Life Sciences, Vol. 23, pp. 431-436 Printed in the U.S.A.
Pergamon Press
DIFFERENTIAL EFFECTS OF DOPAMINE ANTAGONISTS ON PROLACTIN SECRETION FROM CULTURED RAT PITUITARY CELLS Carl Denef and Jean-Jacques Follebouckt Katholieke Universiteit Leuven, Fakulteit der Geneeskunde Afdeling Farmakologie B-3OOO Leuven, Belgium
SUMMARY Various dopamine antagonists, including two novel non-neuroleptic drugs domperidone and halopemide, stimulated apomorphine-suppressed prolactin secretion from cultured rat pituitary cells. The potency of these drugs closely paralleled their rank-order in displacing in vitro H3-halo peridol binding in rat striatum reported by others (IO). Concentrationeffect curves were parallel except those of pimozide and clopimozide which were biphasic : prolactin secretion was stimulated at low concentrations but depressed at concentrations above25nM° When added alone, pimozide and clopimozide, but none of the other drugs tested, also depressed prolactin secretion. The present findings indicate that prolactin secretion from cultured pituitary cells may provide an in vitro test system suitable to differentiate antagonists of dopamine receptors and possibly to distinguish pure from partial antagonists.
Pituitary prolactin secretion is under inhibitory dopaminergic control (I). Dopamine and dopamine agonists suppress prolactin secretion from pituitary in vivo, from the isolated pituitary gland in vitro and from pituitary cells in culture (2,3,4). Neuroleptics, which are competitive antagonists of dopamine receptors, stimulate prolactin secretion in vivo and block the inhibition by dopamine agonists of prolactin secretion from pituitary in vitro (4,5,6). Presumably, prolactin cells have receptors of dopamine agonists and antagonists and this is substantiated by studies demonstrating the presence of stereospecific binding sites for both H3-dopamine and the neuroleptic H3-halo peridol in the pituitary (4,7). These binding sites appear to have certain properties in common with those in rat striatum, although extensive comparative investigations have not been made (7). The present research was intended to determine the potency of a series of dopamine antagonists in reversing the apomorphine-depressed prolactin secretion from cultured rat pituitary cells and to compare their potency with their affinity reported by others to bind to dopamine receptors of rat striatum in vitro (8,9,]0). Particular attention was paid to pimozide and two novel dopamine antagonists, the non-neuroleptic psychoactive drug halopemide (]],12) and the anti-emetic domperidone (13), which all have a pharmacological profile quite different from other dopamine antagonists (11,]2,13,14,15). METHODS Pimozide, clopimozide, butyrophenones, halopemide, domperidone (all obtained from Janssen Pharmaceutica, Belgium), butaclamol (Ayerst), sulpiride (Delagrange) were dissolved in IO % ethanol in O . O 1 N HCI and further diluted in IO % ethanol in 0.9 % saline. All solutions were freshly prepared. Apo0300-9653/78/0807-0431502.00/0 Copyright (c) 1978 Pergamon Press
432
Dopamine Antagonists and Prolactin Cells
Vol. 23, No. 5, 1978
morphine-HCl (Federa, Belgium), chlorpromazine-HC1 (Specia) and perphenazine (Essex, Belgium) were dissolved and diluted in ! % ascorbic acid. NaloxoneHCI (Endo laboratories) was dissolved in 0.9 % saline. Pituitary cells from 200 g female Wistar rats were established in culture as previously described (16), but dishes were not coated with poly-l-lysine. Cells were plated in Falcon multi-wells. About 200,000 cells per dish were used which corresponds to about 1:40 of one gland. Culture medium consisted of Dulbecco's modified Eagle's medium supplemented (16) with fetal calf serum, horse serum, pyruvate, penicillin, streptomycin and O . 0 1 % ascorbic acid. Tests were done on day 5 in culture. The culture medium was removed and the dishes washed with serum free Dulbecco's medium. As shown by others (17) prolactin release is not linear with time after medium change. We found that secretion rates became linear with time after 2 hours of preincubation. Test solutions were added after this preincubation and incubation was for another 4 hours in a humidified C02/air incubator at 370C. After centrifugation at 2,000 rpm for 7 min the media were diluted 10-30 times with 1 % bovine albumin in phosphate buffer pH 7.3 and stored at -250C until assayed. Prolactin was measured with rat-prolactin radioimmunoassay kits distributed by the NIAMDD Pituitary Hormone Distribution Program, Bethesda, Maryland. The amounts of prolactin were expressed in terms of the NIAMDD reference preparation RP-;. Preincubation values were substracted from the values measured at the end of incubation. Treatment effects were tested for significance by analysis of variance. RESULTS Apomorphine effectively suppressed prolactin secretion, the fifty per cent inhibition of release being obtained at 14 nM. At maximal suppression, prolactin levels were 20-25 % of control values. A time study showed that suppression was sustained over the entire incubation period. Dopamine antagonists were first tested for possible direct effects on prolactin secretion (Fig. I). At concentrations from 2.5 nM to ] ~M, phenothiazines, sulpiride, (+)-butaclamol and butyrophenones did not consistently affect spontaneous prolactin release. However, pimozide lowered the secretion in a dose response fashion between 25 nM and I ~M (F-test : p < 0.O1). Its congener
3,0
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I0 -7
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_
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10 -8
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10 -?
Fig.
10"g
10-8
10-s M
1
Effect of various dopamine antagonists on spontaneous prolactin release from cultured pituitary cells. Means of 6 replicates + S.E.M.
Vol. 23, No. 5, 1978
Dopamine Antagonists and Prolactin Cells
433
clopimozide had a similar though less potent effect (p < 0.05). Halopemide and domperidone which both have a 4-piperidino-benzimidazolone moiety like clopimozide were devoid of any suppressive effect on prolactin release. In fact, these compounds had a slight stimulatory effect (F-test, p < 0.02). All dopamine antagonists tested reversed the suppressive effect of apomorphine on prolaetin secretion (Fig. 2). Even at maximal suppression by apomorphine, the effects were at nanomolar concentrations of the antagonists. The order of potency paralleled the order of potency of the same drugs in displacing H3-haloperidol binding in rat striatum (Fig. 3). At low concentrations pimozide and clopimozide elevated prolactin secretion but from about 25 nM secretion was again suppressed. Of the butaclamol isomers only the pharmacologically active (+)-isomer showed activity. One could advance the hypothesis that the intrinsic activity of pimozide to decrease prolactin secretion, if at all specific, could be due to partial agonist activity at the dopamine receptor. Alternatively, the drug might also interact with other receptors such as cholinergic and opiate receptors. At concentrations between O.I nM and ] uM, spiperone, haloperidol and (+)-butaclamol were totally ineffective in blocking the effect of 250 nM pimozide and so were atropine (]O nM) and naloxone (up to 250 nM). DISCUSSION The present investigations confirm earlier findings (3,4) that dopamine antagonists have the ability to block the suppressive effect of dopamine agonists on prolactin release from pituitary cells in culture. By extending such observations on a larger variety of compounds including non-neuroleptic dopamine antagonists, we found an excellent correlation between the rank-order of these drugs in antagonizing the in vitro suppression of prolactin secretion by apomorphine and their affinity, reported by others (10,]3), to compete for H 3haloperidol binding to dopamine receptors of rat striatum in vitro. We therefore propose that the prolactin assay system provides a sensitive in vitro method to study the interaction of dopamine antagonists with their receptors. Importantly, these receptors can be studied in intact cells in which the stimulus-effect coupling can be investigated at the same time. In addition, the system may be most valuable in predicting the affinity of dopamine antagonists for their receptors in the brain. The response of prolactin cells is in clearcut contrast with that of the dopamine-sensitive adenylate cyclase in striatal homogenate (]8). In the latter system several butyrophenones and pimozide have a disproportionately weak activity and their potency does not correlate with their affinity for dopamine receptors. The latter discrepancy has been related to the strong avidity of butyrophenones and pimozide to precipitate in lipophylic constituents of homogenized tissue (]8). Why such phenomenon does not appear to interfere in tile prolactin system is not clear. A possible explanation is that with intact cells and with the small amount of tissue used (about 0.5 mg) adsorption is relatively much smaller. Although there is some evidence from binding studies (7) that receptors of dopamine antagonists may be similar in pituitary and striatum, the present findings suggest certain differences between these two systems. It has been shown that displacement of H3-dopamine (9) or H3-apomorphine (8) binding to striatal membranes requires up to a 100 times higher concentration of dopamine antagonists than displacement of H3-haloperidol binding. In contrast, 50 per cent reversal of apomorphine suppression of prolactin secretion was obtained here with concentrations of dopamine antagonists almost identical to the IC50 values for inhibition of H3-haloperidol binding. This might suggest that the postulated two-state mechanism of the dopamine receptor (9) operates differently in the pituitary.
434
Dopamine Antagonists and Prolactin Cells
Vol. 23, No. 5, 1978
/ ,, I0( E
~
9c
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~
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I
........
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10-9
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10 -s M
Fi$. 2 Disinhibition by dopamine antagonists of apomorphine-suppressed prolactin release from cultured pituitary cells. Per cent disinhibition was calculated from the mean of 6 replicates. Agonist and antagonist were added simultaneously to the culture dish.
1000
OAZaPERONE • CHLO~PROMAZINE
:Z c:
100
O S~LPlRIDE
c
3
~
I'~BUIACLa"°L
10
QHALOPEMID E tCLOPI.OZIOE
o o
~,MOZIOEQ
OHALOPER~OOL
BE~PER,OOL
_u • S~IpERO.E
01
I
ECso
I0
proloctin
secretion
I00
1000
(riM)
Jig. 3 Potency of dopamine antagonists in disinhiblting apomorphine-suppressed prolactin release from cultured pituitary cells plotted against potency in displacing H3-haloperidol binding in rat striatal membranes (data from references 10 and 13). EC50 values are the drug concentrations causing 50 per cent reversal of inhibition of prolactin release induced by 10-7 M apomorphine. EC50 values were determined by log-probit plots (2 experiments) Correlation between the two parameters was highly significant (r = 0.94; p < 0.00]; slope of regression line : 1.]3).
Vol. 23, No. 5, 1978
Dopamine Antagonists and Prolactin Cells
435
Another important finding was that pimozide and its congener clopimozide had an intrinsic inhibitory effect on prolactin secretion. We hypothesized that diphenylbutylpiperidines might be partial agonists of dopamine receptors. Because this intrinsic activity could not be blocked even with high concentrations of neuroleptics devoid of any intrinsic activity in the present system, the above hypothesis was not sustained. One should also consider the possibility that the intrinsic activity of pimozide is due to a membrane disturbance caused by lipophylic adsorption of the drug. However, this seems unlikely since clopimozide was less potent than pimozide but is more lipophylic (19). On the other hand, it is most tempting to speculate that diphenylbutylpiperidines have partial affinity for a pituitary dopamine receptor different from the one activated by apomorphine and blocked by typical neuroleptics. Evidence for the existence of a dopamine receptor not activated by apomorphine or blocked by neuroleptics has already been proposed in the brain (20). ACKNOWLEDGEMENTS We thank Prof. P. De Schepper for critical discussions, Dr. J.E. Leysen for most valuable suggestions and gifts of dopamine antagonists, NIA~.~D for radioimmunoassay kits and R. Dewals andA. De Wolf for technical assistance. REFERENCES I. J. MEITES and J.A. CLEMENS, Vitam.Horm. 30:165-221 (1972). 2. H.G. FLOSS, J.M. CASSADY and J.E. ROBBERS, J.Pharm.Sci. 62:699-715 (1973). 3. R.M. MacLEOD, Frontiers in Neuroendocrinology. Eds. L. ~-~tini and W.F. Ganong, p. 169, Raven Press, New York (1976). 4. M.G. CARON, J. DROUIN, V. RAYMOND, P.A. KELLY and F. LABRIE, Clin.Res. 24: 656 A (1976). 5. J.A. CLEMENS, E.B. SMALSTIG and B.D. SAWYER, Psychopharmacologia 40:123-127 (1974). 6. G. LANGER, E.J. SACHAR, P.H. GRUENandF.S.HALPERN, Nature 266:639-640 (1977). 7. G.M. BROWN, P. SEEMAN and T. LEE, Endocrinology 99:1407-1410 (1976). 8. P. SEEMAN, M. CHAU-WONG, J. TEDESCO and K. WONG, Proc.Natl.Acad.Sci. USA 72:4376-4380 (1975). 9. I. CREESE, D.R. BURT and S.H. SNYDER, Science 192:481-483 (1976). 10. J.E. LEYSEN, C.J.E. NIEMEGEERS, J.P. TOLLENAERE and P.M. LADURON, Nature in press (1978). 11. N. ROMBAUT, H. DE POORTER and J. BRUGMANS, Xlth C.I.N.P. Congress, Vienna, July 9-16 (1978). 12. F.C. COLPAERT, C.J.E. NIEMEGEERS and P.M. LADURON, Xlth C.I.N.P. Congress, Vienna, July 9-16 (]978). 13. A.J. REYNTJENS, C.J.E. NIEMEGEERS, J.M. VAN NEUTEN, P. LADURON, J. HEYKANTS, K.H.L. SCHELLEKENS and P.A.J. JANSSEN, Arzneimittel-Forsch. submitted (1978) 14. A. WOUTERS, Compte Rendu du Congr~s de Psychiatrie et de Neurologie de langue Fran~aise, Session 67, Bruxelles, p. 561 (1969). 15. R.M. PINDER, R.N. BROGDEN, P.R. SAWYER, T.M. SPEIGHT, R. SPENCER and G.S. AVERY, Drugs 12:1-40 (1976). 16. C. DENEF, E. HAUTEKEETE and L. RUBIN, Science 194:848-851 (1976). 17. J.C. GROSHONG, G.E. MILO and W.B. MALARKEY, Life Sci. 20:1821-1828 (1977). 18. Y.C. CLEMENT-CORMIER, J.W. KEBABIAN, G.L. PETZOLD and R--~GREENGARD, Proc. Natl.Acad.Sci. USA 71:1113-1117 (1974). 19. P. LADURON, J.Pharm.Pharmac. 28:250-251 (1976). 20. A.R. COOLS and J.M. VAN ROSSUM, Psychopharmacologia 4--5:243-254 (1976).
Pergamon Press
DIFFERENTIAL EFFECTS OF DOPAMINE ANTAGONISTS ON PROLACTIN SECRETION FROM CULTURED RAT PITUITARY CELLS Carl Denef and Jean-Jacques Follebouckt Katholieke Universiteit Leuven, Fakulteit der Geneeskunde Afdeling Farmakologie B-3OOO Leuven, Belgium
SUMMARY Various dopamine antagonists, including two novel non-neuroleptic drugs domperidone and halopemide, stimulated apomorphine-suppressed prolactin secretion from cultured rat pituitary cells. The potency of these drugs closely paralleled their rank-order in displacing in vitro H3-halo peridol binding in rat striatum reported by others (IO). Concentrationeffect curves were parallel except those of pimozide and clopimozide which were biphasic : prolactin secretion was stimulated at low concentrations but depressed at concentrations above25nM° When added alone, pimozide and clopimozide, but none of the other drugs tested, also depressed prolactin secretion. The present findings indicate that prolactin secretion from cultured pituitary cells may provide an in vitro test system suitable to differentiate antagonists of dopamine receptors and possibly to distinguish pure from partial antagonists.
Pituitary prolactin secretion is under inhibitory dopaminergic control (I). Dopamine and dopamine agonists suppress prolactin secretion from pituitary in vivo, from the isolated pituitary gland in vitro and from pituitary cells in culture (2,3,4). Neuroleptics, which are competitive antagonists of dopamine receptors, stimulate prolactin secretion in vivo and block the inhibition by dopamine agonists of prolactin secretion from pituitary in vitro (4,5,6). Presumably, prolactin cells have receptors of dopamine agonists and antagonists and this is substantiated by studies demonstrating the presence of stereospecific binding sites for both H3-dopamine and the neuroleptic H3-halo peridol in the pituitary (4,7). These binding sites appear to have certain properties in common with those in rat striatum, although extensive comparative investigations have not been made (7). The present research was intended to determine the potency of a series of dopamine antagonists in reversing the apomorphine-depressed prolactin secretion from cultured rat pituitary cells and to compare their potency with their affinity reported by others to bind to dopamine receptors of rat striatum in vitro (8,9,]0). Particular attention was paid to pimozide and two novel dopamine antagonists, the non-neuroleptic psychoactive drug halopemide (]],12) and the anti-emetic domperidone (13), which all have a pharmacological profile quite different from other dopamine antagonists (11,]2,13,14,15). METHODS Pimozide, clopimozide, butyrophenones, halopemide, domperidone (all obtained from Janssen Pharmaceutica, Belgium), butaclamol (Ayerst), sulpiride (Delagrange) were dissolved in IO % ethanol in O . O 1 N HCI and further diluted in IO % ethanol in 0.9 % saline. All solutions were freshly prepared. Apo0300-9653/78/0807-0431502.00/0 Copyright (c) 1978 Pergamon Press
432
Dopamine Antagonists and Prolactin Cells
Vol. 23, No. 5, 1978
morphine-HCl (Federa, Belgium), chlorpromazine-HC1 (Specia) and perphenazine (Essex, Belgium) were dissolved and diluted in ! % ascorbic acid. NaloxoneHCI (Endo laboratories) was dissolved in 0.9 % saline. Pituitary cells from 200 g female Wistar rats were established in culture as previously described (16), but dishes were not coated with poly-l-lysine. Cells were plated in Falcon multi-wells. About 200,000 cells per dish were used which corresponds to about 1:40 of one gland. Culture medium consisted of Dulbecco's modified Eagle's medium supplemented (16) with fetal calf serum, horse serum, pyruvate, penicillin, streptomycin and O . 0 1 % ascorbic acid. Tests were done on day 5 in culture. The culture medium was removed and the dishes washed with serum free Dulbecco's medium. As shown by others (17) prolactin release is not linear with time after medium change. We found that secretion rates became linear with time after 2 hours of preincubation. Test solutions were added after this preincubation and incubation was for another 4 hours in a humidified C02/air incubator at 370C. After centrifugation at 2,000 rpm for 7 min the media were diluted 10-30 times with 1 % bovine albumin in phosphate buffer pH 7.3 and stored at -250C until assayed. Prolactin was measured with rat-prolactin radioimmunoassay kits distributed by the NIAMDD Pituitary Hormone Distribution Program, Bethesda, Maryland. The amounts of prolactin were expressed in terms of the NIAMDD reference preparation RP-;. Preincubation values were substracted from the values measured at the end of incubation. Treatment effects were tested for significance by analysis of variance. RESULTS Apomorphine effectively suppressed prolactin secretion, the fifty per cent inhibition of release being obtained at 14 nM. At maximal suppression, prolactin levels were 20-25 % of control values. A time study showed that suppression was sustained over the entire incubation period. Dopamine antagonists were first tested for possible direct effects on prolactin secretion (Fig. I). At concentrations from 2.5 nM to ] ~M, phenothiazines, sulpiride, (+)-butaclamol and butyrophenones did not consistently affect spontaneous prolactin release. However, pimozide lowered the secretion in a dose response fashion between 25 nM and I ~M (F-test : p < 0.O1). Its congener
3,0
1.C
2£
z
O.=.
i
I
1
I
10 `9
I0 -8
I0 -7
10-6M
_
i
[
10 -8
t
I0 "s M
10 -?
Fig.
10"g
10-8
10-s M
1
Effect of various dopamine antagonists on spontaneous prolactin release from cultured pituitary cells. Means of 6 replicates + S.E.M.
Vol. 23, No. 5, 1978
Dopamine Antagonists and Prolactin Cells
433
clopimozide had a similar though less potent effect (p < 0.05). Halopemide and domperidone which both have a 4-piperidino-benzimidazolone moiety like clopimozide were devoid of any suppressive effect on prolactin release. In fact, these compounds had a slight stimulatory effect (F-test, p < 0.02). All dopamine antagonists tested reversed the suppressive effect of apomorphine on prolaetin secretion (Fig. 2). Even at maximal suppression by apomorphine, the effects were at nanomolar concentrations of the antagonists. The order of potency paralleled the order of potency of the same drugs in displacing H3-haloperidol binding in rat striatum (Fig. 3). At low concentrations pimozide and clopimozide elevated prolactin secretion but from about 25 nM secretion was again suppressed. Of the butaclamol isomers only the pharmacologically active (+)-isomer showed activity. One could advance the hypothesis that the intrinsic activity of pimozide to decrease prolactin secretion, if at all specific, could be due to partial agonist activity at the dopamine receptor. Alternatively, the drug might also interact with other receptors such as cholinergic and opiate receptors. At concentrations between O.I nM and ] uM, spiperone, haloperidol and (+)-butaclamol were totally ineffective in blocking the effect of 250 nM pimozide and so were atropine (]O nM) and naloxone (up to 250 nM). DISCUSSION The present investigations confirm earlier findings (3,4) that dopamine antagonists have the ability to block the suppressive effect of dopamine agonists on prolactin release from pituitary cells in culture. By extending such observations on a larger variety of compounds including non-neuroleptic dopamine antagonists, we found an excellent correlation between the rank-order of these drugs in antagonizing the in vitro suppression of prolactin secretion by apomorphine and their affinity, reported by others (10,]3), to compete for H 3haloperidol binding to dopamine receptors of rat striatum in vitro. We therefore propose that the prolactin assay system provides a sensitive in vitro method to study the interaction of dopamine antagonists with their receptors. Importantly, these receptors can be studied in intact cells in which the stimulus-effect coupling can be investigated at the same time. In addition, the system may be most valuable in predicting the affinity of dopamine antagonists for their receptors in the brain. The response of prolactin cells is in clearcut contrast with that of the dopamine-sensitive adenylate cyclase in striatal homogenate (]8). In the latter system several butyrophenones and pimozide have a disproportionately weak activity and their potency does not correlate with their affinity for dopamine receptors. The latter discrepancy has been related to the strong avidity of butyrophenones and pimozide to precipitate in lipophylic constituents of homogenized tissue (]8). Why such phenomenon does not appear to interfere in tile prolactin system is not clear. A possible explanation is that with intact cells and with the small amount of tissue used (about 0.5 mg) adsorption is relatively much smaller. Although there is some evidence from binding studies (7) that receptors of dopamine antagonists may be similar in pituitary and striatum, the present findings suggest certain differences between these two systems. It has been shown that displacement of H3-dopamine (9) or H3-apomorphine (8) binding to striatal membranes requires up to a 100 times higher concentration of dopamine antagonists than displacement of H3-haloperidol binding. In contrast, 50 per cent reversal of apomorphine suppression of prolactin secretion was obtained here with concentrations of dopamine antagonists almost identical to the IC50 values for inhibition of H3-haloperidol binding. This might suggest that the postulated two-state mechanism of the dopamine receptor (9) operates differently in the pituitary.
434
Dopamine Antagonists and Prolactin Cells
Vol. 23, No. 5, 1978
/ ,, I0( E
~
9c
E
g. o
R(
c
•o=
?(
g o
K
r. 2 =
~
20
~ m P
I
........
d~
10-10
10-9
lO-S
10 -7
10 -s M
Fi$. 2 Disinhibition by dopamine antagonists of apomorphine-suppressed prolactin release from cultured pituitary cells. Per cent disinhibition was calculated from the mean of 6 replicates. Agonist and antagonist were added simultaneously to the culture dish.
1000
OAZaPERONE • CHLO~PROMAZINE
:Z c:
100
O S~LPlRIDE
c
3
~
I'~BUIACLa"°L
10
QHALOPEMID E tCLOPI.OZIOE
o o
~,MOZIOEQ
OHALOPER~OOL
BE~PER,OOL
_u • S~IpERO.E
01
I
ECso
I0
proloctin
secretion
I00
1000
(riM)
Jig. 3 Potency of dopamine antagonists in disinhiblting apomorphine-suppressed prolactin release from cultured pituitary cells plotted against potency in displacing H3-haloperidol binding in rat striatal membranes (data from references 10 and 13). EC50 values are the drug concentrations causing 50 per cent reversal of inhibition of prolactin release induced by 10-7 M apomorphine. EC50 values were determined by log-probit plots (2 experiments) Correlation between the two parameters was highly significant (r = 0.94; p < 0.00]; slope of regression line : 1.]3).
Vol. 23, No. 5, 1978
Dopamine Antagonists and Prolactin Cells
435
Another important finding was that pimozide and its congener clopimozide had an intrinsic inhibitory effect on prolactin secretion. We hypothesized that diphenylbutylpiperidines might be partial agonists of dopamine receptors. Because this intrinsic activity could not be blocked even with high concentrations of neuroleptics devoid of any intrinsic activity in the present system, the above hypothesis was not sustained. One should also consider the possibility that the intrinsic activity of pimozide is due to a membrane disturbance caused by lipophylic adsorption of the drug. However, this seems unlikely since clopimozide was less potent than pimozide but is more lipophylic (19). On the other hand, it is most tempting to speculate that diphenylbutylpiperidines have partial affinity for a pituitary dopamine receptor different from the one activated by apomorphine and blocked by typical neuroleptics. Evidence for the existence of a dopamine receptor not activated by apomorphine or blocked by neuroleptics has already been proposed in the brain (20). ACKNOWLEDGEMENTS We thank Prof. P. De Schepper for critical discussions, Dr. J.E. Leysen for most valuable suggestions and gifts of dopamine antagonists, NIA~.~D for radioimmunoassay kits and R. Dewals andA. De Wolf for technical assistance. REFERENCES I. J. MEITES and J.A. CLEMENS, Vitam.Horm. 30:165-221 (1972). 2. H.G. FLOSS, J.M. CASSADY and J.E. ROBBERS, J.Pharm.Sci. 62:699-715 (1973). 3. R.M. MacLEOD, Frontiers in Neuroendocrinology. Eds. L. ~-~tini and W.F. Ganong, p. 169, Raven Press, New York (1976). 4. M.G. CARON, J. DROUIN, V. RAYMOND, P.A. KELLY and F. LABRIE, Clin.Res. 24: 656 A (1976). 5. J.A. CLEMENS, E.B. SMALSTIG and B.D. SAWYER, Psychopharmacologia 40:123-127 (1974). 6. G. LANGER, E.J. SACHAR, P.H. GRUENandF.S.HALPERN, Nature 266:639-640 (1977). 7. G.M. BROWN, P. SEEMAN and T. LEE, Endocrinology 99:1407-1410 (1976). 8. P. SEEMAN, M. CHAU-WONG, J. TEDESCO and K. WONG, Proc.Natl.Acad.Sci. USA 72:4376-4380 (1975). 9. I. CREESE, D.R. BURT and S.H. SNYDER, Science 192:481-483 (1976). 10. J.E. LEYSEN, C.J.E. NIEMEGEERS, J.P. TOLLENAERE and P.M. LADURON, Nature in press (1978). 11. N. ROMBAUT, H. DE POORTER and J. BRUGMANS, Xlth C.I.N.P. Congress, Vienna, July 9-16 (1978). 12. F.C. COLPAERT, C.J.E. NIEMEGEERS and P.M. LADURON, Xlth C.I.N.P. Congress, Vienna, July 9-16 (]978). 13. A.J. REYNTJENS, C.J.E. NIEMEGEERS, J.M. VAN NEUTEN, P. LADURON, J. HEYKANTS, K.H.L. SCHELLEKENS and P.A.J. JANSSEN, Arzneimittel-Forsch. submitted (1978) 14. A. WOUTERS, Compte Rendu du Congr~s de Psychiatrie et de Neurologie de langue Fran~aise, Session 67, Bruxelles, p. 561 (1969). 15. R.M. PINDER, R.N. BROGDEN, P.R. SAWYER, T.M. SPEIGHT, R. SPENCER and G.S. AVERY, Drugs 12:1-40 (1976). 16. C. DENEF, E. HAUTEKEETE and L. RUBIN, Science 194:848-851 (1976). 17. J.C. GROSHONG, G.E. MILO and W.B. MALARKEY, Life Sci. 20:1821-1828 (1977). 18. Y.C. CLEMENT-CORMIER, J.W. KEBABIAN, G.L. PETZOLD and R--~GREENGARD, Proc. Natl.Acad.Sci. USA 71:1113-1117 (1974). 19. P. LADURON, J.Pharm.Pharmac. 28:250-251 (1976). 20. A.R. COOLS and J.M. VAN ROSSUM, Psychopharmacologia 4--5:243-254 (1976).
