Naunyn-Schmiedeberg's

Naunyn-Schmiedeberg'sArch. Pharmacol.307, 223-226 (1979)

Archivesof Pharmacology 9 by Springer-Verlag1979

Specific Labelling of Postsynaptic ~1 Adrenoceptors in Rat Heart Ventricle by 3H-WB 4101 Rita Raisman, M. Briley, and S. Z. Langer Biology Department Synth61abo (L.E.R.S.), 58, Rue de la Glaci6re, F-75013 Paris, France

Summary. The c~-adrenoceptor ligand, 3H-WB 4101 binds to a single population of independent sites in the rat heart ventricle. These sites are not affected by chemical sympathectomy with 6-hydroxydopamine and thus appear to be located postsynaptically, The relative order of potencies for displacement of 3H-WB 4101 binding: prazosin > phentolamine > yohimbine, is characteristic of that of the cq type of adrenoceptors. In heart ventricle 3H WB-4101 thus seems to label specifically postsynaptically located cq-adrenoceptors. The significance of the specificity of this ligand is discussed with relevance to its use in the central nervous system. Key words: ~-Adrenoceptors - Receptor binding 3H-WB 4101 - Pre- and postsynaptic receptors - 6Hydroxydopamine.

Introduction Pharmacological differences exist between the presynaptic ~-adrenoceptor that regulates noradrenaline release and the postsynaptic ~-adrenoceptor which mediates the response of the effector organ in the peripheral nervous system (Dubocovich and Langer, 1974; Cubeddu et al., 1974; Starke et al., 1974, 1975). Based on the differences in affinities for agonists as well as for antagonists the subclassification of c~adrenoceptors into ~, and % types was proposed (Langer, 1974). The ergot alkaloid 3H-dihydroergocryptine (DHE) has been used to label c~-adrenoceptors in the central nervous system and in a variety of peripheral tissues (Williams and Lefkowitz, 1978a) including the heart (Williams and Lefkowitz, 1978b). Using the binding of Send offprint requests to S. Z. Langer at above address

3H-DHE we have demonstrated that a large proportion of the ~-adrenoceptor population in the rat heart ventricle was destroyed after chemical sympathectomy with 6-hydroxydopamine (Briley et al., 1979; Story et al., 1979). This first direct demonstration that ~adrenoceptors are located on noradrenergic nerve terminals showed that 3H-DHE binds to both pre- and postsynaptic receptors. Another c~-adrenoceptorligand 3H-WB 410 l (2-[(2',6'dimethoxy)phenoxyethylamino] methylbenzodioxan), which, to date, has only been used in the central nervous system (for example, Greenberg et al., 1976), has been shown pharmacologically to have a great selectivity for postsynaptic or ~l-adrenoceptors. In the rat vas deferens its postsynaptic alpha-receptor blocking activity has been reported to be between several hundred (Butler and Jenkinson, 1978), and several thousand-fold (Kapur and Mottram, 1978) greater than its presynaptic blocking activity. In the central nervous system 3H-WB 4101 has been used to label the so-called "antagonist" site of ~-adrenergic receptors in contrast to 3H-clonidine which was considered to label the so-called "agonist" site (Greenberg et al., 1976). In this paper we demonstrate that in rat heart ventricle aH-WB 4101 labels exclusively postsynaptic %-adrenoceptors and we suggest that the "agonist/antagonist site" interpretation of the differences between 3H-clonidine and 3H-WB 4101 (Greenberg et al., 1976) may be equally interpreted by the ~q/% receptor selectivity of the two ligands.

Methods Male rats (200-250g) were killed by decapitation, the hearts immediately removed and the ventricles dissected. Membrane fragments were prepared from homogenates by differential centrifugation in 5 mM Tris/HC1, 0.1 mM MgC12 and 0.25 M sucrose, as described by Alexander et al. (1975).

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Fig. 1. Typical Scatchard plot of 3H-WB 4101 binding to membranes from rat heart ventricle. Membranes were prepared from rat heart ventricle by differential centrifugation. The binding of 3H-WB 4101 was measured at 6 concentrations from 0.1 - 20 nM. Specific binding was defined as that displaced by 10gM phentolamine. The line is fitted by linear regression (correlation coefficient, r = 0.893)

The binding was measured by incubating aliquots of 180 pi of the membrane suspension (containing 120 mg original wet tissue weight) in 50mM Tris/HCl, 1 mM MgCI z with 3H-WB 4101 (The Radiochemical Centre, Amersham, U.K., specific activity 20.0 Ci/mmole) and other drugs where appropriate in a total volume of 250 gl for 15 min at 30~ C with continuous shaking (conditions which have been shown to result in an equilibrium in the brain (Davis et al., 1978) and which we have confirmed is the same in the heart). Following incubation, two 100~Jl aliquots were diluted with 5 ml ice-cold buffer (50raM Tris/HCl, i mM MgC12) and then rapidly filtered through Whatman GF/C glassfibre filters. The filters were washed with two 5 ml aliquots of ice-cold buffer and dried at 110 ~ C. The radioactivity was counted in toluene, PPO (4 g/l), POPOP (0.6 g/l). Total binding was in the range of 150-2,000cpm/filter. Specific binding was defined as that displaced by 10 JaM phentolamine and represented 55 ~o of the total binding at 5 nM 3H-WB 4101. Chemical sympathectomy was performed by giving two injections of 6-hydroxydopamine (6-OHDA) (50mg/kg i.v.) at 24h intervals 2 weeks prior to sacrifice. This treatment resulted, within 24 h of the second injection, in a reduction of 70 - 80 % in the ability of atria to accumulate 3H-noradrenaline, indicating degeneration of the cardiac noradrenergic nerve terminals. Drugs were obtained from the following sources: 6-hydroxydopamine, Sigma; prazosin, Pfizer; phentolamine, Ciba-Geigy; yohimbine, Sigma.

Results The specific binding of 3H-WB 4101 to ventricle membranes showed a single component by Scatchard analysis (Fig. 1) with an apparent dissociation constant,

(M)

Fig.2. Displacement of 3H-WB 410i binding in rat heart ventricle by c~-adrenoceptor antagonists. The binding was measured as described in Fig. 1 at 6nM 3H-WB 4101 in the presence of various concentrations of prazosin (O), phentolamine (11) and yohimbine (A). The displacement of 3H-WB 4101 by the drugs is expressed as the percentage reduction in specific binding. Ordinate: percent inhibition of 3H-WB 4101 binding. Abscissa: concentration of c~-adrenoceptor antagonists. Each point is the mean of three independent determinations

Table 1. Characteristics of 3H-WB 4101 binding to rat heart ventricle membranes after 6-hydroxydopamine treatment. Membranes were prepared from heart ventricles taken from control animals and those treated with 6-OHDA. Specific binding was measured at 6 to 8 concentrations of 3H-WB 4101 between 0.1 and 20.0 nM. The affinity constants (Kd) and maximal binding (Bmax) were calculated by Scatchard analysis. The values given are the mean + S.E.M. ; Nis the number of separate experiments, each determined from a Scatchard plot using 6 concentrations in duplicate

Control 6-OHDA

N

Kd (aM)

Bm~~ (fmoles/g original wet tissue weight)

4 5

0.94 +_0.30 1.29 _+0.90

90.1 _+ 18.0 70.3 4- 16.5

Kd, of 0.94 _ 0.30 nM (n = 4) and maximal binding, Bmax, of 90.1 _+ 18.0 fmoles/g original wet tissue weight, indicating a single population of binding sites. Inhibition of aH-WB 4101 binding by the eadrenoceptor antagonists, prazosin, phentolamine and yohimbine, showed a pattern expected for the ~1 type of adrenoceptors namely, prazosin being more potent than phentolamine which in turn is more active than yohimbine (Fig.2). The ICso values (concentration required to inhibit 50 % of the specific binding) were : prazosin, 10 nM; phentolamine, 70nM; yohimbine, 1,000 nM. Destruction of the noradrenergic nerve endings by chemical sympathectomy with 6-OHDA did not change significantly either the Kd o r Brna,, (Table 1) indicating that the 3H-WB 4101 binding sites are not located on noradrenergic nerve terminals.

R. Raisman et al. : 3H-WB 4101 Labels Postsynaptic ~l-Adrenoceptors

Discussion

3H-DHE has been shown to label both pre- and postsynaptic ~-adrenoceptors in rat heart ventricle (Briley et al., 1979; Story et al., 1979). The presynaptic component, which represented about 60 % of the total aadrenoceptor population, was destroyed after degeneration of noradrenergic nerve endings by pretreatment with 6-OHDA. Pharmacologically the a-adrenoceptor antagonist, WB 4101, is highly selective for the a t or postsynaptic adrenergic receptors in the peripheral nervous system (Butler and Jenkinson, 1978; Kapur and Mottram, 1978). We thus set out to determine if 3H-WB 4101 could be used as a specific a t or postsynaptic ligand. As already demonstrated for the central nervous system (Greenberg et al., 1976) the specific binding of 3H-WB 4101 is to a single population of noninteracting sites. Inhibition of the binding of this 3Hligand by three a-adrenoceptor antagonists showed that their inhibition was directly related to their affinities for the postsynaptic ~-adrenoceptors (Langer, 1979). Furthermore, following treatment with 6OHDA which has been demonstrated to destroy the noradrenergic nerve terminals of the rat heart ventricle with a loss of 60 % of the 3H-DHE binding sites, the specific binding of 3H-WB 4101 remained unchanged. Thus in the peripheral nervous system 3H-WB 4101 would appear to bind specifically to postsynaptically located al-adrenoceptors. The results of the various studies using this ligand in the central nervous system (Davis et al., 1978 ; Skolnick et al., 1978; U'Prichard et al., 1978; Rosenblatt et al., 1979) should thus be interpreted with consideration for its al-adrenoceptor specificity. In particular the conclusions concerning agonist- and antagonist-preferring sites based on the comparison between the binding of 3H-clonidine, an a 2 selective agonist, and the binding of 3H-WB 4101 (Greenberg et al., 1976, 1978; U'Prichard et al., 1977; Peroutka et al., 1978) should be revised, as U'Prichard and Snyder (1979) have in fact themselves recently suggested, in favour of the more established subdivision between al and a 2 adrenoceptor sites. This subdivision in the central nervous system may not necessarily imply the pre- and postsynaptic locations found in the peripheral nervous system. In support of this view we have recently found a 3.5 fold greater maximal binding when using 3H-DHE than when using 3H-WB 4101 to label c~-adrenoceptors in the rat cerebral cortex (Briley, M., and Langer, S. Z., unpublished observations). Similar results have been recently published by U'Prichard and Snyder (1979). It was recently pointed out that the subclassification into a 1 and a z adrenoceptors is mainly based on pharmacological differences in their relative affinities for agonists and

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antagonists and implies neither location nor function of the a-adrenoceptor (Starke and Langer, 1979).

References Alexander, R. W., Williams, L. T., Lefkowitz, R. J. : Identification of cardiac fl-adrenergic receptors by (-) 3H-alprenolol binding. Proc. Natl. Acad. Sci. USA 72, 1564-1568 (1975) Briley, M. S., Langer, S. Z., Story, D. E.: Binding studies on eadrenoceptors and muscarinic cholinoceptors in rat heart ventricle: effect of chemical sympathectomy. Br. J. Pharmacol. 66, 90P (1979) Butler, M., Jenkinson, D. J.: Blockade by WB 4101 of c~adrenoceptors in the rat vas deferens and guinea-pig taenia caeci. Eur. J. Pharmacol. 52, 303-311 (1978) Cubeddu, L. X., Barnes, E. M., Langer, S. Z., Weiner, N. : Release of norepinephrine and dopamine-fl-hydroxylase by nerve stimulation. I. role of neuronal and extranem'onal uptake and of alpha presynaptic receptors. J. Pharmacol. Exp, Ther. 190, 431-450 (1974) Davis, J. N., Arnett, C. D., Hoyler, E., Stalvey, L. P., Daly, J. W., Skolnick, P. : Brain ~-adrenergic receptors: comparison of 3HWB 4101 binding with norepinephrine-stimulated cyclic AMP accumulation in rat cerebral cortex. Brain Res. 159, 125-135 (1978) Dubocovich, M. L., Langer, S. Z. : Negative feed-back regulation of noradrenaline release by nerve stimulation in the perfused cat's spleen: differences in potency of phenoxybenzamine in blocking the pre- and post-synaptic adrenergic receptors. J. Physiol. (Loud.) 237, 505-519 (1974) Greenberg, D. A., U'Prichard, D. C., Snyder, S. H. : ~-Noradrenergic receptor binding in mammalian brain: Differential labelling of agonist and antagonist states. Life Sci, 19, 6 9 - 7 6 (1976) Greenberg, D. A., U'Prichard, D. C., Sheehan, P., Snyder, S. H. : ~Noradrenergic receptors in the brain: differential effects of sodium on binding of 3H-agonists and 3H-antagonists. Brain Res. 140, 378-384 (1978) Kaput, H., Mottram, D. R. : A comparative study on the pre- and post-synaptic alpha blocking activity of a series of benzodioxanes. Biochem. Pharmacol. 27, 1879-1880 (1978) Langer, S. Z.: Presynaptic Regulation of Catecholamine Release. Biochem. Pharmacol. 23, 1793-1800 (1974) Langer, S. Z. : Presynaptic Adrenoceptors and regulation of release. In: The Release of Catecholamines from Adrenergic Neurons, (D. M. Paton, ed.), pp. 5 9 - 85. New York: Pergamon Press 1979 Peroutka, S. J., Greenberg, D. A., U'Prichard, D. C., Snyder, S. H. : Regional variations in alpha-adrenergic receptor interactions of 3H-dihydroergokryptine in calf brain: hnplications for a twosite model of alpha-receptor function. Mol. Pharmacol. 14, 403-412 (1978) Rosenblatt, J. E., Pert, C. B., Tallman, J. F., Pert, A., Bunney, W. E. : The effect of imipramine and lithium on c~- and fi-receptor binding in rat brain. Brain Res. 160, 186-191 (1979) Skolnick, P., Stalvey, L. P., Daly, J. W., Hoyler, E., Davis, J. N.: Binding of c~- and fl-adrenergic ligands to cerebral cortical membranes: Effect of 6-hydroxydopamine treatment and relationship to the responsiveness of cyclic AMP-generating systems in two rat strains. Eur. J. Pharmacol. 47, 201-210 (1978) Starke, K., Langer, S. Z.: A note on terminology for presynaptic receptors. In: Presynaptic Receptors, pp. 1 - 4 , (S. Z. Langer, K. Starke, and M. L. Dubocovich, eds.). Oxford: Pergamon Press /979 Starke, K., Montel, H., Gayk, W., Merker, R. : Comparison of the effects of clonidine on pre- and postsynaptic adrenoceptors in

226 rabbit pulmonary artery. Naunyn-Schmiedeberg's Arch. Pharmacol. 285, 133-150 (1974) Starke, K., Borowski, E., Endo, T. : Preferential blockade of presynaptic c~-adrenoceptors by yohimbine. Eur. J. Pharmacol. 34, 385-388 (1975) Story, D. F., Briley, M. S., Langer, S. Z. : Chemical sympathectomy with 6-hydroxydopamine decreases e-adrenoceptor binding of 3H-dihydroergocryptine to rat heart ventricular membranes but muscarinic cholinoceptor binding of 3H-quinuclidinyl benzilate remains unaltered. Eur. J. Pharmacol. 1979, in press U'Prichard, D. C., Snyder, S. H. : Distinct e-noradrenergic receptors differentiated by binding and physiological relationships. Life Sci. 24, 79-88 0979) U'Prichard, D. C., Greenberg, D. A., Snyder, S. H.: Binding characteristics ofa radiolabeled agonist and antagonist at central nervous system alpha-noradrenergic receptors. Mol. Pharmacol. 13, 454-473 (1977)

Naunyn-Schmiedeberg's Arch. Pharmacol. 307 (1979) U'Prichard, D. C., Greenberg, D. A., Sheehan, P. P., Snyder, S. H. : Tricyclic antidepressants: Therapeutic properties and affinity for ~-noradrenergic receptor binding sites in the brain. Science 199, 197-198 (1978) Williams, L. T., Lefkowitz, R. J. : Identification and study of c~adrenergic receptors by radioligand binding techniques. In: Receptor Binding Studies in Adrenergic Pharmacology, pp. 53 82 (L. T. Williams and R. J. Lefkowitz, eds.). New York: Raven Press 1978a Williams, R. S., Lefkowitz, R. J. : Alpha-adrenergic receptors in rat myocardium. Identification by binding of 3H-dihydroergocryptine. Circ. Res. 43, 721 727 (1978b)

Received March 5/Accepted April 30, 1979

Specific labelling of postsynaptic alpha 1 adrenoceptors in rat heart ventricle by 3H-WB 4101.

Naunyn-Schmiedeberg's Naunyn-Schmiedeberg'sArch. Pharmacol.307, 223-226 (1979) Archivesof Pharmacology 9 by Springer-Verlag1979 Specific Labelling...
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