European Journal of Pharmacology, 55 (1979) 341--343
341
© Elsevier]North-Holland Biomedical Press
Rapid communication GUANINE NUCLEOTIDES REGULATE ANTERIOR PITUITARY DOPAMINE RECEPTORS DAVID R. SIBLEY and IAN CREESE *
Department of Neurosciences, MOO8, University of California, San Diego, La Jolla, California 92093, U.S.A. Received 21 March 1979, accepted 22 March 1979
3H-Spiroperidol labels dopamine receptors in the anterior pituitary which exhibit micromolar affinities for classic dopamine agonists (Creese et al., 1977). These dopamine receptors are distinguished from the dopamine receptors responsible for control of prolactin release which exhibit nanomolar affinity for dopamine agonists (Caron et al., 1978). A dopamine-sensitive adenylate cyclase has recently been described in the pituitary which exhibits micromolar affinity for dopamine (Ahn et al., 1979). Guanine nucleotides, particularly GTP, selectively regulate agonist but not antagonist sensitivity of a number of hormone and neurotransmitter receptors linked to adenylate cyclase. We now report that guanine nucleotides regulate dopamine agonist affinity for 3H-spiroperidol binding sites in anterior pituitary. Homogenates of frozen bovine anterior pituitary (40 vols w/v), prepared in 50 mM Tris-HC1 buffer (pH 7.7 at 25°C) with a Tekmar homogenizer, were centrifuged (5 rain, 500 ×g) to remove connective tissue. The supernatant was centrifuged twice (10 min, 50,000 × g) at 4°C with resuspension in fresh buffer. Final resuspension (100 vois w/v) was in Tris buffer containing 0.1% ascorbic acid, 120 mM NaC1, 5 mM KC1, 1 mM CaC12 and 1 mM MgC12, pH 7.1 at 37°C. After incubation (15 min, 37°C) with 3H-spiroperidol (26 Ci/mmol., New England Nuclear), unlabeUed drugs and nucleotides, samples were filtered
* To whom correspondence should be addressed.
rapidly under vacuum over Whatman GF/B filters, and rinsed rapidly with 3 × 5 ml ice cold Tris-HC1 buffer. Radioactivity trapped on the filters was counted by liquid scintillation spectroscopy. Nonspecific binding of 3Hspiroperidol was determined in the presence of 1/~M (+)-butaclamol. In typical experiments with 0.7 nM ~Hspiroperidol total binding was 2506 + 75 cpm (n = 20) and nonspecific binding was 491 +_ 26 cpm (n = 20). At 0.1 mM, the maximal concentration examined, the nucleotides GTP, GDP, GMP, ATP, ADP and AMP had no effect on total or nonspecific 3H-spiroperidol binding. However, the ability of agonists to displace 3H-spiroperidol binding was markedly influenced by some guanine nucleotides. GTP and GDP (0.1 raM) reduced the apparent affinity of dopamine, apomorphine and ADTN by approximately 6--11 fold for 3H-spiroperidol binding sites (table la). GTP was maximally effective at 0.1 mM with an ECs0 of 10/~M. Other nucleotides were ineffective in reducing the potency of dopamine in displacing 3H-spiroperidol binding (table lb). GTP did not influence the ability of the antagonists chlorpromazine or haloperidol to displace 3H-spiroperidol binding. Bromocriptine was also uninfluenced by GTP. Striatal dopamine receptors have been characterized by 3H-spiroperidol binding and a dopamine-sensitive adenylate cyclase. The different pharmacological specificities of these two markers suggests that each may label distinct dopamine receptors. Kainic acid
342 TABLE 1 Guanine nucleotide regulation of pituitary dopamine receptors. ICs0'S were d e t e r m i n e d b y l o g - p r o b i t analysis o f drug d i s p l a c e m e n t s o f 0.7 n M 3H-spiroperidol in t h e p r e s e n c e or a b s e n c e o f 0.1 m M GTP. D a t a are p r e s e n t e d as m e a n +- S.E.M. o f 3 o r 4 i n d e p e n d e n t replications.
(a) GTP effects on drug displacement of 3H-spiroperidol binding Drug
ICs0, nM
Shift
Control Dopamine (2)-ADTN ] Apomorphine Haloperidol Chlorpromazine Bromocriptine
12 0 0 0 1 460 255 13.6 140 12
+GTP ± 2100 + 230 ± 21 ± 0.7 ± 17 ± 0.7
130 000 14 3 0 0 1 550 13.6 140 12
± 13 0 0 0 ± 2 800 ± 340 ± 0.3 ± 23 ± 2.3
10.8 9.8 6.1 1.0 1.0 1.0
(b) Nucleotide specificity N u c l e o t i d e (0.1 raM)
% I n h i b i t i o n 3H-spiroperidol b i n d i n g b y 10 p M d o p a m i n e (n)
Control GTP GDP GMP ATP ADP AMP
48 20 17 45 38 43 46
± 2 -+ 1 ± 3 ± 1 ± 2 ± 2 ± 3
(7) (7) (4) (4) (4) (4) (4)
I 2-Amino-6,7-dihydroxy-l,2,3,4-tetrahydronaphthalene.
lesions have clearly distinguished two classes of striatal dopamine receptor, one linked to adenylate cyclase on intrinsic neurons and regulated by GTP; the other localized on presynaptic cortico-striate terminals, not linked to adenylate cyclase and not regulated by GTP (Creese et al., 1979). The finding of GTP regulation of agonist affinity for 3Hspiroperidol binding sites in pituitary suggests that these binding sites are associated with adenylate cyclase and similar to the receptors on intrinsic striatal neurons. These binding sites would appear to be associated with dopamine receptors which are distinct from those involved in the control of prolactin release, which does not appear to be a cyclic AMP mediated response (Kebabian and Calne,
1979). This hypothesis is reinforced by the finding that GTP does not regulate the affinity of bromocriptine for 3H-spiroperidol binding sites in the pituitary although it is a more potent agonist than dopamine in regulating prolactin release. Bromocriptine is, however, an antagonist of the striatal dopamine-sensitive adenylate cyclase where its affinity for 3H-spiroperidol binding sites is also insensitive to GTP. These data suggest that bovine anterior pituitary contains two distinct dopamine receptors, one not associated with adenylate cyclase which controls prolactin release, the other adenylate cyclase linked and regulated by GTP with, as of yet, unknown physiological function.
343
Acknowledgements We thank S.H. Snyder for consultation, F. Lopez for technical support and D. Taitano for manuscript preparation. This research was supported by an Institutional grant from UCSD and a grant from the Pharmaceutical Manufacturer's Association. Ian Creese is an Alfred P. Sloan Research Fellow, and David Sibley is supported a PHS training grant GM02267.
References Ahn, H.S., E. Gardner and M.H. Makman, 1979, Anterior pituitary adenylate cyclase: stimulation
by dopamine and other monoamines, European J. Pharmacol. 5 3 , 3 1 3 . Caron, M.G., M. Beaulieu, V. Raymond, B. Gagn6, J. Drouin, R. Lefkowitz and F. Labrie, 1978, Dopamine receptors in the anterior pituitary gland, J. Biol. Chem. 253, 2244. Creese, I., R. Schneider and S.H. Snyder, 1977, 3Hspiroperidol labels dopamine receptors in pituitary and brain, European J. Pharmacol. 46, 377. Creese, I., T. Usdin and S.H. Snyder, 1979, Guanine nueleotides distinguish two dopamine receptors, Nature (in press). Kebabian, J.W. and D.B. Calne, 1979, Multiple receptors for dopamine, Nature 277, 93.
341
© Elsevier]North-Holland Biomedical Press
Rapid communication GUANINE NUCLEOTIDES REGULATE ANTERIOR PITUITARY DOPAMINE RECEPTORS DAVID R. SIBLEY and IAN CREESE *
Department of Neurosciences, MOO8, University of California, San Diego, La Jolla, California 92093, U.S.A. Received 21 March 1979, accepted 22 March 1979
3H-Spiroperidol labels dopamine receptors in the anterior pituitary which exhibit micromolar affinities for classic dopamine agonists (Creese et al., 1977). These dopamine receptors are distinguished from the dopamine receptors responsible for control of prolactin release which exhibit nanomolar affinity for dopamine agonists (Caron et al., 1978). A dopamine-sensitive adenylate cyclase has recently been described in the pituitary which exhibits micromolar affinity for dopamine (Ahn et al., 1979). Guanine nucleotides, particularly GTP, selectively regulate agonist but not antagonist sensitivity of a number of hormone and neurotransmitter receptors linked to adenylate cyclase. We now report that guanine nucleotides regulate dopamine agonist affinity for 3H-spiroperidol binding sites in anterior pituitary. Homogenates of frozen bovine anterior pituitary (40 vols w/v), prepared in 50 mM Tris-HC1 buffer (pH 7.7 at 25°C) with a Tekmar homogenizer, were centrifuged (5 rain, 500 ×g) to remove connective tissue. The supernatant was centrifuged twice (10 min, 50,000 × g) at 4°C with resuspension in fresh buffer. Final resuspension (100 vois w/v) was in Tris buffer containing 0.1% ascorbic acid, 120 mM NaC1, 5 mM KC1, 1 mM CaC12 and 1 mM MgC12, pH 7.1 at 37°C. After incubation (15 min, 37°C) with 3H-spiroperidol (26 Ci/mmol., New England Nuclear), unlabeUed drugs and nucleotides, samples were filtered
* To whom correspondence should be addressed.
rapidly under vacuum over Whatman GF/B filters, and rinsed rapidly with 3 × 5 ml ice cold Tris-HC1 buffer. Radioactivity trapped on the filters was counted by liquid scintillation spectroscopy. Nonspecific binding of 3Hspiroperidol was determined in the presence of 1/~M (+)-butaclamol. In typical experiments with 0.7 nM ~Hspiroperidol total binding was 2506 + 75 cpm (n = 20) and nonspecific binding was 491 +_ 26 cpm (n = 20). At 0.1 mM, the maximal concentration examined, the nucleotides GTP, GDP, GMP, ATP, ADP and AMP had no effect on total or nonspecific 3H-spiroperidol binding. However, the ability of agonists to displace 3H-spiroperidol binding was markedly influenced by some guanine nucleotides. GTP and GDP (0.1 raM) reduced the apparent affinity of dopamine, apomorphine and ADTN by approximately 6--11 fold for 3H-spiroperidol binding sites (table la). GTP was maximally effective at 0.1 mM with an ECs0 of 10/~M. Other nucleotides were ineffective in reducing the potency of dopamine in displacing 3H-spiroperidol binding (table lb). GTP did not influence the ability of the antagonists chlorpromazine or haloperidol to displace 3H-spiroperidol binding. Bromocriptine was also uninfluenced by GTP. Striatal dopamine receptors have been characterized by 3H-spiroperidol binding and a dopamine-sensitive adenylate cyclase. The different pharmacological specificities of these two markers suggests that each may label distinct dopamine receptors. Kainic acid
342 TABLE 1 Guanine nucleotide regulation of pituitary dopamine receptors. ICs0'S were d e t e r m i n e d b y l o g - p r o b i t analysis o f drug d i s p l a c e m e n t s o f 0.7 n M 3H-spiroperidol in t h e p r e s e n c e or a b s e n c e o f 0.1 m M GTP. D a t a are p r e s e n t e d as m e a n +- S.E.M. o f 3 o r 4 i n d e p e n d e n t replications.
(a) GTP effects on drug displacement of 3H-spiroperidol binding Drug
ICs0, nM
Shift
Control Dopamine (2)-ADTN ] Apomorphine Haloperidol Chlorpromazine Bromocriptine
12 0 0 0 1 460 255 13.6 140 12
+GTP ± 2100 + 230 ± 21 ± 0.7 ± 17 ± 0.7
130 000 14 3 0 0 1 550 13.6 140 12
± 13 0 0 0 ± 2 800 ± 340 ± 0.3 ± 23 ± 2.3
10.8 9.8 6.1 1.0 1.0 1.0
(b) Nucleotide specificity N u c l e o t i d e (0.1 raM)
% I n h i b i t i o n 3H-spiroperidol b i n d i n g b y 10 p M d o p a m i n e (n)
Control GTP GDP GMP ATP ADP AMP
48 20 17 45 38 43 46
± 2 -+ 1 ± 3 ± 1 ± 2 ± 2 ± 3
(7) (7) (4) (4) (4) (4) (4)
I 2-Amino-6,7-dihydroxy-l,2,3,4-tetrahydronaphthalene.
lesions have clearly distinguished two classes of striatal dopamine receptor, one linked to adenylate cyclase on intrinsic neurons and regulated by GTP; the other localized on presynaptic cortico-striate terminals, not linked to adenylate cyclase and not regulated by GTP (Creese et al., 1979). The finding of GTP regulation of agonist affinity for 3Hspiroperidol binding sites in pituitary suggests that these binding sites are associated with adenylate cyclase and similar to the receptors on intrinsic striatal neurons. These binding sites would appear to be associated with dopamine receptors which are distinct from those involved in the control of prolactin release, which does not appear to be a cyclic AMP mediated response (Kebabian and Calne,
1979). This hypothesis is reinforced by the finding that GTP does not regulate the affinity of bromocriptine for 3H-spiroperidol binding sites in the pituitary although it is a more potent agonist than dopamine in regulating prolactin release. Bromocriptine is, however, an antagonist of the striatal dopamine-sensitive adenylate cyclase where its affinity for 3H-spiroperidol binding sites is also insensitive to GTP. These data suggest that bovine anterior pituitary contains two distinct dopamine receptors, one not associated with adenylate cyclase which controls prolactin release, the other adenylate cyclase linked and regulated by GTP with, as of yet, unknown physiological function.
343
Acknowledgements We thank S.H. Snyder for consultation, F. Lopez for technical support and D. Taitano for manuscript preparation. This research was supported by an Institutional grant from UCSD and a grant from the Pharmaceutical Manufacturer's Association. Ian Creese is an Alfred P. Sloan Research Fellow, and David Sibley is supported a PHS training grant GM02267.
References Ahn, H.S., E. Gardner and M.H. Makman, 1979, Anterior pituitary adenylate cyclase: stimulation
by dopamine and other monoamines, European J. Pharmacol. 5 3 , 3 1 3 . Caron, M.G., M. Beaulieu, V. Raymond, B. Gagn6, J. Drouin, R. Lefkowitz and F. Labrie, 1978, Dopamine receptors in the anterior pituitary gland, J. Biol. Chem. 253, 2244. Creese, I., R. Schneider and S.H. Snyder, 1977, 3Hspiroperidol labels dopamine receptors in pituitary and brain, European J. Pharmacol. 46, 377. Creese, I., T. Usdin and S.H. Snyder, 1979, Guanine nueleotides distinguish two dopamine receptors, Nature (in press). Kebabian, J.W. and D.B. Calne, 1979, Multiple receptors for dopamine, Nature 277, 93.
