, should cause less sedation and inhibition of saliva secretion than clonidine. How is this assumption of the imidazoline hypothesis supported by experimental observations in animals and humans? 11.1. Animal experiments 11.1.1. Clonidine Sedation of animals by clonidine was recognized early (Hoefke & Kobinger, 1966). Inhibition of the firing of locus coeruleus neurons was first observed by Svensson et al. in 1975. Systemically administered clonidine also inhibits the release of noradrenaline in the hippocampus and the medial prefrontal cortex, brain regions that receive their noradrenergic input from the locus coeruleus (Abercrombie et al., 1988; Gresch et al., 1995; Sacchetti et al., 1999). Several studies showed that clonidine causes hypotension and inhibits the firing of locus coeruleus neurons or lowers noradrenaline release in projection regions of the locus coeruleus at the same doses. Thus, in the rat, the hypotensive doses of clonidine are within the 1 – 30 mg kg 1 range (i.v.; see Table 1). The ED50 value of clonidine for reduction of the firing rate of locus coeruleus neurons in rats, 6– 7 mg kg 1 (i.v.), falls into this range (Dresse & Scuve´e-Moreau, 1986; Engberg & Eriksson, 1991). Clonidine 10 mg kg 1 (i.p.) also lowers the extracellular noradrenaline concentration in the hippocampus of the rat (Sacchetti et al., 1999). Simultaneous effects of clonidine on sympathetic regulatory centres and the locus coeruleus were seen in conscious rabbits using neuroanatomical techniques (Li & Dampney, 1995). The expression of the Fos protein, a marker of neuronal activation, was determined immunohistochemically. Infusion of sodium nitroprusside led to Fos expression in the RVLM, the locus coeruleus, and other brain stem nuclei. Injection of clonidine (7 –30 mg kg 1, i.v.) suppressed the appearance of Fos in the RVLM, as well as in the locus coeruleus. No preferential effect of clonidine in the RVLM was seen. In contrast to the above observations, one group found that low doses of clonidine selectively cause sympathoinhi-
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
bition (Tibirica et al., 1989). In anaesthetized normotensive rats, 2, 5, and 10 mg kg 1 of clonidine (i.v.) lowered the blood pressure and the concentration of 3,4-dihydroxyphenylacetic acid (DOPAC) in the NRL. DOPAC was thought to reflect the activity of catecholaminergic neurons involved in sympathetic regulation because its concentration correlated with blood pressure changes. In contrast to the DOPAC changes in the NRL, clonidine had no effect on the DOPAC concentration in the locus coeruleus up to 10 mg kg 1. Only the high dose of 50 mg kg 1 lowered the DOPAC concentration in the locus coeruleus. The selective action of clonidine in the NRL, a region containing imidazoline-binding sites, was explained by a preferential effect on imidazoline receptors. It should be noted that DOPAC is a suboptimal indicator of neuronal activity, most of all because its source is not clarified. It may derive from newly synthesized dopamine, before the amine is taken up into vesicles, in noradrenergic and adrenergic neurons (Tibirica et al., 1989; Lambas-Senas et al., 1990). However, it may also be produced in dopaminergic neurons from newly synthesized dopamine or dopamine taken up by the dopamine carrier. Interestingly, the pattern of effects of clonidine was quite different in anaesthetized SHR (Tibiric¸a et al., 1992): 10 mg kg 1 of clonidine (i.v.) markedly lowered blood pressure, without changing the DOPAC concentration in the NRL. The authors concluded that in SHR, the site of action of clonidine is not in the NRL and that a2-adrenoceptors are involved in the sympathoinhibition. This conclusion was supported by previous results of the group: in SHR, the hypotensive effect of intracisternally applied clonidine was sensitive to low doses of intracisternally injected yohimbine (Tibirica et al., 1988). A remarkable discrepancy in the observations of two leading groups in the field becomes apparent here. One group did not observe imidazoline receptor-mediated effects in the medulla oblongata of SHR (Tibirica et al., 1988, 1992). In contrast, another group repeatedly obtained results supporting the role of imidazoline receptors in the hypotension elicited by clonidine and moxonidine in SHR (Haxhiu et al., 1994; Buccafusco et al., 1995). In summary, in the majority of the experimental models, clonidine simultaneously lowers blood pressure, inhibits neuronal activity in cardiovascular centres, and inhibits activity of locus coeruleus neurons. The one notable exception is the study in which DOPAC was used as a marker of neuronal activity. As mentioned above, DOPAC may not be an appropriate marker for neuronal activity in the NRL and the locus coeruleus. 11.1.2. Rilmenidine The sedative effect of rilmenidine was already described in the first publication dealing with the mechanism of the hypotensive action of this drug (Laubie et al., 1985). The sedation, measured as the loss of the righting reflex in chickens, appeared at doses ( 5 mg kg 1, s.c.) higher
21
than those necessary to elicit hypotension, and was of short duration. In the same study, clonidine also caused sedation after doses ( 62 mg kg 1, s.c.) higher than those necessary to elicit hypotension, and the maximal duration of sedation was longer than after rilmenidine. The effect of rilmenidine on the firing rate of locus coeruleus neurons was studied by one group (Laubie et al., 1985; Dresse & Scuve´e-Moerau, 1986). Intravenously administered rilmenidine inhibited the firing of locus coeruleus neurons in anaesthetized rats with an ED50 = 350 mg kg 1. This value is identical to the doses of rilmenidine that cause hypotension and bradycardia in anaesthetized rats (ED50 = 250 and 350 mg kg 1, respectively) (Gomez et al., 1991; and see Table 1 for further hypotensive doses of rilmenidine). To our knowledge, the effect of rilmenidine on noradrenaline concentration in the extracellular space in the brain has not been determined in animals with intact a2-adrenoceptors. Simultaneous effects of rilmenidine on sympathetic regulatory centres and the locus coeruleus were seen in conscious rabbits by measuring the expression of the Fos protein (Li & Dampney, 1995). Rilmenidine (150 – 300 mg kg 1, i.v.) suppressed the synthesis of the Fos protein in the RVLM and the locus coeruleus. No preferential effect in the RVLM was seen. Moreover, rilmenidine inhibited locus coeruleus neurons as strongly as clonidine. In addition, rilmenidine markedly suppressed the expression of the Fos protein in the NTS. Such an effect is not predicted by the imidazoline hypothesis for a selective imidazoline agonist. Rilmenidine-evoked changes in neuronal activity were also determined by measuring DOPAC (Tibiric¸a et al., 1991b), and, like in the case of clonidine, the results of these experiments contradict the results of other studies. In anaesthetized normotensive rats, 0.3 and 1.5 mg kg 1 of rilmenidine (i.v.) lowered blood pressure and the concentration of DOPAC in the NRL. In contrast, the DOPAC concentration in the locus coeruleus decreased only after a dose of 15 mg kg 1. It was calculated that rilmenidine possesses 50-fold selectivity for decreasing blood pressure and neuronal activity in the NRL versus decreasing neuronal activity in the locus coeruleus. In line with these observations, the same group showed that intravenously administered rilmenidine, at doses of 0.3 and 1 mg kg 1, does not change the increase in the voltammetrically measured electrochemical signal in the hypothalamus (attributed to noradrenaline) evoked by electrical stimulation (10 Hz) of the ventral noradrenergic pathway (Suaud-Chagny et al., 1992). At doses of 3 and 10 mg kg 1, rilmenidine even increased the signal. In contrast, clonidine (50 mg kg 1; i.v.) reduced the evoked noradrenergic signal. The authors concluded in this latter study that ‘‘. . .rilmenidine, at clinically relevant doses, is not active on central a2-adrenoceptors. . ..’’ In the experiments of Tibiric¸a et al. (1991b) and SuaudChagny et al. (1992), rilmenidine ( 7.5 mg kg 1 i.v.) had no effect on the DOPAC concentration in the locus coeruleus and the evoked noradrenaline release in the hypothalamus. If the voltametric measurements faithfully reflected
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B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
activity of noradrenergic neurons in the locus coeruleus and the hypothalamus, a conclusion on the brain penetration of rilmenidine could be made. Rilmenidine, at i.v. doses of 100 –300 mg kg 1, presynaptically inhibits noradrenaline release from postganglionic sympathetic neurons in the
pithed rat (see Section 12.1 and Table 6). The receptors mediating slowing of locus coeruleus neurons and presynaptic inhibition of noradrenaline release from central nervous neurons and peripheral sympathetic neurons are identical, predominantly a2A/D-adrenoceptors in the rat
Table 6 Peripheral presynaptic inhibition by clonidine-like drugs in whole animal preparations with an artificial sympathetic tone Species Rat
Experimental model Pithed animal; spinal electrical stimulation Pithed animal; spinal electrical stimulation Pithed animal; spinal electrical stimulation
Rat Rat
Rat
Pithed animal; spinal electrical stimulation
Rat
Pithed animal; spinal electrical stimulation Pithed animal; spinal electrical stimulation
Rat
Rat
Pithed animal; spinal electrical stimulation
Rat Rat (SHR)
Rabbit Rabbit
Rabbit
Cat
Cat Dog
Dog
1
Pithed animal; spinal electrical stimulation Pithed animal; spinal electrical stimulation Pithed animal; spinal electrical stimulation Pithed animal; spinal electrical stimulation
Pithed animal; spinal electrical stimulation Urethane anaesthesia; electrical stimulation of the lumbar sympathetic chain Pithed animal; spinal electrical stimulation Pentobarbitone anaesthesia; electrical stimulation of the cardioaccelerator nerves Pentobarbitone anaesthesia; electrical stimulation of the hepatic nerves
Agonist (route of application)
Effect Inhibition of the increase in heart Inhibition of the increase in heart Inhibition of the increase in heart
evoked rate evoked rate evoked rate
Inhibition of the evoked increase in heart rate and blood pressure Inhibition of the evoked increase in heart rate Inhibition of the evoked increase in the plasma noradrenaline concentration Inhibition of the evoked increase in blood pressure Inhibition of the evoked increase in heart rate and blood pressure Inhibition of the evoked increase in heart rate Inhibition of the evoked increase in the plasma noradrenaline concentration Decrease in total body noradrenaline spillover Decrease in the evoked increase in plasma noradrenaline concentration and heart rate Decrease in the evoked increase in blood pressure and heart rate Inhibition of the evoked increase in hindquarter perfusion pressure
Effective doses1
Reference 1
Clonidine (i.v.)
ED50 = 5 mg kg
Clonidine (i.v.)
ED50 1.6 mg kg
Clonidine (i.v.) UK14304 (i.v.) B-HT920 (i.v.) B-HT933 (i.v.) Clonidine (i.v.) Rilmenidine (i.v.)
ED50 = 1.6 mg kg ED50 = 1.5 mg kg ED50 = 2.8 mg kg ED50 = 38 mg kg 10 mg kg 1 300 mg kg 1
Rilmenidine (i.v.)
ED50 53 mg kg
Clonidine (i.v.)
ED50 = 14 mg kg
1
ED50 = 22 mg kg
1
Pichler & Kobinger, 1978 1
1
de Jonge et al., 1981a van Meel et al., 1981
1 1 1
Laubie et al., 1985
1
van Zwieten et al., 1986 Szemeredi et al., 1988
Clonidine (i.v.) Rilmenidine (i.v.)
10 – 30 mg kg 1 100 – 300 mg kg
Moxonidine (i.v.) Clonidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.) Moxonidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.) Moxonidine (i.v.) UK14304 (i.v.)
ED50 = 60 mg kg 1 ED50 = 18 mg kg 1 0.35 – 24 mg kg 1 min 1 17 – 150 mg kg 1 min 1 1.4 – 83 mg kg 1 min 1 1 mg kg 1 min 1 (< 30 mg kg 1 total dose) 100 – 1000 mg kg 1 10 – 100 mg kg 1 3 – 30 mg kg 1
Clonidine (i.v.) Rilmenidine (i.v.)
15 – 1000 mg kg 30 – 1000 mg kg
Guanabenz (i.v.)
32 – 320 mg kg
Inhibition of the evoked increase in heart rate Inhibition of the evoked increase in noradrenaline concentration in the coronary sinus
Guanfacine (i.v.)
6 – 60 mg kg
Clonidine (i.v.)
15 mg kg
1
Yamaguchi et al., 1977
Inhibition of the evoked increase in noradrenaline concentration in the hepatic vein Inhibition of the evoked increase in vascular resistance
Clonidine (i.v.)
20 mg kg
1
Yamaguchi, 1982
Several of the ED50 values are approximate values read from the dose – response curves in the references.
1
Koenig-Berard et al., 1988 1
1
Armah, 1988 Ha¨user et al., 1995
Majewski et al., 1983 Urban et al., 1995b
Pompermayer et al., 1999
1
1
Baum et al., 1970
Scholtysik et al., 1975
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
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(see above in this section and Section 12.1). In the presence of a peripheral effect of rilmenidine, the missing central effect would indicate poor diffusion of the drug through the blood-brain-barrier. The conclusion from the studies in which central neuronal activity was determined electrically or by measuring Fos protein expression is that systemically administered rilmenidine does not possess selectivity for central sympathoinhibitory receptors, inhibition of locus coeruleus neurons occurs simultaneously. Opposed to these observations are the findings of voltammetric measurements. As pointed out in the case of clonidine (Section 11.1.1), one reason for the discrepancy may be that DOPAC does not faithfully reflect neuronal activity in the NRL and locus coeruleus. Moreover, the release of noradrenaline in the hypothalamus was evoked by stimulation at the high frequency of 10 Hz (Suaud-Chagny et al., 1992). Presynaptic inhibition is weak under this condition (see Starke, 1987).
were compared (Fillastre et al., 1988). The two drugs lowered blood pressure equally. After 2 weeks of treatment, rilmenidine caused sedation in 5% and dry mouth in 12% of the patients (data approximately read from the graphs in Fillastre et al. [1988]), and the frequency of these effects did not change with the duration of treatment. In the clonidinetreated group, the frequency of sedation was 13% and the incidence of dry mouth was 30% after 2 weeks of treatment. The frequency of the clonidine-evoked ‘‘dry mouth’’ did not change with time. The sedative effect of clonidine decreased with time, and 6 weeks after the beginning of therapy, there was no significant difference between the sedative effects of rilmenidine and clonidine. Rilmenidine increases the plasma concentration of growth hormone (unpublished observation mentioned by Verbeuren et al. [1990]). This effect is most probably attributable to activation of a2-adrenoceptors in the hypothalamus (see Devesa et al., 1991; Bertherat et al., 1995; Mu¨ller et al., 1995).
11.1.3. Moxonidine The sedative effect of moxonidine was mentioned in the first publication describing the sympathoinhibitory and general pharmacological properties of moxonidine (Armah et al., 1988). It decreased the locomotor activity of mice with an ED50 value of 7.5 mg kg 1 (s.c.). The corresponding ED50 value for clonidine was 0.35 mg kg 1 (s.c.). Armah et al. (1988) also reported that moxonidine decreases salivation in rabbits by 70% after 100 mg kg 1 (i.v.). This dose is similar to doses causing hypotension in the rabbit (see Table 1). To our knowledge, the effects of moxonidine on the firing rate of locus coeruleus neurons or on the concentration of noradrenaline in the extracellular space in the brain have not been determined.
11.2.3. Moxonidine The incidence of sedation and dry mouth in hypertensive patients receiving long-term moxonidine treatment is 5– 8% and 8– 9%, respectively (for meta-analysis of clinical pharmacological trials, see Schachter et al., 1998). There are three controlled studies in which moxonidine was compared with clonidine. In a double-blind crossover study on 20 hypertensive patients, clonidine (0.3 mg/day) and moxonidine (0.3 mg/day), each administered for 2 weeks, lowered blood pressure equally (Pla¨nitz, 1984). Moxonidine caused sedation (15% vs. 60%) and dry mouth (20% vs. 75%) less frequently than clonidine. In the second study, a doubleblind multicentre study, equal doses of clonidine (0.36 mg/ day) and moxonidine (0.37 mg/day) were administered for 6 weeks to 122 and 30 hypertensive patients, respectively (Pla¨nitz, 1987). The two drugs lowered blood pressure equally. The incidence of sedation was similar in the moxonidine- and clonidine-treated groups (13% vs. 17%), but dry mouth was observed less frequently in moxonidine-treated patients than in clonidine-treated patients (20% vs. 47%). In the third study (MacPhee et al., 1992), on 9 normotensive volunteers, a single oral dose of moxonidine (0.2 mg) lowered blood pressure less than a single oral dose of clonidine (0.2 mg). Moxonidine also caused less sedation and less inhibition of saliva flow than clonidine. Since the hypotensive effects of the two drugs differed, it is difficult to compare the frequencies of their side effects. Moxonidine (0.3 mg orally) increases the plasma concentration of growth hormone for at least 2 hr (Bamberger et al., 1995). Summarizing, the newer antihypertensive drugs rilmenidine and moxonidine were compared with clonidine in four studies, using doses that are equally hypotensive. After acute administration and 2 weeks after the beginning of treatment, rilmenidine and moxonidine elicit less sedation and dry mouth than clonidine. After 6 weeks of treatment,
11.2. Studies in humans 11.2.1. Clonidine It was shown in acute studies on normotensive volunteers that clonidine simultaneously leads to hypotension, sedation, and dry mouth (Dollery et al., 1976; Davies et al., 1977). The side effects are also present in patients treated with clonidine for a long time. However, their frequency, especially that of sedation, decreases with time (e.g., Ferder et al., 1987; Fillastre et al., 1988). 11.2.2. Rilmenidine In an acute study on 10 normotensive volunteers, rilmenidine, in addition to lowering blood pressure, caused sedation and dry mouth (Weerasuriya et al., 1984). Rilmenidine was also compared with clonidine in this study. At small equihypotensive doses, rilmenidine inhibited saliva production less than clonidine, and the sedative effect of rilmenidine was also less intensive. In one double-blind multicentric study on 333 hypertensive patients, the effects of rilmenidine and clonidine, administered for 6 weeks,
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only the frequency of dry mouth differs in the different treatment groups. It is interesting that in increasing the plasma concentration of growth hormone, rilmenidine and moxonidine elicit an effect attributed to activation of central a2-adrenoceptors.
12. Contribution of presynaptic inhibition of transmitter release from postganglionic sympathetic neurons to the overall reduction of sympathetic tone produced by clonidine-like drugs
(see Table 1). As an example, the ED50 value of clonidine for inhibition of the electrically evoked bradycardia in pithed rats is 1.6 mg kg 1 (i.v.) (Table 6) (de Jonge et al., 1981a), very similar to the ED50 values of clonidine for lowering blood pressure and heart rate in anaesthetized rats (2.6 and 1.3 mg kg 1 [i.v.], respectively) (Table 1; de Jonge et al., 1981a). Similarly, at doses of 100 – 300 mg kg 1 (i.v.), rilmenidine inhibits the electrically evoked increases in blood pressure and heart rate in pithed rats (Table 6) (Laubie et al., 1985; Koenig-Berard et al., 1988), and the ED50 values of rilmenidine for lowering blood pressure and heart rate in
In most textbooks and reviews, clonidine-like drugs are classified as centrally acting antihypertensive agents, reflecting the opinion that the sole, or most important, site of action of these drugs is located in the CNS (e.g., Kobinger & Pichler, 1990; Verbeuren et al., 1990; Ernsberger et al., 1997; Bousquet & Feldman, 1999). I believe, however, that inhibition of transmitter release from postganglionic sympathetic neurons is an important component in the action of these drugs, and the evidence for the peripheral presynaptic effect is discussed in this section. 12.1. Peripheral presynaptic inhibition in experimental models with an artificial sympathetic tone Activation of presynaptic a2-autoreceptors inhibits transmitter release from postganglionic sympathetic neurons in almost all isolated cardiovascular tissues that have been studied (for reviews, see Starke, 1977, 1987; Langer, 1981; Starke et al., 1989). The subtype of the a2-adrenoceptor predominantly involved in peripheral presynaptic inhibition is a2A or a2A/D (Starke et al., 1995; Trendelenburg et al., 1997). However, recent observations indicate a role also for a2C-adrenoceptors (Altman et al., 1999; Trendelenburg et al., 1999). Table 6 lists experiments with clonidine-like drugs in whole animal models. An artificial sympathetic tone was created, either by electrical stimulation of the entire sympathetic outflow in pithed animals or of some sympathetic nerve trunks in anaesthetized animals. A constant observation in these studies is that clonidine-like drugs inhibit the electrically evoked responses, i.e., the increase in blood pressure or vascular resistance, the increase in heart rate, and the increase in the plasma noradrenaline concentration. The effect is elicited by clonidine, guanfacine, and guanabenz, but also by the more recently developed drugs rilmenidine and moxonidine. The effect was observed in all species studied: rats, rabbits, dogs, and cats. The most likely mechanism is presynaptic inhibition of transmitter release from postganglionic sympathetic neurons. It is very important that the doses eliciting peripheral presynaptic inhibition of neuro-effector transmission (see Table 6) are essentially identical with the doses that cause central nervous sympathoinhibition, hypotension, and bradycardia in conscious or anaesthetized animals
Fig. 4. Comparison of the effects of rilmenidine and moxonidine on mean arterial pressure (MAP) and RSNA in conscious rabbits and on the plasma noradrenaline concentration (PL-NA) and heart rate (HR) in pithed rabbits. In pithed rabbits, either the entire sympathetic outflow (in experiments in which PL-NA was determined) or only the postganglionic sympathetic nerves of the heart (in experiments in which the HR response was studied) were stimulated. Values are expressed as percentages of the initial values preceding drug administration (PRE). Intravenously administered moxonidine and rilmenidine significantly (stars not shown for sake of clarity) lowered the blood pressure and the firing rate of renal sympathetic nerves in conscious rabbits. They also significantly (stars not shown for sake of clarity) inhibited the electrically evoked increases in the PL-NA and HR in pithed rabbits. The figure demonstrates that in the case of both agonists, the peripheral presynaptic effects occur at the same doses that cause central inhibition of sympathetic nerve activity and decrease of blood pressure in conscious animals. It is very likely that the peripheral presynaptic inhibition contributes to the overall reduction of sympathetic tone, and, therefore, to the decrease in blood pressure in conscious rabbits. Data from Urban et al. (1994, 1995a, 1995b).
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anaesthetized rats are 250 and 350 mg kg 1 (i.v.), respectively (Table 1) (Gomez et al., 1991). The identity of doses eliciting peripheral effects in pithed animals and overall sympathoinhibition in intact animals was also shown for rabbits. In Fig. 4, the effects of rilmenidine and moxonidine in pithed and conscious rabbits are compared. In pithed rabbits with electrically stimulated sympathetic outflow, rilmenidine and moxonidine inhibit the electrically evoked increase in the plasma noradrenaline concentration and heart rate. Both effects reflect the peripherally elicited decrease in noradrenaline release from sympathetic neurons. In conscious rabbits, rilmenidine and moxonidine inhibit the firing of renal sympathetic nerves (an indication of central sympathoinhibition) and lower blood pressure (an indication of the overall reduction of sympathetic tone) (Fig. 4). The identity of doses causing peripheral and central sympathoinhibition and overall sympathoinhibition is evident. It may be added that the doses of rilmenidine and moxonidine causing hypotension and central sympathoinhibition in our experiments are identical with the doses causing hypotension and bradycardia in other experiments in rabbits (Table 1) (Armah et al., 1988; Head & Burke, 1991). Since peripheral presynaptic inhibition in animals with artificial sympathetic tone occurs exactly at the same doses that produce central sympathoinhibition and hypotension in intact animals, it is reasonable to assume that the peripheral effect also occurs in intact animals and contributes to the overall reduction of sympathetic tone. 12.2. Peripheral presynaptic inhibition under physiological conditions It is difficult to detect the peripheral presynaptic effect of clonidine-like drugs in vivo during ongoing physiological sympathetic impulse traffic because the drugs simultaneously inhibit sympathetic nerve firing by a central mechanism. It is difficult to determine which portion of the overall sympathoinhibition is attributable to the peripheral action and which is attributable to the central action. Two approaches were used to analyze the role of the peripheral presynaptic inhibition. In one approach, the effect of the pharmacological tools was restricted either to the central or to the peripheral compartment. Sannajust et al. (1992b) compared the effects of intracerebroventricularly and intravenously administered clonidine and rilmenidine in conscious SHR. The drugs lowered blood pressure much less after central administration than after systemical administration. Accordingly, the authors concluded that the findings ‘‘question the involvement of a major central site of action for these antihypertensive a2-adrenoceptor agonists.’’ In the experiments of Brown and Harland (1984) in rats, clonidine was administered systemically and idazoxan was administered intracerebroventricularly. Peripheral presynaptic effects of clonidine became evident from the finding that idazoxan
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prevented the clonidine-evoked hypotension, but not the clonidine-evoked decrease in heart rate and the plasma noradrenaline concentration. In similar experiments in rabbits, we showed that the decrease in heart rate and plasma noradrenaline concentration caused by intravenously injected moxonidine was resistant to intracisternally administered yohimbine (Urban et al., 1995a). A peripheral presynaptic effect of systemically administered clonidine was also shown in humans with the help of MK-467, a peripherally acting a2-adrenoceptor antagonist (Warren et al., 1991). Interesting are the findings obtained with St-91, an analogue of clonidine acting selectively in the periphery. In conscious rats, it elicits hypotension (Gutkowska et al., 1997), indicating that peripheral presynaptic inhibition alone is capable of lowering blood pressure. However, in anaesthetized sheep, St-91 increases systemic vascular resistance and blood pressure (Celly et al., 1999). In two experiments in humans, the effects of clonidine were restricted to the peripheral nervous system, and no sympathoinhibition was observed. In the study of Kiowski et al. (1985), clonidine was infused locally into the brachial artery, and it did not change noradrenaline release in the forearm. The value of this finding is diminished by the oversimplified method of calculation of noradrenaline release: spillover was calculated by multiplying the venoarterial noradrenaline difference with forearm blood flow (for measurement of organ specific catecholamine spillover, see Esler et al., 1990). Kooner et al. (1991) studied the effects of systemically administered clonidine in tetraplegic patients. Since clonidine did not change the increase in blood pressure and skin vascular resistance evoked by bladder stimulation, the authors argued against a peripheral presynaptic inhibition by clonidine. Another approach for identifying peripheral presynaptic action in intact animals is by measuring the sympathetic nerve firing rate, noradrenaline release, and blood pressure simultaneously. Garty et al. (1990) compared the effect of intravenously administered clonidine on the firing rate of renal sympathetic nerves with the effect on renal noradrenaline spillover. Clonidine lowered renal noradrenaline spillover, and this effect was solely attributable to the centrally elicited decrease in renal sympathetic nerve firing. There was no indication of peripheral presynaptic inhibition. It must be noted that sympathetic nerves were probably firing at very high rates in the anaesthetized rats studied by Garty et al. (1990) (arterial plasma noradrenaline level > 1 ng mL 1). It is difficult to detect peripheral presynaptic inhibition under this condition since presynaptic inhibition becomes weak when axon terminals are stimulated at high frequencies (see Starke, 1977, 1987). In a study in anaesthetized rabbits, clonidine was administered systemically, and the effect on the firing rate of renal sympathetic nerves and the plasma noradrenaline concentration was determined (Szabo et al., 1989). Clonidine lowered the arterial plasma noradrenaline concentration disproportionally more than was expected from the decrease in renal nerve firing rate, and this was
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considered as an indication of a peripheral presynaptic action. A weak point of this latter study is that renal sympathetic nerve firing was compared with the arterial plasma noradrenaline concentration, which reflects noradrenaline release from all sympathetically innervated tissues. Wallin and Frisk-Holmberg (1981) observed in humans that clonidine can lower blood pressure without inhibiting muscle sympathetic nerve activity, and suggested that clonidine ‘‘influences sympathetic outflow by a combination of central and peripheral effects.’’ 12.3. Presynaptic imidazoline receptors Molderings et al. (1991) and Go¨thert and Molderings (1991) described that certain imidazoline derivatives (e.g., clonidine, idazoxan, cirazoline, BDF6143) and guanidine derivatives (e.g., aganodine) inhibit electrically evoked noradrenaline release from the rabbit pulmonary artery and aorta, an inhibition that could not be explained by activation of a2-adrenoceptors. Since then, this observation was extended to the rabbit heart (Fuder & Schwarz, 1993), human atrium and pulmonary artery (Likungu et al., 1996; Molderings et al., 1997), and rat aorta and vena cava (Molderings & Go¨thert, 1998). The existence of a presynaptic imidazoline receptor was postulated. The presynaptic imidazoline receptor is distinguished from the a2-adrenoceptor by its relative insensitivity to rauwolscine (Molderings et al., 1991). The presynaptic imidazoline receptor is not similar to the I1 and I2 imidazoline-binding sites (Molderings & Go¨thert, 1995). Recent observations indicate a mutual interaction between the presynaptic imidazoline receptor and the CB1 cannabinoid receptor (Molderings et al., 1999). It should be noted that under stimulation conditions that do not activate a2-autoinhibition, the function of the presynaptic imidazoline receptor is not apparent (Gaiser et al., 1999). Can the peripheral presynaptic imidazoline receptor play a role in the sympathoinhibition elicited by clonidine-like antihypertensive drugs? The answer is negative. The drugs used in therapy are either not effective at the presynaptic imidazoline receptor (moxonidine and rilmenidine) (Molderings et al., 1991, 2000) or have an effect only at concentrations that are much higher than therapeutic plasma concentrations (clonidine) (Molderings et al., 1991). 12.4. Penetration of clonidine-like drugs into the brain Clonidine and its chemical relatives are often described as lipophilic drugs. Due to their lipophilicity, these drugs should easily penetrate into the brain, where they could reach higher concentrations than in the plasma. Such a distribution pattern would support a preferential central sympathoinhibitory effect. The octanol/buffer partition coefficient of clonidine is in the range 2.7 –8.1 (Kobinger & Pichler, 1975; de Jonge et al., 1981a; Armah et al., 1988), and that of moxonidine is
0.88 (Armah et al., 1988). The corresponding coefficient for rilmenidine is not known. For comparison, the octanol/water partition coefficient of a strongly lipophilic drug, D9-tetrahydrocannabinol, is 12091 (Thomas et al., 1990). It is clear, therefore, that clonidine and moxonidine are only very moderately lipophilic. Endothelial cells of brain microvessels possess a carrier for clonidine. The role of this carrier in the passage of clonidine through the blood-brain barrier, however, is not known (Huwyler et al., 1997). The distribution pattern of intravenously administered clonidine is in accord with its moderate lipophilicity. Thus, ‘‘at the peak of the hypotensive response less than 2% of the injected dose is present in the brain and at least equal concentrations of the drug are found in most peripheral tissues’’ (Conway & Jarrott, 1980). Accordingly, the authors conclude that ‘‘the possibility of peripheral mechanisms contributing to the hypotensive effect cannot be dismissed.’’ Considering that rilmenidine and moxonidine are regarded as centrally acting drugs, it is surprising that, to our knowledge, the concentrations of these drugs in the brain after peripheral application have not been determined. Since there are no data on the penetration of rilmenidine and moxonidine into the brain, a hypothesis may be allowed: rilmenidine and moxonidine penetrate less efficiently into the brain than clonidine. This can be expected for moxonidine, since its octanol/buffer partition coefficient is lower than that of clonidine (see above). The results of two in vivo functional studies are well-compatible with the above hypothesis. First, in anaesthetized rats, intravenously applied clonidine and rilmenidine inhibited the firing of locus coeruleus neurons; clonidine (ED50 = 5.5 mg kg 1) was 64-fold more potent than rilmenidine (ED50 = 350 mg kg 1) (Dresse & Scuve´e-Moreau, 1986). After iontophoretic application directly into the locus coeruleus, clonidine (ED50 = 463 nA iontophoretic current) was only 5-fold more potent than rilmenidine (ED50 = 2500 nA current) (Dresse & Scuve´e-Moreau, 1986). (Admittedly, the strength of iontophoretic current is a suboptimal surrogate for doses of the centrally applied drugs.) Second, in anaesthetized rabbits, intravenously applied clonidine (ED20 = 10 mg kg 1; the dose lowering blood pressure by 20%) and moxonidine (ED20 = 30 mg kg 1) lowered the blood pressure, and clonidine was 3-fold more potent than moxonidine (Armah, 1987). The potency relationship reversed after intracisternal application, moxonidine (ED20 = 0.1 mg kg 1) being 5-fold more potent than clonidine (ED20 = 0.5 mg kg 1). Thus, the relative potencies of rilmenidine and moxonidine versus clonidine increased if the drugs were administered directly into the CNS, an observation well compatible with the hypothesis that rilmenidine and moxonidine penetrate into the brain less efficiently than clonidine. A selective peripheral action of rilmenidine would be indicated also by the observation that rilmenidine, at doses 7.5 mg kg 1 (i.v.), does not inhibit noradrenaline release in the locus coeruleus and hypothalamus (Tibiric¸a et al., 1991b; Suaud-Chagny et al., 1992; see Section 11.1.2).
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I conclude that the pharmacokinetic evidence for a preferential central nervous action of clonidine-like drugs is weak or does not exist. Thus, there is good reason to assume that the peripheral presynaptic inhibition occurs simultaneously with the central sympathoinhibition. If moxonidine and rilmenidine indeed penetrate less efficiently into the brain than clonidine, then the weight of peripheral presynaptic inhibition in the overall reduction of sympathetic tone is expected to increase. In addition, the frequency and intensity of central nervous side effects, most of all sedation, are expected to decrease. It is tempting to speculate that the improved effect/side effect profiles of rilmenidine and moxonidine in humans (see Section 11.2) are simply due to the fact that these drugs penetrate less efficiently into the brain than clonidine. 12.5. Summary of the role of peripheral presynaptic inhibition It was shown in the preceding sections that clonidine-like drugs cause presynaptic inhibition in peripheral models with artificial sympathetic tone. The doses were identical with the doses causing sympathoinhibition, hypotension, and bradycardia in intact animals. Peripheral presynaptic inhibition was shown in several studies in intact animals and humans. The failure to demonstrate peripheral presynaptic inhibition in some experiments on animals and humans with ongoing sympathetic impulse traffic is probably due to unsuitable experimental conditions and methods. Moreover, there is no reason to believe that these drugs, through selective accumulation in the brain, elicit effects preferentially in the CNS. Therefore, I suggest that the overall effect of clonidinelike drugs on the cardiovascular system develops as the sum of three primary actions. One action is, undoubtedly, central sympathoinhibition. The second component of the sympathoinhibition is peripheral inhibition of transmitter release from postganglionic sympathetic axons. Interestingly, the two inhibitory effects ‘‘cooperate’’ well. As a consequence of the central sympathoinhibition, the firing rate of sympathetic axons decreases. Presynaptic inhibition, which is frequency-dependent, becomes especially powerful at low nerve firing rates. These two inhibitory actions counteract the third effect of clonidine-like drugs, direct vasoconstriction mediated by a2-adrenoceptors on vascular smooth muscle cells and, finally, blood pressure decreases. Peripheral presynaptic inhibition alone is capable of preventing the blood pressure increase due to vasoconstriction. However, additional central sympathoinhibition is necessary to produce blood pressure decrease.
13. Conclusion Finally, after the critical evaluation of experimental data, the observations that support or are compatible with the imidazoline mode of action are presented in Table 7. Table 8
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Table 7 Observations that support or are compatible with the imidazoline mode of action – In humans, the new ‘‘selective’’ imidazoline-binding site ligands, rilmenidine and moxonidine, lower blood pressure like clonidine. After short-term administration, they cause sedation and inhibit saliva secretion less than clonidine. During long-term therapy, the new drugs cause dry mouth less frequently than clonidine. – In two studies, rilmenidine had no agonist activity at human a2-adrenoceptors. – Some potent a2-adrenoceptor agonists, such as guanabenz and guanfacine, do not cause hypotension when they are injected into the RVLM or applied intracisternally. – In a few studies, the mixed I1/a2 antagonists idazoxan and efaroxan were more effective against rilmenidine and moxonidine than pure a2-adrenoceptor antagonists that produced equal a2-adrenoceptor blockade. – The hypotensive effect of moxonidine, injected into the RVLM of anaesthetized mice, persists in animals in which the a2A/D-adrenoceptor is genetically defect.
summarizes the observations that do not support the imidazoline mode of action and observations that do not fit the predictions of the imidazoline hypothesis. Several of these observations directly support a role for a2-adrenoceptors. Thus, many predictions of the imidazoline hypothesis are not fulfilled, and the uncertainties of the hypothesis seem to outweigh the observations that support it. Therefore, I believe that the pattern of effects of clonidine-like drugs should be explained within the framework of a2-adrenoceptors. The a2-adrenoceptors are generally well-characterized. They are suitably localized in cardiovascular centres and sympathetic neurons to cause sympathoinhibition. The mechanisms of the a2-adrenoceptor-mediated sympathoinhibition in the medulla oblongata and in the sympathetic nervous system are well-characterized. One hypothesis suggests that a2-adrenoceptors and I1 imidazoline receptors coexist and cooperate. Accordingly, both pure a2-adrenoceptor agonists and I1 imidazoline receptor agonists are capable of lowering sympathetic tone by a primary action in the RVLM. The a2-adrenoceptor agonists directly inhibit presympathetic RVLM neurons. The I1 imidazoline agonists increase the release of catecholamines in the RVLM, and the catecholamines, in turn, depress presympathetic RVLM neurons by activating a2-adrenoceptors. This hypothesis can explain the frequent observation that a2-adrenoceptor antagonists counteract the effects of imidazoline hypotensive drugs. However, the key element of this hypothesis — imidazolines increase the release of catecholamines in the RVLM — has not been verified experimentally. Considering this and the uncertainties of the imidazoline hypothesis, I still suggest that the effects of imidazoline antihypertensive drugs should be explained with the primary activation of a2-adrenoceptors. Why then do rilmenidine and moxonidine inhibit saliva secretion and cause sedation in humans less than clonidine? There is no convincing answer. It seems that research
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Table 8 Observations that do not support the imidazoline mode of action and that do not fit the predictions of the imidazoline hypothesis – No selective imidazoline receptor agonist or antagonist has been identified during the 17 years of the existence of the imidazoline hypothesis. AGN192403 proved to be I1-selective in radioligand-binding experiments, but it has no effect on RVLM neurons and on blood pressure. No functional studies have been carried out with another selective ligand, S23230. Different groups have found strongly differing affinity values of key agonists and antagonists for I1-binding sites. For lack of reliable tools, the conclusions of many in vivo studies are fragile. – Despite the intensive work in several laboratories, no I1 imidazoline receptor protein has been identified. Recently, a protein (IRAS-1) with I1 imidazoline-binding capability was discovered. IRAS-1 mRNA is not distributed in the brain as expected for the I1 imidazoline receptor. It is not clear whether the IRAS-1 molecule can function as a pharmacological receptor, carrying out the functions expected from the I1 imidazoline receptor – The activity of spinally projecting sympathoexcitatory RVLM neurons is inhibited in vitro by catecholamine agonists and moxonidine via a2-adrenoceptors, and there is no indication for involvement of I1 imidazoline receptors. The mechanism of the inhibition is clarified and agrees well with the distribution of a2-adrenoceptors in the RVLM. – It is not true that only drugs with affinity for I1 imidazoline-binding sites lower blood pressure when injected into the RVLM. Catecholamines also produce sympathoinhibition. – The hypotensive and sympathoinhibitory effects of clonidine, rilmenidine, and moxonidine were antagonized in many experiments by selective a2-adrenoceptor antagonists. The failure in several experiments to block effects of clonidine-like drugs by a2-adrenoceptor antagonists is likely to be due to insufficient dosing of the antagonists. In several experiments, the mixed I1/a2 antagonists idazoxan and efaroxan had no I1 antagonistic effects that surpassed their a2-blocking actions. – Systemically injected clonidine, rilmenidine, and moxonidine do not lower blood pressure in conscious mice with genetically defect a2A-adrenoceptors. – It is not true that selective I1 imidazoline agonists selectively lower blood pressure, causing only few a2-adrenoceptor-mediated effects. Simultaneous inhibition of locus coeruleus neurons was shown with electrophysiological and neuroanatomical methods. The selective sympathoinhibition in animal experiments is only supported by studies in which DOPAC or noradrenaline were measured with voltammetry. Rilmenidine and moxonidine increase the plasma concentration of growth hormone in humans, an effect attributed to activation of a2-adrenoceptors. – SHR are used to demonstrate I1 imidazoline receptor-mediated hypotension by some researchers, whereas others believe that such a mechanism does not operate in these animals. – There is substantial evidence for activation of peripheral presynaptic a2-adrenoceptors on axon terminals of postganglionic sympathetic neurons by clonidine-like drugs. The peripheral presynaptic inhibition very likely contributes to the overall sympathoinhibition.
concentrated too much on the imidazoline mode of action, and search for alternative explanations was not pursued intensively enough. As pointed out, the diffusion of rilmenidine and moxonidine into the CNS has not been systemically studied. It is conceivable that they cross the blood-brain barrier less readily than clonidine. If this were true, then their sympathoinhibitory action would rely to a greater extent on peripheral presynaptic inhibition than the sympathoinhibitory action of clonidine. They would also evoke centrally evoked side effects less frequently. The affinity and efficacy of these drugs at functional subtypes of a2-adrenoceptors in native tissues has also not been systemically determined. It may be that selective agonist activity at one of the subtypes turns rilmenidine and moxonidine into effective antihypertensive agents with few side effects. The highly intriguing finding that rilmenidine has no agonist activity at the human a2A-adrenoceptor may be the first important observation in this direction. The affinity and efficacy for postjunctional a1-adrenoceptor subtypes should also be systemically studied. The explanation for the improved effect/side effect profile of rilmenidine and moxonidine may be a hitherto not discovered affinity of these drugs for a neurotransmitter receptor, neurotransmitter transporter, neurotransmitter metabolizing enzyme, or ion channel. It may also turn out that the mechanism of action is not the same in the case of rilmenidine and moxonidine. Research in this field is worth pursuing, since, as mentioned in Section 1, this group of drugs has the unique ability to decrease pathologically elevated sympathetic tone.
Acknowledgements This study was supported by the Deutsche Forschungsgemeinschaft (Sz 72/2-3). I thank Dr. Klaus Starke for his critical comments on the manuscript. The help of Ilka Wallmichrath in the preparation of this manuscript is gratefully acknowledged.
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Solomon, R. E., Brody, M. J., & Gebhart, G. F. (1989). Pharmacological characterization of alpha adrenoceptors involved in the antinociceptive and cardiovascular effects of intrathecally administered clonidine. J Pharmacol Exp Ther 251, 27 – 38. Starke, K. (1977). Regulation of noradrenaline release by presynaptic receptor systems. Rev Physiol Biochem Pharmacol 77, 1 – 124. Starke, K. (1987). Presynaptic a-autoreceptors. Rev Physiol Biochem Pharmacol 107, 74 – 146. Starke, K., Go¨thert, M., & Kilbinger, H. (1989). Modulation of neurotransmitter release by presynaptic a2-autoreceptors. Physiol Rev 69, 864 – 989. Starke, K., Trendelenburg, A. U., & Limberger, N. (1995). Presynaptic alpha2-adrenoceptors: subtype determination. Pharmacol Commun 6, 99 – 108. Suaud-Chagny, M. F., Mermet, C., Tibiric¸a, E., Bousquet, P., & Gonon, F. (1992). Does rilmenidine act in vivo on central a2-adrenoceptors modulating noradrenaline release? Eur J Pharmacol 213, 305 – 307. Summers, R. J., & McMartin, L. R. (1993). Adrenoceptors and their second messenger systems. J Neurochem 60, 10 – 23. Sun, M.-K. (1996). Pharmacology of reticulospinal vasomotor neurons in cardiovascular regulation. Pharmacol Rev 48, 465 – 494. Sun, M.-K., & Guyenet, P. G. (1986). Effect of clonidine and g-aminobutyric acid on the discharges of medullo-spinal sympathoexcitatory neurons in the rat. Brain Res 368, 1 – 17. Sun, M.-K., & Reis, D. J. (1995). GABA-mediated inhibition of pacemaker neurons of rostral ventrolateral medulla by clonidine in vitro. Eur J Pharmacol 276, 291 – 296. Surprenant, A., Horstman, D. A., Akbarali, H., & Limbrid, L. E. (1992). A point mutation of the a2-adrenoceptor that blocks coupling to potassium but not calcium currents. Science 257, 977 – 980. Svensson, T. H., Bunney, B. S., & Aghajanian, G. K. (1975). Inhibition of both noradrenergic and serotonergic neurons in brain by the a-adrenergic agonist clonidine. Brain Res 92, 291 – 306. Swedberg, K., Bergh, C.-H., Dickstein, K., McNay, J., & Steinberg, M. (2000). The effects of moxonidine, a novel imidazoline, on plasma norepinephrine in patients with congestive heart failure. J Am Coll Cardiol 35, 398 – 404. Szabo, B., & Urban, R. (1995). Mechanism of sympathoinhibition by imidazolines. Ann N Y Acad Sci 763, 552 – 565. Szabo, B., & Urban, R. (1997). Role of I1 imidazoline receptors in the sympathoinhibiton produced by intracisternally administered rilmenidine and moxonidine. Arzneimittelforschung 47, 1009 – 1015. Szabo, B., Hedler, L., & Starke, K. (1989). Peripheral presynaptic and central effects of clonidine, yohimbine and rauwolscine on the sympathetic nervous system in rabbits. Naunyn Schmiedebergs Arch Pharmacol 340, 648 – 657. Szabo, B., Karsch, V., & Starke, K. (1992a). Effects of inhibitors of neuronal uptake of 5-HT on sympathetic cardiovascular regulation. J Cardiovasc Pharmacol 20, 99 – 107. Szabo, B., Schramm, A., & Starke, K. (1992b). Effect of yohimbine on renal sympathetic nerve activity and renal noradrenaline spillover in anesthetized rabbits. J Pharmacol Exp Ther 260, 780 – 788. Szabo, B., Urban, R., & Starke, K. (1993). Sympathoinhibition by rilmenidine in conscious rabbits: involvement of a2-adrenoceptors. Naunyn Schmiedebergs Arch Pharmacol 348, 593 – 600. Szabo, B., Fro¨hlich, R., & Illes, P. (1996). No evidence for functional imidazoline receptors on locus coeruleus neurons. Naunyn Schmiedebergs Arch Pharmacol 353, 557 – 563. Szemeredi, K., Zukowska-Grojec, Z., Bagdy, G., Hill, J., & Kopin, I. J. (1988). Evidence for a direct peripheral effect of clonidine on the norepinephrine release in vivo in pithed rats. Eur J Pharmacol 145, 251 – 255. Talley, E. M., Rosin, D. L., Lee, A., Guyenet, P. G., & Lynch, K. R. (1996). Distribution of a2A-adrenergic receptor-like immunoreactivity in the rat central nervous system. J Comp Neurol 372, 111 – 134. Thomas, B. F., Compton, D. R., & Martin, B. R. (1990). Characterization of the lipophilicity of natural and synthetic analogs of D9-tetrahydrocan-
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B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35 Vayssettes-Courchay, C., Bouysset, F., Cordi, A. A., Laubie, M., & Verbeuren, T. J. (1996). A comparative study of the reversal by different a2-adrenoceptor antagonists of the central sympatho-inhibitory effect of clonidine. Br J Pharmacol 117, 587 – 593. Verbeuren, T. J., Jordaens, F. H., Zonnekeyn, L. L., & Herman, A. G. (1986). Effect of the amino-oxazoline derivative S-3341 on pre- and postjunctional a-adrenoceptors in isolated blood vessels. Arch Int Pharmacodyn Ther 284, 38 – 52. Verbeuren, T. J., Xuan, A. T. D., Koenig-Be´rard, E., & Vitou, P. (1990). Rilmenidine. Cardiovasc Drug Rev 8, 56 – 70. Wallin, B. G., & Frisk-Holmberg, M. (1981). The antihypertensive mechanism of clonidine in man. Evidence against a generalized reduction of sympathetic activity. Hypertension 3, 340 – 346. Wang, H., Regunathan, S., Ruggiero, D. A., & Reis, D. J. (1993). Production and characterization of antibodies specific for the imidazoline receptor protein. Mol Pharmacol 43, 509 – 515. Warren, J. B., Dollery, C. T., Sciberras, D., & Goldberg, M. R. (1991). Assessment of MK-467, a peripheral a2-adrenergic receptor antagonist, with intravenous clonidine. Clin Pharmacol Ther 50, 71 – 77. Weerasuriya, K., Shaw, E., & Turner, P. (1984). Preliminary clinical pharmacological studies of S3341, a new hypotensive agent, and comparison with clonidine in normal males. Eur J Clin Pharmacol 27, 281 – 286. Wenzel, R. R., Spieker, L., Qui, S., Shaw, S., Lu¨scher, T. F., & Noll, G.
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(1998). I1-Imidazoline agonist moxonidine decreases sympathetic nerve activity and blood pressure in hypertensives. Hypertension 32, 1022 – 1027. Winter, J. C., & Rabin, R. A. (1992). Yohimbine as a serotonergic agent: evidence from receptor binding and drug discrimination. J Pharmacol Exp Ther 263, 682 – 689. Yamaguchi, N. (1982). Evidence supporting the existence of presynaptic a-adrenoceptors in the regulation of endogenous noradrenaline release upon hepatic sympathetic nerve stimulation in the dog liver in vivo. Naunyn Schmiedebergs Arch Pharmacol 321, 177 – 184. Yamaguchi, N., de Champlain, J., & Nadeau, R. A. (1977). Regulation of norepinephrine release from cardiac sympathetic fibers in the dog by presynaptic a- and b-receptors. Circ Res 41, 108 – 117. Zandberg, P., de Jong, W., & de Wied, D. (1979). Effect of catecholaminereceptor stimulating agents on blood pressure after local application in the nucleus tractus solitarii of the medulla oblongata. Eur J Pharmacol 55, 43 – 56. Zhu, Q.-M., Lesnick, J. D., Jasper, J. R., MacLennan, S. J., Dillon, M. P., Eglen, R. M., & Blue, D. R., Jr. (1999). Cardiovascular effects of rilmenidine, moxonidine and clonidine in conscious wild-type and D79N a2A -adrenoceptor transgenic mice. Br J Pharmacol 126, 1522 – 1530.
Associate editor: H. Bo¨nisch
Imidazoline antihypertensive drugs: a critical review on their mechanism of action Bela Szabo* Institut fu¨r Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universita¨t, Albertstrasse 25, D-79104 Freiburg i. Br., Germany
Abstract It was long thought that the prototypical centrally acting antihypertensive drug clonidine lowers sympathetic tone by activating a2-adrenoceptors in the brain stem. Supported by the development of two new centrally acting drugs, rilmenidine and moxonidine, the imidazoline hypothesis evolved recently. It assumes the existence of a new group of receptors, the imidazoline receptors, and attributes the sympathoinhibition to activation of I1 imidazoline receptors in the medulla oblongata. This review analyzes the mechanism of action of clonidine-like drugs, with special attention given to the imidazoline hypothesis. Two conclusions are drawn. The first is that the arguments against the imidazoline hypothesis outweigh the observations that support it and that the sympathoinhibitory effects of clonidine-like drugs are best explained by activation of a2-adrenoceptors. The second conclusion is that this class of drugs lowers sympathetic tone not only by a primary action in cardiovascular regulatory centres in the medulla oblongata. Peripheral presynaptic inhibition of transmitter release from postganglionic sympathetic neurons contributes to the overall sympathoinhibition. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Imidazoline receptor; Blood pressure; Presynaptic receptor; Rostral ventrolateral medulla; Sympathetic nervous system; a2-Adrenoceptor Abbreviations: AGN192403, 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane; CHO, Chinese hamster ovary; DOPAC, 3,4-dihydroxyphenylacetic acid; GABA, g-aminobutyric acid; HEK, human embryonic kidney; 5-HT, 5-hydroxytryptamine; IRAS, imidazoline receptor-antisera-selected; IRP, imidazoline receptor protein; NRL, nucleus reticularis lateralis (mostly used as a synonym for RVLM); NTS, nucleus tractus solitarii; RSNA, renal sympathetic nerve activity; RVLM, rostral ventrolateral medulla; S23230, (-)-(5-[2-methyl-phenoxy-methyl]-1,3-oxazolin-2-yl)amine; SHR, spontaneously hypertensive rats; SK&F86466, 6-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benzazepine HCl
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 2. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Mode of action of clonidine-like drugs: two hypotheses . . . 3.1. The a2-adrenoceptor hypothesis . . . . . . . . . . . . 3.2. The imidazoline hypothesis . . . . . . . . . . . . . . 4. Characterization of drugs used in imidazoline cardiovascular binding studies . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Agonists . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Selective a2-adrenoceptor agonists . . . . . . 4.1.2. Selective I1 imidazoline receptor agonists . . 4.2. Antagonists . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Selective a2-adrenoceptor antagonists . . . . 4.2.2. Selective I1 imidazoline receptor antagonists . 5. The central nervous site of action of clonidine-like drugs . . 5.1. Action in the rostral ventrolateral medulla. . . . . . .
* Tel.: +49-761-203-5312; fax: +49-761-203-5318. E-mail address: [email protected] (B. Szabo). 0163-7258/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 3 - 7 2 5 8 ( 0 1 ) 0 0 1 7 0 - X
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5.2. Action in the nucleus tractus solitarii . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Action in the spinal cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characterization of a2-adrenoceptors and I1 imidazoline-binding sites . . . . . . . . . . . . . . . 6.1. a2-Adrenoceptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1. Molecular identity, second messenger mechanisms . . . . . . . . . . . . . . . . . 6.1.2. Distribution in cardiovascular regulatory centres . . . . . . . . . . . . . . . . . . 6.2. I1 imidazoline receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1. Molecular identity, second messenger mechanisms . . . . . . . . . . . . . . . . . 6.2.2. Distribution in cardiovascular regulatory centres . . . . . . . . . . . . . . . . . . Action of clonidine-like drugs on cardiovascular regulatory nuclei in vitro . . . . . . . . . . . . Correlation between sympathoinhibition and affinity for I1 imidazoline-binding sites and a2-adrenoceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Cats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. Rabbits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4. Monkeys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interaction of agonists with antagonists in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. Studies supporting involvement of a2-adrenoceptors. . . . . . . . . . . . . . . . . . . . 9.2. Studies supporting involvement of I1 receptors . . . . . . . . . . . . . . . . . . . . . . Experiments in genetically modified animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . Relationship between sympathoinhibition and other a2-adrenoceptor-mediated effects. . . . . . . 11.1. Animal experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1.1. Clonidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1.2. Rilmenidine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1.3. Moxonidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2. Studies in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.1. Clonidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.2. Rilmenidine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.3. Moxonidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contribution of presynaptic inhibition of transmitter release from postganglionic sympathetic neurons to the overall reduction of sympathetic tone produced by clonidine-like drugs . . . . . . 12.1. Peripheral presynaptic inhibition in experimental models with an artificial sympathetic tone 12.2. Peripheral presynaptic inhibition under physiological conditions . . . . . . . . . . . . . . 12.3. Presynaptic imidazoline receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4. Penetration of clonidine-like drugs into the brain . . . . . . . . . . . . . . . . . . . . . . 12.5. Summary of the role of peripheral presynaptic inhibition . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Increased release of transmitter from sympathetic neurons — increased sympathetic tone — is an early link in the pathogenesis of essential hypertension in many patients (see Esler & Kaye, 1998). Two groups of the available antihypertensive drugs prevent increased activation of adrenoceptors in cardiovascular tissues, the b- and a1-adrenoceptor antagonists. Reducing transmitter release from sympathetic neurons, i.e., reducing sympathetic tone, is a logical approach, in a certain sense a causal therapy, to lower blood pressure. Clonidine-like antihypertensive drugs lower sympathetic tone. In addition to the older drugs, clonidine, a-methyldopa, guanfacine, and guanabenz, two drugs were introduced into therapy recently, rilmenidine and moxonidine (see Fig. 1 for chemical structures of the drugs). A renaissance of this class of drugs can be observed, which is also supported by new ideas regarding their mechanism of
10 11 11 11 11 11 12 12 12 13 14 14 14 15 15 15 15 18 19 20 20 20 21 23 23 23 23 23 24 24 25 26 26 27 27 28 28
action. The interest in these drugs is also increasing because they may be beneficial in cardiac failure (Manolis et al., 1995; Azevedo et al., 1999; Swedberg et al., 2000) and because they possess analgesic – anaesthetic properties (Pertovaara, 1993). While it is generally accepted that clonidine-like drugs lower sympathetic tone, several hypotheses have been developed to explain the mechanism. The aim of this review is to critically analyze these hypotheses.
2. History A short history of the pharmacology of these drugs is given at first to show the development of ideas. The discovery of imidazolines goes back to 1939. Scientists at Ciba in Basel in Switzerland wanted to obtain new drugs by combining the phenylethylamine moiety of adrenaline with the imidazole structure of histamine (Hartmann & Isler,
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
3
Fig. 1. Structure of agonists and antagonists used in imidazoline cardiovascular research. I1, affinity for I1-binding sites; a2, affinity for a2-adrenoceptors.
1939). They obtained the a-adrenoceptor antagonist tolazoline. It turned out to possess therapeutically useful vasodilating properties. Another of the discovered drugs, naphazoline, is used even today to relieve nasal congestion. In an attempt to produce a new nasal decongestant, the chemist H. Sta¨hle synthesized the compound St155 at Boehringer Ingelheim in Germany in 1962. Unexpectedly, St155 caused strong sedation, and this effect was essential for recognizing that a new class of compounds had been discovered. St155, later named clonidine, also lowered blood pressure and heart rate and inhibited saliva secretion (Hoefke & Kobinger, 1966; see also Klingspohr, 1983). Clonidine turned out to be the prototype of centrally acting antihypertensive drugs. Other centrally acting antihypertensive drugs followed: guanabenz (Baum et al., 1970), guan-
facine (Scholtysik et al., 1975), rilmenidine (Laubie et al., 1985), and moxonidine (Armah, 1987; Armah et al., 1988). The development of a-methyldopa followed a different course, which will not be outlined here. It is actually the oldest centrally acting antihypertensive drug (Oates et al., 1960). During the 20 years following the discovery of clonidine, it was shown that clonidine and its relatives decrease blood pressure by causing central sympathoinhibition and bradycardia. It was thought that activation of a2-adrenoceptors in the medulla oblongata is responsible for the central sympathoinhibition (for reviews, see Schmitt, 1977; Kobinger, 1978; Van Zwieten et al., 1984; Kobinger & Pichler, 1990). In 1984, Bousquet et al. hypothesized a new mechanism of action for clonidine-like drugs. It was suggested that a new receptor, the imidazoline receptor,
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B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
Table 1 Hypotensive and sympathoinhibitory doses of systemically administered clonidine-like drugs Species
Anaesthesia
Effect
Rat
Pentobarbitone
Decrease in blood pressure Decrease in heart rate Decrease in blood pressure and the catecholamine oxidation peak in the NRL Decrease in blood pressure and heart rate Decrease in blood pressure and heart rate
Rat
Pentobarbitone
Rat Rat
Pentobarbitone Pentobarbitone
Rat (SHR) Rat Rat
Decrease in blood pressure and heart rate Urethane Pentobarbitone
Rat (SHR) Pentobarbitone Conscious Rat (SHR) Urethane Mouse Conscious
Rabbit
Rabbit Rabbit Rabbit
Rabbit
Cat Cat Dog Humans Humans Humans
Humans
Humans Humans
Decrease in blood pressure Decrease in heart rate Decrease in blood pressure and the catecholamine oxidation peak in the NRL Decrease in blood pressure and heart rate Decrease in blood pressure and heart rate Decrease in blood pressure and heart rate Decrease in blood pressure and heart rate Decrease in blood pressure and heart rate Decrease in blood pressure and heart rate
Conscious
Decrease in blood pressure Decrease in heart rate Decrease in blood pressure Decrease in heart rate Pentobarbitone Decrease in blood pressure Pentobarbitone Decrease in blood pressure and heart rate Conscious Decrease in blood pressure, heart rate, renal sympathetic nerve activity, and plasma noradrenaline concentration Conscious Decrease in blood pressure, heart rate, renal sympathetic nerve activity, and plasma noradrenaline concentration Urethane/chloralose Decrease in the firing rate of splanchnic sympathetic nerves Urethane Decrease in the firing rate of splanchnic and renal sympathetic nerves Chloralose/urethane Decrease in blood pressure and heart rate Conscious Decrease in blood pressure and sympathetic nerve activity Conscious Decrease in blood pressure and plasma noradrenaline concentration Conscious Decrease in blood pressure, sympathetic nerve activity, plasma noradrenaline concentration and vascular resistance Conscious Decrease in blood pressure, heart rate, sympathetic nerve activity, and plasma noradrenaline concentration Conscious Decrease in blood pressure, sympathetic nerve activity, and calf vascular resistance Conscious Decrease in blood pressure and heart rate
Agonist (route of application) Clonidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.) Rilmenidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.) Rilmenidine (i.v.)
Effective doses1
Reference
ED50 2.6 mg kg ED50 1.3 mg kg 2 – 10 mg kg 1
1
100 – 1000 mg kg 300 – 1000 mg kg 3 – 30 mg kg 1 100 – 1000 mg kg 3 – 30 mg kg 1 ED50 = 250 mg kg ED50 = 350 mg kg 300 – 1500 mg kg
1
de Jonge et al., 1981a
1
Tibirica et al., 1989 van Zwieten et al., 1986 Koenig-Berard et al., 1988
1
1
1
Gomez et al., 1991
1 1
Tibiric¸a et al., 1991b
Moxonidine (i.v.) Rilmenidine (i.v.) Rilmenidine (i.v.)
3 – 30 mg kg 1 30 – 300 mg kg 1 3 – 30 mg kg 1 30 – 300 mg kg 1 40 mg kg 1 30 – 300 mg kg 1 300 – 1000 mg kg 1 100 – 1000 mg kg 1 ED50 7 mg kg 1 ED50 14 mg kg 1 ED50 180 mg kg 1 ED50 210 mg kg 1 10 – 100 mg kg 1 30 – 1000 mg kg 1 100 – 1000 mg kg 1
Moxonidine (i.v.)
3 – 100 mg kg
Guanfacine (i.v.)
ED50 = 83 mg kg
Clonidine (i.v.)
10 – 100 mg kg
Clonidine (i.v.) Clonidine (i.v.)
1 – 100 mg kg 100 – 275 mg
Clonidine (i.v.)
200 mg
Warren et al., 1991
Clonidine (p.o.)
300 mg
Muzi et al., 1992
Clonidine (i.v.) Rilmenidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.) Moxonidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.) Moxonidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.)
1
Haxhiu et al., 1994 Zhu et al., 1999
Head & Burke, 1991
Armah et al., 1988 Feldman et al., 1990 Szabo et al., 1993; Urban et al., 1994 Urban et al., 1995a
1
1
Sannajust et al., 1992b
1
Scholtysik et al., 1975 Haeusler, 1976 Hoefke & Kobinger, 1966 Wallin & Frisk-Holmberg, 1981
Moxonidine (p.o.) 400 mg
Wenzel et al., 1998
Moxonidine (p.o.) 200 – 400 mg
Greenwood et al., 2000
Rilmenidine (p.o.) 1000 – 2000 mg
Weerasuriya et al., 1984
The table gives an overview of the effective hypotensive and sympathoinhibitory doses of the drugs for comparison with doses eliciting other effects. The aim was not to give a complete list of all studies in which hypotensive and sympathoinhibitory effects are described. 1 Several of the ED50 values are approximate values read from the dose – response curves in the references.
should be made responsible for the central sympathoinhibition caused by clonidine (Bousquet et al., 1984). This suggestion was supported by observations that clonidine and its chemical relatives bind to specific imidazoline-binding sites, in addition to a2-adrenoceptors, in the medulla oblong-
ata. The two new antihypertensive drugs rilmenidine and moxonidine were shown to have high affinity and moderate selectivity for imidazoline-binding sites, and their sympathoinhibitory effects were attributed to activation of these imidazoline receptors.
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
It turned out that the imidazoline-binding sites are heterogeneous. At least three groups of binding sites were classified. The medullary binding sites mediating the sympathoinhibitory effects of clonidine-like drugs were termed I1; their molecular identity has not been clarified yet. This review deals with the role of I1 imidazoline receptors in cardiovascular regulation. A second major binding site, I2 receptor or idazoxan-preferring receptor, is characterized by high affinity for idazoxan and low affinity for clonidine. I2-binding sites are widely distributed in the CNS and in peripheral tissues. Most I2-binding sites are located on the monoamine oxidase A and B enzymes in the outer membrane of the mitochondria, and binding to these sites allosterically modulates the enzymes. The pharmacology of I2 receptors has been reviewed by Parini et al. (1996) and Eglen et al. (1998) (see also Raddatz et al., 2000). A third binding site, recently named I3, was identified in pancreatic b cells; the activation of these sites increases insulin secretion. Imidazolines can directly interact with the ionconducting pore component (Kir6.2) of the ATP-sensitive K + channel, and this may be the major mechanism of the enhanced insulin secretion (Proks & Ashcroft, 1997; Rustenbeck et al., 1997; for a review, see Morgan et al., 1999). In 1984, Atlas and Burstein (1984a, 1984b) isolated a substance from calf brain that displaced radiolabelled clonidine from its binding sites. Accordingly, the compound was named clonidine-displacing substance. A search was started to identify the chemical identity of this putative endogenous ligand of the imidazoline receptor. In 1994, agmatine was identified as an endogenous ligand of the imidazoline binding sites (Li et al., 1994). It is, however, not the only clonidine-displacing substance (see, e.g., Parker et al., 1999). The pharmacology of the endogenous ligands of imidazoline receptors has been reviewed recently (Regunathan & Reis, 1996; Eglen et al., 1998; Reis & Regunathan, 2000).
3. Mode of action of clonidine-like drugs: two hypotheses Clonidine-like drugs cause sympathoinhibition, i.e., they reduce the release of sympathetic transmitter in peripheral tissues. As a result, vascular resistance, heart rate, force of cardiac contraction, and blood pressure decrease. Enhancement of vagal tone also contributes to the cardiac effects of the drugs. Table 1 gives examples of the cardiovascular effects in different species. Regarding the mechanism of the central sympathoinhibition elicited by clonidine-like drugs, two different hypotheses have been developed: the a2-adrenoceptor hypothesis and the imidazoline hypothesis. 3.1. The 2-adrenoceptor hypothesis According to the older a2-adrenoceptor hypothesis, a2-adrenoceptors mediate all effects of clonidine. Blood pressure reduction is due to activation of sympathoinhibitory a2-adrenoceptors in the medulla oblongata. The side
5
effects — the most disturbing of which are sedation and inhibition of saliva secretion — are also elicited by activation of a2-adrenoceptors. If this hypothesis is true, then it is difficult to separate the desired and unwanted effects of clonidine-like drugs. The a2-adrenoceptor hypothesis was the original explanation of the mode of action of clonidinelike drugs (for reviews, see Schmitt, 1977; Kobinger, 1978; Kobinger & Pichler, 1990; Van Zwieten et al., 1984), and this view still enjoys considerable support (for reviews, see Guyenet et al., 1995; Guyenet, 1997; Eglen et al., 1998). 3.2. The imidazoline hypothesis According to the more recent imidazoline hypothesis, imidazoline I1 receptors in the rostral ventrolateral medulla (RVLM) are important for the sympathoinhibitory action of clonidine, and the role of I1 receptors is particularly prominent in the case of rilmenidine and moxonidine. The side effects of clonidine — sedation and dry mouth — are mediated by a2-adrenoceptors. Rilmenidine and moxonidine cause only few a2-adrenoceptor-mediated side effects because they are selective for I1 receptors. If the imidazoline hypothesis is true, then I1-selective drugs, devoid of the typical side effects of other centrally acting antihypertensive drugs, could be widely used in antihypertensive therapy. The imidazoline mode of action is supported by several recent reviews (Ernsberger et al., 1995; Ernsberger & Haxhiu, 1997; Bousquet & Feldman, 1999; Head & Burke, 2000a). The following major arguments are used to support the imidazoline hypothesis. (1) a2-Adrenoceptor agonists without affinity for I1-binding sites do not cause sympathoinhibition when they are microinjected into the RVLM. In contrast, drugs devoid of affinity for a2-adrenoceptors, but with affinity for I1 receptors, lower blood pressure. (2) Antagonists possessing affinity for I1 receptors block the cardiovascular effects of I1 imidazoline agonists more effectively than pure a2-adrenoceptor antagonists do. (3) Selective I1 imidazoline agonists lower blood pressure without simultaneously eliciting a2-adrenoceptor-mediated effects. These arguments will be discussed in detail in Sections 8– 11.
4. Characterization of drugs used in imidazoline cardiovascular research in radioligand binding studies The chemical structures of important agonists and antagonists are shown in Fig. 1. Radioligand binding experiments played an essential role in the development of the imidazoline hypothesis. The affinities of key drugs for a2-adrenoceptors and I1-binding sites are given in Tables 2 and 3. Imidazoline I1-binding sites were identified and studied in a number of tissues, e.g., in the ventral medulla oblongata of humans, cows, and rabbits; in bovine and rat corpus striatum; in rat kidney; in the bovine adrenal medulla; in PC12 cells; and in human platelets. They were labeled by [3H]clonidine, [3H]para-aminoclonidine, [3H]para-iodoclonidine, or
6
Table 2 Affinities of agonists1 that are used in cardiovascular studies for a2-adrenoceptors2 and I1 imidazoline-binding sites3 Clonidine Radioligand
Species
Tissue
a2
I1
Rilmenidine
Moxonidine
a2
a2
I1
Guanabenz a2
I1
Guanfacine I1
a2
I1
a-Methyl-noradrenaline
Noradrenaline
a2
a2
I1
I1
Reference
2105
Ernsberger et al., 1987
120,000
Ernsberger et al., 1990
> 106
Ernsberger et al., 1993 Molderings et al., 1993 Czerwiec et al., 1996 Felsen et al., 1994 Piletz & Sletten, 1993 Piletz et al., 1996
Ki or IC50 (nM)4 Cattle
[3H]pAminoclonidine
Cattle
[3H]Clonidine [3H]Clonidine [3H]Clonidine [3H]Clonidine [125I]p-Iodoclonidine [125I]p-Iodoclonidine
Cattle Cattle Cattle Dog Humans Humans
[3H]Clonidine
Humans Humans Humans Human
3
[ H]Clonidine [3H]Clonidine
Humans Humans
[3]MK-912
[3H]Clonidine [3H]Clonidine (I1) [3H]Yohimbine (a2) [3H]p-Iodoclonidine [3H]Rilmenidine [3]MK-912
Humans Humans Humans Rabbit Rabbit
Ventrolateral medulla (I1) Cortex (a2) Ventrolateral medulla (l1) Cortex (a2) Ventrolateral medulla Adrenal medulla Striatum Prostata Platelet Platelet (I1) Transfected CHO cells (a2A) (a2B) (a2C) NRL (I1) Cortex (a2) NRL Ventrolateral medulla Transfected HEK 293 cells (a2A) (a2B) (a2C) Ventral medulla Brain
255 6
PC12 cells Brain Brain cortex (a2A/D) (a2B) Kidney (a2C)
77 > 106
1 5 4
1 15 220
180
6 84 590
75
1600 55 55
9 31 9
59
36 43 13 112
33
2 78 5000 0.7 0.7 4
13 10 16
11 7
122 89,000
360 >10,000 428 240
2
35 35
2 3 1
160
2500
19
16 8 5
191
97 163 5 122
135 105 412
> 10
22 > 10 593
>20,000
5
Bricca et al., 1989b
5
Bricca et al., 1989a Bricca et al., 1994
28 5
> 10 >20,000
5600
Jasper et al., 1998; Zhu et al., 1999 62 69 135 6
1585 1738 4677 6
6
15 371
Rat Rat Guinea pig
2505
6
4266 > 10,000 > 10,000 81
22 282 447
93 1380 3890 16
>20,000
11
2427
3631 2630 2884 49
>20,000
1995 2754 1349 22
1.3
2371 6
19 147 155
713 5950 2800
Bricca et al., 1993 Hamilton et al., 1991
1065 8
0.8
>20,000
0.8 2860 1950 7700
12 460 160
17
17
Separovic et al., 1996 King et al., 1992 Uhlen et al., 1995
18 1850 834
1
The term agonist refers to the interaction with a2-adrenoceptors.
2
In most of the studies, it was not determined which subtype of the a2-adrenoceptors is involved in the binding (a2A, a2A/D, a2B, or a2C). The a2A/D denomination refers to the species ortholog of the a2A-adrenoceptor found in rats, mice, and guinea pigs.
3
Catecholamine-insensitive binding sites. Since the I1 nomenclature was developed later, the binding sites are not called I1 in several publications.
4
Only IC50 values are reported in several publications, but they are close to Ki values because the radioligands were used at concentrations close to their dissociation constants.
5
Estimation of affinity for I1-binding sites is compromised by the presence of a2-adrenoceptors.
6
Possesses affinity for a2-adrenoceptors and I1-binding sites, but exact affinities were not determined.
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
[3H]pAminoclonidine
Table 3 Affinities of antagonists1 that are used in cardiovascular studies for a2-adrenoceptors2 and I1 imidazoline-binding sites3 Idazoxan Radioligand
Species
Tissue
a2
Efaroxan I1
a2
Yohimbine I1
a2
SK&F86466 I1
a2
Phentolamine I1
a2
I1
Reference
425
Ernsberger et al., 1987 Ernsberger et al., 1990
Ki or IC50 (nM)4 [3H]p-Aminoclonidine 3
[ H]p-Aminoclonidine 3
[3H]Clonidine [3H]Clonidine [3H]Clonidine [3H]Clonidine (I1) [3H]Yohimbine (a2) [3H]p-Iodoclonidine [3H]RX821002 [3H]RX821002
[3H]Rilmenidine [3]MK-912
[3H]Yohimbine or [3H]rauwolscine
Cattle Cattle Cattle Dog Humans Humans
Humans Humans Rabbit Rabbit Rat Rat Humans Rat Rat Rat Guinea pig
Humans Rat Opossum
Ventrolateral medulla (I1) Cortex (a2) Ventrolateral medulla (I1) Cortex (a2) Ventrolateral medulla Adrenal medulla Prostata Platelet Platelet (I1) Transfected CHO cells (a2A) (a2B) (a2C) NRL Ventrolateral medulla Ventrolateral medulla Brain PC12 cells RINm5F cells (a2A/D) Platelet (a2A) Cerebral cortex (a2A/D) Lung (a2B) Brain Brain cortex (a2A/D) (a2B) Kidney (a2C) HT29 cells (a2A) Lung (a2B) OK cell (a2C)
32005 179
11
186
93,000
61
84 19
0.11
103 5 1020 1255
12 20 235
20 120
52 52
441,000 441,000
3055 1027
Bricca et al., 1989a Bricca et al., 1994 Bricca et al., 1993 Hamilton et al., 1991
1600 239
10 10 178
51 34 86
33 152 1216 1.5
11,500 568 22
> 20,000 21,810
6 180
2 10 5 2 17 10 2 145 20 2 5 0.6
11
Haxhiu et al., 1994 Molderings et al., 1993 Felsen et al., 1994 Piletz & Sletten, 1993 Piletz et al., 1996
148
5 4 1 4
2570 104 3 30 7 41 176 6 0.6 1 0.2
10 13 11 11
2 2 3
7 24 12 27 9
6 4 10
Separovic et al., 1996 Remaury & Paris, 1992 Renouard et al., 1994
King et al., 1992 Uhlen et al., 1995
Blaxall et al., 1991
1
The term antagonist refers to the interaction with a2-adreceptors.
2
In most of the studies, it was not determined which subtype of the a2-adrenoceptors is involved in the binding (a2A, a2A/D, a2B, or a2C). The a2A/D denomination refers to the species ortholog of the a2A-adrenoceptor found in rats, mice, and guinea pigs.
3
Catecholamine-insensitive-binding sites. Since the I1 nomenclature was developed later, the binding sites are not called I1 in several publications.
4
Only IC50 values are reported in several publications, but they are close to Ki values because the radioligands were used at concentrations close to their dissociation constants.
5
Estimation of affinity for I1-binding sites is compromised by the presence of a2-adrenoceptors.
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
[ H]Clonidine [3H]Clonidine [3H]Clonidine [125I]p-Iodoclonidine [125I]p-Iodoclonidine
Cattle
7
8
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
[3H]rilmenidine. The a2-adrenoceptors were labelled by the same ligands, and in some studies by [3H]yohimbine, [3H]MK-912, or [3H]RX821002 (methoxy-idazoxan). There are several contradictions between the data, and it is especially difficult to identify compounds that are selective for I1 imidazoline receptors. In a few cases, affinities for identified subtypes of a2-adrenoceptors are available. They are given for sake of completeness, but are not discussed systemically. 4.1. Agonists 4.1.1. Selective 2-adrenoceptor agonists In most studies, the catecholamines noradrenaline and a-methylnoradrenaline possess appreciable affinity for a2-adrenoceptors and very low affinity for I1-binding sites (Table 2). In fact, the low affinity of catecholamines for I1-binding sites belongs to the definition of these sites. Guanabenz and guanfacine possess high affinity for a2-adrenoceptors (except in the study of Hamilton et al., 1991). In the study of Ernsberger et al. (1993), guanabenz and guanfacine possess very low affinity for I1-binding sites, and accordingly, these guanidine compounds should be selective a2-adrenoceptor agonists. However, in several other studies, a fairly high affinity for I1-binding sites was found (Bricca et al., 1989b; Hamilton et al., 1991; Piletz et al., 1996). UK14304 (not shown in Table 2) has high affinity for a2-adrenoceptors (Ki = 1– 2 nM) (Piletz et al., 1991; Bricca et al., 1993). Its affinity for I1-binding sites was very low in the rabbit brain stem (Ki = 1531 nM) (Bricca et al., 1993), but high, comparable with its affinity for a2-adrenoceptors, in human platelets (Ki = 2 nM) (Piletz et al., 1991). It recently was shown that medetomidine (not shown in Table 2) possesses high affinity for a2-adrenoceptors (Ki = 3 nM), but low affinity for I1-binding sites (Ki = 385 –14600 nM) (Piletz & Sletten, 1993; Ernsberger et al., 1997). Thus, the catecholamines noradrenaline and a-methylnoradrenaline are undoubtedly selective for a2-adrenoceptors. Their use in vivo, however, is limited. In addition to a2-adrenoceptors, they activate, of course, other adrenoceptors. Also, their pharmacokinetic behaviour (e.g., failure to pass the blood –brain barrier; uptake by several neuronal and extraneuronal transporters) renders them unsuitable for many kinds of experiments. The radioligand binding data on UK14304, guanabenz, and guanfacine are far from being unequivocal. At the moment, medetomidine seems to be the agonist of choice if an a2-selective agonist has to be used. 4.1.2. Selective I1 imidazoline receptor agonists The key drug for developing the imidazoline hypothesis was clonidine. Its affinity for a2-adrenoceptors is in the low nanomolar range in most experiments. It also possesses affinity for I1-binding sites. In some studies, this affinity is slightly higher; in other studies, it is lower than its affinity
for a2-adrenoceptors. Altogether, clonidine is not a selective I1 agonist. Rilmenidine possesses affinity for a2-adrenoceptors (except in the study of Hamilton et al., 1991) and I1-binding sites. Only the data of Ernsberger et al. (1993) and Hamilton et al. (1991) indicate selectivity for I1-binding sites. In addition to rilmenidine, moxonidine is claimed to be selective for I1 imidazoline-binding sites. It is clear from the data in Table 2 that moxonidine possesses appreciable affinity for a2-adrenoceptors. In two studies, the affinity of moxonidine was somewhat higher for I1-binding sites than for a2-adrenoceptors (Ernsberger et al., 1993; Piletz et al., 1996). However, in the studies of Bricca et al. (1993, 1994), moxonidine had practically no affinity for I1 imidazoline-binding sites. It is remarkable that the data from different laboratories on one of the two supposedly selective I1 agonists differ so dramatically. Rilmenidine and moxonidine had rather high affinity for human a2A-, a2B-, and a2C-adrenoceptors expressed in Chinese hamster ovary (CHO) cells (Piletz et al., 1996). The affinities of these drugs for the human a2-adrenoceptor subtypes were much lower in transfected human embryonic kidney (HEK) 293 cells (Jasper et al., 1998; Zhu et al., 1999), and the reason for the discrepancy is not known. The low affinity of rilmenidine and moxonidine for the a2A/D-, a2B-, and a2C-adrenoceptors of the guinea pig (Uhlen et al., 1995) is also remarkable. King et al. (1992) studied the binding of [3H]rilmenidine to membranes prepared from rat cerebral cortex and determined two binding sites. Rilmenidine was bound with high affinity (Kd = 17 nM; Ki = 6 nM) to a2-adrenoceptors. The other, low-affinity binding site showed some similarity with the idazoxan or I2-binding sites observed in many studies. Such binding sites are not thought to participate in the cardiovascular effects of clonidine-like drugs. Association of rilmenidine with I1-binding sites was not observed. In a corresponding paper (King et al., 1995), the distribution of [3H]rilmenidine-binding sites in the brain was studied. [ 3H]Rilmenidine labelled a2-adrenoceptors throughout the brain, notably also in the locus coeruleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus, and the RVLM — areas that may be involved in the central cardiovascular effects and side effects of rilmenidine. [3H]Rilmenidine also labelled the I2-like binding sites, already seen in the cortex (see above), in many brain regions. Notably, the I2-like binding was not preferential in regions of the medulla oblongata thought to be involved in the cardiovascular effects of rilmenidine. It is altogether surprising that using one of the two ‘‘selective’’ I1 ligands, rilmenidine, no specific I1 binding was observed in the RVLM or elsewhere in the brain. Radioligand binding studies unequivocally show that rilmenidine and moxonidine possess affinity for a2-adrenoceptors. In vitro functional studies indicate that moxonidine is an agonist at a2A-adrenoceptors (notably also at the human a2A-adrenoceptor). Moxonidine inhibits electrically evoked noradrenaline release in the rabbit pulmonary artery
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
and human atrium (Molderings et al., 1991, 2000), stimulates incorporation of [35S]GTPgS in HEK 293 cells transfected with the human a2A-adrenoceptor (Zhu et al., 1999), and elicits contraction in the dog saphenous vein (Zhu et al., 1999). However, moxonidine did not activate human a2B- and a2C-adrenoceptors expressed in HEK 293 cells (Zhu et al., 1999). The behaviour of rilmenidine in functional studies is less clear. Thus, rilmenidine inhibits electrically evoked noradrenaline release in the rabbit pulmonary artery (Verbeuren et al., 1986; Molderings et al., 2000), elicits contraction in the dog saphenous vein (Verbeuren et al., 1986; Marsault et al., 1996), and stimulates incorporation of [35S]GTPgS in HEK 293 cells transfected with the mouse a2A/D-adrenoceptor, indicating agonist activity at a2A- and a2A/D-adrenoceptors in rabbits, dogs, and mice. In contrast, the a2A-adrenoceptors mediating inhibition of electrically evoked contractions of the pig tail artery and urinary bladder (Ali et al., 1998) and of electrically evoked noradrenaline release from sympathetic nerve endings of the human heart were not activated by rilmenidine (Molderings et al., 2000). In these preparations, rilmenidine behaved as an antagonist of a2A-adrenoceptors. In HEK 293 cells transfected with human a2A-, a2B-, and a2C-adrenoceptors, rilmenidine displayed affinity for the a2-binding sites without stimulating incorporation of [35S]GTPgS (Jasper et al., 1998). This pattern of effect also supports an antagonist character of rilmenidine at human a2-adrenoceptors. The pharmacological profile of rilmenidine is made even more complex by the observation that rilmenidine is an antagonist of a1-adrenoceptors in a rat vascular preparation (Cario-Toumaniantz et al., 1998). Two compounds with high selectivity for I1 imidazolinebinding sites were recently synthesized: 2-endo-amino3-exo-isopropylbicyclo[2.2.1]heptane (AGN192403) (Munk et al., 1996) and (-)-(5-[2-methyl-phenoxy-methyl]-1,3oxazolin-2-yl)amine (S23230) (Barrot et al., 2000). AGN192403 possesses high affinity for the I1-binding sites of the bovine ventrolateral medulla labelled with [3H]clonidine (Ki = 42 nM). The affinities for the human a2A-, rat a2B-, and human a2C-adrenoceptors expressed in CHO cells were much lower (Ki > 20,000 nM for all three receptor subtypes). S23230 has high affinity for the I1-binding sites in bovine adrenals labelled with [3H]clonidine (Ki = 6 nM). The affinity of this drug for a2-adrenoceptors in the bovine cerebral cortex is much lower (Ki = 7400 nM). Summarizing the data on the I1 binding profile of drugs, it must be said that no agonists exist with unequivocally proven selectivity for I1-binding sites. A selectivity ratio of about 30 was shown for rilmenidine and moxonidine in only one study (Ernsberger et al., 1993). In several other papers, only a marginal selectivity for I1-binding sites (selectivity ratio = 3 –4) (Piletz et al., 1996) or low or very low affinity for I1-binding sites was reported. Moxonidine proved to be an agonist of a2-adrenoceptors in all species studied. In contrast, rilmenidine was an agonist of a2-adrenoceptors in some species, but an antagonist in others, including humans.
9
AGN192403 behaves in binding studies as a selective ligand. It seems, however, that this compound has no cardiovascular effects (see Sections 7, 8.3, and 8.4). It is not known whether the other selective I1 ligand, S23230, elicits cardiovascular effects expected for an I1 imidazoline receptor agonist or antagonist. 4.2. Antagonists The affinities of five antagonists used in imidazoline cardiovascular research are shown in Table 3. Three (idazoxan, efaroxan, and phentolamine) possess an imidazoline ring, whereas two (yohimbine and 6-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benzazepine HCl [SK&F86466]) have different chemical structures. It is clear that all five drugs are rather potent a2-adrenoceptor antagonists. 4.2.1. Selective 2-adrenoceptor antagonists It was repeatedly found that the affinity of yohimbine and SK&F86466 for I1-binding sites is much lower than their affinity for a2-adrenoceptors. Therefore, these drugs can be considered as a2-selective antagonists. In some cardiovascular studies, the imidazoline derivative methoxy-idazoxan (also called RX821002; not shown in Table 3) was used as an a2-selective antagonist. Methoxy-idazoxan is a high-affinity a2 antagonist (Ki or Kd = 2 –10 nM) (Langin et al., 1989; Trendelenburg et al., 1996; Ernsberger et al., 1997). Since its affinity for I1-binding sites is much lower (Ki = 400 nM) (Ernsberger et al., 1997), this compound can also be considered a2-selective. 4.2.2. Selective I1 imidazoline receptor antagonists The three antagonists with an imidazoline structure — idazoxan, efaroxan, and phentolamine — possess, in addition to their affinity for a2-adrenoceptors, affinity for I1-binding sites. The affinity of idazoxan for I1-binding sites is generally lower than its affinity for a2-adrenoceptors (except in the study of Hamilton et al. [1991]). Phentolamine does not differentiate between a2-adrenoceptors and I1-binding sites. The behaviour of efaroxan is not clear. According to Haxhiu et al. (1994), it is highly selective for I1-binding sites (173-fold). This selectivity is due to the very high affinity of efaroxan for the I1-binding sites (Ki = 0.11 nM). However, much lower I1 affinity values were found by Piletz and Sletten (1993), Piletz et al. (1996), and Separovic et al. (1996). Summarizing the state of knowledge on antagonists, it can be concluded that yohimbine, SK&F86466, and methoxyidazoxan are reasonably selective for a2-adrenoceptors. In contrast, the evidence for the I1 selectivity of efaroxan is not convincing. Thus, we do not possess I1-selective antagonists. At the end of the section describing the drugs used in imidazoline cardiovascular research (Section 4), it must be critically remarked that only very few reliable drugs are available for use in in vivo experiments to determine involvement of I1 imidazoline receptors or a2-adrenocep-
10
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
tors. Thus, as pointed out above, no selective I1 imidazoline receptor agonists or antagonists have been identified. Moreover, very few binding data have been obtained on tissues of rats and rabbits, despite the fact that almost all in vivo experiments have been carried out in these species. The results of experiments carried out on rats and rabbits are regularly interpreted using the binding data of rilmenidine, moxonidine, idazoxan, and efaroxan determined on bovine tissues. Such extrapolation of data to other species is questionable because the receptors can markedly differ in various species. It is known, e.g., that the susceptibility of the a2A-adrenoceptor to agonists and antagonists differs markedly in rats, rabbits, and humans (see Trendelenburg et al., 1996; Molderings et al., 2000). It would be necessary to determine the binding and functional characteristics of the key imidazoline and a2 drugs at I1 imidazoline receptors and a2A-, a2B-, and a2C-adrenoceptors. Within one species, binding and functional studies on I1 imidazoline receptors and a2A-, a2B-, and a2C-adrenoceptors should be carried out under identical conditions. Without such comprehensive sets of data for rats, rabbits, and humans, the meaning of many in vivo observations remains unclear.
5. The central nervous site of action of clonidine-like drugs Clonidine-like drugs are often classified as ‘‘centrally acting antihypertensive drugs,’’ reflecting the view that they lower sympathetic nerve firing by a primary action in the CNS. An action in the medulla oblongata is supported, e.g., by the observations that clonidine-like drugs lower sympathetic tone after administration of low doses into the vertebral artery (e.g., Timmermans et al., 1981; van Zwieten et al., 1986) or into the cisterna cerebellomedullaris (e.g., Kobinger, 1967; Chan et al., 1996; Bock et al., 1999). In this section, the mechanism of action within the CNS will be discussed. Peripheral mechanisms can also contribute to the reduction of sympathetic tone. It will be discussed in detail in Section 12 that clonidine and its relatives can lower the amount of transmitter released per action potential from postganglionic sympathetic neurons by activating inhibitory presynaptic receptors. 5.1. Action in the rostral ventrolateral medulla The RVLM (it is alternatively called subretrofacial nucleus, ventrolateral medullary pressor area, or nucleus reticularis lateralis [NRL]1) is the final relay station of the baroreceptor reflex pathway in the brain. It contains the perikarya of sympathoexcitatory neurons projecting to sym1
The nomenclature is not uniform. Most often, NRL is used as a synonym for RVLM. In some publications, however, NRL refers to a nucleus caudal of the RVLM. In this review, we will keep the RVLM and NRL denominations, as they were used in the original references.
pathetic preganglionic neurons in the intermediolateral column of the spinal cord. Many RVLM neurons synthesize adrenaline, and these neurons constitute the C1 adrenergic cell group. The RVLM is considered as the primary site of action of clonidine-like drugs (for reviews, see Dampney, 1994; Ernsberger et al., 1995; Guyenet et al., 1995; Reis, 1996; Sun, 1996; Guyenet, 1997). The high sensitivity of the ventral surface of the medulla oblongata to locally applied clonidine was shown by Bousquet and Guertzenstein in 1973. The opinion that clonidine-like drugs lower sympathetic tone by a principal action in the RVLM is based on a set of observations made first by Punnen et al. (1987). They showed that clonidine lowers blood pressure in rats when it is microinjected into the RVLM, and this effect is prevented by microinjection of the imidazoline a2-adrenoceptor antagonist idazoxan into the RVLM. Idazoxan also prevented the hypotensive effect of systemically administered clonidine. The effects of systemically administered clonidine were identical in animals decerebrated at the level of the midbrain, indicating that structures lying rostrally, most importantly the hypothalamus, are not necessary for the hypotensive effect. Finally, Punnen et al. (1987) excluded a role of the nucleus tractus solitarii (NTS) in the sympathoinhibition produced by systemically administered clonidine by showing that inactivation of the NTS by lidocaine does not change the hypotensive action of clonidine. The observations of Punnen et al. (1987) have since been repeated by several groups. In addition, it was shown that systemically and locally microapplied clonidine inhibits the activity of sympathoexcitatory RVLM neurons (Sun & Guyenet, 1986; Haselton & Guyenet, 1989; Clement & McCall, 1991; Allen & Guyenet, 1993). A role of the RVLM in the sympathoinhibition is also supported by the observation that systemically administered clonidine and rilmenidine suppress the expression of c-fos mRNA and Fos protein in this nucleus (Li & Dampney, 1995; El-Mas & Abdel-Rahman, 2000). Importantly, it has been shown that the more recently synthesized drugs rilmenidine and moxonidine also elicit sympathoinhibition if they are microinjected into the RVLM (Feldman et al., 1990; Gomez et al., 1991; Haxhiu et al., 1994; Head & Burke, 1998, 2000b; Tolentino-Silva et al., 2000; Mayorov & Head, 2001). One group observed that clonidine caused hypotension and bradycardia, not only when it was microinjected into the RVLM, but also when it was administered into the caudal ventrolateral medulla in rats (McAuley et al., 1989). A vasodepression was also seen after injection of clonidine into the gigantocellular depressor area in the medulla of the rat (Aicher & Drake, 1999). In the cat, the medullary site of action of clonidine is lying caudal from the RVLM, in the rostral part of the NRL (Gatti et al., 1988). 5.2. Action in the nucleus tractus solitarii The NTS is the region in the medulla oblongata in which axons of baroreceptor and chemoreceptor afferents termi-
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nate. The catecholamines noradrenaline and a-methylnoradrenaline cause hypotension and bradycardia when they are microinjected into the NTS, and the effects can be blocked by a2-adrenoceptor antagonists (e.g., Zandberg et al., 1979; Kubo & Misu, 1981; Head & Burke, 1998). The NTS was long considered to be the major site of action of clonidine-like drugs in the medulla (for a review, see Kobinger & Pichler, 1990). However, microinjection of clonidine and rilmenidine into the NTS elicited no or only a moderate hypotension (Zandberg et al., 1979; Gomez et al., 1991). This fact and the observation that lesion of the NTS does not greatly change the hypotensive effect of systemically administered clonidine led to the opinion that the NTS is not the primary medullary site of action of clonidine-like drugs (for a review, see Ernsberger et al., 1995). It was shown recently, however, that rilmenidine lowers blood pressure, heart rate, and sympathetic nerve firing rate when it is microinjected into the NTS of rabbits (Head & Burke, 1998). It is also a recent observation that neuronal activity in the NTS, measured as expression of c-fos mRNA and Fos protein, was inhibited by systemically administered clonidine and rilmenidine (Li & Dampney, 1995; El-Mas & Abdel-Rahman, 2000). 5.3. Action in the spinal cord Clonidine and several other a2-adrenoceptor agonists were also administered intrathecally at thoracal and lumbar levels. Clonidine (Connor et al., 1981; Kubo et al., 1987; Solomon et al., 1989; Eisenach & Tong, 1991; Filos et al., 1994), guanabenz (Kubo et al., 1988), B-HT920 (Kubo et al., 1987), and dexmedetomidine (Eisenach et al., 1994) lowered blood pressure and, under certain experimental conditions, also heart rate. The effects were antagonized by intrathecally injected a2-adrenoceptor antagonists (Connor et al., 1981; Kubo et al., 1987, 1988; Solomon et al., 1989; Eisenach & Tong, 1991). In several of these studies, it was verified that the effects of the a2-adrenoceptor agonists were due to a direct spinal action and not to actions elsewhere, after diffusion of the drugs into the systemic circulation. Spinal sympathoinhibition occurs also after intracerebroventricular administration of rilmenidine (Sannajust et al., 1992a). Moreover, sympathoinhibition at the level of the spinal cord was suggested to play a role after systemic administration of clonidine in spontaneously hypertensive rats (SHR) (Tibiric¸a et al., 1992). Adrenaline hyperpolarizes some of the sympathetic preganglionic neurons in isolated spinal cord slices, an effect that is sensitive to a2-adrenoceptor blockade (Miyazaki et al., 1989). Thus, activation of a2-adrenoceptors in the spinal cord causes sympathoinhibition. It is not clear, however, whether the spinal effect contributes to the overall sympathoinhibition caused by systemically administered clonidine-like drugs. In summary, clonidine-like drugs lower sympathetic tone and blood pressure when they are locally applied into the
11
RVLM, NTS, and spinal cord. The involvement of the RVLM in the sympathoinhibition produced by systemically applied drugs has been demonstrated, whereas the involvement of the NTS and spinal cord cannot be excluded. Of course, the effectiveness of clonidine-like drugs in the CNS does not exclude simultaneous peripheral actions on sympathetic neurons. 6. Characterization of A2-adrenoceptors and I1 imidazoline-binding sites 6.1. 2-Adrenoceptors 6.1.1. Molecular identity, second messenger mechanisms Three genetically defined subtypes of the a2-adrenoceptor have been identified: a2A, a2B, and a2C (Bylund et al., 1994; Docherty, 1998). Due to minor differences in the amino acid sequence, the a2A-adrenoceptors of humans, rabbits, and pigs differ pharmacologically from the a2A-adrenoceptors of rats, mice, guinea pigs, and cows. The ortholog receptor found in rats, mice, guinea pigs, and cows is denominated in this review as the a2A/D-adrenoceptor. The intracellular signal transmission mechanisms of the a2-adrenoceptors are well-characterized. These receptors typically couple to Gi- and Go-proteins, and this can lead to inhibition of adenylate cyclase and to changes in the open probability of K + channels and voltage-dependent Ca2 + channels (e.g., Summers & McMartin, 1993; Hein & Kobilka, 1995; Docherty, 1998; Goldstein, 1998). 6.1.2. Distribution in cardiovascular regulatory centres The distribution of the mRNA and the receptor protein of a2-adrenoceptor subtypes in the pons, medulla, and spinal cord is well-characterized. mRNA for the a2A/D-adrenoceptor predominated in regions relevant for cardiovascular regulation (Nicholas et al., 1993, 1996). Thus, a2A/D-mRNA was observed in the locus coeruleus, A5 pontine nucleus, RVLM, dorsal motor nucleus of the vagus, nucleus ambiguus, and intermediolateral column of the spinal cord. The distribution of the appropriate receptor protein, visualized immunohistochemically, is very similar. Labelling was seen in the locus coeruleus, A5 pontine nucleus, NTS, dorsal motor nucleus of the vagus, and the RVLM (Talley et al., 1996). The great majority of the tyrosine hydroxylase positive cells in the locus coeruleus and the C1 cell group of the RVLM contained the a2A/D-receptor protein (Rosin et al., 1993). In addition, many a2A/D-receptors were identified as presynaptic heteroreceptors located on terminals of non-adrenergic afferent axons in the vicinity of RVLM/C1 cells (Milner et al., 1999). The distribution of the a2C-adrenoceptor protein is similar to the distribution of the a2A/D-adrenoceptor, i.e., it is found in the locus coeruleus, dorsal motor nucleus of the vagus, the RVLM, and intermediolateral column of the spinal cord (Rosin et al., 1996).
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In summary, the molecular structure and second messenger mechanisms of the a2-adrenoceptors are well-characterized. The a2-adrenoceptors are appropriately located in the medulla to influence the function of cardiovascular regulatory centres. 6.2. I1 imidazoline receptors 6.2.1. Molecular identity, second messenger mechanisms The molecular structure of the I1 imidazoline receptor has not been identified. Two antibodies have played an important role in the search for the receptor protein. One antibody was raised against an imidazoline receptor protein (IRP antibody) that was affinity purified on columns labelled with paraaminoclonidine or idazoxan (Wang et al., 1993). Another antibody was raised as a secondary antibody (anti-idiotypic antibody) against a primary antibody that was directed against idazoxan (Bennai et al., 1996). Solubilized proteins from extracts of different tissues were immunolabelled using one of these antibodies. Immunoreactive proteins of different sizes were detected in the rat brain (45 kDa) (Escriba´ et al., 1995), human brain (43 kDa) (Bennai et al., 1996), bovine adrenomedullary cells (31 kDa, 70 kDa, 90 kDa) (Wang et al., 1993; Ivanov et al., 1998a), human platelets (33 kDa, 95 kDa) (Ivanov et al., 1998a), and MEG-01 cells (33 kDa, 85 kDa) (Ivanov et al., 1998a). An 85-kDa protein was recognized by both antibodies in human and rat brain and PC12 cells (Ivanov et al., 1998c). Since the density of this latter protein in different brain regions correlated with the density of I1-binding sites, it was considered to be a candidate for the full-length I1 IRP. It was suggested that the smaller proteins observed in previous studies were proteolytic fragments of this 85-kDa protein. The IRP antibody and the anti-idiotypic antibody were also used to isolate a cDNA clone from a human hippocampal cDNA expression library. In the first step, a 1.8-kbp cDNA clone (5A-1B) was sequenced, corresponding to a 66-kDa protein, but this was apparently not the full-length cDNA (Ivanov et al., 1998b). Recently, the full-length cDNA was determined to be a 5.1-kbp molecule, corresponding to a 1504 amino acid protein (Piletz et al., 2000). The latter cDNA was named imidazoline receptor-antiseraselected (IRAS) cDNA and the protein product, IRAS-1 protein. Transfection of the IRAS cDNA into CHO cells increased the density of I1-binding sites in these cells, and rilmenidine and moxonidine had high affinity, Ki = 10 and 14 nM, respectively, for these binding sites (Piletz et al., 2000). The sequence analysis of the IRAS cDNA indicated that the product is not a G-protein-coupled receptor. Sequence homologies were found with chromogranins, cytokine receptors, cytochrome P450 enzymes, and the ryanodine receptor (Piletz et al. 2000). Thus, IRAS-1 protein seems to be an I1 imidazoline-binding site. It is not clear, however, whether it is a receptor capable of inducing cellular changes attributed, until now, to I1 receptors.
Only a limited number of observations are available on the intracellular transmission mechanisms of the putative I1 imidazoline receptor. The I1 imidazoline-binding sites are enriched in cellular membranes, and the binding of paraiodoclonidine is inhibited by Gpp(NH)p. These observations led to the suggestion that they represent plasma membrane-bound G-protein-coupled receptors (Ernsberger & Shen, 1997). Imidazoline agonists, like clonidine, have no effect on basal and forskolin-stimulated cyclic AMP levels, do not activate phosphatidylinositol-sensitive phospholipase C, and affect the influx of Ca2 + ions into cells only at very high concentrations (Regunathan et al., 1990, 1991; Liedtke et al., 1993). In PC12 cells, moxonidine stimulates, at the rather high concentrations of 0.1 –1 mM, phosphatidylcholine-sensitive phospholipase C, releasing diacylglyceride and choline phosphate (Separovic et al., 1996, 1997). Moreover, the hypotensive effect of moxonidine microinjected into the RVLM of rats is prevented by pretreatment with an inhibitor of phosphatidylcholine-sensitive phospholipase C (Separovic et al., 1997). The identified second messenger mechanisms do not seem to fit the properties of the IRAS-1 protein, which was identified as an I1 imidazoline-binding site (see above). 6.2.2. Distribution in cardiovascular regulatory centres The distribution of I1 imidazoline-binding sites in the medulla oblongata was studied with autoradiography using [125I]para-iodoclonidine as label (Ernsberger et al., 1995). The RVLM was strongly labelled, whereas only weak labelling was seen in the NTS. Surprisingly, a rather strong diffuse labelling was seen on the entire cross-sectional area of the rostral medulla. The binding of [125I]para-iodoclonidine in the bovine RVLM was shown to be located on (pre)synaptic membranes (Heemskerk et al., 1998). In one immunohistochemical study (Ruggiero et al., 1998), the IRP antibody (Wang et al., 1993; see Section 6.2.1.) was used to identify IRPs. The IRP antibody recognizes both I1- and I2-binding sites. Labelling was seen in the NTS, dorsal motor nucleus of the vagus, nucleus ambiguus, RVLM, and intermediolateral column of the thoracic spinal cord. Labelling of these regions was not preferential, the IRP antibody labelled many structures throughout the brain. In situ hybridization and quantitative northern blot analysis revealed an mRNA distribution pattern in the CNS that does not strongly correlate with the known distribution of I1-binding sites. High concentrations of 5A-1B mRNA (the truncated form of IRAS) were seen, e.g., in the pituitary, cerebellum, cortex, hippocampus, and extrapyramidal motor nuclei, and lower concentrations in the medulla oblongata (Ivanov et al., 1998b). In summary, the molecular identity of the I1 imidazoline receptor has not been clarified. Until now, only one protein (IRAS-1) was identified that could be an I1 imidazolinebinding site. The distribution of this protein does not fit the expected preferential localization of I1 imidazoline receptors in the medulla oblongata. Knowing the amino acid sequence
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
of IRAS-1, it is difficult to assign a function to this protein. It is also not obvious how IRAS-1 could lead to the generation of the second messengers ascribed to the putative I1 imidazoline receptor.
7. Action of clonidine-like drugs on cardiovascular regulatory nuclei in vitro The effect of clonidine-like drugs on RVLM neurons in rat brain slices has been analyzed in a few studies. In one study, clonidine lowered the firing rate of RVLM pacemaker neurons by hyperpolarizing the cells (Sun & Reis, 1995). Since the hyperpolarization was blocked by bicuculline and tetrodotoxin, it was suggested that clonidine acted indirectly, by enhancing the synaptic release of g-aminobutyric acid (GABA). Noradrenaline (in the presence of
13
prazosin) and UK14304 can also hyperpolarize and inhibit RVLM neurons. These effects are sensitive to a2-adrenoceptor blockade by yohimbine (Hayar et al., 1997). In several experiments, the effects of imidazoline and catecholamine drugs on identified spinally projecting sympathoexcitatory RVLM neurons were studied (Li et al., 1995, 1998; Hayar & Guyenet, 1999, 2000). The spinally projecting neurons were identified with the help of a retrogradely transported fluorescent label that had been injected into the spinal cord in vivo several days before killing the animals for brain slice preparation. a-Methylnoradrenaline and moxonidine inhibited the spontaneous firing of bulbospinal RVLM neurons, whereas the I1 imidazoline-binding site ligand AGN192403 was without effect (Li et al., 1995; Hayar & Guyenet, 2000; see Fig. 2). Noradrenaline and moxonidine also inhibited glutamatergic and GABAergic synaptic transmission between afferent axons and RVLM
Fig. 2. Interactions between moxonidine (Mox), AGN192403 (AGN), and SK&F86466 (SKF) in rat brain slices. Bulbospinal presympathetic neurons in the RVLM were studied with the patch – clamp technique. A: Moxonidine (10 mM) lowers the firing frequency of a neuron, and the effect is fully reversed by SK&F86466 (10 mM). B: In the presence of SK&F86466 (10 mM), moxonidine (10 mM) and AGN192403 (10 mM) do not affect the firing rate of RVLM neurons, whereas the GABAB receptor agonist baclofen (10 mM) causes strong inhibition. C: Moxonidine (10 mM) elicits an outward current in RVLM neurons, and the effect is completely prevented by SK&F86466 (10 mM). D: Moxonidine (10 mM) inhibits electrical stimulation-evoked excitatory postsynaptic currents (EPSCs) in an RVLM neuron, and the effect is antagonized by SK&F86466 (10 mM). The figure demonstrates that moxonidine inhibits sympathoexcitatory neurons in the RVLM. The antagonism by SK&F86466 indicates involvement of a2-adrenoceptors; the I1 imidazoline receptor ligand AGN192403 is without effect. Moxonidine suppressed RVLM neurons most likely by evoking an outward current (i.e., hyperpolarization) and by inhibiting the excitatory input of the neurons. Adapted from Hayar and Guyenet (2000), with kind permission of the authors.
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neurons by a presynaptic mechanism (Hayar & Guyenet, 1999, 2000; see Fig. 2). The effects of a-methylnoradrenaline, noradrenaline, and moxonidine were antagonized by the selective a2-adrenoceptor antagonists methoxy-idazoxan and SK&F86466 (see Fig. 2). In addition, noradrenaline and UK14304 inhibited high voltage-activated Ca2 + channels in RVLM neurons (Li et al., 1998). The somadendritic and presynaptic a2-adrenoceptor-mediated effects in the RVLM agree well with the localization of a2-adrenoceptors, which were observed in the soma of RVLM neurons and in the vicinity of RVLM neurons on axon terminals (see Section 6.1.2). In summary, catecholamine and imidazoline agonists inhibit the activity of bulbospinal sympathoexcitatory RVLM neurons. Direct inhibition of RVLM neurons, inhibition of their excitatory input, and enhancement of their inhibitory input may all contribute to the inhibition of activity. The involvement of a2-adrenoceptors in these effects is verified. In contrast, there was no indication for an involvement of I1 imidazoline receptors.
8. Correlation between sympathoinhibition and affinity for I1 imidazoline-binding sites and A2-adrenoceptors The observation of Bousquet et al. (1984) that drugs possessing an imidazoline structure, but devoid of affinity for a2-adrenoceptors, lower blood pressure when they are injected into the NRL (used as a synonym of RVLM) was the starting point of the imidazoline hypothesis. Since then, it has been claimed repeatedly that the sympathoinhibitory action of drugs injected into the RVLM depends on their affinity for imidazoline receptors. It was even assumed that selective a2-adrenoceptor agonists do not cause sympathoinhibition. The experimental evidence behind this important component of the imidazoline hypothesis is reviewed in Sections 8.1 –8.4. 8.1. Cats The imidazoline hypothesis emerged from the work of Bousquet et al. (1984) in cats. It was shown that three imidazolines — clonidine (an a2-adrenoceptor agonist), cirazoline (an a1-adrenoceptor agonist and an a2 antagonist), and St-587 (an a1-adrenoceptor agonist) — lowered blood pressure when they were microinjected into the NRL. In contrast, the a2-adrenoceptor agonist a-methylnoradrenaline had no effect. It was inferred that affinity for a2-adrenoceptors was not important for the sympathoinhibition. Instead, an imidazoline chemical structure was necessary to elicit hypotension. It recently was shown that cirazoline, at least in the rabbit, is a strong partial agonist at a2-adrenoceptors (EC50 = 40 nM; Emax = 80%) (Gaiser et al., 1999). If this is also true in the cat, then there is only one drug in the experiments of Bousquet et al. (1984) that lowered blood pressure without being an a2-adrenoceptor agonist, St-587. Remarkably,
St-587 did not lower blood pressure when injected into the vertebral artery of cats (De Jonge et al., 1981b), although many clonidine-like drugs lower blood pressure after such an application (e.g., van Zwieten et al., 1986). The behaviour of guanfacine, an a2-adrenoceptor agonist displaying low affinity for I1 sites in the majority of radioligand binding studies (see Table 2), fits the predictions of the imidazoline hypothesis. Thus, guanfacine did not change the blood pressure and heart rate of cats when it was topically administered to the ventral surface of the medulla in the vicinity of the RVLM. In contrast, clonidine, applied at the same site, elicited marked effects (Scholtysik et al., 1975). 8.2. Rats Several drugs with affinity for I1 receptors and a2-adrenoceptors were microinjected at one fixed and identical dose (1 nmol/injection site) into the RVLM of rats (Ernsberger et al., 1990). Most of the drugs lowered blood pressure. Drugs with high affinity for I1 receptors (clonidine, para-aminoclonidine) produced the strongest hypotensive response, whereas selective a2 -adrenoceptor agonists (noradrenaline, adrenaline, and a-methylnoradrenaline) had only weak effects. There was a correlation between the affinities of the drugs for I1-binding sites and the magnitude of the hypotensive response, whereas there was no correlation between the affinity for a2-adrenoceptors and the hypotensive response. In a similar study (Buccafusco et al., 1995), dose – response curves for the hypotensive effect of a series of intracisternally administered agonists were constructed. The selective a2-adrenoceptor agonist guanfacine had no effect on blood pressure. There was a correlation between the affinity of the drugs for I1-binding sites and their potency to produce hypotension, but no such correlation existed if the affinity of the drugs for a2-adrenoceptors was considered. The correlation between I1 affinity and hypotensive response and the missing correlation between a2 affinity and hypotension was considered as support for the imidazoline mode of action (Ernsberger et al., 1990; Buccafusco et al., 1995). Some shortcomings are apparent, however, in both studies. Only a few drugs were included that possess I1 affinity and cause substantial hypotension (clonidine, para-aminoclonidine, oxymetazoline, and cimetidine in Ernsberger et al. [1990]; moxonidine, clonidine, lofexidine, and ICI106207 in Buccafusco et al. [1995]). Importantly, the correlation between I1 affinity and the hypotensive effect is less than impressive for these respective four drugs. Another shortcoming is that affinity for a2-adrenoceptors of the catecholamines noradrenaline, adrenaline, and a-methylnoradrenaline was compared with their in vivo efficacy. Compared with their affinity in cell free binding assays, the catecholamines will appear less potent under in vivo conditions because they are subject to neuronal uptake and metabolism. In addition, their interaction with a1- and b-adrenoceptors will greatly modify their effects in vivo.
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a-Methylnoradrenaline and noradrenaline, microinjected into the RVLM, were effective in several studies. Thus, a-methylnoradrenaline lowered blood pressure and heart rate, and the maximal effect appeared after 10 nmol/injection site (Granata et al., 1986). The 10-nmol dose of a-methylnoradrenaline is similar to the doses that lowered blood pressure after injection into the NTS, a region in which the sympathoinhibitory role of a2-adrenoceptors is generally accepted (De Jong & Nijkamp, 1976; Zandberg et al., 1979; Kubo & Misu, 1981). In another study, a-methylnoradrenaline applied by microiontophoresis in the RVLM inhibited the activity of a set of bulbospinal sympathoexcitatory neurons. The same set of neurons was also inhibited by locally and systemically administered clonidine (Allen & Guyenet, 1993). Noradrenaline also caused hypotension when it was injected into the RVLM of rats (Sinha et al., 1985). Thus, catecholamines, which possess only very low affinity for I1-binding sites, consequently lower blood pressure when they are microinjected into the RVLM, probably by activating a2-adrenoceptors. 8.3. Rabbits Microinjection of a-methylnoradrenaline into the RVLM of rabbits lowered blood pressure, heart rate, and the firing rate of renal sympathetic nerves. The blood pressure – renal sympathetic nerve activity (RSNA) reflex curve was shifted to the right, and the maximum of the curve was depressed (Head & Burke, 1998, 2000b). The hypotensive doses of a-methylnoradrenaline, microinjected into the RVLM or into the NTS, were identical. Injection of AGN192403, the drug that possesses high affinity and high selectivity for I1-binding sites (see Section 4.1.2), directly into the CNS of the rabbit, elicited no cardiovascular effects (Munk et al., 1996). 8.4. Monkeys Intravenous injection of AGN192403 did not change the blood pressure of monkeys, and it also did not antagonize the hypotension produced by clonidine (Munk et al., 1996). In summary, a2-adrenoceptor agonists lower blood pressure when they are injected into the RVLM of rats and rabbits. The role of a2-adrenoceptors in the RVLM of cats is less clear; however, there are too few observations to exclude a role for a2-adrenoceptors. It is not clear why guanfacine is not active when it is administered intracisternally or to the surface of the ventral medulla; it may penetrate into the brain only poorly. It is very remarkable that the first drug, AGN192403, which in radioligand binding studies proved to be selective for I1-binding sites, has no effect on blood pressure, neither when administered alone nor when combined with clonidine. The suggestion that there is no a2-adrenoceptor-mediated sympathoinhibition in the RVLM and that the effects of clonidine-like drugs correlate with the affinity of the drugs for I1-binding sites is not sufficiently supported by experimental observations.
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9. Interaction of agonists with antagonists in vivo Cardiovascular experiments with antagonists on anaesthetised or conscious animals are essential for determining the receptors primarily activated by clonidine-like drugs. Table 4 shows studies that support the role of a2-adrenoceptors in the sympathoinhibition produced by clonidine-like drugs. In contrast, Table 5 compiles studies in which the authors concluded that I1 receptors are involved in the sympathoinhibition. These studies are now critically evaluated. 9.1. Studies supporting involvement of 2-adrenoceptors As outlined in the Section 4.2.1 (see also Table 3), affinity for a2-adrenoceptors and lack of affinity for I1 receptors was verified for three antagonists: yohimbine, SK&F86466, and methoxy-idazoxan. Antagonism by these three a2-selective drugs of the sympathoinhibitory and hypotensive effects of clonidine, rilmenidine and moxonidine, supports involvement of a2-adrenoceptors in these effects. Yohimbine was already used in 1973 by Schmitt et al. to analyze the cardiovascular effects of clonidine (when, of course, the imidazoline hypothesis did not yet exist). Table 4 lists the studies in which yohimbine, SK&F86466, and methoxy-idazoxan were effective against clonidine, rilmenidine, and moxonidine. Involvement of a2-adrenoceptors was shown in several species in anaesthetized and also in conscious states. Several routes of drug administration were used: agonists were injected intravenously, into the vertebral artery, into the lateral cerebral ventricle, and into the cisterna cerebellomedullaris. Antagonists were frequently administered by the same way as the agonists, but, in a few cases, systemically administered agonists were combined with intracerebrally applied antagonists. Several types of interactions between agonists and antagonists were tested in order to study the role of a2-adrenoceptors. (1) In the simplest experiments, the a2-adrenoceptor antagonist counteracted the effect of clonidine-like drugs (Schmitt et al., 1973; Bolme et al., 1974; Farsang et al., 1980; Timmermans et al., 1981; Johansson et al., 1981; McConnaughey & Ingenito, 1982; van Zwieten et al., 1986; Tibirica et al., 1988; Allen & Guyenet, 1993; Szabo et al., 1993; Szabo & Urban, 1995). (2) In more complex designs, it was shown that yohimbine and SK&F86466 are as potent against rilmenidine and moxonidine as against the selective a2-adrenoceptor agonist UK14304 (Urban et al., 1994, 1995a). (3) Finally, it was shown that yohimbine and SK&F86466 were at least as effective against clonidine, rilmenidine, and moxonidine as the mixed I1/a2 antagonists idazoxan and efaroxan (Hieble & Kolpak, 1993; Vayssettes-Courchay et al., 1996; Bock et al., 1999). In our opinion, the studies of Hieble and Kolpak (1993) and Bock et al. (1999) convincingly show that a2-adrenoceptors are involved in the hypotensive actions of clonidine,
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Table 4 In vivo studies with agonists and antagonists supporting involvement of a2-adrenoceptors in the hypotension Species
Anaesthesia
Measured parameters
Rat Rat (SHR) Rat (SHR) Rat (SHR)
Chloralose Pentobarbitone Pentobarbitone a-Chloralose
Blood Blood Blood Blood
Rat
Pentobarbitone
Blood pressure, heart rate, sympathetic nerve activity
Rat
Urethane
Rat
Urethane
Firing rate of RVLM neurons Blood pressure, heart rate
Rabbit
Conscious
Rabbit
Conscious
Rabbit
Conscious
Rabbit
Conscious
Dog Cat
pressure, heart rate pressure, heart rate pressure, heart rate pressure
Agonist (route of application)
Antagonist (route of application)
Clonidine (i.v.) Clonidine (i.v.) Clonidine (i.c.v.) Clonidine (i.v.) Guanabenz (i.v.) Clonidine (i.v.)
Yohimbine (i.v.) Yohimbine (i.p.) Yohimbine (i.c.v.) Idazoxan (i.v.) SK&F86466 (i.v.) Idazoxan (i.v.) Methoxy-idazoxan (i.v.) Yohimbine (i.v.) Yohimbine (i.v.)
Bolme et al., 1974 Farsang et al., 1980 Tibirica et al., 1988 Hieble & Kolpak, 1993
Methoxy-idazoxan (gigantocellular depressor area microinjection) Yohimbine (i.v.) Methoxy-idazoxan (i.v.)
Aicher & Drake, 1999
Rilmenidine (i.v.) UK14304 (i.v.)
SK&F86466 (i.v.)
Urban et al., 1994
Clonidine (i.v.) Clonidine (gigantocellular depressor area microinjection)
Rilmenidine (i.v.)
Reference
Vayssettes-Courchay et al., 1996
Allen & Guyenet, 1993
Szabo et al., 1993; Szabo & Urban, 1995
Moxonidine (i.v.) UK14304 (i.v.)
Yohimbine (i.v.) Yohimbine (i.c.m.)
Urban et al., 1995a
Rilmenidine (i.c.m.) Moxonidine (i.c.m.) a-Methyldopa (i.c.m.) Clonidine (vertebral artery)
Cat
Chloralose + urethane a-Chloralose
Blood pressure, heart rate, sympathetic nerve activity Blood pressure
Clonidine (i.v.) Clonidine (i.c.m.) Clonidine (vertebral artery)
Cat
Pentobarbitone
Blood pressure, heart rate
Cat
Chloralose
Blood pressure
Cat
Conscious
Blood pressure, heart rate
Clonidine (vertebral and carotid artery) Rilmenidine (vertebral artery) Clonidine (i.c.v.)
Yohimbine (i.c.m.) SK&F86466 (i.c.m.) Efaroxan (i.c.m.) Yohimbine (vertebral artery) Yohimbine (i.v.) Yohimbine (i.c.m.) Yohimbine (vertebral artery) Rauwolscine (vertebral artery) Yohimbine (i.v.)
Bock et al., 1999
Pentobarbitone
Blood pressure, heart rate, sympathetic nerve activity, plasma noradrenaline concentration Blood pressure, heart rate, sympathetic nerve activity, plasma noradrenaline concentration Blood pressure, heart rate, sympathetic nerve activity, plasma noradrenaline concentration Blood pressure, heart rate, plasma noradrenaline concentration Blood pressure
Cat
Conscious
Blood pressure, heart rate
Clonidine (i.v.)
Sea gull
Pentobarbitone
Blood pressure, heart rate
Clonidine (i.v.)
Yohimbine (vertebral artery) Yohimbine (i.c.v.) Efaroxan (i.c.v.) Yohimbine (i.c.v.) Efaroxan (i.c.v.) Yohimbine (i.v.)
Schmitt et al., 1973
Timmermans et al., 1981
McConnaughey & Ingenito, 1982 van Zwieten et al., 1986 Ally, 1997 Ally, 1998 Johansson et al., 1981
i.c.m., injection into the cisterna cerebellomedullaris; i.c.v., injection into a lateral cerebral ventricle.
rilmenidine, and moxonidine. What is more, there was no indication of a role for I1 receptors in these studies. Hieble and Kolpak (1993) first determined i.v. doses of idazoxan and SK&F86466 that caused equal blockade of a2-adrenoceptors in pithed rats. The interaction of i.v.-administered antagonists, at the determined doses, with i.v.-administered clonidine was then studied in anaesthetized rats: idazoxan and SK&F86466 were equieffective at blocking the hypotensive effect of clonidine. This result suggests that the entire effect of idazoxan is attributable to blockade of
a2-adrenoceptors. An additional effect of idazoxan, due to blockade of I1 receptors, could not be seen. In the study of Bock et al. (1999), all drugs were administered intracisternally in conscious rabbits. a-Methyldopa, rilmenidine, and moxonidine lowered blood pressure, heart rate, and the plasma noradrenaline concentration. Efaroxan, yohimbine, and SK&F86466 were administered after the agonists. The hypotensive effects of all three agonists were completely antagonized by all three antagonists (see Fig. 3). The mixed I1/a2 antagonist efaroxan was the most potent of the three
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
17
Table 5 In vivo studies with agonists and antagonists in which the authors conclude that imidazoline receptors are involved in the hypotension Species
Anaesthesia
Measured parameters
Rat Rat
Pentobarbitone Pentobarbitone
Rat
Urethane
Blood pressure, heart rate Blood pressure, heart rate DOPAC concentration in RVLM and LC Blood pressure
Rat
Urethane
Blood pressure
Rat
Conscious
Rat (SHR)
Urethane
Blood pressure, heart rate, sympathetic nerve activity Blood pressure, heart rate
Rat
Urethane
Blood pressure
Mouse
Urethane
Blood pressure, heart rate
Rabbit
Pentobarbitone
Blood pressure, heart rate
Rabbit
Conscious
Blood pressure, heart rate
Rabbit
Conscious
Blood pressure, heart rate
Rabbit
Conscious
Rabbit
Urethane
Blood pressure, heart rate, plasma noradrenaline concentration Blood pressure, heart rate, sympathetic nerve activity
Agonist (route of application)
Antagonist (route of application)
Clonidine (i.c.v.) Clonidine (i.v.)
Yohimbine (i.c.v.) Yohimbine (i.c.m.) Idazoxan (i.c.m.)
Tibirica et al., 1988 Tibiric¸a et al., 1991a
Clonidine (RVLM microinjection) Rilmenidine (i.v.)
Idazoxan (RVLM microinjection) SK&F86466 (RVLM microinjection) SK&F86466 (RVLM microinjection) Idazoxan (RVLM microinjection) Idazoxan (i.c.m.) Yohimbine (i.c.m.) Efaroxan (RVLM microinjection) SK&F86466 (RVLM microinjection) Yohimbine (i.v.) Idazoxan (i.v.) Efaroxan (RVLM microinjection) SK&F86466 (RVLM microinjection) Idazoxan (i.c.m.) Yohimbine (i.c.m.) Idazoxan (i.c.m.) RX821002 (i.c.m.)
Ernsberger et al., 1990
Rilmenidine (i.v.) Rilmenidine (i.c.m.) Moxonidine (RVLM microinjection; i.v.) Clonidine (i.v.) Moxonidine (RVLM microinjection; i.v.) Rilmenidine (i.c.m.) Rilmenidine (i.c.m.) a-Methyldopa (i.c.m.) Rilmenidine (i.c.m.) Moxonidine (i.c.m.) Clonidine (i.c.m.) a-Methyldopa (i.c.m.) Rilmenidine (i.c.m.) Moxonidine (i.c.m.) Guanabenz (i.c.m.) Rilmenidine (RVLM, NTS microinjection)
Reference
Gomez et al., 1991 Mayorov et al., 1993 Haxhiu et al., 1994 Mao & Abdel-Rahman, 1996 Tolentino-Silva et al., 2000 Feldman et al., 1990 Sannajust & Head, 1994
Efaroxan (i.c.m.) RX821002 (i.c.m.)
Chan et al., 1996
Yohimbine (i.c.m.) Efaroxan (i.c.m.)
Szabo & Urban, 1997
Idazoxan (RVLM, NTS microinjection) RX821002 (RVLM, NTS microinjection)
Head & Burke, 1998, 2000b
a-Methylnoradrenaline (RVLM, NTS NTS microinjection) i.c.m., injection into the cisterna cerebellomedullaris; i.c.v., injection into a lateral cerebral ventricle; LC, locus coeruleus.
antagonists against rilmenidine and moxonidine. At first glance, this would suggest a role for I1 receptors in the effects of rilmenidine and moxonidine. However, efaroxan was the most potent of the three antagonists also against a-methyldopa, the active metabolite of which, a-methylnoradrenaline, acts as an a2-adrenoceptor agonist. The data show that selective a2-adrenoceptor antagonists are capable of fully antagonizing the effects of rilmenidine and moxonidine. In addition, the antagonism by the mixed I1/a2 antagonist efaroxan could be fully attributed to blockade of a2-adrenoceptors. An additional effect of efaroxan, due to blockade of I1 receptors, was not seen. It has been argued repeatedly that a2 antagonists do not counteract the effects of agonists by blocking the same primary receptor that is activated by the agonists. Instead, they produce ‘‘functional’’ antagonism; e.g., systemically administered antagonists could inhibit transmission between sympathetic neurons and vascular smooth muscles by blocking vascular a1- and a2-adrenoceptors. In this situation, a centrally elicited sympathoinhibition by clonidine-
like drugs would not be accompanied by hypotension (see Ernsberger & Haxhiu, 1997). Such a ‘‘functional’’ antagonism by systemically administered yohimbine, SK&F86466, and methoxy-idazoxan is unlikely, since these antagonists possess relatively low affinity for a1-adrenoceptors and they did not cause major blood pressure changes when they were administered alone in the above mentioned studies. Moreover, in some studies, systemically administered yohimbine, SK&F86466, and methoxy-idazoxan antagonized the inhibition of sympathetic nerve activity, the inhibition of the firing of RVLM neurons, and the reduction in plasma noradrenaline concentration produced by clonidine, rilmenidine, and moxonidine (Schmitt et al., 1973; Allen & Guyenet, 1993; Szabo et al., 1993; Urban et al., 1994, 1995a; Szabo & Urban, 1995; Vayssettes-Courchay et al., 1996). Experiments in which yohimbine was used as an a2 antagonist were criticized because yohimbine may also interfere with 5-hydroxytryptamine (5-HT) receptors (Ernsberger & Haxhiu, 1997). Indeed, yohimbine possesses rel-
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B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
et al., 1994). However, in cardiovascular studies, it does not elicit responses that are consistent with an effect at 5-HT1A receptors (compare, e.g., Szabo et al., 1992b with Ramage & Fozard, 1987 and Szabo et al., 1992a). It should be noted that idazoxan, the antagonist used in the majority of in vivo studies supporting a role of I1 imidazoline receptors, has an affinity for 5-HT1A receptors comparable with that of yohimbine (Ki = 62 nM) (Kawai et al., 1994). The third type of functional antagonism was suggested by Head et al. (Sannajust & Head, 1994; Chan et al., 1996; Head et al., 1998; Head & Burke, 2000a): clonidine-like drugs primarily activate I1 receptors in the medulla oblongata, but medullary a2-adrenoceptors participate in the chain of events finally leading to sympathoinhibition. a2 Antagonists would produce functional antagonism of the effects of clonidine-like drugs acting downstream from the I1 receptor. This hypothesis diminishes the discriminative power of all experiments in which selective a2 antagonists were used to determine whether a2-adrenoceptors are the primary sites of action of clonidine-like drugs. It should be noted that essential elements of this hypothesis have remained unverified. 9.2. Studies supporting involvement of I1 receptors
Fig. 3. Interactions between the agonists a-methyldopa (AMD), rilmenidine (RIL), and moxonidine (MOX) and the antagonists efaroxan (EFA), yohimbine (YOH), and SK&F86466 (SKF) in conscious rabbits. All drugs were administered into the cisterna magna (i.c.m.). At first, animals were pretreated with a-methyldopa (400 mg kg 1), rilmenidine (10 mg kg 1), or moxonidine (0.3 mg kg 1). The three agonists lowered blood pressure equally by 12 mm Hg (not shown), and the blood pressure remained at the hypotensive values in animals not receiving antagonists (not shown). Administration of efaroxan, yohimbine, and SK&F86466 dose-dependently and significantly increased blood pressure in animals pretreated with the agonists. The antagonists had no significant effects in animals that had not been pretreated with agonists (not shown). The figure demonstrates that the hypotensive effects of rilmenidine and moxonidine were antagonized by all three antagonists. Notably, the selective a2-adrenoceptor antagonists yohimbine and SK&F86466 were capable of causing full antagonism. Efaroxan, which is suggested to be a selective I1 imidazoline receptor antagonist, was 3 - to 10 - fold more potent against rilmenidine and moxonidine than yohimbine and SK&F86466. In my opinion, this is not due to the affinity of efaroxan for I1 receptors, but due to its high affinity for a2-adrenoceptors: efaroxan was the most potent of the three antagonists also against a-methyldopa, which is thought to activate a2-adrenoceptors after its conversion into a-methylnoradrenaline. Significant blood pressure increase evoked by the antagonists: * P < 0.05. Data from Bock et al. (1999).
atively high affinity for 5-HT1A-binding sites (Ki = 29 – 74 nM) and elicits behavioural effects similar to those of 5-HT1A receptor agonists (Winter & Rabin, 1992; Kawai
As pointed out in Section 4.2.2 (see also Table 3), no I1-selective antagonists are available. For lack of better drugs, idazoxan and efaroxan were used in the studies that concluded that I1 receptors are the primary sites of action of clonidine-like drugs (Table 5). Both drugs are a2-adrenoceptor antagonists, with additional affinity for I1-binding sites. Several kinds of experimental designs were used. In the study of Tibirica et al. (1988), yohimbine was ineffective against clonidine, and this was taken as an indication of the involvement of imidazoline receptors in the effects of clonidine. In other studies, the selective a2 antagonists yohimbine and SK&F86466 were compared with idazoxan and efaroxan. The effects of clonidine, rilmenidine, and moxonidine were considered to be mediated by I1 receptors, if idazoxan and efaroxan were more effective than yohimbine or SK&F86466 at antagonizing the hypotensive and sympathoinhibitory effects of clonidine, rilmenidine, and moxonidine (Ernsberger et al., 1990; Feldman et al., 1990; Gomez et al., 1991; Tibiric¸a et al., 1991a; Mayorov et al., 1993; Haxhiu et al., 1994; Mao & AbdelRahman, 1996; Tolentino-Silva et al., 2000). These studies have a major shortcoming. It was not experimentally verified that at the given doses, yohimbine and SK&F86466, on the one hand, and idazoxan and efaroxan, on the other hand, caused equal a2-adrenoceptor blockade. In other words, the greater effectivity of idazoxan and efaroxan than that of yohimbine and SK&F86466 at antagonizing the effects of clonidine, rilmenidine, and moxonidine may have been due to more complete blockade of a2-adrenoceptors and not to blockade of I1 receptors. For example, idazoxan is 10- to 20fold more potent than yohimbine at the a2A/D-adrenoceptor
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
(Table 3; Remaury & Paris, 1992; Renouard et al., 1994; Uhlen et al., 1995). a2A/D is the most abundant a2-adrenoceptor subtype in cardiovascular regulatory centres of rats. Idazoxan antagonized more effectively than yohimbine (the two antagonists were administered at equimolar doses) the effects of clonidine (Tibiric¸a et al., 1991a) and rilmenidine (Mayorov et al., 1993). These findings probably reflect the difference in affinity of the two antagonists for the a2A/D-adrenoceptor. In addition to their high affinity for a2-adrenoceptors, the imidazoline antagonists may penetrate into brain tissue more easily than the non-imidazoline antagonists, and this may enhance their effectivity in vivo. The effects of clonidine, rilmenidine, and moxonidine, administered into the cisterna cerebellomedullaris of conscious rabbits, were analyzed in three studies (Sannajust & Head, 1994; Chan et al., 1996; Szabo & Urban, 1997). These agonists were compared with guanabenz and a-methyldopa. The interaction with intracisternally administered yohimbine, methoxy-idazoxan, idazoxan, and efaroxan was also tested. A constant finding of these studies was that the a2-adrenoceptor antagonists yohimbine and methoxy-idazoxan always antagonized the effects of clonidine, rilmenidine, and moxonidine. In the study of Sannajust and Head (1994), methoxy-idazoxan was 10-fold more potent against a-methyldopa than against rilmenidine, suggesting a different mechanism of action for the two agonists. In the experiments of Chan et al. (1996) and Szabo and Urban (1997), the mixed I1/a2 antagonist efaroxan was more effective than the a2-adrenoceptor antagonists methoxyidazoxan and yohimbine (efaroxan, methoxy-idazoxan, and yohimbine were administered at doses that produced equal blockade of a2-adrenoceptors) against rilmenidine and moxonidine. Accordingly, it was concluded that I1 receptors were likely to be involved in the effects of rilmenidine and moxonidine. Interestingly, Chan et al. (1996) concluded that clonidine acts mainly through a2-adrenoceptors. It should be critically noted that the conclusions of these studies, in favour of involvement of I1 receptors, rely on 3- to 10-fold potency differences of antagonists. This value may be too low for firm conclusions. In two recent studies in anaesthetized rabbits, rilmenidine and the antagonists methoxy-idazoxan and idazoxan were microinjected into the RVLM (Head & Burke, 1998, 2000b). Rilmenidine elicited hypotension and inhibited RSNA. In addition, the blood pressure –RSNA reflex curve was shifted to the left, and the maximum of the curve was depressed. The effects of rilmenidine on blood pressure and the blood pressure –RSNA reflex curve were counteracted by methoxy-idazoxan and idazoxan. Since idazoxan had no effect against a-methylnoradrenaline, it was suggested that rilmenidine acted by activating I1 receptors. It is remarkable that in these two studies, idazoxan antagonized I1 receptor-mediated effects; yet, at the same dose, it did not counteract a2-adrenoceptor-mediated effects. From the results of binding studies (Table 3), one would expect
19
that idazoxan blocks a2-adrenoceptors more readily than I1 receptors. The findings of the in vivo agonist/antagonist interaction experiments can be summarized as follows. In the majority of experiments, pure a2-adrenoceptor antagonists were capable of preventing or abolishing the hypotensive and sympathoinhibitory effects of imidazoline antihypertensive drugs. In some experiments, only the mixed I1/a2 antagonists idazoxan and efaroxan produced full blockade of the hypotensive effects. It is doubtful, however, whether the doses of the applied selective a2-adrenoceptor antagonists were sufficient. Only three studies showed that idazoxan and efaroxan were more effective than selective a2-adrenoceptor antagonists and that this difference was not due to more complete blockade of a2-adrenoceptors by idazoxan and efaroxan.
10. Experiments in genetically modified animals Mice with genetically modified a2-adrenoceptors were also used to clarify the mechanism of action of clonidinelike drugs. In some studies, the D79N mouse strain was used (MacMillan et al., 1996; Zhu et al., 1999; TolentinoSilva et al., 2000). In these mutant mice, the coupling of the a2A/D-adrenoceptor to certain K + channels is disrupted (Surprenant et al., 1992). Other studies used mice in which the a2A/D-adrenoceptor (Altman et al., 1999), the a2B-adrenoceptor, or the a2C-adrenoceptor was deleted (Link et al., 1996). Compared with control mice, the hypotensive action of UK14304 and dexmedetomidine, injected into the carotid artery, was lost in D79N mice (MacMillan et al., 1996). The hypotensive effect of dexmedetomidine was also lost in a2A/D-adrenoceptor knockout mice (Altman et al., 1999). UK14304 and dexmedetomidine are selective a2-adrenoceptor agonists without affinity for I1 receptors (see Section 4.1.1). These results indicate, therefore, that activation of a2A/D-adrenoceptors can cause hypotension; they do not help to answer the question whether a2-adrenoceptors are involved in the action of I1 imidazoline agonists, such as clonidine, rilmenidine, and moxonidine. The hypotensive action of clonidine, rilmenidine, and moxonidine, administered i.v. in conscious animals, was lost in D79N mice (Zhu et al., 1999). In contrast to these results, the hypotensive effect of moxonidine, microinjected directly into the RVLM, was preserved in anaesthetized D79N mice (Tolentino-Silva et al., 2000). The authors of this latter study believe that the effect of moxonidine is mediated by I1 imidazoline receptors. They suggest that the effect is more pronounced in anaesthetized animals and with direct application of moxonidine into the RVLM, as compared with systemical administration in conscious animals. Still, the difference between the observations of Zhu et al. (1999) and Tolentino-Silva et al. (2000) remains unexplained.
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B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
The hypotensive effect of dexmedetomidine was also studied in mice in which the a2B-adrenoceptor or the a2C-adrenoceptor was deleted (Link et al., 1996). Deletion of the a2C-receptor had no influence on the hypotensive effect, whereas in a2B-deficient mice, the hypotension was potentiated. The enhanced hypotension was attributed to the missing a2B-adrenoceptor-mediated vascular contraction. Fairbanks and Wilcox (1999) observed an interesting non-cardiovascular effect of moxonidine, and the results may be relevant to cardiovascular studies. Moxonidine caused spinal antinociception in mice. This effect persisted in D79N mice, and could be antagonized by the selective a2-adrenoceptor antagonist SK&F86466. The authors concluded that the effect of moxonidine was mediated by a2B- or a2C-adrenoceptors. Thus, the experiments in genetically modified animals did not really help to answer the question of the role of a2-adrenoceptors in the effects of I1 imidazoline receptor agonists. Either agonists without affinity for I1 receptors were used, or the results of the different studies are contradictory.
11. Relationship between sympathoinhibition and other A2-adrenoceptor-mediated effects Sedation and inhibition of saliva secretion are the major side effects of centrally acting antihypertensive drugs. Sedation was indeed the effect that led to the discovery of the prototype of this class of drugs, clonidine (see Section 2). The sedative effect is attributed to a2-adrenoceptor-mediated inhibition of the activity of noradrenergic neurons in the locus coeruleus (De Sarro et al., 1987; Correa-Sales et al., 1992; Scheinin & Schwinn, 1992). The a2-adrenoceptor responsible for the inhibition of locus coeruleus neurons and sedation was recently identified as the a2A/D receptor (Chiu et al., 1995; Mizobe et al., 1996; No¨renberg et al., 1997; Mateo & Meana, 1999; see also Sallinen et al., 1997). One study showed inhibition of locus coeruleus neurons mediated by a2B- or a2C-adrenoceptors (Arima et al., 1998). The imidazoline antihypertensive drugs clonidine (IC50 = 6 nM), rilmenidine (IC50 = 127 nM), and moxonidine (IC50 = 49 nM) inhibit the firing of locus coeruleus neurons in rat brain slices, and the effects are blocked by the selective a2-adrenoceptor antagonist SK&F86466 (Szabo et al., 1996). The mechanism of the inhibition of salivation by clonidine-like drugs is less clear. While a2-adrenoceptors mediate this effect, the subtype of the involved a2-adrenoceptor is not known yet. Experimental data support action at three different sites. (1) Clonidine-like drugs can inhibit salivation, with a primary action in the CNS, probably in the brain stem (Green et al., 1979; Warren et al., 1991). (2) Saliva secretion can be lowered by inhibition of acetylcholine release from postganglionic cholinergic neurons in the salivary glands (Montastruc et al., 1989; Izumi et al., 1995). (3) Finally, it was suggested that clonidine-like drugs inhibit saliva secre-
tion by direct action on the secretory cells in the salivary glands (Kaniucki et al., 1984; Lung, 1994, 1998). An important component of the imidazoline hypothesis deals with the relationship between sympathoinhibition and a2-adrenoceptor-mediated effects. It is suggested that the drugs that cause sympathoinhibition by activation of a2-adrenoceptors, i.e., guanabenz, guanfacine, and a-methyldopa, simultaneously elicit the a2-adrenoceptor-mediated side effects of sedation and dry mouth. In contrast, the imidazoline agonists rilmenidine and moxonidine, and some say even clonidine, cause sympathoinhibition, without eliciting a2-adrenoceptor-mediated effects. a2-Adrenoceptormediated effects would appear only after higher doses of these drugs. Importantly, the new drugs rilmenidine and moxonidine, due to their improved I1/a2 selectivity, should cause less sedation and inhibition of saliva secretion than clonidine. How is this assumption of the imidazoline hypothesis supported by experimental observations in animals and humans? 11.1. Animal experiments 11.1.1. Clonidine Sedation of animals by clonidine was recognized early (Hoefke & Kobinger, 1966). Inhibition of the firing of locus coeruleus neurons was first observed by Svensson et al. in 1975. Systemically administered clonidine also inhibits the release of noradrenaline in the hippocampus and the medial prefrontal cortex, brain regions that receive their noradrenergic input from the locus coeruleus (Abercrombie et al., 1988; Gresch et al., 1995; Sacchetti et al., 1999). Several studies showed that clonidine causes hypotension and inhibits the firing of locus coeruleus neurons or lowers noradrenaline release in projection regions of the locus coeruleus at the same doses. Thus, in the rat, the hypotensive doses of clonidine are within the 1 – 30 mg kg 1 range (i.v.; see Table 1). The ED50 value of clonidine for reduction of the firing rate of locus coeruleus neurons in rats, 6– 7 mg kg 1 (i.v.), falls into this range (Dresse & Scuve´e-Moreau, 1986; Engberg & Eriksson, 1991). Clonidine 10 mg kg 1 (i.p.) also lowers the extracellular noradrenaline concentration in the hippocampus of the rat (Sacchetti et al., 1999). Simultaneous effects of clonidine on sympathetic regulatory centres and the locus coeruleus were seen in conscious rabbits using neuroanatomical techniques (Li & Dampney, 1995). The expression of the Fos protein, a marker of neuronal activation, was determined immunohistochemically. Infusion of sodium nitroprusside led to Fos expression in the RVLM, the locus coeruleus, and other brain stem nuclei. Injection of clonidine (7 –30 mg kg 1, i.v.) suppressed the appearance of Fos in the RVLM, as well as in the locus coeruleus. No preferential effect of clonidine in the RVLM was seen. In contrast to the above observations, one group found that low doses of clonidine selectively cause sympathoinhi-
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
bition (Tibirica et al., 1989). In anaesthetized normotensive rats, 2, 5, and 10 mg kg 1 of clonidine (i.v.) lowered the blood pressure and the concentration of 3,4-dihydroxyphenylacetic acid (DOPAC) in the NRL. DOPAC was thought to reflect the activity of catecholaminergic neurons involved in sympathetic regulation because its concentration correlated with blood pressure changes. In contrast to the DOPAC changes in the NRL, clonidine had no effect on the DOPAC concentration in the locus coeruleus up to 10 mg kg 1. Only the high dose of 50 mg kg 1 lowered the DOPAC concentration in the locus coeruleus. The selective action of clonidine in the NRL, a region containing imidazoline-binding sites, was explained by a preferential effect on imidazoline receptors. It should be noted that DOPAC is a suboptimal indicator of neuronal activity, most of all because its source is not clarified. It may derive from newly synthesized dopamine, before the amine is taken up into vesicles, in noradrenergic and adrenergic neurons (Tibirica et al., 1989; Lambas-Senas et al., 1990). However, it may also be produced in dopaminergic neurons from newly synthesized dopamine or dopamine taken up by the dopamine carrier. Interestingly, the pattern of effects of clonidine was quite different in anaesthetized SHR (Tibiric¸a et al., 1992): 10 mg kg 1 of clonidine (i.v.) markedly lowered blood pressure, without changing the DOPAC concentration in the NRL. The authors concluded that in SHR, the site of action of clonidine is not in the NRL and that a2-adrenoceptors are involved in the sympathoinhibition. This conclusion was supported by previous results of the group: in SHR, the hypotensive effect of intracisternally applied clonidine was sensitive to low doses of intracisternally injected yohimbine (Tibirica et al., 1988). A remarkable discrepancy in the observations of two leading groups in the field becomes apparent here. One group did not observe imidazoline receptor-mediated effects in the medulla oblongata of SHR (Tibirica et al., 1988, 1992). In contrast, another group repeatedly obtained results supporting the role of imidazoline receptors in the hypotension elicited by clonidine and moxonidine in SHR (Haxhiu et al., 1994; Buccafusco et al., 1995). In summary, in the majority of the experimental models, clonidine simultaneously lowers blood pressure, inhibits neuronal activity in cardiovascular centres, and inhibits activity of locus coeruleus neurons. The one notable exception is the study in which DOPAC was used as a marker of neuronal activity. As mentioned above, DOPAC may not be an appropriate marker for neuronal activity in the NRL and the locus coeruleus. 11.1.2. Rilmenidine The sedative effect of rilmenidine was already described in the first publication dealing with the mechanism of the hypotensive action of this drug (Laubie et al., 1985). The sedation, measured as the loss of the righting reflex in chickens, appeared at doses ( 5 mg kg 1, s.c.) higher
21
than those necessary to elicit hypotension, and was of short duration. In the same study, clonidine also caused sedation after doses ( 62 mg kg 1, s.c.) higher than those necessary to elicit hypotension, and the maximal duration of sedation was longer than after rilmenidine. The effect of rilmenidine on the firing rate of locus coeruleus neurons was studied by one group (Laubie et al., 1985; Dresse & Scuve´e-Moerau, 1986). Intravenously administered rilmenidine inhibited the firing of locus coeruleus neurons in anaesthetized rats with an ED50 = 350 mg kg 1. This value is identical to the doses of rilmenidine that cause hypotension and bradycardia in anaesthetized rats (ED50 = 250 and 350 mg kg 1, respectively) (Gomez et al., 1991; and see Table 1 for further hypotensive doses of rilmenidine). To our knowledge, the effect of rilmenidine on noradrenaline concentration in the extracellular space in the brain has not been determined in animals with intact a2-adrenoceptors. Simultaneous effects of rilmenidine on sympathetic regulatory centres and the locus coeruleus were seen in conscious rabbits by measuring the expression of the Fos protein (Li & Dampney, 1995). Rilmenidine (150 – 300 mg kg 1, i.v.) suppressed the synthesis of the Fos protein in the RVLM and the locus coeruleus. No preferential effect in the RVLM was seen. Moreover, rilmenidine inhibited locus coeruleus neurons as strongly as clonidine. In addition, rilmenidine markedly suppressed the expression of the Fos protein in the NTS. Such an effect is not predicted by the imidazoline hypothesis for a selective imidazoline agonist. Rilmenidine-evoked changes in neuronal activity were also determined by measuring DOPAC (Tibiric¸a et al., 1991b), and, like in the case of clonidine, the results of these experiments contradict the results of other studies. In anaesthetized normotensive rats, 0.3 and 1.5 mg kg 1 of rilmenidine (i.v.) lowered blood pressure and the concentration of DOPAC in the NRL. In contrast, the DOPAC concentration in the locus coeruleus decreased only after a dose of 15 mg kg 1. It was calculated that rilmenidine possesses 50-fold selectivity for decreasing blood pressure and neuronal activity in the NRL versus decreasing neuronal activity in the locus coeruleus. In line with these observations, the same group showed that intravenously administered rilmenidine, at doses of 0.3 and 1 mg kg 1, does not change the increase in the voltammetrically measured electrochemical signal in the hypothalamus (attributed to noradrenaline) evoked by electrical stimulation (10 Hz) of the ventral noradrenergic pathway (Suaud-Chagny et al., 1992). At doses of 3 and 10 mg kg 1, rilmenidine even increased the signal. In contrast, clonidine (50 mg kg 1; i.v.) reduced the evoked noradrenergic signal. The authors concluded in this latter study that ‘‘. . .rilmenidine, at clinically relevant doses, is not active on central a2-adrenoceptors. . ..’’ In the experiments of Tibiric¸a et al. (1991b) and SuaudChagny et al. (1992), rilmenidine ( 7.5 mg kg 1 i.v.) had no effect on the DOPAC concentration in the locus coeruleus and the evoked noradrenaline release in the hypothalamus. If the voltametric measurements faithfully reflected
22
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
activity of noradrenergic neurons in the locus coeruleus and the hypothalamus, a conclusion on the brain penetration of rilmenidine could be made. Rilmenidine, at i.v. doses of 100 –300 mg kg 1, presynaptically inhibits noradrenaline release from postganglionic sympathetic neurons in the
pithed rat (see Section 12.1 and Table 6). The receptors mediating slowing of locus coeruleus neurons and presynaptic inhibition of noradrenaline release from central nervous neurons and peripheral sympathetic neurons are identical, predominantly a2A/D-adrenoceptors in the rat
Table 6 Peripheral presynaptic inhibition by clonidine-like drugs in whole animal preparations with an artificial sympathetic tone Species Rat
Experimental model Pithed animal; spinal electrical stimulation Pithed animal; spinal electrical stimulation Pithed animal; spinal electrical stimulation
Rat Rat
Rat
Pithed animal; spinal electrical stimulation
Rat
Pithed animal; spinal electrical stimulation Pithed animal; spinal electrical stimulation
Rat
Rat
Pithed animal; spinal electrical stimulation
Rat Rat (SHR)
Rabbit Rabbit
Rabbit
Cat
Cat Dog
Dog
1
Pithed animal; spinal electrical stimulation Pithed animal; spinal electrical stimulation Pithed animal; spinal electrical stimulation Pithed animal; spinal electrical stimulation
Pithed animal; spinal electrical stimulation Urethane anaesthesia; electrical stimulation of the lumbar sympathetic chain Pithed animal; spinal electrical stimulation Pentobarbitone anaesthesia; electrical stimulation of the cardioaccelerator nerves Pentobarbitone anaesthesia; electrical stimulation of the hepatic nerves
Agonist (route of application)
Effect Inhibition of the increase in heart Inhibition of the increase in heart Inhibition of the increase in heart
evoked rate evoked rate evoked rate
Inhibition of the evoked increase in heart rate and blood pressure Inhibition of the evoked increase in heart rate Inhibition of the evoked increase in the plasma noradrenaline concentration Inhibition of the evoked increase in blood pressure Inhibition of the evoked increase in heart rate and blood pressure Inhibition of the evoked increase in heart rate Inhibition of the evoked increase in the plasma noradrenaline concentration Decrease in total body noradrenaline spillover Decrease in the evoked increase in plasma noradrenaline concentration and heart rate Decrease in the evoked increase in blood pressure and heart rate Inhibition of the evoked increase in hindquarter perfusion pressure
Effective doses1
Reference 1
Clonidine (i.v.)
ED50 = 5 mg kg
Clonidine (i.v.)
ED50 1.6 mg kg
Clonidine (i.v.) UK14304 (i.v.) B-HT920 (i.v.) B-HT933 (i.v.) Clonidine (i.v.) Rilmenidine (i.v.)
ED50 = 1.6 mg kg ED50 = 1.5 mg kg ED50 = 2.8 mg kg ED50 = 38 mg kg 10 mg kg 1 300 mg kg 1
Rilmenidine (i.v.)
ED50 53 mg kg
Clonidine (i.v.)
ED50 = 14 mg kg
1
ED50 = 22 mg kg
1
Pichler & Kobinger, 1978 1
1
de Jonge et al., 1981a van Meel et al., 1981
1 1 1
Laubie et al., 1985
1
van Zwieten et al., 1986 Szemeredi et al., 1988
Clonidine (i.v.) Rilmenidine (i.v.)
10 – 30 mg kg 1 100 – 300 mg kg
Moxonidine (i.v.) Clonidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.) Moxonidine (i.v.) Clonidine (i.v.) Rilmenidine (i.v.) Moxonidine (i.v.) UK14304 (i.v.)
ED50 = 60 mg kg 1 ED50 = 18 mg kg 1 0.35 – 24 mg kg 1 min 1 17 – 150 mg kg 1 min 1 1.4 – 83 mg kg 1 min 1 1 mg kg 1 min 1 (< 30 mg kg 1 total dose) 100 – 1000 mg kg 1 10 – 100 mg kg 1 3 – 30 mg kg 1
Clonidine (i.v.) Rilmenidine (i.v.)
15 – 1000 mg kg 30 – 1000 mg kg
Guanabenz (i.v.)
32 – 320 mg kg
Inhibition of the evoked increase in heart rate Inhibition of the evoked increase in noradrenaline concentration in the coronary sinus
Guanfacine (i.v.)
6 – 60 mg kg
Clonidine (i.v.)
15 mg kg
1
Yamaguchi et al., 1977
Inhibition of the evoked increase in noradrenaline concentration in the hepatic vein Inhibition of the evoked increase in vascular resistance
Clonidine (i.v.)
20 mg kg
1
Yamaguchi, 1982
Several of the ED50 values are approximate values read from the dose – response curves in the references.
1
Koenig-Berard et al., 1988 1
1
Armah, 1988 Ha¨user et al., 1995
Majewski et al., 1983 Urban et al., 1995b
Pompermayer et al., 1999
1
1
Baum et al., 1970
Scholtysik et al., 1975
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
23
(see above in this section and Section 12.1). In the presence of a peripheral effect of rilmenidine, the missing central effect would indicate poor diffusion of the drug through the blood-brain-barrier. The conclusion from the studies in which central neuronal activity was determined electrically or by measuring Fos protein expression is that systemically administered rilmenidine does not possess selectivity for central sympathoinhibitory receptors, inhibition of locus coeruleus neurons occurs simultaneously. Opposed to these observations are the findings of voltammetric measurements. As pointed out in the case of clonidine (Section 11.1.1), one reason for the discrepancy may be that DOPAC does not faithfully reflect neuronal activity in the NRL and locus coeruleus. Moreover, the release of noradrenaline in the hypothalamus was evoked by stimulation at the high frequency of 10 Hz (Suaud-Chagny et al., 1992). Presynaptic inhibition is weak under this condition (see Starke, 1987).
were compared (Fillastre et al., 1988). The two drugs lowered blood pressure equally. After 2 weeks of treatment, rilmenidine caused sedation in 5% and dry mouth in 12% of the patients (data approximately read from the graphs in Fillastre et al. [1988]), and the frequency of these effects did not change with the duration of treatment. In the clonidinetreated group, the frequency of sedation was 13% and the incidence of dry mouth was 30% after 2 weeks of treatment. The frequency of the clonidine-evoked ‘‘dry mouth’’ did not change with time. The sedative effect of clonidine decreased with time, and 6 weeks after the beginning of therapy, there was no significant difference between the sedative effects of rilmenidine and clonidine. Rilmenidine increases the plasma concentration of growth hormone (unpublished observation mentioned by Verbeuren et al. [1990]). This effect is most probably attributable to activation of a2-adrenoceptors in the hypothalamus (see Devesa et al., 1991; Bertherat et al., 1995; Mu¨ller et al., 1995).
11.1.3. Moxonidine The sedative effect of moxonidine was mentioned in the first publication describing the sympathoinhibitory and general pharmacological properties of moxonidine (Armah et al., 1988). It decreased the locomotor activity of mice with an ED50 value of 7.5 mg kg 1 (s.c.). The corresponding ED50 value for clonidine was 0.35 mg kg 1 (s.c.). Armah et al. (1988) also reported that moxonidine decreases salivation in rabbits by 70% after 100 mg kg 1 (i.v.). This dose is similar to doses causing hypotension in the rabbit (see Table 1). To our knowledge, the effects of moxonidine on the firing rate of locus coeruleus neurons or on the concentration of noradrenaline in the extracellular space in the brain have not been determined.
11.2.3. Moxonidine The incidence of sedation and dry mouth in hypertensive patients receiving long-term moxonidine treatment is 5– 8% and 8– 9%, respectively (for meta-analysis of clinical pharmacological trials, see Schachter et al., 1998). There are three controlled studies in which moxonidine was compared with clonidine. In a double-blind crossover study on 20 hypertensive patients, clonidine (0.3 mg/day) and moxonidine (0.3 mg/day), each administered for 2 weeks, lowered blood pressure equally (Pla¨nitz, 1984). Moxonidine caused sedation (15% vs. 60%) and dry mouth (20% vs. 75%) less frequently than clonidine. In the second study, a doubleblind multicentre study, equal doses of clonidine (0.36 mg/ day) and moxonidine (0.37 mg/day) were administered for 6 weeks to 122 and 30 hypertensive patients, respectively (Pla¨nitz, 1987). The two drugs lowered blood pressure equally. The incidence of sedation was similar in the moxonidine- and clonidine-treated groups (13% vs. 17%), but dry mouth was observed less frequently in moxonidine-treated patients than in clonidine-treated patients (20% vs. 47%). In the third study (MacPhee et al., 1992), on 9 normotensive volunteers, a single oral dose of moxonidine (0.2 mg) lowered blood pressure less than a single oral dose of clonidine (0.2 mg). Moxonidine also caused less sedation and less inhibition of saliva flow than clonidine. Since the hypotensive effects of the two drugs differed, it is difficult to compare the frequencies of their side effects. Moxonidine (0.3 mg orally) increases the plasma concentration of growth hormone for at least 2 hr (Bamberger et al., 1995). Summarizing, the newer antihypertensive drugs rilmenidine and moxonidine were compared with clonidine in four studies, using doses that are equally hypotensive. After acute administration and 2 weeks after the beginning of treatment, rilmenidine and moxonidine elicit less sedation and dry mouth than clonidine. After 6 weeks of treatment,
11.2. Studies in humans 11.2.1. Clonidine It was shown in acute studies on normotensive volunteers that clonidine simultaneously leads to hypotension, sedation, and dry mouth (Dollery et al., 1976; Davies et al., 1977). The side effects are also present in patients treated with clonidine for a long time. However, their frequency, especially that of sedation, decreases with time (e.g., Ferder et al., 1987; Fillastre et al., 1988). 11.2.2. Rilmenidine In an acute study on 10 normotensive volunteers, rilmenidine, in addition to lowering blood pressure, caused sedation and dry mouth (Weerasuriya et al., 1984). Rilmenidine was also compared with clonidine in this study. At small equihypotensive doses, rilmenidine inhibited saliva production less than clonidine, and the sedative effect of rilmenidine was also less intensive. In one double-blind multicentric study on 333 hypertensive patients, the effects of rilmenidine and clonidine, administered for 6 weeks,
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only the frequency of dry mouth differs in the different treatment groups. It is interesting that in increasing the plasma concentration of growth hormone, rilmenidine and moxonidine elicit an effect attributed to activation of central a2-adrenoceptors.
12. Contribution of presynaptic inhibition of transmitter release from postganglionic sympathetic neurons to the overall reduction of sympathetic tone produced by clonidine-like drugs
(see Table 1). As an example, the ED50 value of clonidine for inhibition of the electrically evoked bradycardia in pithed rats is 1.6 mg kg 1 (i.v.) (Table 6) (de Jonge et al., 1981a), very similar to the ED50 values of clonidine for lowering blood pressure and heart rate in anaesthetized rats (2.6 and 1.3 mg kg 1 [i.v.], respectively) (Table 1; de Jonge et al., 1981a). Similarly, at doses of 100 – 300 mg kg 1 (i.v.), rilmenidine inhibits the electrically evoked increases in blood pressure and heart rate in pithed rats (Table 6) (Laubie et al., 1985; Koenig-Berard et al., 1988), and the ED50 values of rilmenidine for lowering blood pressure and heart rate in
In most textbooks and reviews, clonidine-like drugs are classified as centrally acting antihypertensive agents, reflecting the opinion that the sole, or most important, site of action of these drugs is located in the CNS (e.g., Kobinger & Pichler, 1990; Verbeuren et al., 1990; Ernsberger et al., 1997; Bousquet & Feldman, 1999). I believe, however, that inhibition of transmitter release from postganglionic sympathetic neurons is an important component in the action of these drugs, and the evidence for the peripheral presynaptic effect is discussed in this section. 12.1. Peripheral presynaptic inhibition in experimental models with an artificial sympathetic tone Activation of presynaptic a2-autoreceptors inhibits transmitter release from postganglionic sympathetic neurons in almost all isolated cardiovascular tissues that have been studied (for reviews, see Starke, 1977, 1987; Langer, 1981; Starke et al., 1989). The subtype of the a2-adrenoceptor predominantly involved in peripheral presynaptic inhibition is a2A or a2A/D (Starke et al., 1995; Trendelenburg et al., 1997). However, recent observations indicate a role also for a2C-adrenoceptors (Altman et al., 1999; Trendelenburg et al., 1999). Table 6 lists experiments with clonidine-like drugs in whole animal models. An artificial sympathetic tone was created, either by electrical stimulation of the entire sympathetic outflow in pithed animals or of some sympathetic nerve trunks in anaesthetized animals. A constant observation in these studies is that clonidine-like drugs inhibit the electrically evoked responses, i.e., the increase in blood pressure or vascular resistance, the increase in heart rate, and the increase in the plasma noradrenaline concentration. The effect is elicited by clonidine, guanfacine, and guanabenz, but also by the more recently developed drugs rilmenidine and moxonidine. The effect was observed in all species studied: rats, rabbits, dogs, and cats. The most likely mechanism is presynaptic inhibition of transmitter release from postganglionic sympathetic neurons. It is very important that the doses eliciting peripheral presynaptic inhibition of neuro-effector transmission (see Table 6) are essentially identical with the doses that cause central nervous sympathoinhibition, hypotension, and bradycardia in conscious or anaesthetized animals
Fig. 4. Comparison of the effects of rilmenidine and moxonidine on mean arterial pressure (MAP) and RSNA in conscious rabbits and on the plasma noradrenaline concentration (PL-NA) and heart rate (HR) in pithed rabbits. In pithed rabbits, either the entire sympathetic outflow (in experiments in which PL-NA was determined) or only the postganglionic sympathetic nerves of the heart (in experiments in which the HR response was studied) were stimulated. Values are expressed as percentages of the initial values preceding drug administration (PRE). Intravenously administered moxonidine and rilmenidine significantly (stars not shown for sake of clarity) lowered the blood pressure and the firing rate of renal sympathetic nerves in conscious rabbits. They also significantly (stars not shown for sake of clarity) inhibited the electrically evoked increases in the PL-NA and HR in pithed rabbits. The figure demonstrates that in the case of both agonists, the peripheral presynaptic effects occur at the same doses that cause central inhibition of sympathetic nerve activity and decrease of blood pressure in conscious animals. It is very likely that the peripheral presynaptic inhibition contributes to the overall reduction of sympathetic tone, and, therefore, to the decrease in blood pressure in conscious rabbits. Data from Urban et al. (1994, 1995a, 1995b).
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
anaesthetized rats are 250 and 350 mg kg 1 (i.v.), respectively (Table 1) (Gomez et al., 1991). The identity of doses eliciting peripheral effects in pithed animals and overall sympathoinhibition in intact animals was also shown for rabbits. In Fig. 4, the effects of rilmenidine and moxonidine in pithed and conscious rabbits are compared. In pithed rabbits with electrically stimulated sympathetic outflow, rilmenidine and moxonidine inhibit the electrically evoked increase in the plasma noradrenaline concentration and heart rate. Both effects reflect the peripherally elicited decrease in noradrenaline release from sympathetic neurons. In conscious rabbits, rilmenidine and moxonidine inhibit the firing of renal sympathetic nerves (an indication of central sympathoinhibition) and lower blood pressure (an indication of the overall reduction of sympathetic tone) (Fig. 4). The identity of doses causing peripheral and central sympathoinhibition and overall sympathoinhibition is evident. It may be added that the doses of rilmenidine and moxonidine causing hypotension and central sympathoinhibition in our experiments are identical with the doses causing hypotension and bradycardia in other experiments in rabbits (Table 1) (Armah et al., 1988; Head & Burke, 1991). Since peripheral presynaptic inhibition in animals with artificial sympathetic tone occurs exactly at the same doses that produce central sympathoinhibition and hypotension in intact animals, it is reasonable to assume that the peripheral effect also occurs in intact animals and contributes to the overall reduction of sympathetic tone. 12.2. Peripheral presynaptic inhibition under physiological conditions It is difficult to detect the peripheral presynaptic effect of clonidine-like drugs in vivo during ongoing physiological sympathetic impulse traffic because the drugs simultaneously inhibit sympathetic nerve firing by a central mechanism. It is difficult to determine which portion of the overall sympathoinhibition is attributable to the peripheral action and which is attributable to the central action. Two approaches were used to analyze the role of the peripheral presynaptic inhibition. In one approach, the effect of the pharmacological tools was restricted either to the central or to the peripheral compartment. Sannajust et al. (1992b) compared the effects of intracerebroventricularly and intravenously administered clonidine and rilmenidine in conscious SHR. The drugs lowered blood pressure much less after central administration than after systemical administration. Accordingly, the authors concluded that the findings ‘‘question the involvement of a major central site of action for these antihypertensive a2-adrenoceptor agonists.’’ In the experiments of Brown and Harland (1984) in rats, clonidine was administered systemically and idazoxan was administered intracerebroventricularly. Peripheral presynaptic effects of clonidine became evident from the finding that idazoxan
25
prevented the clonidine-evoked hypotension, but not the clonidine-evoked decrease in heart rate and the plasma noradrenaline concentration. In similar experiments in rabbits, we showed that the decrease in heart rate and plasma noradrenaline concentration caused by intravenously injected moxonidine was resistant to intracisternally administered yohimbine (Urban et al., 1995a). A peripheral presynaptic effect of systemically administered clonidine was also shown in humans with the help of MK-467, a peripherally acting a2-adrenoceptor antagonist (Warren et al., 1991). Interesting are the findings obtained with St-91, an analogue of clonidine acting selectively in the periphery. In conscious rats, it elicits hypotension (Gutkowska et al., 1997), indicating that peripheral presynaptic inhibition alone is capable of lowering blood pressure. However, in anaesthetized sheep, St-91 increases systemic vascular resistance and blood pressure (Celly et al., 1999). In two experiments in humans, the effects of clonidine were restricted to the peripheral nervous system, and no sympathoinhibition was observed. In the study of Kiowski et al. (1985), clonidine was infused locally into the brachial artery, and it did not change noradrenaline release in the forearm. The value of this finding is diminished by the oversimplified method of calculation of noradrenaline release: spillover was calculated by multiplying the venoarterial noradrenaline difference with forearm blood flow (for measurement of organ specific catecholamine spillover, see Esler et al., 1990). Kooner et al. (1991) studied the effects of systemically administered clonidine in tetraplegic patients. Since clonidine did not change the increase in blood pressure and skin vascular resistance evoked by bladder stimulation, the authors argued against a peripheral presynaptic inhibition by clonidine. Another approach for identifying peripheral presynaptic action in intact animals is by measuring the sympathetic nerve firing rate, noradrenaline release, and blood pressure simultaneously. Garty et al. (1990) compared the effect of intravenously administered clonidine on the firing rate of renal sympathetic nerves with the effect on renal noradrenaline spillover. Clonidine lowered renal noradrenaline spillover, and this effect was solely attributable to the centrally elicited decrease in renal sympathetic nerve firing. There was no indication of peripheral presynaptic inhibition. It must be noted that sympathetic nerves were probably firing at very high rates in the anaesthetized rats studied by Garty et al. (1990) (arterial plasma noradrenaline level > 1 ng mL 1). It is difficult to detect peripheral presynaptic inhibition under this condition since presynaptic inhibition becomes weak when axon terminals are stimulated at high frequencies (see Starke, 1977, 1987). In a study in anaesthetized rabbits, clonidine was administered systemically, and the effect on the firing rate of renal sympathetic nerves and the plasma noradrenaline concentration was determined (Szabo et al., 1989). Clonidine lowered the arterial plasma noradrenaline concentration disproportionally more than was expected from the decrease in renal nerve firing rate, and this was
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B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
considered as an indication of a peripheral presynaptic action. A weak point of this latter study is that renal sympathetic nerve firing was compared with the arterial plasma noradrenaline concentration, which reflects noradrenaline release from all sympathetically innervated tissues. Wallin and Frisk-Holmberg (1981) observed in humans that clonidine can lower blood pressure without inhibiting muscle sympathetic nerve activity, and suggested that clonidine ‘‘influences sympathetic outflow by a combination of central and peripheral effects.’’ 12.3. Presynaptic imidazoline receptors Molderings et al. (1991) and Go¨thert and Molderings (1991) described that certain imidazoline derivatives (e.g., clonidine, idazoxan, cirazoline, BDF6143) and guanidine derivatives (e.g., aganodine) inhibit electrically evoked noradrenaline release from the rabbit pulmonary artery and aorta, an inhibition that could not be explained by activation of a2-adrenoceptors. Since then, this observation was extended to the rabbit heart (Fuder & Schwarz, 1993), human atrium and pulmonary artery (Likungu et al., 1996; Molderings et al., 1997), and rat aorta and vena cava (Molderings & Go¨thert, 1998). The existence of a presynaptic imidazoline receptor was postulated. The presynaptic imidazoline receptor is distinguished from the a2-adrenoceptor by its relative insensitivity to rauwolscine (Molderings et al., 1991). The presynaptic imidazoline receptor is not similar to the I1 and I2 imidazoline-binding sites (Molderings & Go¨thert, 1995). Recent observations indicate a mutual interaction between the presynaptic imidazoline receptor and the CB1 cannabinoid receptor (Molderings et al., 1999). It should be noted that under stimulation conditions that do not activate a2-autoinhibition, the function of the presynaptic imidazoline receptor is not apparent (Gaiser et al., 1999). Can the peripheral presynaptic imidazoline receptor play a role in the sympathoinhibition elicited by clonidine-like antihypertensive drugs? The answer is negative. The drugs used in therapy are either not effective at the presynaptic imidazoline receptor (moxonidine and rilmenidine) (Molderings et al., 1991, 2000) or have an effect only at concentrations that are much higher than therapeutic plasma concentrations (clonidine) (Molderings et al., 1991). 12.4. Penetration of clonidine-like drugs into the brain Clonidine and its chemical relatives are often described as lipophilic drugs. Due to their lipophilicity, these drugs should easily penetrate into the brain, where they could reach higher concentrations than in the plasma. Such a distribution pattern would support a preferential central sympathoinhibitory effect. The octanol/buffer partition coefficient of clonidine is in the range 2.7 –8.1 (Kobinger & Pichler, 1975; de Jonge et al., 1981a; Armah et al., 1988), and that of moxonidine is
0.88 (Armah et al., 1988). The corresponding coefficient for rilmenidine is not known. For comparison, the octanol/water partition coefficient of a strongly lipophilic drug, D9-tetrahydrocannabinol, is 12091 (Thomas et al., 1990). It is clear, therefore, that clonidine and moxonidine are only very moderately lipophilic. Endothelial cells of brain microvessels possess a carrier for clonidine. The role of this carrier in the passage of clonidine through the blood-brain barrier, however, is not known (Huwyler et al., 1997). The distribution pattern of intravenously administered clonidine is in accord with its moderate lipophilicity. Thus, ‘‘at the peak of the hypotensive response less than 2% of the injected dose is present in the brain and at least equal concentrations of the drug are found in most peripheral tissues’’ (Conway & Jarrott, 1980). Accordingly, the authors conclude that ‘‘the possibility of peripheral mechanisms contributing to the hypotensive effect cannot be dismissed.’’ Considering that rilmenidine and moxonidine are regarded as centrally acting drugs, it is surprising that, to our knowledge, the concentrations of these drugs in the brain after peripheral application have not been determined. Since there are no data on the penetration of rilmenidine and moxonidine into the brain, a hypothesis may be allowed: rilmenidine and moxonidine penetrate less efficiently into the brain than clonidine. This can be expected for moxonidine, since its octanol/buffer partition coefficient is lower than that of clonidine (see above). The results of two in vivo functional studies are well-compatible with the above hypothesis. First, in anaesthetized rats, intravenously applied clonidine and rilmenidine inhibited the firing of locus coeruleus neurons; clonidine (ED50 = 5.5 mg kg 1) was 64-fold more potent than rilmenidine (ED50 = 350 mg kg 1) (Dresse & Scuve´e-Moreau, 1986). After iontophoretic application directly into the locus coeruleus, clonidine (ED50 = 463 nA iontophoretic current) was only 5-fold more potent than rilmenidine (ED50 = 2500 nA current) (Dresse & Scuve´e-Moreau, 1986). (Admittedly, the strength of iontophoretic current is a suboptimal surrogate for doses of the centrally applied drugs.) Second, in anaesthetized rabbits, intravenously applied clonidine (ED20 = 10 mg kg 1; the dose lowering blood pressure by 20%) and moxonidine (ED20 = 30 mg kg 1) lowered the blood pressure, and clonidine was 3-fold more potent than moxonidine (Armah, 1987). The potency relationship reversed after intracisternal application, moxonidine (ED20 = 0.1 mg kg 1) being 5-fold more potent than clonidine (ED20 = 0.5 mg kg 1). Thus, the relative potencies of rilmenidine and moxonidine versus clonidine increased if the drugs were administered directly into the CNS, an observation well compatible with the hypothesis that rilmenidine and moxonidine penetrate into the brain less efficiently than clonidine. A selective peripheral action of rilmenidine would be indicated also by the observation that rilmenidine, at doses 7.5 mg kg 1 (i.v.), does not inhibit noradrenaline release in the locus coeruleus and hypothalamus (Tibiric¸a et al., 1991b; Suaud-Chagny et al., 1992; see Section 11.1.2).
B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
I conclude that the pharmacokinetic evidence for a preferential central nervous action of clonidine-like drugs is weak or does not exist. Thus, there is good reason to assume that the peripheral presynaptic inhibition occurs simultaneously with the central sympathoinhibition. If moxonidine and rilmenidine indeed penetrate less efficiently into the brain than clonidine, then the weight of peripheral presynaptic inhibition in the overall reduction of sympathetic tone is expected to increase. In addition, the frequency and intensity of central nervous side effects, most of all sedation, are expected to decrease. It is tempting to speculate that the improved effect/side effect profiles of rilmenidine and moxonidine in humans (see Section 11.2) are simply due to the fact that these drugs penetrate less efficiently into the brain than clonidine. 12.5. Summary of the role of peripheral presynaptic inhibition It was shown in the preceding sections that clonidine-like drugs cause presynaptic inhibition in peripheral models with artificial sympathetic tone. The doses were identical with the doses causing sympathoinhibition, hypotension, and bradycardia in intact animals. Peripheral presynaptic inhibition was shown in several studies in intact animals and humans. The failure to demonstrate peripheral presynaptic inhibition in some experiments on animals and humans with ongoing sympathetic impulse traffic is probably due to unsuitable experimental conditions and methods. Moreover, there is no reason to believe that these drugs, through selective accumulation in the brain, elicit effects preferentially in the CNS. Therefore, I suggest that the overall effect of clonidinelike drugs on the cardiovascular system develops as the sum of three primary actions. One action is, undoubtedly, central sympathoinhibition. The second component of the sympathoinhibition is peripheral inhibition of transmitter release from postganglionic sympathetic axons. Interestingly, the two inhibitory effects ‘‘cooperate’’ well. As a consequence of the central sympathoinhibition, the firing rate of sympathetic axons decreases. Presynaptic inhibition, which is frequency-dependent, becomes especially powerful at low nerve firing rates. These two inhibitory actions counteract the third effect of clonidine-like drugs, direct vasoconstriction mediated by a2-adrenoceptors on vascular smooth muscle cells and, finally, blood pressure decreases. Peripheral presynaptic inhibition alone is capable of preventing the blood pressure increase due to vasoconstriction. However, additional central sympathoinhibition is necessary to produce blood pressure decrease.
13. Conclusion Finally, after the critical evaluation of experimental data, the observations that support or are compatible with the imidazoline mode of action are presented in Table 7. Table 8
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Table 7 Observations that support or are compatible with the imidazoline mode of action – In humans, the new ‘‘selective’’ imidazoline-binding site ligands, rilmenidine and moxonidine, lower blood pressure like clonidine. After short-term administration, they cause sedation and inhibit saliva secretion less than clonidine. During long-term therapy, the new drugs cause dry mouth less frequently than clonidine. – In two studies, rilmenidine had no agonist activity at human a2-adrenoceptors. – Some potent a2-adrenoceptor agonists, such as guanabenz and guanfacine, do not cause hypotension when they are injected into the RVLM or applied intracisternally. – In a few studies, the mixed I1/a2 antagonists idazoxan and efaroxan were more effective against rilmenidine and moxonidine than pure a2-adrenoceptor antagonists that produced equal a2-adrenoceptor blockade. – The hypotensive effect of moxonidine, injected into the RVLM of anaesthetized mice, persists in animals in which the a2A/D-adrenoceptor is genetically defect.
summarizes the observations that do not support the imidazoline mode of action and observations that do not fit the predictions of the imidazoline hypothesis. Several of these observations directly support a role for a2-adrenoceptors. Thus, many predictions of the imidazoline hypothesis are not fulfilled, and the uncertainties of the hypothesis seem to outweigh the observations that support it. Therefore, I believe that the pattern of effects of clonidine-like drugs should be explained within the framework of a2-adrenoceptors. The a2-adrenoceptors are generally well-characterized. They are suitably localized in cardiovascular centres and sympathetic neurons to cause sympathoinhibition. The mechanisms of the a2-adrenoceptor-mediated sympathoinhibition in the medulla oblongata and in the sympathetic nervous system are well-characterized. One hypothesis suggests that a2-adrenoceptors and I1 imidazoline receptors coexist and cooperate. Accordingly, both pure a2-adrenoceptor agonists and I1 imidazoline receptor agonists are capable of lowering sympathetic tone by a primary action in the RVLM. The a2-adrenoceptor agonists directly inhibit presympathetic RVLM neurons. The I1 imidazoline agonists increase the release of catecholamines in the RVLM, and the catecholamines, in turn, depress presympathetic RVLM neurons by activating a2-adrenoceptors. This hypothesis can explain the frequent observation that a2-adrenoceptor antagonists counteract the effects of imidazoline hypotensive drugs. However, the key element of this hypothesis — imidazolines increase the release of catecholamines in the RVLM — has not been verified experimentally. Considering this and the uncertainties of the imidazoline hypothesis, I still suggest that the effects of imidazoline antihypertensive drugs should be explained with the primary activation of a2-adrenoceptors. Why then do rilmenidine and moxonidine inhibit saliva secretion and cause sedation in humans less than clonidine? There is no convincing answer. It seems that research
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B. Szabo / Pharmacology & Therapeutics 93 (2002) 1–35
Table 8 Observations that do not support the imidazoline mode of action and that do not fit the predictions of the imidazoline hypothesis – No selective imidazoline receptor agonist or antagonist has been identified during the 17 years of the existence of the imidazoline hypothesis. AGN192403 proved to be I1-selective in radioligand-binding experiments, but it has no effect on RVLM neurons and on blood pressure. No functional studies have been carried out with another selective ligand, S23230. Different groups have found strongly differing affinity values of key agonists and antagonists for I1-binding sites. For lack of reliable tools, the conclusions of many in vivo studies are fragile. – Despite the intensive work in several laboratories, no I1 imidazoline receptor protein has been identified. Recently, a protein (IRAS-1) with I1 imidazoline-binding capability was discovered. IRAS-1 mRNA is not distributed in the brain as expected for the I1 imidazoline receptor. It is not clear whether the IRAS-1 molecule can function as a pharmacological receptor, carrying out the functions expected from the I1 imidazoline receptor – The activity of spinally projecting sympathoexcitatory RVLM neurons is inhibited in vitro by catecholamine agonists and moxonidine via a2-adrenoceptors, and there is no indication for involvement of I1 imidazoline receptors. The mechanism of the inhibition is clarified and agrees well with the distribution of a2-adrenoceptors in the RVLM. – It is not true that only drugs with affinity for I1 imidazoline-binding sites lower blood pressure when injected into the RVLM. Catecholamines also produce sympathoinhibition. – The hypotensive and sympathoinhibitory effects of clonidine, rilmenidine, and moxonidine were antagonized in many experiments by selective a2-adrenoceptor antagonists. The failure in several experiments to block effects of clonidine-like drugs by a2-adrenoceptor antagonists is likely to be due to insufficient dosing of the antagonists. In several experiments, the mixed I1/a2 antagonists idazoxan and efaroxan had no I1 antagonistic effects that surpassed their a2-blocking actions. – Systemically injected clonidine, rilmenidine, and moxonidine do not lower blood pressure in conscious mice with genetically defect a2A-adrenoceptors. – It is not true that selective I1 imidazoline agonists selectively lower blood pressure, causing only few a2-adrenoceptor-mediated effects. Simultaneous inhibition of locus coeruleus neurons was shown with electrophysiological and neuroanatomical methods. The selective sympathoinhibition in animal experiments is only supported by studies in which DOPAC or noradrenaline were measured with voltammetry. Rilmenidine and moxonidine increase the plasma concentration of growth hormone in humans, an effect attributed to activation of a2-adrenoceptors. – SHR are used to demonstrate I1 imidazoline receptor-mediated hypotension by some researchers, whereas others believe that such a mechanism does not operate in these animals. – There is substantial evidence for activation of peripheral presynaptic a2-adrenoceptors on axon terminals of postganglionic sympathetic neurons by clonidine-like drugs. The peripheral presynaptic inhibition very likely contributes to the overall sympathoinhibition.
concentrated too much on the imidazoline mode of action, and search for alternative explanations was not pursued intensively enough. As pointed out, the diffusion of rilmenidine and moxonidine into the CNS has not been systemically studied. It is conceivable that they cross the blood-brain barrier less readily than clonidine. If this were true, then their sympathoinhibitory action would rely to a greater extent on peripheral presynaptic inhibition than the sympathoinhibitory action of clonidine. They would also evoke centrally evoked side effects less frequently. The affinity and efficacy of these drugs at functional subtypes of a2-adrenoceptors in native tissues has also not been systemically determined. It may be that selective agonist activity at one of the subtypes turns rilmenidine and moxonidine into effective antihypertensive agents with few side effects. The highly intriguing finding that rilmenidine has no agonist activity at the human a2A-adrenoceptor may be the first important observation in this direction. The affinity and efficacy for postjunctional a1-adrenoceptor subtypes should also be systemically studied. The explanation for the improved effect/side effect profile of rilmenidine and moxonidine may be a hitherto not discovered affinity of these drugs for a neurotransmitter receptor, neurotransmitter transporter, neurotransmitter metabolizing enzyme, or ion channel. It may also turn out that the mechanism of action is not the same in the case of rilmenidine and moxonidine. Research in this field is worth pursuing, since, as mentioned in Section 1, this group of drugs has the unique ability to decrease pathologically elevated sympathetic tone.
Acknowledgements This study was supported by the Deutsche Forschungsgemeinschaft (Sz 72/2-3). I thank Dr. Klaus Starke for his critical comments on the manuscript. The help of Ilka Wallmichrath in the preparation of this manuscript is gratefully acknowledged.
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