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Presynaptically acting catecholamines bind to α2-adrenoceptors labelled by 3H-clonidine
European Journal of Pharmacology, 67 (1980) 187--192 © Elsevier/North-Holland Biomedical Press
187
PRESYNAPTICALLY ACTING CATECHOLAMINES BIND TO a2-ADRENOCEPTORS LABELLED BY 3H-CLONIDINE MILT TITELER* and PHILIP SEEMAN
Department of Pharmacology, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Received 20 February 1980, revised MS received 20 June 1980, accepted 30 June 1980
M. TITELER and P. SEEMAN, Presynaptically acting catecholamines bind to o~-adrenoceptors labelled by 3H-clonidine, European J. Pharmacol. 67 (1980) 187--192. It is known that certain catecholamine congeners can decelerate the heart rate by inhibiting the prejunctional sympathetic neurones to the atrium. The present study was done to determine whether this presynaptic action might be associated with either dopamine receptors or ~-adrenergic receptors. The effects of 10 catecholamine congeners were tested on the specific binding of 0.2 nM 3H-WB-4101 and 0.2 nM 3H-clonidine to calf frontal cortex homogenates, and that of 2 nM 3H-apomorphine to calf caudate nucleus homogenates. The drugs tested were apomorphine, the aminotetralin M-7, 3 dialkylated dopamine congeners, and 5 congeners of octahydrobenzo(f~-quinoline. The ICs0 values (concentrations for 50% inhibition of binding) ranged from 400 to 28 000 nM for °H-WB-4101, from 3 to 270 nM for 3H-apomorphine, and from 9 to 1000 nM for ~H-clonidine. Only the ICs0 values for ~H-clonidine binding correlated with the in vitro ICs0 values for inhibiting atrial acceleration (data from Long et al., 1975, 1979). These findings suggest that 3 H-clonidine appears to bind to the same site in brain (~2 -adrenoceptor) on which catecholaminergic drugs act to produce cardiodeceleration. Octahydrobenzoquinolines Cardiac deceleration
Aminotetralin
Adrenergic receptors
1. Introduction Evidence has been accumulating over the past few years that drugs and neurotransmitters can act at presynaptic catecholamine receptors as well as at postsynaptic catecholamine receptors. A series of catecholamine derivatives have recently been synthesized and assayed for their ability to inhibit presynaptically the cardio-acceleration produced by electrically stimulating the sympathetic nerves to the heart (Long et al., 1975; Cannoh et al., 1978; Long et al., 1979; Ilhan et al., 1976). The present study was undertaken to investigate the site of action of these drugs in producing this effect on the heart; i.e. we wanted to determine whether the effect was being * To whom correspondence should be sent.
Dopamine receptors
mediated through presynaptic dopamine receptors or via presynaptic ~-adrenoceptors. 3H-Apomorphine has been postulated to label a high-affinity dopamine receptor (Titeler et al., 1978), while 3H-WB-4101 and ~Hclonidine (U'Prichard et al., 1977) appear to label ~-adrenergic receptors in mammalian brain. The effects of the various dopaminergic congeners were tested on the binding of these 3H-ligands and the results were correlated with their biological potencies in the intact tissue.
2. Materials and methods
2.1. Preparation of calf caudate homogenates The experiments were done on crude homogenates of calf caudate, prepared as
188
described (Titeler et ai., 1978). Fresh calf brain caudates were removed within 2 h after death, pooled, sliced into small cubes, and suspended in buffer at an approximate concent~ation of 50 mg (wet weight)/ml of buffer (15 mM Tris-HC1, pH 7.4/5 mM Na2EDTA/1.1 mM ascorbate/12.5 pM nialamide). The frontal cortex grey matter was gently scraped off the brain and treated in the same manner as the caudate. Further details may be found in the References in Titeler et al. (197~).
2.2. Binding of ~H-WB-4101 ~H-WB-4101 (13 Ci/mmol) was purchased from New England Nuclear Corp. (Boston, MA}. The ~H-WB-4101 binding assays were done in glass test tubes (12 × 75 mm), in which the following aliquots were placed (using Eppendorf-Brinkmann pipettes with polypropylene tips), 0.2 ml of 3H-WB-4101 (final concentration, 0.2 nM); 0.2 ml of brain homogenate (always added last and containing 0.35 mg of protein); and 0.2 ml of buffer or 0.1 ml of buffer and 0.1 ml of drug. Specific binding was defined by the amount of ~H-WB-4101 binding displaced by 1 t~M phentolamine. Each determination was performed in quintuplicate. After the samples were incubated for 60 min {22°C), a 0.5 ml aliquot was removed (polypropylene pipette tip) from the mixture and filtered under reduced pressure through a glass fiber filter (GF/B; Whatman; 24 mm diameter) on a Millipore stainless steel mesh support; the filtration took less than 1 sec. The filter was then washed once with 10 ml of buffer. The wash buffer was delivered by gravity from a syringe repipette over a period of 4 sec. The filters were not blotted or dried but were placed directly into liquid scintillation vials, 8 ml of Aquasol (New England Nuclear Corp.) were added, and the samples were assayed for ~H (42% efficiency) after storage at 4°C for at least 6 h to allow temperature equilibration and to permit the glass fiber filters to become uniformly translucent.
M. TITELER ET AL.
2.3. Binding of ~H-clonidine 3H-Clonidine ( 26.7 Ci/mmole) was generously donated by Dr. W.D. Bechtel of Boehringer Ingelheim. The 3H-clonidine binding assay was done as above with the following modifications. Specific binding was defined as the amount of 0.2 nM ~H-clonidine binding displaced by 100 nM clonidine as this concentration of clonidine reduced binding to the same level as excess phentolamine or dihydroergocryptine. The total incubation volume was 1.8 ml, contained 0.5 mg protein, and was applie~i directly to the filters. The rinse consisted of two 5 ml washes with buffer.
2.4. Binding of 3H-apomorphine Binding of 3H-apomorphine (at 2 nM) was done as previously outlined (see in Titeler et al., 1978) and was defined as that which was displaceable by 200 nM apomorphine.
2.5. Drugs The hydrobromides of M-7 (or 5, 6-dihydroxy-2-N,N-dimethylaminotet~alin), the N,NTABLE 1 Structures of congeners derived from octahydrobenzo(f)quinoline.
OH
4aN/R
TL-224 TL-137 GJH-171 GJH-166 TL-121
R
10b-4a ring juncture
H H CH3 CH3 C~H s
cis trans cis trans trans
3H-CLONIDINE R E C E P T O R S
189
TABLE 2 ICs0 values for atrial deceleration. The 3H-clonidine (0.2 riM) and 3H-WB-4101 (0.2 nM) assays were done on calf frontal cortex h o m o g e n a t e and the 3H-apomorphine (2 nM) assays were done on calf caudate h o m o g e n a t e . The n u m b e r at the b o t t o m of the c o l u m n s are correlation coefficients determined by linear regression using the least squares m e t h o d .
ICs0 (nM) 1 for atrial inhibition GJH-166 TL-121 M-7 TL-224 GJH-171 TL-137 N,N-Dimethyldopamine Apomorphine
3 H-clonidine
2.6 ( 0 . 1 - - 6 ) 2`3 3.5 ( 1 . 9 - - 6 . 4 ) 2 20 s,6 80 ( 2 0 - - 1 2 2 ) 2 45 ( 1 0 - - 8 0 ) 2'3 64 ( 4 5 - - 4 3 0 ) 2 70 ( 4 0 - - 1 5 0 ) 4's 43 ( 7 - - 7 3 ) 4'~ 580 ( 3 0 0 - - 1 2 0 0 ) 2 340 ( 2 3 0 - - 5 7 0 ) 4 420 (190--2700) 4
N,N-Diethyldopamine N,N-Dipropyldopamine
3H-apo
3H-WB-4101
7.7 3.1 13 270 260 33 10.5
1000 400 2000 4000 6500 8000 28 0 0 0
300 800 1000
3.8 70 100
1000 9000 7500
r = 0.86
r = 0.42
17 9.4 20 36 95 350 250
r = 0.54
~ Concentration for 50% inhibition of the atrio-accelerator response to 2 Hz transmural stimulation of cat atrium in vitro. : Long et al., 1 9 7 9 . 3 Sharabi et al., 1976. 4 Cannon et al., 1 9 7 8 . 8 Ilhan et al., 1 9 7 6 . 6 Long et al., 1975.
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ICso for ATRIAL DECELERATION (moles/L)
Fig. 1. Correlations between the ICs0 values of various dopaminergic congeners on the specific binding of 3Hclonidine (A) and the specific binding of 3H-apomorphine (B) to calf brain (frontal cortex for 3H-clonidine; caudate nucleus for 3H-apomorphine) homogenate and their ICs0 values on the electrically stimulated cat atria preparation. The atrial data are from Long et al., 1975, 1979; Cannon et al., 1978; Ilhan et al., 1976; Sharabi et al., 1976. The atrial value for apomorphine was first reported to be 43 n M (Ilhan et al., 1976; Cannon et al,, 1978), but more recently reported to be 580 n M (Long et al., 1979); both values are included in fig.1. The specific binding of 0.2 n M 3H-clonidine was defined as that displaceable by I p M phentolamine, while that for 2 n M 3H-apomorphine was defined as that displaceable by 200 n M apomorphine.
190 dialkyl-substituted dopamines, and the congeners of octahydrobenzo(f)-quinoline (see table 1) were generously donated by Drs. J. Long and J.G. Cannon, University of Iowa.
3. Results Table 2 lists the ICs0 values of the congeners obtained by assaying the effects of these drugs on three putative catecholamine receptor binding sites. Also listed are the ICs0 values of the congeners in inhibiting the electrically stimulated atrial acceleration of the cat atrium (Long et al., 1975; Cannon et al., 1978; Long et al., 1979). In general, the congeners displayed a high affinity for both the 3H-apomorphine binding site and the ~Hclonidine binding site. They displayed much lower affinities for the -~'H-WB-4101 binding site. The correlation coefficients at the bottom of the ICs0 columns (table 2) were derived by comparing these values with the ICs0 values listed in the first column by means of a linear regression using the least squares method. The Hill coefficient (nH) for clonidine and phentolamine competing against ~H-clonidine binding were close to unity, 0.98 and 0.92, respectively; the n H values for four agonists, however, were rather low: 0.62 for TL-121, 0.60 for TL-224, 0.61 for GJH-171 and 0.69 for apomorphine. 4. Discussion
Fig. 1 correlates the ICs0 values for binding sites with the ICs0 values for these drugs in inhibiting the atrial accelerator response (Long et al., 1975; Cannon et al., 1978; Long et al., 1979). Only the 3H-clonidine data (fig. 1A) correlated significantly with the biological potencies. Although some of these drugs were potent at the ~H-apomorphine binding site (fig. 1B) their biological potencies did not correlate strongly with their in vitro affinities for this binding site. This represents a revision of our original interpretation of these data,
M. TITELER ET AL. since we had previously found a correlation between the ICso values for these congeners and the ICs0 values for atrial deceleration (Titeler et al., 1978). That correlation, however, had used the value of 43 nM for atrial deceleration of apomorphine (Ilhan et al., 1976; Cannon et al., 1978). A more recent value for atrial deceleration by apomorphine is reported to be 580 nM (Long et al., 1979), and the inclusion of this ICs0 value abolishes the correlation with the 3H-apomorphine data (fig. 1B). There was no obvious reason for the differing v.alues for apomorphine (Dr. J.P. Long, personal communication). It is worth noting that if some of these compounds are partial agonists and others need only activate a small percentage of the receptors to produce half-maximal effect, the absence of a correlation between the ICs0 values on the tissue and those on binding would be understandable. Although there was a good correlation between the 3H-clonidine ICs0 values and the biological ICs0 values (fig. 1A), the concentrations were not equimolar. The competition curves of these dopamine congeners for the ~H-clonidine binding site were 'shallow'; that is, they did not follow the theoretically predicted shape of a direct competition curve. The Hill coefficients ranged between 0.60 and 0.69. Phentolamine and clonidine, however, did produce competition curves with Hill .coefficients close to unity. The shallow competition curves produced by these catecholamine derivatives thus result in apparent ICs0 values that are higher than the ICs0 values produced by a competition curve with a Hill coefficient of 1. This effect results in an upward shift in the correlation line in fig. 1A. A past issue had been whether the high affinity 3H-clonidine binding site represented an agonist conformation of the post-synaptic a-adrenergic receptor (U'Prichard et al., 1977) or an a2-adrenergic receptor (U'Prichard et al., 1978; Titeler et al., 1978; U'Prichard and Snyder, 1979). The fact that the 3H-clonidine binding data in the present study correlate with the presynaptic cardiac concentrations of the catecholamine congeners suggests that
~H-CLONIDINE RECEPTORS
these congeners could act on presynaptic adrenoceptors in the heart. Thus, although these correlative data (fig.1A) do not provide any direct support for the presynaptic localization of 3H-clonidine binding sites in the brain, they do provide evidence for the pharmacological identity of 3H-clonidine sites with presynaptic a2-receptors. Although no 3H-clonidine binding has been detected in rat heart (U'Prichard and Snyder, 1979), this does not necessarily indicate that such sites do not exist in this tissue. Homogenates of whole heart could greatly dilute the densities of such sites, making detection impossible. Further studies on heart tissue are necessary to clarifythis point. Recently, a report of two high-affinity sites for 3H-clonidine has been published (U'Prichard and Snyder, 1978). The results from the present experiments indicate that it is unlikely that in the present assay system that more than one specific binding site for ~H-clonidine was being labelled. All the congeners competed for specific binding with parallel slopes. Thus, if there were two sites they had identical relative affinitiesfor these series of drugs. Furthermore, phentolamine and clonidine competed for the binding site with Hill coefficients very close to I and reduced the specific binding to the same baseline levels. Therefore, if two sites were being labelled, they had the same affinitiesfor both phentolamine and clonidine. (At higher concentrations of ~H-clonidine there is another specific binding site detectable; manuscript in preparation). O n the other hand, the shallow displacement curves of the dopaminergic congeners in inhibiting 3H-clonidine binding (with n H = 0.6) does suggest that ~H-clonidine m a y label two high affinity sites (U'Prichard and Snyder, 1978). The effect of these congeners was also studied on ~H-spiperone binding (to be published). There was no correlation with their biological potencies in inhibiting cardioacceleration. Acknowledgements W e thank Dr. J. Long, Dr. J.G. Cannon (University
191 of Iowa) and Dr. W,D. Bechtel (Boehringer Ingelheim) for their generous donations of drug samples. We thank Mr. J. Tedesco for his kind assistance. Excellent technical aid was given by Mrs. Anna Banaszuk and Mrs. Joan Dumas. Supported by the Medical Research Council of Canada, the Ontario Medical Health Foundation and the Hospital for Sick Children Foundation (Toronto).
References Cannon, J.G., F.-L. Hsu, J.P. Long, J.R. Flynn, B. Costall and R.J. Naylor, 1978, Preparation and biological actions of some symmetrically N,N-disubstituted dopamines, J. Med. Chem. 21,248. llhan, M., J.P. Long and J.G. Cannon, 1976, Effects of some dopamine analogs and haloperidol on response to stimulation of adrenergic nerves using cat atria in vitro, Arch. Int. Pharmacodyn. 219, 193. Long, J.P., S. Heintz, J.G. Cannon and K. Lira, 1975, Inhibition of the sympathetic nervous system by 5,6-dihydroxy-2-dimethylaminotetralin (M-7), apomorphine, and dopamine, J. Pharmacol. Exp. Ther. 192,336. Long, J.P., D.B. Rusterholz, J.R. Flynn and J.G. Cannon, 1979, Inhibitory receptors on the adrenergic nerve terminal, Proc. Syrup. Peripheral Dopamine Receptors (Strasbourg) (in press). Sharabi, F.M., J.P. Long, J.G. Cannon and G.J. Hatheway, 1974, Inhibition of the sympathetic nervous system by a series of heterocyclic congeners of dopamine, J. Pharmacol. Exp. Ther. 199, 630. Titeler, M., J. Tedesco and P. Seeman, 1978, Selective labeling of presynaptic receptors by 3H-dopamine, 3H-apomorphine and 3H-clonidine; labeling of post-synaptic sites by 3H-neuroleptics, Life Sci. 23,587. U'Prichard, D.C., M.E. Charness, D. Robertson and S.H. Snyder, 1978, Prazosin: differential affinities for two populations of (~-noradrenergic receptor binding sites, European J. Pharmacol. 50, 87. U'Prichard, D.C., D.A. Greenberg and S.H. Snyder, 1977, Binding characteristics of a radiolabeled agonist and antagonist at central nervous system alpha noradrenergic receptors, Mol. Pharmacol. 13, 454. U'Prichard, D. and S.H. Snyder, 1978, Two ~Hclonidine o~-noradrenergic sites in brain with differential response to 6-0H dopamine treatment, Soc. Neurosci. Abstr. 4,523. U'Prichard, D.C. and S.H. Snyder, 1979, Distinct (x-noradrenergic receptors differentiated by binding and physiological relationships, Life Sci. 24, 79.
187
PRESYNAPTICALLY ACTING CATECHOLAMINES BIND TO a2-ADRENOCEPTORS LABELLED BY 3H-CLONIDINE MILT TITELER* and PHILIP SEEMAN
Department of Pharmacology, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Received 20 February 1980, revised MS received 20 June 1980, accepted 30 June 1980
M. TITELER and P. SEEMAN, Presynaptically acting catecholamines bind to o~-adrenoceptors labelled by 3H-clonidine, European J. Pharmacol. 67 (1980) 187--192. It is known that certain catecholamine congeners can decelerate the heart rate by inhibiting the prejunctional sympathetic neurones to the atrium. The present study was done to determine whether this presynaptic action might be associated with either dopamine receptors or ~-adrenergic receptors. The effects of 10 catecholamine congeners were tested on the specific binding of 0.2 nM 3H-WB-4101 and 0.2 nM 3H-clonidine to calf frontal cortex homogenates, and that of 2 nM 3H-apomorphine to calf caudate nucleus homogenates. The drugs tested were apomorphine, the aminotetralin M-7, 3 dialkylated dopamine congeners, and 5 congeners of octahydrobenzo(f~-quinoline. The ICs0 values (concentrations for 50% inhibition of binding) ranged from 400 to 28 000 nM for °H-WB-4101, from 3 to 270 nM for 3H-apomorphine, and from 9 to 1000 nM for ~H-clonidine. Only the ICs0 values for ~H-clonidine binding correlated with the in vitro ICs0 values for inhibiting atrial acceleration (data from Long et al., 1975, 1979). These findings suggest that 3 H-clonidine appears to bind to the same site in brain (~2 -adrenoceptor) on which catecholaminergic drugs act to produce cardiodeceleration. Octahydrobenzoquinolines Cardiac deceleration
Aminotetralin
Adrenergic receptors
1. Introduction Evidence has been accumulating over the past few years that drugs and neurotransmitters can act at presynaptic catecholamine receptors as well as at postsynaptic catecholamine receptors. A series of catecholamine derivatives have recently been synthesized and assayed for their ability to inhibit presynaptically the cardio-acceleration produced by electrically stimulating the sympathetic nerves to the heart (Long et al., 1975; Cannoh et al., 1978; Long et al., 1979; Ilhan et al., 1976). The present study was undertaken to investigate the site of action of these drugs in producing this effect on the heart; i.e. we wanted to determine whether the effect was being * To whom correspondence should be sent.
Dopamine receptors
mediated through presynaptic dopamine receptors or via presynaptic ~-adrenoceptors. 3H-Apomorphine has been postulated to label a high-affinity dopamine receptor (Titeler et al., 1978), while 3H-WB-4101 and ~Hclonidine (U'Prichard et al., 1977) appear to label ~-adrenergic receptors in mammalian brain. The effects of the various dopaminergic congeners were tested on the binding of these 3H-ligands and the results were correlated with their biological potencies in the intact tissue.
2. Materials and methods
2.1. Preparation of calf caudate homogenates The experiments were done on crude homogenates of calf caudate, prepared as
188
described (Titeler et ai., 1978). Fresh calf brain caudates were removed within 2 h after death, pooled, sliced into small cubes, and suspended in buffer at an approximate concent~ation of 50 mg (wet weight)/ml of buffer (15 mM Tris-HC1, pH 7.4/5 mM Na2EDTA/1.1 mM ascorbate/12.5 pM nialamide). The frontal cortex grey matter was gently scraped off the brain and treated in the same manner as the caudate. Further details may be found in the References in Titeler et al. (197~).
2.2. Binding of ~H-WB-4101 ~H-WB-4101 (13 Ci/mmol) was purchased from New England Nuclear Corp. (Boston, MA}. The ~H-WB-4101 binding assays were done in glass test tubes (12 × 75 mm), in which the following aliquots were placed (using Eppendorf-Brinkmann pipettes with polypropylene tips), 0.2 ml of 3H-WB-4101 (final concentration, 0.2 nM); 0.2 ml of brain homogenate (always added last and containing 0.35 mg of protein); and 0.2 ml of buffer or 0.1 ml of buffer and 0.1 ml of drug. Specific binding was defined by the amount of ~H-WB-4101 binding displaced by 1 t~M phentolamine. Each determination was performed in quintuplicate. After the samples were incubated for 60 min {22°C), a 0.5 ml aliquot was removed (polypropylene pipette tip) from the mixture and filtered under reduced pressure through a glass fiber filter (GF/B; Whatman; 24 mm diameter) on a Millipore stainless steel mesh support; the filtration took less than 1 sec. The filter was then washed once with 10 ml of buffer. The wash buffer was delivered by gravity from a syringe repipette over a period of 4 sec. The filters were not blotted or dried but were placed directly into liquid scintillation vials, 8 ml of Aquasol (New England Nuclear Corp.) were added, and the samples were assayed for ~H (42% efficiency) after storage at 4°C for at least 6 h to allow temperature equilibration and to permit the glass fiber filters to become uniformly translucent.
M. TITELER ET AL.
2.3. Binding of ~H-clonidine 3H-Clonidine ( 26.7 Ci/mmole) was generously donated by Dr. W.D. Bechtel of Boehringer Ingelheim. The 3H-clonidine binding assay was done as above with the following modifications. Specific binding was defined as the amount of 0.2 nM ~H-clonidine binding displaced by 100 nM clonidine as this concentration of clonidine reduced binding to the same level as excess phentolamine or dihydroergocryptine. The total incubation volume was 1.8 ml, contained 0.5 mg protein, and was applie~i directly to the filters. The rinse consisted of two 5 ml washes with buffer.
2.4. Binding of 3H-apomorphine Binding of 3H-apomorphine (at 2 nM) was done as previously outlined (see in Titeler et al., 1978) and was defined as that which was displaceable by 200 nM apomorphine.
2.5. Drugs The hydrobromides of M-7 (or 5, 6-dihydroxy-2-N,N-dimethylaminotet~alin), the N,NTABLE 1 Structures of congeners derived from octahydrobenzo(f)quinoline.
OH
4aN/R
TL-224 TL-137 GJH-171 GJH-166 TL-121
R
10b-4a ring juncture
H H CH3 CH3 C~H s
cis trans cis trans trans
3H-CLONIDINE R E C E P T O R S
189
TABLE 2 ICs0 values for atrial deceleration. The 3H-clonidine (0.2 riM) and 3H-WB-4101 (0.2 nM) assays were done on calf frontal cortex h o m o g e n a t e and the 3H-apomorphine (2 nM) assays were done on calf caudate h o m o g e n a t e . The n u m b e r at the b o t t o m of the c o l u m n s are correlation coefficients determined by linear regression using the least squares m e t h o d .
ICs0 (nM) 1 for atrial inhibition GJH-166 TL-121 M-7 TL-224 GJH-171 TL-137 N,N-Dimethyldopamine Apomorphine
3 H-clonidine
2.6 ( 0 . 1 - - 6 ) 2`3 3.5 ( 1 . 9 - - 6 . 4 ) 2 20 s,6 80 ( 2 0 - - 1 2 2 ) 2 45 ( 1 0 - - 8 0 ) 2'3 64 ( 4 5 - - 4 3 0 ) 2 70 ( 4 0 - - 1 5 0 ) 4's 43 ( 7 - - 7 3 ) 4'~ 580 ( 3 0 0 - - 1 2 0 0 ) 2 340 ( 2 3 0 - - 5 7 0 ) 4 420 (190--2700) 4
N,N-Diethyldopamine N,N-Dipropyldopamine
3H-apo
3H-WB-4101
7.7 3.1 13 270 260 33 10.5
1000 400 2000 4000 6500 8000 28 0 0 0
300 800 1000
3.8 70 100
1000 9000 7500
r = 0.86
r = 0.42
17 9.4 20 36 95 350 250
r = 0.54
~ Concentration for 50% inhibition of the atrio-accelerator response to 2 Hz transmural stimulation of cat atrium in vitro. : Long et al., 1 9 7 9 . 3 Sharabi et al., 1976. 4 Cannon et al., 1 9 7 8 . 8 Ilhan et al., 1 9 7 6 . 6 Long et al., 1975.
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ICso for ATRIAL DECELERATION (moles/L)
Fig. 1. Correlations between the ICs0 values of various dopaminergic congeners on the specific binding of 3Hclonidine (A) and the specific binding of 3H-apomorphine (B) to calf brain (frontal cortex for 3H-clonidine; caudate nucleus for 3H-apomorphine) homogenate and their ICs0 values on the electrically stimulated cat atria preparation. The atrial data are from Long et al., 1975, 1979; Cannon et al., 1978; Ilhan et al., 1976; Sharabi et al., 1976. The atrial value for apomorphine was first reported to be 43 n M (Ilhan et al., 1976; Cannon et al,, 1978), but more recently reported to be 580 n M (Long et al., 1979); both values are included in fig.1. The specific binding of 0.2 n M 3H-clonidine was defined as that displaceable by I p M phentolamine, while that for 2 n M 3H-apomorphine was defined as that displaceable by 200 n M apomorphine.
190 dialkyl-substituted dopamines, and the congeners of octahydrobenzo(f)-quinoline (see table 1) were generously donated by Drs. J. Long and J.G. Cannon, University of Iowa.
3. Results Table 2 lists the ICs0 values of the congeners obtained by assaying the effects of these drugs on three putative catecholamine receptor binding sites. Also listed are the ICs0 values of the congeners in inhibiting the electrically stimulated atrial acceleration of the cat atrium (Long et al., 1975; Cannon et al., 1978; Long et al., 1979). In general, the congeners displayed a high affinity for both the 3H-apomorphine binding site and the ~Hclonidine binding site. They displayed much lower affinities for the -~'H-WB-4101 binding site. The correlation coefficients at the bottom of the ICs0 columns (table 2) were derived by comparing these values with the ICs0 values listed in the first column by means of a linear regression using the least squares method. The Hill coefficient (nH) for clonidine and phentolamine competing against ~H-clonidine binding were close to unity, 0.98 and 0.92, respectively; the n H values for four agonists, however, were rather low: 0.62 for TL-121, 0.60 for TL-224, 0.61 for GJH-171 and 0.69 for apomorphine. 4. Discussion
Fig. 1 correlates the ICs0 values for binding sites with the ICs0 values for these drugs in inhibiting the atrial accelerator response (Long et al., 1975; Cannon et al., 1978; Long et al., 1979). Only the 3H-clonidine data (fig. 1A) correlated significantly with the biological potencies. Although some of these drugs were potent at the ~H-apomorphine binding site (fig. 1B) their biological potencies did not correlate strongly with their in vitro affinities for this binding site. This represents a revision of our original interpretation of these data,
M. TITELER ET AL. since we had previously found a correlation between the ICso values for these congeners and the ICs0 values for atrial deceleration (Titeler et al., 1978). That correlation, however, had used the value of 43 nM for atrial deceleration of apomorphine (Ilhan et al., 1976; Cannon et al., 1978). A more recent value for atrial deceleration by apomorphine is reported to be 580 nM (Long et al., 1979), and the inclusion of this ICs0 value abolishes the correlation with the 3H-apomorphine data (fig. 1B). There was no obvious reason for the differing v.alues for apomorphine (Dr. J.P. Long, personal communication). It is worth noting that if some of these compounds are partial agonists and others need only activate a small percentage of the receptors to produce half-maximal effect, the absence of a correlation between the ICs0 values on the tissue and those on binding would be understandable. Although there was a good correlation between the 3H-clonidine ICs0 values and the biological ICs0 values (fig. 1A), the concentrations were not equimolar. The competition curves of these dopamine congeners for the ~H-clonidine binding site were 'shallow'; that is, they did not follow the theoretically predicted shape of a direct competition curve. The Hill coefficients ranged between 0.60 and 0.69. Phentolamine and clonidine, however, did produce competition curves with Hill .coefficients close to unity. The shallow competition curves produced by these catecholamine derivatives thus result in apparent ICs0 values that are higher than the ICs0 values produced by a competition curve with a Hill coefficient of 1. This effect results in an upward shift in the correlation line in fig. 1A. A past issue had been whether the high affinity 3H-clonidine binding site represented an agonist conformation of the post-synaptic a-adrenergic receptor (U'Prichard et al., 1977) or an a2-adrenergic receptor (U'Prichard et al., 1978; Titeler et al., 1978; U'Prichard and Snyder, 1979). The fact that the 3H-clonidine binding data in the present study correlate with the presynaptic cardiac concentrations of the catecholamine congeners suggests that
~H-CLONIDINE RECEPTORS
these congeners could act on presynaptic adrenoceptors in the heart. Thus, although these correlative data (fig.1A) do not provide any direct support for the presynaptic localization of 3H-clonidine binding sites in the brain, they do provide evidence for the pharmacological identity of 3H-clonidine sites with presynaptic a2-receptors. Although no 3H-clonidine binding has been detected in rat heart (U'Prichard and Snyder, 1979), this does not necessarily indicate that such sites do not exist in this tissue. Homogenates of whole heart could greatly dilute the densities of such sites, making detection impossible. Further studies on heart tissue are necessary to clarifythis point. Recently, a report of two high-affinity sites for 3H-clonidine has been published (U'Prichard and Snyder, 1978). The results from the present experiments indicate that it is unlikely that in the present assay system that more than one specific binding site for ~H-clonidine was being labelled. All the congeners competed for specific binding with parallel slopes. Thus, if there were two sites they had identical relative affinitiesfor these series of drugs. Furthermore, phentolamine and clonidine competed for the binding site with Hill coefficients very close to I and reduced the specific binding to the same baseline levels. Therefore, if two sites were being labelled, they had the same affinitiesfor both phentolamine and clonidine. (At higher concentrations of ~H-clonidine there is another specific binding site detectable; manuscript in preparation). O n the other hand, the shallow displacement curves of the dopaminergic congeners in inhibiting 3H-clonidine binding (with n H = 0.6) does suggest that ~H-clonidine m a y label two high affinity sites (U'Prichard and Snyder, 1978). The effect of these congeners was also studied on ~H-spiperone binding (to be published). There was no correlation with their biological potencies in inhibiting cardioacceleration. Acknowledgements W e thank Dr. J. Long, Dr. J.G. Cannon (University
191 of Iowa) and Dr. W,D. Bechtel (Boehringer Ingelheim) for their generous donations of drug samples. We thank Mr. J. Tedesco for his kind assistance. Excellent technical aid was given by Mrs. Anna Banaszuk and Mrs. Joan Dumas. Supported by the Medical Research Council of Canada, the Ontario Medical Health Foundation and the Hospital for Sick Children Foundation (Toronto).
References Cannon, J.G., F.-L. Hsu, J.P. Long, J.R. Flynn, B. Costall and R.J. Naylor, 1978, Preparation and biological actions of some symmetrically N,N-disubstituted dopamines, J. Med. Chem. 21,248. llhan, M., J.P. Long and J.G. Cannon, 1976, Effects of some dopamine analogs and haloperidol on response to stimulation of adrenergic nerves using cat atria in vitro, Arch. Int. Pharmacodyn. 219, 193. Long, J.P., S. Heintz, J.G. Cannon and K. Lira, 1975, Inhibition of the sympathetic nervous system by 5,6-dihydroxy-2-dimethylaminotetralin (M-7), apomorphine, and dopamine, J. Pharmacol. Exp. Ther. 192,336. Long, J.P., D.B. Rusterholz, J.R. Flynn and J.G. Cannon, 1979, Inhibitory receptors on the adrenergic nerve terminal, Proc. Syrup. Peripheral Dopamine Receptors (Strasbourg) (in press). Sharabi, F.M., J.P. Long, J.G. Cannon and G.J. Hatheway, 1974, Inhibition of the sympathetic nervous system by a series of heterocyclic congeners of dopamine, J. Pharmacol. Exp. Ther. 199, 630. Titeler, M., J. Tedesco and P. Seeman, 1978, Selective labeling of presynaptic receptors by 3H-dopamine, 3H-apomorphine and 3H-clonidine; labeling of post-synaptic sites by 3H-neuroleptics, Life Sci. 23,587. U'Prichard, D.C., M.E. Charness, D. Robertson and S.H. Snyder, 1978, Prazosin: differential affinities for two populations of (~-noradrenergic receptor binding sites, European J. Pharmacol. 50, 87. U'Prichard, D.C., D.A. Greenberg and S.H. Snyder, 1977, Binding characteristics of a radiolabeled agonist and antagonist at central nervous system alpha noradrenergic receptors, Mol. Pharmacol. 13, 454. U'Prichard, D. and S.H. Snyder, 1978, Two ~Hclonidine o~-noradrenergic sites in brain with differential response to 6-0H dopamine treatment, Soc. Neurosci. Abstr. 4,523. U'Prichard, D.C. and S.H. Snyder, 1979, Distinct (x-noradrenergic receptors differentiated by binding and physiological relationships, Life Sci. 24, 79.