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Influence of serotonergic and adrenergic antagonists on TRH-induced prolactin release in the monkey
Pergamon Press
Life Sciences Vol . 22, pp . 439-444 Printed in the 1T .S .A .
INFLUENCE OF SEROTONERGIC AND ADRENERGIC ANTAGONISTS ON TRH-INDUCED PROLACTIN RELEASE IN THE MONKEY Richard R . Gala, Judith A . Peters, David R . Pieper, and Marappa G . Subramanian Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201 . (Received in final form September 29, 1977) Summary The influence of methysergide, cyproheptadine and SQ 10,631 (serotonergic receptor blockers) at the dose of 35 jig/kg, 50 ug/kg and 5 mg/kg, respectively, and propranolol, phentolamine and phenoxybenzamine (adrenergic receptor blockers) at the dose of 1 mg/kg on TRHinduced prolactin release was studied in sexually mature female monkeys . The serotonergic antagonists had no effect on TRH-induced prolactin release . Both ß and a adrenergic antagonists gave a similar potentiation of the TRH-induced prolactin response but only phenoxybenzamine plus TRH was statistically different (P < 0 .05) from TRH alone . The effect of the adrenergic receptor blockers is believed to be due to actions on dopamine receptors . The regulation of prolactin is under the control of a hypothalamic inhibiting factor (1) . Considerable evidence indicates that this factor may be dopamine (2-4) . There is also evidence that a prolactin stimulatory factor is present in the hypothalamus (5,6) and that thyrotropin releasing hormone (TRH) releases prolactin as well as thryotropin (7) . We have observed in rats and monkeys that high doses of TRH induce lower increments of prolactin release than do low doses (8,9) . One possible explanation for this is that the high doses of TRH stimulate biogenic amines that are inhibitory to prolactin release . The present study was undertaken to test this possibility using sexually mature female monkeys injected with serotonergic and adrenergic receptor blockers followed by a high dose of TRH . Methods A total of 5 sexually mature regularly cycling female monkeys (3 Macaca mulatta and 2 Macaca fascicularis) were utilized in this study . The animals were sampled at weekly intervals under ketamine anesthesia which does not influence basal prolactin levels (9-11) . Blood samples were obtained from either the femoral or tail vein using an indwelling 25 gauge butterfly catheter . Following an initial blood sample (0 .6 ml), either serotonergic or adrenergic antagonists were injected . Fifteen minutes later a second blood sample was obtained and 1 ug/kg of TRH was injected intravenously through the catheter . Subsequent blood samples were obtained 15, 30 and 45 minutes after TRH administration . All experiments were performed between 1300 and 1600 hours and the blood allowed to clot overnight at 3°C . The recovered serum was frozen and I Supported by NIH General Research Support Grant No . RR 5384 to Wayne State University School of Medicine . 439
440
TRH-Induced Prolactin Release
METHYSERGIDE
CYPROHEPTADINE
Vol . 22, No . 6, 1978
S010631
a
c v t u
S su0 n. E N
Time (min)
FIG . 1 Influence of serotonergic receptor antagonists on TRH-induced prolactin release . A . Methysergide ; B . Cyproheptadine ; C . SQ 10,631 (2 chloro2'-[3-(dimethylamino) propyl] thio-cinnamanilide HC1) . All drugs were administered intravenously . Each point represents the results from 5 monkeys . The vertical lines represent the standard error of the mean . stored at -20°C until assayed for prolactin . Serum prolactin levels were determined at two dilutions in duplicate using a primate prolactin radioimmunoassay (12) . The data is expressed as ng/ml change from the initial serum prolactin value when only a single drug was used and from the second prolactin value (15 minutes after the initial blood sample) whenever two drugs were examined . The data was assessed statistically using a two-way analysis of variance (13) with computer assistance and is presented with means and the standard error of the mean . The serotonergic receptor antagonists were : methysergide maleate, 35 u9/kg ; cyproheptadine HCl, 50 jig/kg ; and SQ 10,631 (2-chloro-2'-[3-(dimethylamino) propyl] thio-cinnamanilide, HC1), 5 mg/kg . The adrenergic receptor antagonists were propranolol HC1, 1 mg/kg ; phentolamine HCl,1 mg/kg ; and phenoxybenzamine HC1, 1 mg/kg . All drugs were dissolved in 50% ethanol-saline solution except propranolol which was dissolved in saline . The administration of vehicle alone had no effect on basal prolactin levels . There was no carry-over effect of the drugs from one week to the next on basal prolactin levels .
Vol . 22, No . 6, 1978
PROPRANOLOL /3
TRH-Induced Prolactin Release
PHENTOLAMINE
or
441
PHENOD(YBENZAw11NE or
c0u É W g u E
fA41
FIG . 2 Influence of adrenergic receptor antagonists on TRH-induced prolactin release . A . Propranolol, ß blocker ; B . Phentolamine, a blocker ; C . Phenoxybenzamine, a blocker . Each point represents the results from 5 monkeys . All drugs were administered intravenously . The vertical lines represent the standard error of the mean . Results The prior administration of serotonergic receptor blocking drugs had little to no effect on TRH-induced prolactin release (Fig . 1) . The administration of adrenergic receptor blockers resulted in a potentiation of the TRH-induced prolactin release (Fig . 2) . Statistical analysis of the data, however, indicated that only phenoxybenzamine plus TRH was statistically significant (P < 0 .05) from TRH administration alone . Discussion The administration of methysergide and cyproheptadine alone resulted in a slight increase in serum prolactin and was in agreement with our previous observations (12) . The lack of an effect on prolactin release of adrenergic receptor blockers at the dose used was also in agreement with our previous observations (12) and those of others (14) . The lack of an effect of cyproheptadine on TRH-induced prolactin release confirms a previous clinical publication (15) but does not agree with a second clinical report in which the drug attenuated the TRH-induced prolactin release
442
TRH-Induced Prolactin Release
Vol . 22, No . 6, 1978
(16) . The reason for the decreased response by the latter investigators is not apparent .
The slightly higher prolactin level induced by TRH when animals were previously injected with adrenergic receptor blockers suggests that high TRH doses stimulate the adrenergic system which in turn blunts TRH-induced prolactin release. The difficulty with this interpretation lies in the observation that both the B blocker propranolol, and the two a blockers, phentolamine and phenoxybenzamine, resulted in a similar potentiation of prolactin release to TRH . In most biological systems there are distinctly different responses to the two classes of adrenergic drugs (17) . We have observed that when these drugs are administered at high doses they all induce some degree of prolactin release (12) . It has been reported that propranolol is capable of dopaminergic blockade in some systems but the exact mechanism is not understood (17) . MacLeod and Lehmeyer have observed that phentolamine could partially block the suppressing effect of dopamine added to AP incubations but propranolol could not (2) . Others have observed in sheep that phenoxybenzamine potentiated arginine induced prolactin release (18) . In view of the recent findings of two functionally and pharmacologically distinct dopamine receptors in the brain of the rat (19) and our findings here and elsewhere (12) of a and ß adrenergic blockers increasing prolactin release in a similar manner, it is suggested that one class of adrenergic blockers (a) may have an action on blocking pituitary dopamine receptors which is only apparent with induced prolactin release and the second class of blockers (ß) may have its action on the central nervous system because of its reported lack of effect directly on the pituitary . Acknowledgements The authors would like to thank Mrs . Cynthia Van De Walle for her expert technical assistance in the performance of the prolactin radioimmunoassay and the National Institute for Arthritis, Metabolism and Digestive Diseases for supplying us with the human prolactin used for iodination and standards. The authors appreciate receiving as a gift the following drugs : methysergide maleate, Sandoz Pharmaceuticals, Hanover, NJ ; cyproheptadine HCl, Merck, Sharp and Dohme Res . Labs ., Rahway, NJ ; propranolol HC1, Ayerst Labs, Inc ., New York, NY ; phentolamine HC1, Ciba Pharmaceutical Co ., Summit, NJ ; phenoxybenzamine HC1, Smith, Kline and French Labs, Philadelphia, PA ; and TRH, Abbott Laboratories, North Chicago, IL . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 .
J . MEITES and J .A . CLEMENS, Vitamins Hormones 30 165-221 (1972) . R.M . MacLEOD and J .E . LEHMEYER, n cr no o 94 1077-1085 (1974) . C .J . SHARR and J .A . CLEMENS, EndocrinnoJ~ X02-1212 (1974) . B . CHODOROFF, G . CHODOROFF, an R . R. GALA, Ex erient~ia 32 773-774 (1977) . o h sioC.S . NICOLL, R .P . FIORINDO, C .T . MAKENNEE, and J.A . PAR3~UNS, tes, trohic Hormones of the H othalamus : Assay and Chemistr , J . 970) . editor pp . - 0 , ams an ns, Ba t more, R.C . VALVERDE, V . CHIEFFO, and S. REICHLIN, Endocrinology 91 982-993 (1972) . C .Y . BOWERS, H .G . FREISEN, P . HWANG, H .J . GU D , an K . OLKERS, Biochem. Bio h s . Res . Commun . 45 1033-1041 (1971) . D . M. AWSON and R .R . GALA, J . Endocr . 66 151-157 (1975) . R .R . GALA, M.G . SUBRAMANIAN, T.T.-TE-TE-K, and S . JAQUES, Jr ., Horm . Res . 7 118-128 (1976) . R .R . GALA and S. JAQUES, Jr ., Endocr . Res . Commun . _Z 95-108 (1975) . RS, an . JAQUES, Jr ., Experientia R .R . GALA, M.G . SUBRAMANIAN, J. A . 32 941-942 (1976) . R .R . GALA, M.G . SUBRAMANIAN, J.A . PETERS, and D.R . PIEPER, Life Sci . 20 631-638 (1977) .
Vol . 22, No . 6, 1978 13 .
TRH-Induced Prolactin Release
443
W .J . DIXON and F .J . MASSEY, Jr ., Introduction to Statistical Analysis (p . 163) 2nd Edition, McGraw-Hill, N (1968) . 14 . J .A . BOARD, R .J . FIERRO, A .J . WASSERMAN, and A .S . BHATNAGAR, Am . J . Obst . & G n . 127 285-287 (1977) . 15 . .FERRARI, A . PARACCHI, M . RONDENA, P . BECK-PECCOZ, and G . FAGLIA, Clin . Endocr . 5 575-578 (1976) . 16 .0 .EC GGÉ, A .D . ROGOL, M .M . VARMA, and R .M . BLIZZARD, J . Clin . Endocr . Metab . 4 4 210-213 (1977) . 17 . . iTICKURSON and B . COLLIER, The Pharmacological Basis of Thera eutics L .S . Goodman and A . Gilman, eds . 5th edition pp . 533-564 Macmillan Co ., London (1976) . 18 . S .L . DAVIS and M .L . BORGER, Endocrinology 92 303-309 (1973) . 19 . A .R . COOLS, Nonstriatal Domaine Neurons, É. Costa and G .L . Gessa, eds . _Adv . Biochem . Psychopharm . Vol . 6 pp . 215-226) Raven Press, NY (1977) .
Life Sciences Vol . 22, pp . 439-444 Printed in the 1T .S .A .
INFLUENCE OF SEROTONERGIC AND ADRENERGIC ANTAGONISTS ON TRH-INDUCED PROLACTIN RELEASE IN THE MONKEY Richard R . Gala, Judith A . Peters, David R . Pieper, and Marappa G . Subramanian Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201 . (Received in final form September 29, 1977) Summary The influence of methysergide, cyproheptadine and SQ 10,631 (serotonergic receptor blockers) at the dose of 35 jig/kg, 50 ug/kg and 5 mg/kg, respectively, and propranolol, phentolamine and phenoxybenzamine (adrenergic receptor blockers) at the dose of 1 mg/kg on TRHinduced prolactin release was studied in sexually mature female monkeys . The serotonergic antagonists had no effect on TRH-induced prolactin release . Both ß and a adrenergic antagonists gave a similar potentiation of the TRH-induced prolactin response but only phenoxybenzamine plus TRH was statistically different (P < 0 .05) from TRH alone . The effect of the adrenergic receptor blockers is believed to be due to actions on dopamine receptors . The regulation of prolactin is under the control of a hypothalamic inhibiting factor (1) . Considerable evidence indicates that this factor may be dopamine (2-4) . There is also evidence that a prolactin stimulatory factor is present in the hypothalamus (5,6) and that thyrotropin releasing hormone (TRH) releases prolactin as well as thryotropin (7) . We have observed in rats and monkeys that high doses of TRH induce lower increments of prolactin release than do low doses (8,9) . One possible explanation for this is that the high doses of TRH stimulate biogenic amines that are inhibitory to prolactin release . The present study was undertaken to test this possibility using sexually mature female monkeys injected with serotonergic and adrenergic receptor blockers followed by a high dose of TRH . Methods A total of 5 sexually mature regularly cycling female monkeys (3 Macaca mulatta and 2 Macaca fascicularis) were utilized in this study . The animals were sampled at weekly intervals under ketamine anesthesia which does not influence basal prolactin levels (9-11) . Blood samples were obtained from either the femoral or tail vein using an indwelling 25 gauge butterfly catheter . Following an initial blood sample (0 .6 ml), either serotonergic or adrenergic antagonists were injected . Fifteen minutes later a second blood sample was obtained and 1 ug/kg of TRH was injected intravenously through the catheter . Subsequent blood samples were obtained 15, 30 and 45 minutes after TRH administration . All experiments were performed between 1300 and 1600 hours and the blood allowed to clot overnight at 3°C . The recovered serum was frozen and I Supported by NIH General Research Support Grant No . RR 5384 to Wayne State University School of Medicine . 439
440
TRH-Induced Prolactin Release
METHYSERGIDE
CYPROHEPTADINE
Vol . 22, No . 6, 1978
S010631
a
c v t u
S su0 n. E N
Time (min)
FIG . 1 Influence of serotonergic receptor antagonists on TRH-induced prolactin release . A . Methysergide ; B . Cyproheptadine ; C . SQ 10,631 (2 chloro2'-[3-(dimethylamino) propyl] thio-cinnamanilide HC1) . All drugs were administered intravenously . Each point represents the results from 5 monkeys . The vertical lines represent the standard error of the mean . stored at -20°C until assayed for prolactin . Serum prolactin levels were determined at two dilutions in duplicate using a primate prolactin radioimmunoassay (12) . The data is expressed as ng/ml change from the initial serum prolactin value when only a single drug was used and from the second prolactin value (15 minutes after the initial blood sample) whenever two drugs were examined . The data was assessed statistically using a two-way analysis of variance (13) with computer assistance and is presented with means and the standard error of the mean . The serotonergic receptor antagonists were : methysergide maleate, 35 u9/kg ; cyproheptadine HCl, 50 jig/kg ; and SQ 10,631 (2-chloro-2'-[3-(dimethylamino) propyl] thio-cinnamanilide, HC1), 5 mg/kg . The adrenergic receptor antagonists were propranolol HC1, 1 mg/kg ; phentolamine HCl,1 mg/kg ; and phenoxybenzamine HC1, 1 mg/kg . All drugs were dissolved in 50% ethanol-saline solution except propranolol which was dissolved in saline . The administration of vehicle alone had no effect on basal prolactin levels . There was no carry-over effect of the drugs from one week to the next on basal prolactin levels .
Vol . 22, No . 6, 1978
PROPRANOLOL /3
TRH-Induced Prolactin Release
PHENTOLAMINE
or
441
PHENOD(YBENZAw11NE or
c0u É W g u E
fA41
FIG . 2 Influence of adrenergic receptor antagonists on TRH-induced prolactin release . A . Propranolol, ß blocker ; B . Phentolamine, a blocker ; C . Phenoxybenzamine, a blocker . Each point represents the results from 5 monkeys . All drugs were administered intravenously . The vertical lines represent the standard error of the mean . Results The prior administration of serotonergic receptor blocking drugs had little to no effect on TRH-induced prolactin release (Fig . 1) . The administration of adrenergic receptor blockers resulted in a potentiation of the TRH-induced prolactin release (Fig . 2) . Statistical analysis of the data, however, indicated that only phenoxybenzamine plus TRH was statistically significant (P < 0 .05) from TRH administration alone . Discussion The administration of methysergide and cyproheptadine alone resulted in a slight increase in serum prolactin and was in agreement with our previous observations (12) . The lack of an effect on prolactin release of adrenergic receptor blockers at the dose used was also in agreement with our previous observations (12) and those of others (14) . The lack of an effect of cyproheptadine on TRH-induced prolactin release confirms a previous clinical publication (15) but does not agree with a second clinical report in which the drug attenuated the TRH-induced prolactin release
442
TRH-Induced Prolactin Release
Vol . 22, No . 6, 1978
(16) . The reason for the decreased response by the latter investigators is not apparent .
The slightly higher prolactin level induced by TRH when animals were previously injected with adrenergic receptor blockers suggests that high TRH doses stimulate the adrenergic system which in turn blunts TRH-induced prolactin release. The difficulty with this interpretation lies in the observation that both the B blocker propranolol, and the two a blockers, phentolamine and phenoxybenzamine, resulted in a similar potentiation of prolactin release to TRH . In most biological systems there are distinctly different responses to the two classes of adrenergic drugs (17) . We have observed that when these drugs are administered at high doses they all induce some degree of prolactin release (12) . It has been reported that propranolol is capable of dopaminergic blockade in some systems but the exact mechanism is not understood (17) . MacLeod and Lehmeyer have observed that phentolamine could partially block the suppressing effect of dopamine added to AP incubations but propranolol could not (2) . Others have observed in sheep that phenoxybenzamine potentiated arginine induced prolactin release (18) . In view of the recent findings of two functionally and pharmacologically distinct dopamine receptors in the brain of the rat (19) and our findings here and elsewhere (12) of a and ß adrenergic blockers increasing prolactin release in a similar manner, it is suggested that one class of adrenergic blockers (a) may have an action on blocking pituitary dopamine receptors which is only apparent with induced prolactin release and the second class of blockers (ß) may have its action on the central nervous system because of its reported lack of effect directly on the pituitary . Acknowledgements The authors would like to thank Mrs . Cynthia Van De Walle for her expert technical assistance in the performance of the prolactin radioimmunoassay and the National Institute for Arthritis, Metabolism and Digestive Diseases for supplying us with the human prolactin used for iodination and standards. The authors appreciate receiving as a gift the following drugs : methysergide maleate, Sandoz Pharmaceuticals, Hanover, NJ ; cyproheptadine HCl, Merck, Sharp and Dohme Res . Labs ., Rahway, NJ ; propranolol HC1, Ayerst Labs, Inc ., New York, NY ; phentolamine HC1, Ciba Pharmaceutical Co ., Summit, NJ ; phenoxybenzamine HC1, Smith, Kline and French Labs, Philadelphia, PA ; and TRH, Abbott Laboratories, North Chicago, IL . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 .
J . MEITES and J .A . CLEMENS, Vitamins Hormones 30 165-221 (1972) . R.M . MacLEOD and J .E . LEHMEYER, n cr no o 94 1077-1085 (1974) . C .J . SHARR and J .A . CLEMENS, EndocrinnoJ~ X02-1212 (1974) . B . CHODOROFF, G . CHODOROFF, an R . R. GALA, Ex erient~ia 32 773-774 (1977) . o h sioC.S . NICOLL, R .P . FIORINDO, C .T . MAKENNEE, and J.A . PAR3~UNS, tes, trohic Hormones of the H othalamus : Assay and Chemistr , J . 970) . editor pp . - 0 , ams an ns, Ba t more, R.C . VALVERDE, V . CHIEFFO, and S. REICHLIN, Endocrinology 91 982-993 (1972) . C .Y . BOWERS, H .G . FREISEN, P . HWANG, H .J . GU D , an K . OLKERS, Biochem. Bio h s . Res . Commun . 45 1033-1041 (1971) . D . M. AWSON and R .R . GALA, J . Endocr . 66 151-157 (1975) . R .R . GALA, M.G . SUBRAMANIAN, T.T.-TE-TE-K, and S . JAQUES, Jr ., Horm . Res . 7 118-128 (1976) . R .R . GALA and S. JAQUES, Jr ., Endocr . Res . Commun . _Z 95-108 (1975) . RS, an . JAQUES, Jr ., Experientia R .R . GALA, M.G . SUBRAMANIAN, J. A . 32 941-942 (1976) . R .R . GALA, M.G . SUBRAMANIAN, J.A . PETERS, and D.R . PIEPER, Life Sci . 20 631-638 (1977) .
Vol . 22, No . 6, 1978 13 .
TRH-Induced Prolactin Release
443
W .J . DIXON and F .J . MASSEY, Jr ., Introduction to Statistical Analysis (p . 163) 2nd Edition, McGraw-Hill, N (1968) . 14 . J .A . BOARD, R .J . FIERRO, A .J . WASSERMAN, and A .S . BHATNAGAR, Am . J . Obst . & G n . 127 285-287 (1977) . 15 . .FERRARI, A . PARACCHI, M . RONDENA, P . BECK-PECCOZ, and G . FAGLIA, Clin . Endocr . 5 575-578 (1976) . 16 .0 .EC GGÉ, A .D . ROGOL, M .M . VARMA, and R .M . BLIZZARD, J . Clin . Endocr . Metab . 4 4 210-213 (1977) . 17 . . iTICKURSON and B . COLLIER, The Pharmacological Basis of Thera eutics L .S . Goodman and A . Gilman, eds . 5th edition pp . 533-564 Macmillan Co ., London (1976) . 18 . S .L . DAVIS and M .L . BORGER, Endocrinology 92 303-309 (1973) . 19 . A .R . COOLS, Nonstriatal Domaine Neurons, É. Costa and G .L . Gessa, eds . _Adv . Biochem . Psychopharm . Vol . 6 pp . 215-226) Raven Press, NY (1977) .