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agonist-preferring α-adrenoceptors or α1α2-adrenoceptors ?
191
European Journal o f Pharmacology, 63 (1980) 19t-194
@)Elsevier/North-Holland Biomedical Press Short communication ANTAGONIST/AGONIST-PREFERRING a-ADRENOCEPTORS OR a,/a2-ADRENOCEPTORS? TOSHIYUKI TANAKA and KLAUS STARKE * Pharmakologisches Institut, Universitiit Freiburg, D-7800 Freiburg, FRG
Received 27 February 1980, accepted 3 March 1980
T. TANAKA and K. STARKE, Antagonist/agonist-preferring O~-adrenoceptors or o~l/o~2-adrenoceptors? , European J. Pharmacol. 63 (1980) 191--194. Yohimbine and some stereoisomeric alkaloids inhibited the binding of 3H-clonidine and 3H-WB-4101 to rat cerebral cortex membranes. Rauwolscine and yohimbine had much higher affinity to the 3H-clonidine than to the 3H-WB-4101 site, whereas the reverse was true for corynanthine. The results indicate that the 3H-clonidine site is an ~2-adrenoceptor and not an agonist-selective site whereas the 3H-WB-4101 site is an ~l-adrenoceptor and not an antagonist-selective site. (~l"Adrenoceptor
~2Adrenoceptor
Yohimbine
1. Introduction Studies on pre- and postsynaptic ~-adrenoceptors of sympathetic neuroeffector junctions have led to the view that ~-adrenoceptors could be divided into ~,- and ~2~subtypes (Langer, 1974; Berthelsen and Pettinger, 1977). For instance, clonidine preferentially activates, and yohimbine preferentially blocks, the ~2-receptor, whereas phenylephrine mainly activates, and 2-[(2',6'
* Address correspondence to: Dr. Klaus Starke, Pharmakologisches Institut, Hermann-Herder-Strasse 5, D-7800 Freiburg, Federal Republic of Germany.
Rauwolscine
Corynanthine
clonidine, the other one by 3H-WB-4101 (U'Prichard et al., 1977). a-Receptor agonists were much more p o t e n t in inhibiting 3Hclonidine than 3H-WB-4101 binding, whereas the antagonists were much more potent in inhibiting 3H-WB-4101 than 3H-clonidine binding. This was originally thought to be the distinguishing feature of the 3H-clonidine and the 3H-WB-4101 site which were therefore also called the agonist a-receptor site and the antagonist s-receptor site, respectively (U'Prichard et al., 1977; Greenberg and Snyder, 1978). More recently it has been proposed that the 3H-WB-4101 site may in fact be the a 1-receptor of the functional studies and the 3H~clonidine site the ~2-receptor, the preference of drugs for one site or the other being unrelated to their agonist or antagonist properties (U'Prichard et al., 1978; see also Miach et al., 1978). There is no published evidence that would decide between these two interpretations of the binding data. We have recently observed in functional experiments that yohimbine diastereomers were good tools for the dif-
192
ferentiation of a~- and a2-adrenoceptors; in particular, rauwolscine was even more selective than yohimbine for a2-receptors (Weitzell et al., 1979). We now report the effect of four stereoisomers on the binding of 3H-clonidine and 3H-WB-4101. Our results are direct evidence against the first and for the second interpretation.
2. Materials and methods Chemicals were obtained from the following sources: 3H-clonidine, specific activity 26.7 Ci/mmol, Boehringer, Ingelheim; 3H-WB-4101, specific activity 25.4 Ci/mmol, New England Nuclear, Dreieich; (--)-noradrenaline base, Hoechst, Frankfurt; rauwolscine hydrochloride, yohimbine hydrochloride and corynanthine, Roth, Karlsruhe; pseudoyohimbine, ICN Pharmaceuticals, Plainview, N.Y. Membranes from cerebral cortex of male rats (200-250 g) were prepared and binding assays performed as described by U'Prichard et al. (1977) with high concentration of (--)noradrenaline to determine nonspecific binding. Ascorbic acid 0.05% was included in incubation mixtures in order to prevent oxidation of noradrenaline. Since 3H-WB-4101 was adsorbed to test tubes, mixtures without membranes were also incubated and counted for radioactivity in order to obtain the total concentration in the incubation fluid. A substantial fraction of the total concentration of 3H-WB-4101 (up to 18%) was b o u n d to the brain membranes. In the case of 3H-clonidine, this fraction was less than 5%. Data were analyzed b y non-linear least-squares curve fitting essentially according to the method of Hancock et al. (1979) which corrects for the decrease in free ligand concentration due to binding to membranes.
3. Results In saturation experiments specific binding of 3H-clonidine (total concentration 0.0625-
T. T A N A K A , K. S T A R K E
32 nM) occurred to a single population of sites with a maximal number Bmax of 242 + 10 fmol/mg protein and a dissociation constant K D of 2.72 + 0.26 nM. Specific binding of 3H-WB-4101 (total concentration 0.0193.22 nM) also was to one population of sites with Bmax of 194 + 3 fmol/mg protein and K D of 0.29 + 0.02 nM (means + S.E.M. of 4 experiments). K D values were similar to those reported previously (U'Prichard et al., 1977). We did not observe two populations of 3H-clonidine binding sites as found recently by U'Prichard et al. (1979) in some b u t not all experiments. Fig. 1 shows that rauwolscine and corynanthine inhibited the binding of b o t h 3H-
100
"
~
"'~.
50
' ~
~,
o
rn I
0
,. I
I
I
I
1
I
10- 9
10-8
10 -7
10 -6
10- 5
10-/,
M
rauwotscine
or
corynanthine
Fig. l . I n h i b i t i o n o f aH-elonidine and 3H-WB-4101 b i n d i n g t o rat cerebral c o r t e x m e m b r a n e s by rauwolscine and c o r y n a n t h i n e . • • E f f e c t o f rauwolscine o n 3H-clonidine b i n d i n g ; o . . . . . . o e f f e c t o f r a u w o l s c i n e o n 3H-WB-4101 b i n d i n g ; • -• e f f e c t o f c o r y n a n t h i n e o n 3H-clonidine binding; []. . . . . . [] e f f e c t o f c o r y n a n t h i n e o n 3H-WB-4101 binding. Abscissa: c o n c e n t r a t i o n o f c o m p e t i t o r ; ordinate: 3H-ligand b o u n d , expressed as p e r c e n t o f total binding in t h e a b s e n c e o f c o m p e t i t o r . Data are f r o m o n e e x p e r i m e n t carried o u t in triplicate. T o t a l c o n c e n t r a t i o n s o f 3H-clonidine a n d 3H-WB-4101 were 0.5 and 0.38 nM, respectively. Curves r e p r e s e n t t h e best fit t o t h e e x p e r i m e n t a l data as calculated b y non-linear least-squares m e t h o d o f curve fitting.
RADIOLABELING OF a~- AND a2-ADRENOCEPTORS
193
TABLE 1 Inhibition of 3H-clonidine and 3H-WB-4101 specific binding by yohimbine stereoisomers. Total concentration of 3H-clonidine was 0.5 nM, total concentrations of 3H-WB-4101 ranged from 0.38 to 0.94 nM. In each experiment, 7--9 concentrations of the stereoisomers were tested, and dissociation constants K i were calculated from concentration-inhibition curves as shown in fig. 1. Values are means of 2 experiments each carried out in triplicate; individual values from the 2 experiments are in parentheses. Competitor
Rauwolscine Yohimbine Corynanthine Pseudoyohimbine
K i for 3H-clonidine site (nM)
K i for 3H-WB-4101 site (nM)
K i 3H-WB-4101 site
41.3 (39.1;43.3) 47.2 (46.3; 48.1) 6200 (5180; 7220) 1955 (1490; 2420)
2225 419 172 2515
53.9 8.9 0.028 1.3
clonidine and 3H-WB-4101. Rauwolscine competed much more strongly with 3H-clonidine than with 3H-WB-4101, whereas the reverse was true for corynanthine. Dissociation constants Ki of the four stereoisomers for the two binding sites are given in table 1. The c o m p o u n d with the highest affinity to the 3H~lonidine site was rauwolscine, closely followed b y yohimbine; pseudoyohimbine had much less, and corynanthine the least affinity. In constrast, corynanthine was the most p o t e n t competitor for 3H-WB-4101 binding, yohimbine and rauwolscine were less potent and pseudoyohimbine was also a weak competitor.
4. Discussion Yohimbine, corynanthine, rauwolscine and pseudoyohimbine are competitive a-adrenoceptor blocking drugs (WeitzeU et al., 1979). As shown b y the Ki ratios in table 1, corynanthine had a 36 times greater affinity to the 3H-WB-4101 than to the 3H-clonidine site. This would be consistent with the idea that antagonists generally bind preferentially to the 3H-WB-4101 site (and agonists to the 3H-clonidine site). Yet, the findings with yohimbine and rauwolscine argue against this view. Yohimbine had 9 times greater affinity to the 3H~clonidine than to the 3H-
(1970; 2480) (407; 431) (156; 188) {2500; 2530)
K i 3H-clonidine site
WB-4101 site. (A ratio of 3.2 was reported b y U'Prichard et al., 1977, and was explained b y the partial agonist character of yohimbine; however, to our knowledge there is no evidence for a partial agonist effect of yohimbine at a-adrenoceptors.) Moreover, rauwolscine had no less than a 54 times greater affinity to the 3H~lonidine site than to the 3H-WB-4101 site. The high selectivity of the antagonist rauwolscine for the 3H-clonidine site rules out the possibility that this site generally prefers agonists. In a study of functional effects of the four isomers on sympathetic neuroeffector transmission in the rabbit pulmonary artery, yohimbine and rauwolscine even more so, displayed a high selectivity for a2~adrenoceptors, whereas corynanthine was selective for a ~-adrenoceptors; pseudoyohimbine had little effect on either adrenoceptor (Weitzell et al., 1979). The affinities o f these antagonists to the 3H~lonidine site as reported here mirror their functional a2-receptor blocking potencies in the pulmonary artery, while the affinities to the 3H-WB-4101 site mirror their functional al-receptor blocking potencies. This close agreement indicates that in brain membranes 3H~lonidine labels selectively not an agonist a-receptor but an a2-adrenoceptor, whereas 3H-4101 labels selectively not an antagonist a-receptor but an a l-adrenoceptor.
194 Acknowledgements We thank :Dr. Hancock and his coworkers from Durham, North Carolina, for the computer program used here for data evaluation. Our work was supported by the Deutsche Forschungsgemeinschaft (SFB 70).
References Berthelsen, S. and W.A. Pettinger, 1977, A functional basis for classification of a-adrenergic receptors, Life Sci. 21,595. Butler, M. and D.H. Jenkinson, 1978, Blockade by WB 4101 of 0~-adrenoceptors in the rat vas deferens and guinea-pig taenia caeci, European J. Pharmacol. 52, 303. Greenberg, D.A. and S.H. Snyder, 1978, Pharmacological properties of (3H)dihydroergokryptine binding sites associated with alpha noradrenergic receptors in rat brain membranes, Mol. Pharmaeol. 14, 38. Hancock, A.A., A.L. DeLean and R.J. Lefkowitz, 1979, Quantitative resolution of beta-adrenergic receptor subtypes by selective ligand binding:
T. TANAKA, K. STARKE application of a computerized model fitting technique, Mol. Pharmacol. 16, 1. Langer, S.Z., 1974, Presynaptic regulation of catecholamine release, Biochem. Pharmacol. 23, 1793. Miach, P.J., J.P. Dausse and P. Meyer, 1978, Direct biochemical demonstration of two types of a-adrenoceptor in rat brain, Nature 274,492. Starke, K., 1977, Regulation of noradrenaline release by presynaptic receptor systems, Rev. Physiol. Biochem. Pharmacol. 77, 1. U'Prichard, D.C., W.D. Bechtel, B.M. Rout and S.H. Snyder, 1979, Multiple apparent alpha-noradrenergic receptor binding sites in rat brain: effect of 6-hydroxydopamine, Mol. Pharmacol. 16, 47. U'Prichard, D.C., M.E. Charness, D. Robertson and S.H. Snyder, 1978, Prazosin: differential affinities for two populations of a-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. Weitzell, R., T. Tanaka and K. Starke, 1979, Preand postsynaptic effects of yohimbine stereoisomers on noradrenergic transmission in the pulmonary artery of the rabbit, Naunym Schmiedeb. Arch. Pharmacol. 308, 127.
European Journal o f Pharmacology, 63 (1980) 19t-194
@)Elsevier/North-Holland Biomedical Press Short communication ANTAGONIST/AGONIST-PREFERRING a-ADRENOCEPTORS OR a,/a2-ADRENOCEPTORS? TOSHIYUKI TANAKA and KLAUS STARKE * Pharmakologisches Institut, Universitiit Freiburg, D-7800 Freiburg, FRG
Received 27 February 1980, accepted 3 March 1980
T. TANAKA and K. STARKE, Antagonist/agonist-preferring O~-adrenoceptors or o~l/o~2-adrenoceptors? , European J. Pharmacol. 63 (1980) 191--194. Yohimbine and some stereoisomeric alkaloids inhibited the binding of 3H-clonidine and 3H-WB-4101 to rat cerebral cortex membranes. Rauwolscine and yohimbine had much higher affinity to the 3H-clonidine than to the 3H-WB-4101 site, whereas the reverse was true for corynanthine. The results indicate that the 3H-clonidine site is an ~2-adrenoceptor and not an agonist-selective site whereas the 3H-WB-4101 site is an ~l-adrenoceptor and not an antagonist-selective site. (~l"Adrenoceptor
~2Adrenoceptor
Yohimbine
1. Introduction Studies on pre- and postsynaptic ~-adrenoceptors of sympathetic neuroeffector junctions have led to the view that ~-adrenoceptors could be divided into ~,- and ~2~subtypes (Langer, 1974; Berthelsen and Pettinger, 1977). For instance, clonidine preferentially activates, and yohimbine preferentially blocks, the ~2-receptor, whereas phenylephrine mainly activates, and 2-[(2',6'
* Address correspondence to: Dr. Klaus Starke, Pharmakologisches Institut, Hermann-Herder-Strasse 5, D-7800 Freiburg, Federal Republic of Germany.
Rauwolscine
Corynanthine
clonidine, the other one by 3H-WB-4101 (U'Prichard et al., 1977). a-Receptor agonists were much more p o t e n t in inhibiting 3Hclonidine than 3H-WB-4101 binding, whereas the antagonists were much more potent in inhibiting 3H-WB-4101 than 3H-clonidine binding. This was originally thought to be the distinguishing feature of the 3H-clonidine and the 3H-WB-4101 site which were therefore also called the agonist a-receptor site and the antagonist s-receptor site, respectively (U'Prichard et al., 1977; Greenberg and Snyder, 1978). More recently it has been proposed that the 3H-WB-4101 site may in fact be the a 1-receptor of the functional studies and the 3H~clonidine site the ~2-receptor, the preference of drugs for one site or the other being unrelated to their agonist or antagonist properties (U'Prichard et al., 1978; see also Miach et al., 1978). There is no published evidence that would decide between these two interpretations of the binding data. We have recently observed in functional experiments that yohimbine diastereomers were good tools for the dif-
192
ferentiation of a~- and a2-adrenoceptors; in particular, rauwolscine was even more selective than yohimbine for a2-receptors (Weitzell et al., 1979). We now report the effect of four stereoisomers on the binding of 3H-clonidine and 3H-WB-4101. Our results are direct evidence against the first and for the second interpretation.
2. Materials and methods Chemicals were obtained from the following sources: 3H-clonidine, specific activity 26.7 Ci/mmol, Boehringer, Ingelheim; 3H-WB-4101, specific activity 25.4 Ci/mmol, New England Nuclear, Dreieich; (--)-noradrenaline base, Hoechst, Frankfurt; rauwolscine hydrochloride, yohimbine hydrochloride and corynanthine, Roth, Karlsruhe; pseudoyohimbine, ICN Pharmaceuticals, Plainview, N.Y. Membranes from cerebral cortex of male rats (200-250 g) were prepared and binding assays performed as described by U'Prichard et al. (1977) with high concentration of (--)noradrenaline to determine nonspecific binding. Ascorbic acid 0.05% was included in incubation mixtures in order to prevent oxidation of noradrenaline. Since 3H-WB-4101 was adsorbed to test tubes, mixtures without membranes were also incubated and counted for radioactivity in order to obtain the total concentration in the incubation fluid. A substantial fraction of the total concentration of 3H-WB-4101 (up to 18%) was b o u n d to the brain membranes. In the case of 3H-clonidine, this fraction was less than 5%. Data were analyzed b y non-linear least-squares curve fitting essentially according to the method of Hancock et al. (1979) which corrects for the decrease in free ligand concentration due to binding to membranes.
3. Results In saturation experiments specific binding of 3H-clonidine (total concentration 0.0625-
T. T A N A K A , K. S T A R K E
32 nM) occurred to a single population of sites with a maximal number Bmax of 242 + 10 fmol/mg protein and a dissociation constant K D of 2.72 + 0.26 nM. Specific binding of 3H-WB-4101 (total concentration 0.0193.22 nM) also was to one population of sites with Bmax of 194 + 3 fmol/mg protein and K D of 0.29 + 0.02 nM (means + S.E.M. of 4 experiments). K D values were similar to those reported previously (U'Prichard et al., 1977). We did not observe two populations of 3H-clonidine binding sites as found recently by U'Prichard et al. (1979) in some b u t not all experiments. Fig. 1 shows that rauwolscine and corynanthine inhibited the binding of b o t h 3H-
100
"
~
"'~.
50
' ~
~,
o
rn I
0
,. I
I
I
I
1
I
10- 9
10-8
10 -7
10 -6
10- 5
10-/,
M
rauwotscine
or
corynanthine
Fig. l . I n h i b i t i o n o f aH-elonidine and 3H-WB-4101 b i n d i n g t o rat cerebral c o r t e x m e m b r a n e s by rauwolscine and c o r y n a n t h i n e . • • E f f e c t o f rauwolscine o n 3H-clonidine b i n d i n g ; o . . . . . . o e f f e c t o f r a u w o l s c i n e o n 3H-WB-4101 b i n d i n g ; • -• e f f e c t o f c o r y n a n t h i n e o n 3H-clonidine binding; []. . . . . . [] e f f e c t o f c o r y n a n t h i n e o n 3H-WB-4101 binding. Abscissa: c o n c e n t r a t i o n o f c o m p e t i t o r ; ordinate: 3H-ligand b o u n d , expressed as p e r c e n t o f total binding in t h e a b s e n c e o f c o m p e t i t o r . Data are f r o m o n e e x p e r i m e n t carried o u t in triplicate. T o t a l c o n c e n t r a t i o n s o f 3H-clonidine a n d 3H-WB-4101 were 0.5 and 0.38 nM, respectively. Curves r e p r e s e n t t h e best fit t o t h e e x p e r i m e n t a l data as calculated b y non-linear least-squares m e t h o d o f curve fitting.
RADIOLABELING OF a~- AND a2-ADRENOCEPTORS
193
TABLE 1 Inhibition of 3H-clonidine and 3H-WB-4101 specific binding by yohimbine stereoisomers. Total concentration of 3H-clonidine was 0.5 nM, total concentrations of 3H-WB-4101 ranged from 0.38 to 0.94 nM. In each experiment, 7--9 concentrations of the stereoisomers were tested, and dissociation constants K i were calculated from concentration-inhibition curves as shown in fig. 1. Values are means of 2 experiments each carried out in triplicate; individual values from the 2 experiments are in parentheses. Competitor
Rauwolscine Yohimbine Corynanthine Pseudoyohimbine
K i for 3H-clonidine site (nM)
K i for 3H-WB-4101 site (nM)
K i 3H-WB-4101 site
41.3 (39.1;43.3) 47.2 (46.3; 48.1) 6200 (5180; 7220) 1955 (1490; 2420)
2225 419 172 2515
53.9 8.9 0.028 1.3
clonidine and 3H-WB-4101. Rauwolscine competed much more strongly with 3H-clonidine than with 3H-WB-4101, whereas the reverse was true for corynanthine. Dissociation constants Ki of the four stereoisomers for the two binding sites are given in table 1. The c o m p o u n d with the highest affinity to the 3H~lonidine site was rauwolscine, closely followed b y yohimbine; pseudoyohimbine had much less, and corynanthine the least affinity. In constrast, corynanthine was the most p o t e n t competitor for 3H-WB-4101 binding, yohimbine and rauwolscine were less potent and pseudoyohimbine was also a weak competitor.
4. Discussion Yohimbine, corynanthine, rauwolscine and pseudoyohimbine are competitive a-adrenoceptor blocking drugs (WeitzeU et al., 1979). As shown b y the Ki ratios in table 1, corynanthine had a 36 times greater affinity to the 3H-WB-4101 than to the 3H-clonidine site. This would be consistent with the idea that antagonists generally bind preferentially to the 3H-WB-4101 site (and agonists to the 3H-clonidine site). Yet, the findings with yohimbine and rauwolscine argue against this view. Yohimbine had 9 times greater affinity to the 3H~clonidine than to the 3H-
(1970; 2480) (407; 431) (156; 188) {2500; 2530)
K i 3H-clonidine site
WB-4101 site. (A ratio of 3.2 was reported b y U'Prichard et al., 1977, and was explained b y the partial agonist character of yohimbine; however, to our knowledge there is no evidence for a partial agonist effect of yohimbine at a-adrenoceptors.) Moreover, rauwolscine had no less than a 54 times greater affinity to the 3H~lonidine site than to the 3H-WB-4101 site. The high selectivity of the antagonist rauwolscine for the 3H-clonidine site rules out the possibility that this site generally prefers agonists. In a study of functional effects of the four isomers on sympathetic neuroeffector transmission in the rabbit pulmonary artery, yohimbine and rauwolscine even more so, displayed a high selectivity for a2~adrenoceptors, whereas corynanthine was selective for a ~-adrenoceptors; pseudoyohimbine had little effect on either adrenoceptor (Weitzell et al., 1979). The affinities o f these antagonists to the 3H~lonidine site as reported here mirror their functional a2-receptor blocking potencies in the pulmonary artery, while the affinities to the 3H-WB-4101 site mirror their functional al-receptor blocking potencies. This close agreement indicates that in brain membranes 3H~lonidine labels selectively not an agonist a-receptor but an a2-adrenoceptor, whereas 3H-4101 labels selectively not an antagonist a-receptor but an a l-adrenoceptor.
194 Acknowledgements We thank :Dr. Hancock and his coworkers from Durham, North Carolina, for the computer program used here for data evaluation. Our work was supported by the Deutsche Forschungsgemeinschaft (SFB 70).
References Berthelsen, S. and W.A. Pettinger, 1977, A functional basis for classification of a-adrenergic receptors, Life Sci. 21,595. Butler, M. and D.H. Jenkinson, 1978, Blockade by WB 4101 of 0~-adrenoceptors in the rat vas deferens and guinea-pig taenia caeci, European J. Pharmacol. 52, 303. Greenberg, D.A. and S.H. Snyder, 1978, Pharmacological properties of (3H)dihydroergokryptine binding sites associated with alpha noradrenergic receptors in rat brain membranes, Mol. Pharmaeol. 14, 38. Hancock, A.A., A.L. DeLean and R.J. Lefkowitz, 1979, Quantitative resolution of beta-adrenergic receptor subtypes by selective ligand binding:
T. TANAKA, K. STARKE application of a computerized model fitting technique, Mol. Pharmacol. 16, 1. Langer, S.Z., 1974, Presynaptic regulation of catecholamine release, Biochem. Pharmacol. 23, 1793. Miach, P.J., J.P. Dausse and P. Meyer, 1978, Direct biochemical demonstration of two types of a-adrenoceptor in rat brain, Nature 274,492. Starke, K., 1977, Regulation of noradrenaline release by presynaptic receptor systems, Rev. Physiol. Biochem. Pharmacol. 77, 1. U'Prichard, D.C., W.D. Bechtel, B.M. Rout and S.H. Snyder, 1979, Multiple apparent alpha-noradrenergic receptor binding sites in rat brain: effect of 6-hydroxydopamine, Mol. Pharmacol. 16, 47. U'Prichard, D.C., M.E. Charness, D. Robertson and S.H. Snyder, 1978, Prazosin: differential affinities for two populations of a-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. Weitzell, R., T. Tanaka and K. Starke, 1979, Preand postsynaptic effects of yohimbine stereoisomers on noradrenergic transmission in the pulmonary artery of the rabbit, Naunym Schmiedeb. Arch. Pharmacol. 308, 127.