The role of imidazoline receptors in blood pressure regulation

Pharmac. Ther.Vol. 54, pp. 231-248, 1992 Printed in Great Britain. All rights reserved

0163-7258/92$15.00 © 1992 Pergamon Press Ltd

Associate Editor: M. J, LEwis

THE ROLE OF IMIDAZOLINE RECEPTORS IN BLOOD PRESSURE REGULATION CARLENE A. HAMILTON Department of Medicine and Therapeutics, Western Infirmary, Glasgow, G I I 6NT, U.K. Abstract--Using the ligands [3H] clonidine and [3H] idazoxan, nonadrenergic imidazoline preferring binding sites have been identified in a range of tissues from several species including man. These sites may represent a new family of receptors. An endogenous ligand and potential clonidine displacing substance has been identified. There is strong evidence for an involvement of the nonadrenergic imidazoline [3H] clonidine labelled sites in the nucleus reticularis lateralis in blood pressure regulation, and some evidence for a role in sodium regulation in the kidney for the [3HI idazoxan labelled sites. Some drugs which were previously thought to act via ~t2-adrenoceptors, may mediate their effects in part via these imidazoline sites.

CONTENTS 1. Background 2. Identification of Imidazoline Preferring Sites 2.1. Sites labelled by [3H] clonidine and analogs 2.2. Sites labelled by [3H] idazoxan 3. Characterization of Imidazoline Preferring Sites 4. Physiological Responses Linked to the Imidazoline Sites 4.1. Blood pressure and heart rate regulation 4.2. Regulation of Na+/H ÷ exchange 4.3. Modulation of noradrenaline release from postganglionic sympathetic nerve endings 4.4. Hormonal regulation 4.4.1. Prolactin secretion 4.4.2. Insulin release 4.5. Effects on gastric ulceration 4.6. Effects related to cerebral ischemia 5. Endogenous Ligands for the Imidazoline Sites--Clonidine Displacing Substance 6. Imidazoline Preferring Drugs and Blood Pressure Regulation 6.1. Central effects 6.2. Renal effects 7. Summary and Conclusions References

231 232 232 234 236 237 237 239 240 240 240 240 240 241 242 243 243 244 244 245

1. B A C K G R O U N D In the 1970s and 1980s n u m e r o u s differences in biological responses between ch-adrenoceptor agonists o f the phenethylamine and imidazoline classes were reported (Ruffolo et al., 1976, 1977, 1983; Bousquet et al., 1984). It was suggested that agonists with imidazoline-like structures acted at a different site on the ct-adrenoceptor to their phenethylamine counterparts (Ruffolo et al., 1977). Note on nomenclature--These nonadrenergic imidazoline preferring sites have been referred to by various names by the different groups working in the field. These include imidazole, imidazoline and imidazoline-guanidinium receptive sites, imidazole, imidazoline and idazoxan or I receptors. For consistency, one term--the imidazoline preferring site has been used throughout in this review. 231

232

C.A. HAMILTON

CI NH

O-CH2 H ClRAZOLINE

~N--N~

CLONIDINE

r N (i..~N

03 ""'-

H UK 14304

IDAZOXAN

cI V

O

NH II " CH "- N ~ N H --C - - NH2

N

A

RILMENIDINE

GUANABENZ

CH2--CH-- CH2"--N."'~1

N

BHT 920

FIG. 1. Some compounds with imidazoline or closely related structures. In 1986 using [3H] p-aminoclonidine (a partial ~2-adrenoceptor agonist) Meeley and colleagues identified nonadrenergic 'imidazoline preferring' sites in bovine brain. Numerous other reports of ct2-adrenergic drugs binding at nonadrenergic imidazoline preferring sites in a range of tissues and species followed and the concept evolved that rather than acting at a separate site on the ~t2-adrenoceptor, these compounds could act at a distinct class of receptor--the imidazoline receptor to produce physiological effects. A number of compounds reported to have affinity for these sites are shown in Fig. 1.

2. IDENTIFICATION OF IMIDAZOLINE PREFERRING SITES 2.1. SITESLABELLEDBY [3H] CLONIDINEAND ANALOGS Nonadrenergic binding sites were first identified using the ligand [3H]p-aminoclonidine in bovine ventrolateral medulla by Meeley and coworkers (1986). They showed that noradrenaline only

Imidazoline receptors and blood pressure

233

1:I i

40

20

-20 -10

-9

-8

-7

-6

-5

-4

Log[] FIG. 2. Inhibition of [3H] p oaminoclonidine binding to distinct subpopulations of sites in ventrolateralla medulla membranes. 0 , Noradrenaline (n = 7); C), clonidine (n = 3). Reprinted from Ernsberger et al. (1987), with permission of the authors and the copyright holder, Elsevier Science Publishers BV, Amsterdam. displaced 70-75% o f the specifically bound ligand although clonidine and phentolamine displaced all specifically bound [3H] p-aminoclonidine (Fig. 2). Further characterization of these nonadrenergic sites demonstrated a relatively high affinity for imidazoles and imidazolines, but a lack of affinity for phenylethylamines such as noradrenaline and alkaloids such as yohimbine (Ernsberger et al., 1987). Nonadrenergic imidazoline preferring sites have also been identified in human brain stem (Bricca et al., 1988), rat brain (Kamisaki et al,, 1990) and kidney (Ernsberger et al., 1988a)

TABLE1. Drug Affinities at Imidazoline Sites Labelled by [SH] Clonidine and [SH] p-Aminoclonidine

Species Cow Rat Rabbit Cow Rabbit Man Rabbit

Tissue Ventrolateral medulla Striatum Kidney Forebrain Ventrolateral medulla Kidney Forebrain Nucleus reticularis lateralis Forebrain Kidney

Ligand

Displacing

PAC

Cimetidine

480 + 88t

PAC CI CI PAC

Cimetidine Cimetidine Cimetidine Guanabenz

6100"~ > 100,000~ > 100,000~ > 1,000,000" II

CI CI C1

Guanabenz Guanabenz Cirazoline

1.5 __+0.2§ 1.3 _+ 1.3 20¶

CI C1

Cirazoline Cirazoline

0.6 _+0.1§ 0.8 + 0.1§

*Results expressed as Ki rather than ICs0. ~'Ernsberger et al. (1987). ~Kamisaki et al. (1990). §Hamilton et al. (1991). [[Ernsberger et al. 0990). ¶Bricca et al. 0989). PAC, p-aminoclonidine; C1, clonidine.

Ics0 (nM)

234

C.A. HAMILTON

loo!

,.,=

0

10

9

8

7

6

5

4

- LOG [C_,OMPETITOFI(M)I FIG. 3. Inhibition of [3H] RX 781094 (idazoxan) binding to basolateral membranes from rabbit kidney. O, Tramazoline; O, tolazoline; O, ( - ) adrenaline; ~, rauwolscine; A, serotonin; A, phenylephrine; n = 3. Reprinted from Coupry et al. (1987), with permission of the authors and the copyright holder, Academic Press, Orlando. and rabbit brain and kidney (Hamilton et al., 1991) using either [3H] p-aminoclonidine or [31-I] clonidine. However, differences between the sites identified by the various groups emerge on comparison of the data (Table 1). In rat kidney nonadrenergic [3H] p-aminoclonidine binding represented 29% of the total specific sites and displayed a high affinity for a number of imidazolines and imidazoles including cimetidine (Ernsberger et al., 1988b). In contrast, in rabbit kidney using [3H] clonidine as the ligand, Hamilton et al. (1991) reported that the majority of sites labelled were nonadrenergic and that the affinity of cimetidine for the site was low. Similar discrepancies emerge when comparing nonadrenergic binding sites in the brain using [3H] clonidine and p-aminoclonidine. Bricca and colleagues (1989) were unable to identify any nonadrenergic binding in rat medulla oblongata using [3H] clonidine as the ligand, whereas Kamisaki et al. (1990) found that approximately one third of the sites in rat medulla oblongata labelled by [3HI p-aminoclonidine were nonadrenergic imidazoline preferring sites. Moreover, the group reported a Ki for cimetidine at this site of 6.11 p M, whereas using the same ligand an IC50 of 480 nu was observed in bovine ventrolateral medulla (Ernsberger et al., 1987). Whether these differences can be related to the ligand ([3H] clonidine vs [3H] p-aminoclonidine), the species or technical differences in assays is not clear. However, the possibility of multiple binding sites cannot be ruled out.

2.2. SITES LABELLEDBY [3H] IDAZOXAN Idazoxan is an ~2-imidazoline type adrenoceptor antagonist. It has been reported to have a higher potency and specificity for 0tE-adrenoceptors than yohimbine and its isomer rauwolscine (Doxey et al., 1984) and has been used to label ~2-adrenoceptors (Howlett et al., 1982). However, in 1987 Coupry and colleagues demonstrated that in addition to labelling ~tE-adrenergic sites, [3H] idazoxan binds to nonadrenergic imidazoline preferring sites in rabbit basolateral membranes (Fig. 3). Adrenaline and rauwolscine displaced less than 25% of specifically bound [3H] idazoxan whereas the imidazolines tramazoline and tolazoline displaced all specifically bound idazoxan. Moreover, whereas [3I-/] idazoxan gave a binding site density of 566 _+ 118 fmol/mg protein Bm,x

Imidazoline receptors and blood pressure

235

TABLE 2. Drug Affinities at Imidazoline Sites Labelled by [3HI Idazoxan

Species

Tissue

Man Rabbit Pig Rat Man Rabbit Rabbit Rabbit Man Rabbit Pig Rat Man Rabbit Rabbit Rabbit

Kidney Kidney Kidney Kidney Liver Liver Urethra Forebrain Kidney Kidney Kidney Kidney Liver Liver Urethra Forebrain

Drug Guanabenz Guanabenz Guanabenz Guanabenz Guanabenz Guanabenz Guanabenz Guanabenz Clonidine Clonidine Clonidine Clonidine Clonidine Clonidine Clonidine Clonidine

Ki (nu) 9.6 + 1.55" 1.8 ___0.5"t" 18 + 5"~: 87§ 172.5 + 50.7L[ 4.7 + 0.711 4.4 ___0.9¶ 1.3 + 0.1 t 2575 + 710t 303 + 311" 10,000 + 3,000":~ > 10,000~ 6763 +_64211 7406 + 12991[ 1265 4- 170¶ 543 _ 80*l

*Results expressed as IC50 rather than Ki. tLachaud-Pettiti et al. (1991). :~Vigne et al. (1989). §Michel et al. (1989). lITesson et al. (1991). ¶Yablonsky and Dausse (1989). ~Hamilton et al. (1991).

for [3H] rauwolscine binding under the same conditions was only 155 + 28 fmol/mg protein consistent with idazoxan labelling additional sites. Reports of [3H] idazoxan binding to nonadrenergic sites in other tissues followed rapidly. In the rabbit nonadrenergic binding to brain (Hamilton et al., 1988), smooth muscle urethra (Yablonsky et al., 1988) and adipocyte membranes (Langin and Lafontan, 1989) was demonstrated. In addition, nonadrenergic binding of [3H] idazoxan to rat and human renal cortical membranes (Michel et al., 1989), human platelets and myometrium (Michel et al., 1989) and guinea pig cerebral cortex (Wikberg, 1989), pig kidney (Vigne et al., 1989), rat brain (Brown et al., 1990) and human and rabbit liver (Tesson et al., 1991) has been shown. Differences appear to exist between the nonadrenergic sites labelled by idazoxan in different species (Table 2). For example, clonidine and phentolamine did not displace [3H] idazoxan from human kidney (Michel et al., 1989) but did from rabbit kidney (Hamilton et al., 1988) and guinea pig brain (Wikberg, 1989). Differences in binding affinity in human and rabbit liver have also been reported for guanabenz and UK 14304 (Tesson et al., 1991), and as for nonadrenergic [3H] clonidine binding the possibility of species heterogeneity has been suggested for nonadrenergic [3HI idazoxan binding (Michel and Insel, 1989). There is also evidence for within species tissue heterogeneity. Wikberg and colleagues (1991) have suggested that subtypes of [3H] idazoxan imidazoline sites can be delineated using stereoisomers of medetomidine in guinea pig tissues. Whereas the affinities of the ( - ) and ( + ) isomers for the [3H] idazoxan binding site were similar in the ileum the affinity of the ( - ) isomer was approximately ten times lower than that of the ( + ) isomer in the cortex. In addition to heterogeneity within [3H] clonidine and [3HI idazoxan binding sites differences in the affinity of a number of compounds between these binding sites has been reported. Whether the nonadrenergic sites labelled by [3H] clonidine and [3H] idazoxan are one and the same or have separate identities and function has been the subject of some controversy (Bousquet, 1989; Michel and Insel, 1989). However, recently Hamilton and colleagues (1991) demonstrated differences in nonadrenergic [3H] clonidine and [3H] idazoxan binding in rabbit forebrain and kidney membranes under identical experimental conditions. On the basis of these studies these authors suggested that a whole family of imidazoline receptors may exist (Hamilton, 1992).

236

C.A. HAMILTON 3. CHARACTERIZATION OF IMIDAZOLINE PREFERRING SITES

In most tissues studied ~2 and imidazoline preferring sites can be separated from each other in a number of ways. The nonadrenergic imidazoline site labelled by [3H] idazoxan has been partially purified and characterised and shown to be distinct from the 0t2-adrenoceptor. An anatomical location of [3H] idazoxan sites distinct from [3H] rauwolscine sites has been reported in rat brain (Boyajian et al., 1987). Using autoradiographic techniques the authors showed that the densest areas for [3H] idazoxan binding appeared over anterior olfactory nuclei, fundus striatum, septum, thalamus, hypothalamus, amygdala, entorhinal cortex, central gray, inferior colliculus, dorsal parabrachial nucleus, locus ceruleus and nucleus of the solitary tract. The most dense [3H] rauwolscine labelling appeared over nucleus caudate-putamen, nucleus accumbens, olfactory tubercle, islands of Colleja, hippocampus, parasubiculum, basolateral amygdaloid nucleus and substantia nigra. The maximum binding values for [3HI idazoxan were consistently higher than those for [3H] rauwolscine and the pharmacological data (Boyajian and Leslie, 1987) was consistent with [3H] rauwolscine labelling adrenergic sites while [3H] idazoxan labelled both adrenergic and imidazoline preferring sites. Further evidence that the 0t2 and imidazoline binding sites labelled by [3H] idazoxan are distinct comes from studies in which COS 7 cells transfected with genes for the ~2C4 and ~2C~0 subtypes were shown to contain no nonadrenergic [3H] idazoxan sites (Michel et aL, 1990). The imidazoline sites labelled by [3HI idazoxan in guinea pig cerebral cortex (Wikberg and Uhlen, 1990) and rabbit kidney (Parini et al., 1989) have been solubilized and could be physically separated from ~2-adrenoceptors, indicating that the ct2 and imidazoline sites labelled by [3HI idazoxan are discrete entities and not different sites as the same protein. The imidazoline site was trypsin sensitive showing it to be a protein not a lipid component of the membrane, and in contrast to ~2-adrenergic sites was not bound to heparin-agarose or lectin resin indicating that it was devoid of complex N linked oligosaccarides (Parini et al., 1989). Unlike ~2-adrenoceptor sites the imidazoline sites labelled by idazoxan may not be sensitive to changes in Na ÷ concentration or GTP and its analogs (Michel et al., 1989). However, GTP and its analogs only modulate agonist binding to the receptor and, as Michel pointed out, it is possible that although UK 14304 (5 bromo-6-(imidazolin-2-ylamino)-quinoxaline) is an agonist at ~2-adrenoceptors it was not acting as an agonist at the imidazoline sites labelled by idazoxan in rat kidney. Additional evidence for a lack of sensitivity to GTP has been obtained recently in that GTP analogs also failed to modulate binding of a number of other compounds including guanabenz, clonidine and cirazoline at the imidazoline site (Zonnenschein et al., 1990). Although the [3HI idazoxan imidazoline preferring site is insensitive to Na ÷ and divalent cations it has been reported to be sensitive to K ÷ (Zonnenschein et al., 1990; Lachaud-Pettiti et al., 1991). In basolateral membranes from human kidney, K + inhibited binding by up to 70% with an ECs0 of 70 mM and acted as a competitive inhibitor (Lachaud-Pettiti et al., 1991). In rat liver 4 aminopyridine, a K ÷ channel blocker in neuronal cells inhibited specific idazoxan binding with an IC50 of 0.34 mM, a concentration which is effective in blocking K + channels. Cs ÷ and NH~- also interfered with [3H] idazoxan binding and it has been suggested that these imidazoline sites might be coupled to K ÷ gating (Zonnenschein et al., 1990). Recently an intracellular location for the imidazoline sites labelled by [3H] idazoxan has been proposed. In human platelets Michel and coworkers (1990) showed that, although imidazoline preferring sites could be detected in crude membrane preparations, they were not found in purified plasma membranes suggesting that these sites were located in an intracellular compartment. An intracellular location for these sites in human renal proximal tubules has also been proposed (Lachaud-Pettiti et al., 1991); while the major localization of 0q-adrenoceptors was in the basolateral membranes, imidazoline sites were present in both basolateral and intracellular membranes. Moreover, in human and rabbit liver the imidazoline preferring site labelled by [3H] idazoxan have been localized to the outer mitochondrial membrane (Tesson et al., 1991). Interestingly, most of the drugs reported to have a high affinity for the nonadrenergic sites labelled by idazoxan are relatively lipophylic and thus should have access to intracellular binding sites.

Imidazoline receptors and blood pressure Clonidine 0.1

1

¢t-MNE 0.1

1

237 ST 587

Cirazollne 10

0.01 0.1

1

3

lo~g

-10

._. -20

-30

7..

FIG. 4. Effects on the mean blood pressure (MAP) of various ct-adrenergic drugs injected bilaterally into the NRL of anesthetised normotensive cats. r = 4; *p < 0.05; **p < 0.01; ***p < 0.001. Reprinted from Bosquet et al. (1984), with permission of the authors and the copyright holder, American Society for Pharmacology and Experimental Therapeutics, Bethesda. 4. PHYSIOLOGICAL RESPONSES LINKED TO THE IMIDAZOLINE SITES If these nonadrenergic imidazoline binding sites are true receptors they should be linked to some physiological response. A number of roles for both the sites labelled by [3H] clonidine and those labelled by [3HI idazoxan have been proposed with varying amounts of supporting evidence as discussed below.

4.1. BLOOD PRESSURE AND HEART RATE REGULATION

There is good evidence that the sites in the brain labelled by [3H] clonidine and [3H] p-aminoclonidine are involved in the regulation of blood pressure. As long ago as 1984 Bousquet and colleagues reported that injection of imidazolines (clonidine, cirazoline and ST 87) caused hypotension when injected into the nucleus reticularis lateralis of anaesthetised cats while the • 2-adrenoceptor selective catecholamine ~ methyl-noradrenaline had no effect (Fig. 4). Subsequently other publications indicated that effects of ~2-adrenoceptor agonists on blood pressure and heart rate might be related to their structure. In 1986 Sasaki and colleagues reported that although injection of both clonidine and guanfacine into the lateral ventricle of anesthetized rats resulted in a decrease in blood pressure and heart rate, only responses to clonidine were blocked by yohimbine. Local injections into the nucleus tractus solitarius (NTS) and ventromedial hypothalamus (VMH) suggested that clonidine was acting on the NTS while guanafacine acted more rostrally on the VMH. King and Pang (1988) suggested that clonidine (imidazolidine) and BHT 920 (thiazoloazepin) had different cardiovascular effects on intracerebroventricular injection, clonidine significantly decreased blood pressure and had a small effect on heart rate, while BHT 920 increased blood pressure and decreased heart rate. Neither the pressor effect of BHT 920 nor the depressor effect of clonidine was abolished by rauwolscine. Although these results suggested that not all the depressor and bradycardic effects of centrally acting antihypertensive drugs are mediated by ~t2-adrenoceptors, no overall pattern was apparent. However, more recently it has been shown that in anesthetized rats the hypotension and bradycardia induced by drugs injected into the rostral ventrolateral medulla correlated with affinity at imidazoline sites labelled by [3H] p-aminoclonidine but not with ct2-adrenoceptor affinity (Ernsberger et al., 1990) (Fig. 5). Furthermore, the imidazole idazoxan selectively reversed the fall in arterial pressure elicited by clonidine

238

C.A. HAMILTON

10Pl~nym ~

PIw~rm ~

o -

o

..............................

N~=~zo~

•

"1,--'-

N~mro=n= HTM

-10

a-MIe~E

-10

•

Kolne~ i NE O

-20

NE

-

-20

Oxymmzoene

g

-30

C~dme

-30

I

p

o

CNnet~e

PAC

/Tc-.

.or - 0.837

r - -0.051

-50 "~-1o

-9

-8

-7

Affinity at Imidazole

-s

-s

binding sites

> -4

,

-10

(pKi)

I

I

I

-9

-8

-7

Affinity

1 i -6

, 4.,,,...J g >4

-5

at 0.2 -receptors (pKi)

FIG. 5. Relationships between vasodepressor potency and binding affinity at imidazole binding sites or c<2-adrenoceptors. Reprinted from Ernsberger et aL (1990), with permission of the authors and the copyright holder, American Society for Pharmacology and Experimental Therapeutics, Bethesda.

whereas the nonimidazole c<2-adrenoceptor antagonist S K F 86466 did not attenuate the hypotensive effect. This work strongly supports the hypothesis that imidazoline receptors in the RVL are involved in blood pressure and heart rate regulation. However, not all the blood pressure lowering effects of noncatechol ct2-adrenoceptor agonists should be attributed to actions at imidazoline sites. In most cases c<2-adrenoceptor activation, particularly in the NTS, will also be important and the relative contribution of ~2 and imidazoline site activation to effects on the cardiovascular system will depend upon the structure of individual compounds.

Inhibition

%

25

20

L

I

15

L

10 5 0

T I~0~ N)

ClRAZOUNE

UK 14304

(4)

(4)

RILMB~IIDINE CIMETIDINE

(2)

(s)

FIG. 6. Effect of imidazoline derivatives on the 22Na uptake by isolated proximal cells from rabbit kidney. Reprinted from Bidet et al. (1990), with permission of the authors and the copyright holder, Elsevier Science Publishers BV, Amsterdam.

Imidazoline receptors and blood pressure

239

/

500-

400.

z

300.

I °. 100-

~1

10"9

!

I

10"8

!

I0"7

|

10-6

!

10-5

I

I0-4

Drug conoemmtlon (mol/I)

FIG. 7. Effectsof imidazolinesand a-adrenoceptor antagonists on the electrically(2H2) evoked [3H] overflow from superfused strips of the rabbit pulmonary artery. (3, Rauwolfscine; Q, BDF 6143; I~, idazoxan; A, phentolamine; n = 4--7. Reprinted from Gothert and Molderings (1991), with permission of the authors and the copyright holder, Springer-Verlag, Heidelberg.

4.2. REGULATIONOF Na+/H + EXCHANGE 22Na influx in rat proximal tubule segments is regulated differently by ct2-adrenoceptor agonists with phenylethylamine and imidazoline structures suggesting that effects are not only mediated via ~t2-adrenoceptors. The potassium-sparing diuretic amiloride and its analogs have been shown to exhibit a high affinity for nonadrenergic sites labelled by [3H] idazoxan in rabbit urethral smooth muscle, rabbit adipocytes and pig kidney membranes (Yablonsky and Dausse, 1989; Langin and Lafontan, 1989; Vigne et al., 1989). These compounds are known to act at the Na+/H + exchanger and sodium pumps, possibly indicating a role for the [3H] idazoxan binding site in Na+/H + regulation. However, the relative affinities of these diuretics at the [3H] idazoxan site did not always correspond to their activity at the Na+/H + exchanger. More direct evidence for the involvement of the imidazoline sites labelled by [3H] idazoxan in sodium exchange in the kidney has come from Bidet and colleagues (1990). In rabbit renal proximal tubule cells they observed an order of potency for inhibition of 22Na influx cirazoline>idazoxan > U K 14304 > rilmenidine >>cimetidine (Fig. 6). This is similar to the potency order previously observed for the inhibition of [3H] idazoxan binding in rabbit renal proximal tubules (Coupry et al., 1987; Parini et al., 1989). Moreover, when the effects of two of these compounds, UK 14304 and rilmenidine were examined in the presence of high concentrations of yohimbine to saturate 0t2-adrenoceptors, inhibition of 22Na uptake was still observed. In addition in the presence of Na ÷ idazoxan and cirazoline inhibited cell realkalinization supporting the hypothesis that imidazoline derivatives decrease Na ÷ influx in the proximal tubule through inhibition of the Na÷/H + antiporter. However, the magnitude of the changes reported was small and the authors suggest that modulation of Na+/H + antiporter activity could be secondary to other effects induced by imidazoline derivatives.

240

C.A. HAMILTON

4.3. MODULATIONOF NORADRENALINERELEASEFROMPOSTGANGLIONIC SYMPATHETICNERVE ENDINGS Electrically evoked tritium overflow from blood vessels previously labelled with [3H] noradrenaline characteristically increases in the presence of ~2-adrenoceptor antagonists due to blockade of the presynaptic g2-adrenoceptors regulating feedback inhibition of noradrenaline release. However, in the presence of a number of g2-adrenoceptors with imidazoline structures bell shaped curves have been observed (Docherty et al., 1982; Gothert and Molderings, 1991) with the imidazolines causing inhibition at higher concentrations (Fig. 7). The inhibition of tritium evoked overflow was not modified by metitepine, atropine, theophylline, dipyridamole or indomethacin and was still observed in the presence of high concentrations of rauwolscine and phenoxybenzamine suggesting that neither ~j- or ~2-adrenoceptors nor 5HT, muscarinic, P~ purinoceptors or prostaglandin receptors are involved in the imidazoline induced inhibition of noradrenaline release (Gothert and Molderings, 1991). No data on binding of either [3H] clonidine or [3H] idazoxan in blood vessels is available, thus, as discussed by Gothert and Molderings, the data do not allow comparison with imidazoline preferring sites identified in other tissues but suggest that presynaptic imidazoline sites may be involved in the regulation of noradrenaline release from sympathetic nerve endings. 4.4. HORMONAL REGULATION 4.4.1. Prolactin Secretion In 1989 Krulich and colleagues reported that whereas yohimbine increased prolactin secretion idazoxan inhibited secretion. One interpretation of these results would be that while yohimbine was acting at ~2-adrenoceptors idazoxan was inhibiting release via actions at imidazoline receptors. However, in a subsequent paper the same authors showed disparate effects of yohimbine and rauwolscine on prolactin release, results which cannot be interpreted in terms of imidazoline receptors (Jurcovicova et al., 1989). 4.4.2. Insulin Release ct2-Adrenoceptors located on the pancreatic B-cell exert inhibitory effects on insulin release (Nakaki et al., 1980; Langer et al., 1983). It has long been known that phentolamine enhances basal insulin release and it was thought that B-cell function was continuously inhibited by a basal adrenergic tone which was overcome by ~2-adrenoceptor blockade. However, it has recently been suggested that the enhancement of insulin release by phentolamine is not related to its ~t-adrenoceptor blocking properties, but to its imidazole structure (Schulz and Hasselblatt, 1989). Rauwolscine and benextramine, both of which are potent ~2-adrenoceptor antagonists, did not stimulate insulin release under the same conditions, whereas antazoline, tolazoline, BDF 8933 (4-fluoro2(imidazoline 2 ylamino) isoindoline-maleinate) and 1 benzylimidazole all of which have imidazole structures did. Not all of those compounds have ~t2-adrenoceptor properties. Antazoline, for example, is predominantly an H, antihistamine drug. Thus effects on insulin release cannot be related to actions at the ~2-adrenoceptor. Moreover, even for imidazoles with affinity for ~2-adrenoceptors there is a dissociation between the concentrations required to antagonize ~2-adrenoceptors on pancreatic islets and potentiate insulin release. Neither the binding of [3H] clonidine nor [3H] idazoxan to imidazoline preferring sites on pancreatic islets has been examined. Therefore currently it is not possible to relate effects on insulin release to binding site affinity. However, the findings of Schulz and Hasselblatt (1989) suggest that insulin release may be modulated by drugs acting at nonadrenergic imidazoline preferring sites. 4.5. EFFECTSON GASTRIC ULCERATION Clonidine has been shown to inhibit gastric ulceration (Kunchandy et al., 1985) or to have a dual action--prevention at low doses and potentiation at higher doses (Del Soldato, 1986). In ethanol-induced gastric lesions not only clonidine but also ct-methylnoradrenaline and guanzabenz

Imidazoline receptors and blood pressure

241

100

80' /-h

10

°

m

\ m

0

I -3

,

I -2 Log

I -

10

,

I -9

,

n -8

,

I -1

,

(dilution)

i

I

,

-7

' "6

.

n -5

Log [Clonidine] FIG. 8. CDS displacement of [3H] p-aminoclonidine binding to membranes from bovine ventrolateral medulla. O, Clonidine; @, CDS; n = 3-5. Reprinted from Meeley et al. (1986), with permission of the authors and the copyright holder, Pergamon Press, Oxford.

CDS

(Units)

1.0

t

f

0.5

0.25

2 min

FIG. 9. Contractile effectof CDS on rat' gastric fundus strip. Reprinted from Felsen et al. (1987), with permission of the authors and the copyright holder, Elsevier Science Publishers BV, Amsterdam.

have a protective effect which is antagonized by yohimbine. However, at higher doses in addition to clonidine, oxymetazoline aggravates lesions, an action which is not antagonized by yohimbine and is not seen with ~-methyldopamine or guanabenz (Bhandare et al., 1991). Such effects are consistent with affinities for the imidazoline site labelled by [3H] idazoxan in human liver (Tesson et al., 1991), but the exact receptor mechanisms and metabolic pathways involved remain to be elucidated.

4.6. EFFECTS RELATED TO CEREBRAL ISCHEMIA It has been proposed that stimulation of adrenoceptors in the brain may modify neural damage associated with cerebral ischemia and the ~:-adrenoceptor antagonist idazoxan has been shown to reduce the magnitude of the delayed neural degeneration elicited by global cerebral ischemia (Yasuda et al., 1978; Gustafson et al., 1989). More recently it has been reported that the weak ct2-adrenoceptor agonist rilmenidine can also reduce focal cerebral ischemia (Maiese et al., 1992). In these studies ischemia was produced by ligation of the middle cerebral artery. Drugs were administered intravenously immediately following occlusion of the artery and the animals killed 24 hr later for examination of the brain. Although both idazoxan and rilmenidine reduced infarct volume in this model the nonimidazoline ct2-adrenoceptor antagonist SKF 86466 (6-Chloro-N-methyl 2345-tetrahydro-l-H-3 benzazepine) had no neuroprotective effect. The authors suggested that occupation of imidazoline receptors either in the ischemic zone or at remote brain sites may be responsible for the neuroprotection of rilmenidine and idazoxan.

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C.A. HAMILTON 5. ENDOGENOUS LIGANDS FOR THE IMIDAZOLINE SITES-CLONIDINE DISPLACING SUBSTANCE

Although imidazoline sites clearly distinct from ~2-adrenoceptor binding sites have been partially purified and a number of functions proposed, endogenous ligands have yet to be positively identified. However, there is one strong candidate--clonidine displacing substance (CDS). CDS was first isolated by Atlas and Burnstein (1984a,b) in rat and bovine brain. The substance was thermostabile, not affected by proteolytic enzymes such as trypsin, papain and pronase, or by boiling in 0.2 HC1 for 5 min. It was devoid of amino acids, highly hydrophobic and had an estimated molecular weight of 500 + 50 Da. Using immunological techniques CDS has been demonstrated in a number of peripheral tissues in the rat, including adrenal gland, gastric fundus, heart, small intestine, kidney, liver, lung, skeletal muscle and serum (Hensley et al., 1989). Using radioligand binding techniques the presence of an endogenous noncatecholamine substance with a high affinity for [3H] idazoxan labelled sites has been confirmed in bovine lung but not medulla (Hussain et al., 1991a,b). Like clonidine and related compounds CDS is able to displace [3H] clonidine and [3H] p-aminoclonidine from both adrenergic and nonadrenergic binding sites in bovine ventrolateral medulla (Fig. 8) (Meeley et al., 1986) and rat brain (Atlas and Burnstein, 1984a; Ernsberger et al., 1988b), to displace [3H] idazoxan from rabbit kidney (Coupry et al., 1989) and rat liver membranes (Zonnenschein et al., 1990) and [3H] rauwolscine from human platelets (Diamant et al., 1987) suggesting that it binds at both ct2-adrenergic and imidazoline sites. However, reported K~ values for displacement varied by nearly 10 fold. This could be attributed to binding of CDS at different ~2 and imidazoline subtypes or differences in CDS prepared by different groups. On injection into the area of the rostral ventrolateral medulla of anesthetized rats CDS, prepared from bovine brain, has been reported to cause a rapid but transient dose related decrease in blood pressure (Meeley et al., 1986). Decreases occurred within 10 sec of injection and returned to preinjection levels within 5 min. However, Bousquet and colleagues (1986) reported that injection of CDS into the nucleus reticularis of cats, in contrast to clonidine, increased blood pressure. There was no significant modification of heart rate. In addition, in both cats and rats pretreatment with CDS prevented the hypotensive and bradycardic effect of clonidine (Bousquet et al., 1987). In all cases CDS was prepared from bovine brain by substantially the same method. However, subtle differences in CDS preparations cannot be ruled out as a cause for discrepancies in the reported properties of the compound. CDS may also be active on smooth muscle. Felsen and colleagues (1987) reported that it had no effect on vascular smooth muscle, but caused a dose related contraction of rat gastric fundi strips, while Diamant and Atlas (1986) demonstrated inhibition of the twitch response of rat vas deferens by CDS (Fig. 9). The former was unaffected by pretreatment with atropine, bradykinin, methysergide, phenoxybenzamine or SKF 86466, a selective nonimidazole ~2-adrenoceptor antagonist. The latter response was blocked by yohimbine and phentolamine suggesting it to be mediated via ~2-adrenoceptors. More recently Atlas and colleagues (Atlas, 1991) reported that an extract of CDS prepared from human serum contracted rat aorta in a manner similar to clonidine suggesting that it may have actions on vascular tissue. CDS modulates platelet aggregation in a manner similar to clonidine (Diamant et al., 1987). On its own it has no effect on aggregation, but it inhibits the primary aggregation to adrenaline and potentiates ADP and collagen induced aggregation in human platelets. Whether these actions are mediated entirely by actions at ct2-adrenoceptors is unclear, but it has recently been shown that [3H] idazoxan labels sites on human platelets which are not recognised by rauwolscine or adrenaline in addition to ct2-adrenoceptor sites (Zonnerschein et al., 1990). CDS has been identified in human serum, and it has been reported that levels are raised in pregnancy induced hypertension (Atlas, 1991). In methanolic extracts prepared from serum of subjects with pregnancy induced hypertension CDS levels were 12.2+ 1.5 units/ml serum compared to 4.2 +0.5 units/ml in normal pregnancy and 4.50 +0.3 units/ml in serum from subjects with chronic hypertension.

Imidazoline receptors and blood pressure Dry Mouth % •

--

243 Drowsiness

%

il':t j i ~ 15ci NS

Day0 Dey14 Day28 Day42

1.0

Dey0 Day14 O~28 Day42

1.0

Day0

Day14 Day28 Day42

Day0

Day14 Oay28 D~4,?.

FIG. 10. Incidence and intensity of dry mouths and drowsinesswith rilmenidineand clonidine. I-I, Rilmenidine (n = 162); m, clonidine (n = 171); ***p <0.001; **p <0.01; *p <0.05. Reprinted from Fillastre et al. (1988), with permission of the authors and the copyright holder, Cahners Publishing Company, Newton.

6. IMIDAZOLINE PREFERRING DRUGS AND BLOOD PRESSURE REGULATION 6.1. CENTRALEFFECTS It has generally been accepted that the central hypotensive effect of clonidine and related drugs is mediated via stimulation of ot2-adrenoceptors within the brain stem (Timmermans and van Zwieten, 1982). However, as discussed earlier these compounds also appear to lower blood pressure via actions on imidazoline sites in the nucleus reticularis lateralis. In addition to lowering blood pressure, clonidine-like compounds cause sedation and dry mouth. Sedation is believed to be a consequence of actions on ~:-adrenoceptors in the locus ceruleus (Kleinlogel et al., 1975; Timmermans et al., 1981). Such effects have been reported to affect the quality of life of patients taking these drugs, impair compliance and restrict their use in antihypertensive treatment (van Zwieten et al., 1984; Croog et al., 1986). Compounds which acted preferentially on the imidazoline sites in the brain stem might therefore be expected to lower blood pressure and not cause the same degree of sedation and other ot2-adrenoceptor mediated effects. In this context the oxazoline, rilmenidine, has a relatively high affinity for imidazoline sites compared to ot2-adrenoceptor sites. Its relative affinity for the nonadrenergic sites labelled by [3H] clonidine compared to adrenergic sites labelled by [3H] yohimbine is approximately 200:1 (Hamilton et al., 1991), whereas the relative affinity of clonidine for the same sites is about 20:1. Rilmenidine has been shown to be effective in lowering blood pressure in a number of animal models including normotensive and spontaneously hypertensive rats (van Zwieten et al., 1986; Jarrott et al., 1988) and sino-aortic denervated dogs (Valet et al., 1988), However, in contrast to clonidine rilmenidine has been reported to have no effect on phenobarbitone induced sleeping time in rats and minimal effects on the righting reflex in chicks (Koenig-Berard et al., 1988) suggesting that it has very little sedative activity. Clinical trials in man suggest that rilmenidine and clonidine are equally effective in reducing blood pressure but that the incidence of sedation and dry mouth are greatly reduced in subjects receiving rilmenidine (Fillastre et al., 1988) (Fig. 10). In studies comparing rilmenidine with methyldopa, comparative control of blood pressure was achieved with significantly less 'weakness', drowsiness, orthostatic dizziness or dry mouth. Such observations would be consistent with the hypothesis that rilmenidine mediates its hypotensive effects mainly via imidazoline sites in the nucleus reticularis lateralis and has a low affinity for ~2-adrenoceptors while clonidine acts both JPT 5413~B

244

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via imidazoline sites and ct2-adrenoceptors in the nucleus tractus solitarius, locus coeruleus and dorsal raphe causing sedation and dry mouth in addition to hypotension. Another potential advantage of rilmenidine over many other centrally acting antihypertensive drugs is that it has not been reported to cause 'clonidine withdrawal syndrome' in man, while in rats the effects of this syndrome are markedly reduced compared to clonidine (Lewis et al., 1987; Sannajust et al., 1989). This also may be related to rilmenidines lack of effect at 0t2-adrenoceptors. It has been reported that down regulation of 0t2-adrenoceptor number occurs in rabbit hindbrain with chronic clonidine but not rilmenidine treatment. This would result in an imbalance between 0t2-adrenoceptor number and agonist levels on drug withdrawal which could contribute to the hypertensive and behavioural effects observed on ceasing treatment. In contrast no down regulation of imidazoline sites has been observed on chronic drug treatment (Hamilton et al., 1990; Hamilton, 1992). Thus imidazoline sites may be less sensitive to down regulation and less likely to be involved in withdrawal reactions. Differences between other centrally acting antihypertensive drugs in their blood pressure lowering and behavioural effects have also been observed. In a comparative study on the central effects of clonidine, BHT 920 (2-amino-6-alkyl-5,6,7,8 tetrahydro-4H-thiazolo-[4,5-d]-azepines) and guanfacine in the monkey the potency of the 3 agonists in lowering blood pressure was clonidine = BHT 920 > guanfacine while for sedation the potency order was clonidine > BHT 920 > guanfacine (Arnsten et al., 1988). It is possible that differences in the relative affinities of the drugs for ~t2-adrenoceptor and imidazoline binding sites contribute to the reported differences in response. 6.2. RENALEFFCTS There is much evidence to suggest abnormalities in renal sodium handling in both human and animal models of hypertension. ~2-Adrenoceptors may play a role in tubular sodium absorption and there are numerous reports of alterations in ot2-adrenoceptor number in the kidney in various animal models of hypertension (Nord et al., 1987; Attari et al., 1989; Stanko and Smyth, 1991). However, many drugs which act at 0t-adrenoceptors also have a high affinity for imidazoline sites. Moreover the density of imidazoline sites is much greater than that of ct2-adrenoceptors in the kidney (Coupry et al., 1987; Hamilton et al., 1991). Imidazoline sites, like the 0t2-adrenoceptor, appear to modulate sodium entry and the activity of the Na+/H ÷ exchanger and may therefore play a role in the regulation of blood pressure. However, while 0~2-adrenoceptor activation enhances sodium influx the ct2-adrenoceptor agonist UK 14304 which has an imidazole structure appears to inhibit influx via actions at imidazoline sites (Bidet et al., 1990). To further complicate matters idazoxan acts as an antagonist on 0~2-adrenoceptors and imidazoline sites in the brain stem but may act as an agonist on renal imidazoline sites. Like the ~2-adrenoceptor agonists cirazoline and UK 14304, it inhibits 22Na+ uptake (Bidet et al., 1990). In addition, chronic treatment with idazoxan has been shown to reduce the number of [3H] idazoxan binding sites in kidney membranes without affecting [3H] yohimbine binding, consistent with agonist down regulation of the imidazoline site in the kidney labelled by [3H] idazoxan (Yakubu et al., 1990). Thus, effects of drugs with a high affinity for imidazoline sites on sodium handling and blood pressure regulation may not be the same as those of ~2-adrenoceptor selective compounds. However, much more work is required before the effects of drugs active at imidazoline sites on renal regulation of blood pressure can be evaluated.

7. SUMMARY AND CONCLUSIONS Nonadrenergic imidazoline preferring sites have been identified using the ligands [3H] idazoxan, [3H] clonidine and its analog [3H] p-aminoclonidine. All these ligands have previously been used to label ~t2-adrenoceptor sites. The ligands [3H] clonidine and [3H] idazoxan may label different sites and it is possible that the nonadrenergic sites represent a new family of receptors. However, much is still unknown about these sites. Receptors have yet to be isolated and fully characterized and second messengers identified.

Imidazoline receptors and blood pressure

245

A potential endogenous ligand for these sites has been identified---clonidine displacing substance and a number of physiological responses linked to these sites postulated. In particular, there is good evidence for an involvement of the nonadrenergic [3H] clonidine labelled sites in the nucleus reticularis lateralis in blood pressure regulation. In addition, the sites in the kidney labelled by [3H] idazoxan may be involved in sodium handling. Some drugs which were previously thought to act via ~2-adrenoceptors may mediate their effects at least in part via activation of imidazoline receptors. Design of more specific drugs which have low affinity for g2-adrenoceptors but act at subpopulations of imidazoline receptors could prove valuable therapeutic agents. Compounds acting specifically on the imidazoline sites in the brain stem might be expected to have good antihypertensive properties with fewer side effects than the majority of centrally acting antihypertensive drugs currently available. Whether or not drugs acting primarily at imidazoline sites have additional therapeutic roles is uncertain and must await further clarification of the role of imidazoline sites.

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