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Stimulation of 3H-apomorphine binding by dopamine and bromocriptine
European Journal of Pharmacology, 61 (1980) 209--211 © Elsevier/North-Holland Biomedical Press
209
Rapid communication STIMULATION OF 3H-APOMORPHINE BINDING BY DOPAMINE AND BROMOCRIPTINE H.A. ROBERTSON
Department of Pharmacology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 Received 10 December 1979, accepted 11 December 1979
Apomorphine is classically recognized as a dopamine agonist but when tested on adenylate cyclase activity, it is often only a partial agonist both in cell-free homogenates and in preparations of intact cells. In other systems (see review by Kebabian and Calne, 1979) apomorphine mimicks dopamine effects. The diverse pharmacological effects of apomorphine suggest that more than one type of receptor exists (Kebabian and Calne, 1979). 3H-Apomorphine has been used at low concentrations as a ligand to label what are presumably presynaptic dopamine receptors (Nagy et al., 1978). While repeating these studies, it was observed that when dopamine itself is used to displace aH-apomorphine bound to membranes isolated from rat striatal and limbic tissues, high concentrations (300/~M) of dopamine, rather than displacing aH-apomorphine as predicted, produced a highly significant and reproducibie increase in 3Hapomorphine binding. Fresh rat brain tissue (striatum and nucleus accumbens) was homogenized in a glass homogenizer with teflon pestle in 20 vol of ice-cold 15 mM Tris--HC1, pH 7.4, containing 5 mM Na2 EDTA, 1.1 mM citric acid and 12.5 #M pargyline. The homogenate was then centrifuged at 40 000 × g for 15 min, and resuspended in the original volume of buffer. This suspension was heated at 37°C for 1 h and frozen overnight (--20 ° C). The preparation was thawed for the binding assays, and centrifuged at 40 000 × g for 15 min at 4 ° C. The pellet was resuspended in buffer using a polytron homogenizer. Ali-
quots (100/11) containing 300 pg protein were incubated at 22°C for 30 min with 900/~1 of 50 mM Tris--HCl, pH 7.8, containing 3 nM 3H-apomorphine (New England Nuclear, spec. act. 38.6 Ci/mmol) and varying concentrations of dopamine (HC1 salt, Sigma Chemical Co.) or bromocriptine (Sandoz). The reaction was terminated by diluting with 2 ml of 50 mM Tris--HC1, pH 7.8, and rapidly filtering on Whatman GF/B glass fibre filters. The filters were washed with 2 × 5 ml of 50 mM Tris--HC1, pH 7.8, and bound radioactivity determined by liquid scintillation counting in 10 ml of biofluor (New England Nuclear). In all the preparations examined, addition of 300 #M dopamine produced more than 1000% increase in aH-apomorphine binding over control. For rat nucleus accumbens, control aH-apomorphine binding was 115 + 4 fmol/mg protein while addition of 300/aM dopamine produced a 1194% increase in binding to 1373 + 29 fmol/mg protein. For rat striatum, the control value was 128 + 4 fmol/ mg protein. Addition of 300 pM dopamine produced a 1066% increase to 1364+ 36 fmol/mg protein. All values are the mean + S.E.M. of 16 determinations. To characterize further this increase in 3H-apomorphine binding, the effect of varying concentrations of dopamine and bromocriptine on 3H-apomorphine binding by rat striatum-nucleus accumbens membranes was examined. Table 1 illustrates a typical experiment. Both dopamine and bromocriptine displaced aH-apomorphine binding in low cencentrations. However, at higher conce~ ¢~°
210 TABLE 1 Effect of dopamine and bromocriptine on the binding of 3H-apomorphine to rat striatal/nucleus accumbens homogenates. 3H-Apomorphine concentration was 3 nM. Values are the mean -+ S.E.M. o f 3 determinations. Concentration (uM)
0 0.5 1 10 50 100 250 1000
3H-apomorphine bound (fmol/mg protein) Dopamine
Bromocriptine
438-+ 47 341-+ 15 264-+ 37 3 2 8 + 15 1873 -+ 101 2 6 7 2 + 20 2643-+ 24 2241 + 16
490+55 385 + 25 376+50 378-+24 544 + 17 854+24 1109-+51 1970 + 61
tions, the effect was a pronounced stimulation of binding. The increase in binding induced by dopamine is sigmoidal and saturable at about 100 pM dopamine. The ECs0 (concentration required to produce a 50% stimulation in binding) is about 50#M. Bromocriptine produced a similar increase in 3H-apomorphine binding (table 1). Other dopamine agonists such as lergotrile and dihydroxyergotamine also stimulate 3H-apomorphine binding. The dopamine-induced 3H-apomorphine binding is not blocked by #molar concentrations of neuroleptics such as haloperidol, (+)-butaclamol, chlorpromazine or pimozide but is competitively reversed by the dopamine agonist epinine (ICs0 about 3 pM). Significantly, addition of 1 #M ascorbate completely abolishes dopamine-stimulated 3Hapomorphine binding. Ascorbate (10 pM) also completely abolishes dopamine stimulation of adenylate cyclase in rat striatal homogenates (Thomas and Zemp, 1977) and endogenous ascorbate may explain the observation that dopamine does not stimulate adenylate cyclase in striatal slices (Palmer et al., 1973). The evidence suggests that apomorphine may bind to two distinct sites on neuronal membranes. In the absence of added dopamine, 3H-apomorphine labels a population of receptors which may be presynaptic (Nagy et
al., 1978). Up to about 30 gM, dopamine displaces 3H-apomorphine from this site. However, above this concentration, dopamine and several other Dl-dopamine agonists stimulate 3H-apomorphine binding to another or to an altered (reduced?) site. This site appears to be an agonist site as dopamine blocking agents such as the neuroleptics do not affect binding to it nor does dopamine stimulate the binding of 3H-spiroperidol to these membrane fragments. The precise nature of this site is unknown but may be of importance in developing an understanding of the actions of such agents as bromocriptine and lergotrile.
Acknowledgements I thank Drs. M. Wilkinson, G.M. McKenzie, T.D. White and P.E. Dresel for stimulating discussion and Marc R. Peterson for excellent technical assistance. This work was supported by the Medical Research Council of Canada.
References Kebabian, J.W. and D.B. Calne, 1979, Multiple receptors for dopamine, Nature 277, 93. Nagy, J.I., T. Lee, P. Seeman and H.C. Fibiger, 1978, Direct evidence for presynaptic and postsynaptic dopamine receptors in brain, Nature 274, 278.
211 Palmer, G.C., F. Sulser and G.A. Robison, 1973, Effects of neurohumoral and adrenergic agents on cyclic AMP levels in various areas of the rat brain in vitro, Neuropharmacology 12,327.
Thomas, T.N. and J.W. Zemp, 1977, Inhibition of dopamine sensitive adenylate cyclase from rat brain striatal homogenates by ascorbic acid, J. Neurochem. 28,663.
209
Rapid communication STIMULATION OF 3H-APOMORPHINE BINDING BY DOPAMINE AND BROMOCRIPTINE H.A. ROBERTSON
Department of Pharmacology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 Received 10 December 1979, accepted 11 December 1979
Apomorphine is classically recognized as a dopamine agonist but when tested on adenylate cyclase activity, it is often only a partial agonist both in cell-free homogenates and in preparations of intact cells. In other systems (see review by Kebabian and Calne, 1979) apomorphine mimicks dopamine effects. The diverse pharmacological effects of apomorphine suggest that more than one type of receptor exists (Kebabian and Calne, 1979). 3H-Apomorphine has been used at low concentrations as a ligand to label what are presumably presynaptic dopamine receptors (Nagy et al., 1978). While repeating these studies, it was observed that when dopamine itself is used to displace aH-apomorphine bound to membranes isolated from rat striatal and limbic tissues, high concentrations (300/~M) of dopamine, rather than displacing aH-apomorphine as predicted, produced a highly significant and reproducibie increase in 3Hapomorphine binding. Fresh rat brain tissue (striatum and nucleus accumbens) was homogenized in a glass homogenizer with teflon pestle in 20 vol of ice-cold 15 mM Tris--HC1, pH 7.4, containing 5 mM Na2 EDTA, 1.1 mM citric acid and 12.5 #M pargyline. The homogenate was then centrifuged at 40 000 × g for 15 min, and resuspended in the original volume of buffer. This suspension was heated at 37°C for 1 h and frozen overnight (--20 ° C). The preparation was thawed for the binding assays, and centrifuged at 40 000 × g for 15 min at 4 ° C. The pellet was resuspended in buffer using a polytron homogenizer. Ali-
quots (100/11) containing 300 pg protein were incubated at 22°C for 30 min with 900/~1 of 50 mM Tris--HCl, pH 7.8, containing 3 nM 3H-apomorphine (New England Nuclear, spec. act. 38.6 Ci/mmol) and varying concentrations of dopamine (HC1 salt, Sigma Chemical Co.) or bromocriptine (Sandoz). The reaction was terminated by diluting with 2 ml of 50 mM Tris--HC1, pH 7.8, and rapidly filtering on Whatman GF/B glass fibre filters. The filters were washed with 2 × 5 ml of 50 mM Tris--HC1, pH 7.8, and bound radioactivity determined by liquid scintillation counting in 10 ml of biofluor (New England Nuclear). In all the preparations examined, addition of 300 #M dopamine produced more than 1000% increase in aH-apomorphine binding over control. For rat nucleus accumbens, control aH-apomorphine binding was 115 + 4 fmol/mg protein while addition of 300/aM dopamine produced a 1194% increase in binding to 1373 + 29 fmol/mg protein. For rat striatum, the control value was 128 + 4 fmol/ mg protein. Addition of 300 pM dopamine produced a 1066% increase to 1364+ 36 fmol/mg protein. All values are the mean + S.E.M. of 16 determinations. To characterize further this increase in 3H-apomorphine binding, the effect of varying concentrations of dopamine and bromocriptine on 3H-apomorphine binding by rat striatum-nucleus accumbens membranes was examined. Table 1 illustrates a typical experiment. Both dopamine and bromocriptine displaced aH-apomorphine binding in low cencentrations. However, at higher conce~ ¢~°
210 TABLE 1 Effect of dopamine and bromocriptine on the binding of 3H-apomorphine to rat striatal/nucleus accumbens homogenates. 3H-Apomorphine concentration was 3 nM. Values are the mean -+ S.E.M. o f 3 determinations. Concentration (uM)
0 0.5 1 10 50 100 250 1000
3H-apomorphine bound (fmol/mg protein) Dopamine
Bromocriptine
438-+ 47 341-+ 15 264-+ 37 3 2 8 + 15 1873 -+ 101 2 6 7 2 + 20 2643-+ 24 2241 + 16
490+55 385 + 25 376+50 378-+24 544 + 17 854+24 1109-+51 1970 + 61
tions, the effect was a pronounced stimulation of binding. The increase in binding induced by dopamine is sigmoidal and saturable at about 100 pM dopamine. The ECs0 (concentration required to produce a 50% stimulation in binding) is about 50#M. Bromocriptine produced a similar increase in 3H-apomorphine binding (table 1). Other dopamine agonists such as lergotrile and dihydroxyergotamine also stimulate 3H-apomorphine binding. The dopamine-induced 3H-apomorphine binding is not blocked by #molar concentrations of neuroleptics such as haloperidol, (+)-butaclamol, chlorpromazine or pimozide but is competitively reversed by the dopamine agonist epinine (ICs0 about 3 pM). Significantly, addition of 1 #M ascorbate completely abolishes dopamine-stimulated 3Hapomorphine binding. Ascorbate (10 pM) also completely abolishes dopamine stimulation of adenylate cyclase in rat striatal homogenates (Thomas and Zemp, 1977) and endogenous ascorbate may explain the observation that dopamine does not stimulate adenylate cyclase in striatal slices (Palmer et al., 1973). The evidence suggests that apomorphine may bind to two distinct sites on neuronal membranes. In the absence of added dopamine, 3H-apomorphine labels a population of receptors which may be presynaptic (Nagy et
al., 1978). Up to about 30 gM, dopamine displaces 3H-apomorphine from this site. However, above this concentration, dopamine and several other Dl-dopamine agonists stimulate 3H-apomorphine binding to another or to an altered (reduced?) site. This site appears to be an agonist site as dopamine blocking agents such as the neuroleptics do not affect binding to it nor does dopamine stimulate the binding of 3H-spiroperidol to these membrane fragments. The precise nature of this site is unknown but may be of importance in developing an understanding of the actions of such agents as bromocriptine and lergotrile.
Acknowledgements I thank Drs. M. Wilkinson, G.M. McKenzie, T.D. White and P.E. Dresel for stimulating discussion and Marc R. Peterson for excellent technical assistance. This work was supported by the Medical Research Council of Canada.
References Kebabian, J.W. and D.B. Calne, 1979, Multiple receptors for dopamine, Nature 277, 93. Nagy, J.I., T. Lee, P. Seeman and H.C. Fibiger, 1978, Direct evidence for presynaptic and postsynaptic dopamine receptors in brain, Nature 274, 278.
211 Palmer, G.C., F. Sulser and G.A. Robison, 1973, Effects of neurohumoral and adrenergic agents on cyclic AMP levels in various areas of the rat brain in vitro, Neuropharmacology 12,327.
Thomas, T.N. and J.W. Zemp, 1977, Inhibition of dopamine sensitive adenylate cyclase from rat brain striatal homogenates by ascorbic acid, J. Neurochem. 28,663.