0013-7227/78/0102-0001$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 102, No. 1 Printed in U.S.A.
Neuropharmacological Regulation of Episodic Growth Hormone and Prolactin Secretion in the Rat JOSEPH B. MARTIN,* DOMINIQUE DURAND,f WENDY GURD, GAIL FAILLE, JUDY AUDET, AND PAUL BRAZEAU^ Division of Neurology, Department of Medicine, The Montreal General Hospital and Department of Neurology and Neurosurgery, The Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada ABSTRACT. The effects on GH and PRL secretion of several pharmacological agents known to modify central neurotransmitter action were determined in unanesthetized male rats. Phenoxybenzamine, an a-adrenergic blocker (5 mg/kg iv), abolished episodic GH secretion and caused elevation of serum PRL levels. Propranolol, a /?-adrenergic blocker (5 mg/kg iv), had no effect on GH secretion and caused a small but significant depression in PRL levels. Parachlorophenylalanine methyl ester, an inhibitor of tryptophan hydroxylase (300-350 mg/kg ip), resulted in significant inhibition of GH pulsatile secretion and suppressed PRL levels. Methysergide hydrogen maleinate (25 mg/kg iv), a ser-
otonin receptor antagonist, also inhibited GH secretion, but produced a transient stimulation in PRL levels. Atropine sulfate (2 mg/kg iv) caused significant suppression in GH secretion, but had no effect on PRL. Picrotoxin, a y-aminobutyric acid antagonist, in a subconvulsive dose of 1-3 mg/kg iv, also depressed GH episodic secretion but did not affect PRL levels. These results indicate that several neurotransmitters, i.e., norepinephrine, serotonin, acetylcholine, and Y-aminobutyric acid, found in high concentration in the hypothalamus, influence GH and PRL secretion. (Endocrinology 102: 106,1978)
T
HERE is considerable evidence that certain central nervous system neurotransmitters, notably dopamine, norepinephrine, and serotonin exert an influence on GH secretion via effects on the brain (see References 1 and 2 for review). Most studies directed toward clarification of the role of these substances have depended upon pharmacological stimulation of GH secretion or block of such stimulation by other pharmacological agents. For example, L-dopa, apomorphine, clonidine, and 5-hydroxytryptophan are reported to stimulate GH secretion in states of low basal secretion in several species (2). On the other hand, GH release in man induced by insulin hypoglycemia, arginine, L-dopa, exercise, and
vasopressin is blocked by prior administration of phentolamine, an a-adrenergic blocker (2), and insulin-induced GH secretion is also abolished by serotonin antagonists (3, 4). In contrast, physiological GH secretion in man, as exemplified by sleep-associated GH release, is not affected by chlorpromazine or a-adrenergic blockade (5), and is variably reported to be unaffected (6) or enhanced (7) by serotonin antagonists. The objective of the present experiments was to evaluate systematically the influence of dopamine, norepinephrine, serotonin, and other central nervous system neurotransmitters in regulation of physiological episodic GH secretion in the unanesthetized male rat. Previous studies from our laboratory have documented that GH secretion in undisturbed male rats is characterized by an ultradian Received April 27, 1977. * Research Associate, Medical Research Council of rhythm with GH surges occurring at 3.0- to Canada. To whom requests for reprints should be ad3.5-h intervals (8-10). Individual bursts of GH dressed. f Present address: Laboratoire de Physiologie, U.E.R. secretion result in rapid increases in plasma des Sciences Pharmaceutiques, 4 avenue de l'Observatoire, levels from undetectable (
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FIG. 2. Effect of phenoxybenzamine on GH secretion in four individual rats. Control injection, given on one sample day, had no effect on episodic GH secretion (O O). Arrows and numbers at top of figure show GH values greater than 300 ng/ml. Phenoxybenzamine resulted in inhibition of GH ( • • ) . Arrows below each abscissa indicate time of injection.
levels returned to basal low values before the second dose. This stimulatory effect on PRL was significant when quantitated by planimetry (Table 1).
GH and PRL data from seven animals given 1 or 3 mg/kg who showed no convulsive effects are presented here. GH secretion was significantly inhibited by picrotoxin, whereas PRL secretion was unaffected (Table 1).
Effects ofatropine on GH and PRL secretion Atropine produced significant inhibition of GH secretion (Fig. 3, bottom, and Table 1), but had no effect on PRL. The suppression in GH was evident immediately after administration of the first dose. Effect ofpicrotoxin on GH and PRL secretion The subconvulsive dose of picrotoxin was determined in preliminary experiments by iv administration of the drug in doses ranging from 0.5-5 mg/kg. A dose of 1 mg/kg had no observable effect, whereas 3 mg/kg induced convulsions in three of seven animals. The
Discussion These studies suggest that several CNS neurotransmitters are involved in the regulation of GH and PRL secretion in the rat. The importance of the present experiments is that they provide data on the role of such substances in physiological hormone secretion as opposed to results obtained from pharmacologically induced responses. The results confirm our earlier hypothesis of the importance of a-adrenergic mechanisms in the regulation of episodic GH secretion (11). a-Adrenergic blockade with phenoxybenzamine was effective in most rats in totally
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MARTIN ET AL.
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FIG. 3. Effects of PCPA, MES, and atropine on GH and PRL secretion. PCPA significantly suppressed secretion of both hormones (cf. Table 1). MES inhibited GH and stimulated PRL. Atropine suppressed GH but had no effect on PRL levels.
abolishing GH secretion, whereas /^-blockade with propranolol had no effect. The suppressive effect of phenoxybenzamine was greater than that induced by any other drug. These findings implicate norepinephrine as a neurotransmitter in hypothalamic control of GH secretion and permit speculation that this substance stimulates growth hormone-releasing factor secretion in species as divergent as rat and primate (15). The present studies do not indicate that ^-blockade enhances GH secretion as has been shown to occur in man (2). The role of dopamine in episodic GH secretion appears to be much less important. Willoughby et al. (16) reported that (+)-butaclamol, a specific, potent dopamine antagonist, resulted in only a minor suppression of GH episodic secretion. Pimozide, another dopamine antagonist, has similar effects (Martin,
Endo ' > 1978 Vol 102 i N o l
unpublished data). In both cases, the effects were much less than those noted with phenoxybenzamine in the present experiments. Serotonin has been* frequently implicated in the regulation of GH secretion. Collu et al (17) reported that intraventricular serotonin caused GH secretion in urethane-anesthetized rats. 5-Hydroxy-tryptophan, the precursor of serotonin, is effective in causing GH release when administered systemically to man (18), monkey (19), and rat (20). Cyproheptadine and MES, both serotonin antagonists, inhibit insulin-induced GH secretion in man (3, 4) and block the rise in GH that follows administration of 5-hydroxy-tryptophan in the rat (20). In the present experiments, administration of PCPA, which causes depletion of serotonin in brain (21), and MES, a serotonin receptor antagonist, both had a marked suppressive effect on episodic GH secretion. These data are consistent with a potentiating role of serotonin in physiological GH secretion and are in agreement with previous reports that postulate a role of serotonin in GH stimulation (17, 18). The present data also suggest that acetylcholine and GABA have stimulatory effects on GH secretion. Atropine, a muscarinic cholinergic blocker, and picrotoxin, a GABA antagonist, both resulted in significant GH suppression. These effects were less than that observed with phenoxybenzamine or PCPA. The role of acetylcholine and GABA, both of which are present in considerable concentration in the hypothalamus, in the regulation of physiological GH secretion has not been previously investigated. The effects of the drugs used in the present experiments on PRL secretion are more difficult to interpret. Phenoxybenzamine and MES both caused highly significant stimulation of PRL secretion, whereas PCPA, propranolol, atropine, and picrotoxin had no effect or produced slight suppression of secretion. The rise in PRL after phenoxybenzamine was remarkably large and sustained, an effect greater than that observed after administration of aMT (11), which results in depletion
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PHARMACOLOGY OF GH AND PRL SECRETION
of both norepinephrine and dopamine, or after administration of dopamine receptor blockers such as butaclamol (16) or pimozide (Martin, unpublished results). Several other studies have shown stimulation of PRL secretion after a-adrenergic blockade. Lawson and Gala (22) reported elevation in plasma PRL in female ovariectomized rats after intra-arterial administration of phenoxybenzamine in a dose of 25 mg/kg but not after 5 or 10 mg/kg. Phenoxybenzamine also elevates PRL in sheep (23). However, there is also evidence that a-adrenergic stimulation enhances PRL secretion. We reported previously that clonidine further augmented elevated PRL levels in male rats treated with aMT (11). Fuxe and Hokfelt (24) have shown that activation of noradrenergic receptors by clonidine decreases activity of tuberoinfundibular dopaminergic neurons. It is possible that phenoxybenzamine may have acted by stimulation of a-adrenergic receptors or by blocking other biogenic amine receptors as suggested by Lawson and Gala (22). Propranolol had a small inhibitory effect on PRL in the present studies. This is in agreement with the report of Lawson and Gala (22), who found only minor effects of /?-adrenergic blockade on PRL secretion. In their studies, high doses (50 mg/kg) elicited a modest rise in PRL. These same workers found no effect after atropine administration in doses as high as 50 mg/kg. Grandison et al. (25) similarly found no effect of atropine on PRL in male rats. Serotonin is generally regarded to act via the CNS to stimulate PRL secretion (13). Intraventricular administration of serotonin (26) or systemic administration of the serotonin precursors, tryptophan or 5-OH tryptophan, is reported to cause elevation of PRL in the rat (27). Conversely, inhibition of 5hydroxytryptophan synthesis by PCPA is reported to reduce baseline PRL levels in the male rat (28), a finding confirmed in the present experiments. PCPA also blocks sucklinginduced PRL release in the female rat (29) and the serotonin blocker MES inhibits the rise in PRL that accompanies estrogen injection (30) or stress in the rat (31). Our finding that MES caused transient elevation in PRL
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is similar to the results reported by Lawson and Gala (22) using the serotonin receptor antagonist methiothepin. Thus, the observed stimulatory effect on PRL might be attributed to initial stimulation of serotonin receptors or to other effects such as inhibition of dopamine receptors. There is no evidence available to either confirm or negate these possibilities. Inhibition of dopamine receptors would seem unlikely since dopamine blockade has minimal inhibitory effects on episodic GH secretion (16), whereas in the present experiments, MES produced marked suppression. The fact that depletion of serotonin with PCPA failed to increase PRL secretion argues against an inhibitory role of serotonin in PRL control and provides additional evidence that the MES effect may have been due to initial serotonin receptor activation. Since GH secretion is inhibited and PRL is released by a variety of stresses in the rat (2, 12, 32), consideration of this possibility is important in interpretation of the results with phenoxybenzamine and MES. Against this possibility are the facts that the rats showed no observable behavioral effects from the drugs, the time course of the effects of MES and phenoxybenzamine is different, and most stress effects on PRL secretion are brief, rather than sustained (33). The effect of the injection technique, per se, can be excluded as a factor since none of the control rats showed a PRL response. Although it is not possible to exclude the role of stress entirely, it is likely that the results observed are due to a direct pharmacological effect. Systemic administration of drugs as used in the present experiments provides no evidence with respect to exact site of action of these agents. Dopamine receptors have been postulated to exist on PRL-secreting cells in pituitary (see Reference 12 for review), but no evidence is available to implicate a direct effect of biogenic amines on pituitary GHsecreting cells. The amines have no effect on GH secretion in pituitary monolayer cultures (W. Vale, personal communication; P. Brazeau, unpublished data). Evidence for a neural site of action in GH regulation was provided in earlier experiments that used electrical
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MARTIN ET AL.
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stimulation of various brain sites in pentobarbital-anesthetized rats. In those experiments, GH release after hypothalamic stimulation was not affected by administration of aMT, whereas GH release induced by stimulation of the hippocampus and basolateral amygdala was completely blocked by prior administration of the drug (34, 35). In further studies, it was shown that phenoxybenzamine, an a-adrenergic blocker, but not propranolol, a (3 blocker, also suppressed amygdaloid-induced GH release. PCPA was shown to prevent amygdaloid-induced GH release but had no effect on hypothalamic-mediated GH stimulation. The interpretation given to these results was that relay of electrically induced GH release from extrahypothalamic to hypothalamic regions was dependent upon a-adrenergic and serotonergic mechanisms, whereas hypothalamic-induced GH release was unaffected by these drugs.
Acknowledgments We are grateful to the Medical Research Council of Canada for financial support. The generous provision of radioimmunoassay materials by the NIAMDD is acknowledged. We thank Tina Crossfield for technical assistance and Miss Debbie Davis for secretarial assistance.
10.
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12. 13. 14. 15. 16.
17. 18.
19. 20. 21. 22.
References 1. Frohman, L. A., and M. E. Stachura, Neuropharmacologic control of neuroendocrine function in man, Metabolism 24: 211, 1975. 2. Martin, J. B., Brain regulation of GH secretion, In Martini, L, and W. F. Ganong (eds.), Frontiers in Neuroendocrinology, vol. 4, Raven Press, New York, 1976, p. 129. 3. Bivens, D. H., H. E. Lebovitz, and J. M. Feldman, Inhibition of hypoglycemia-induced growth hormone secretion by the serotonin antagonists cyproheptadine and methysergide, N EnglJMed289: 236, 1973 4. Smythe, G. A., and L. Lazarus, Suppression of human growth hormone secretion by melatonin and cyproheptadine, J Clin Invest 54: 116, 1974. 5. Takahashi, Y., D. M. Kipnis, and W. H. Daughaday, Growth hormone secretion during sleep, J Clin Invest 47: 2079,1968. 6. Malarkey, W. B., and J. R. Mendell, Failure of a serotonin inhibitor to effect nocturnal GH and prolactin secretion in patients with Duchenne muscular dystrophy, J Clin Endocrinol Metab 43: 889, 1976. 7. Mendelson, W. B., L. S. Jacobs, J. D. Reichman, E. Othmer, P. E. Cryer, B. Trivedi, and W. H. Daughaday, Methysergide: suppression of sleep-related growth hormone secretion, J Clin Invest 56: 690, 1975. 8. Tannenbaum, G. S., and J. B. Martin, Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat, Endocrinology 98: 540, 1976. 9. Willoughby, J. 0., J. B. Martin, P. B. Brazeau, and L. P. Renaud, Pulsatile growth hormone: failure to demonstrate a
23. 24.
25. 26. 27. 28.
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30. 31.
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correlation to sleep phases in the rat, Endocrinology 98: 593, 1976. Tannenbaum, G. S., J. B. Martin, and E. Colle, Ultradian growth hormone rhythm in the rat: effects of feeding, hyperglycemia and insulin-induced hypoglycemia, Endocrinology 99: 270, 1976. Durand, D., J. B. Martin, and P. Brazeau, Evidence for a role of a-adrenergic mechanisms in regulation of episodic growth hormone secretion in the rat, Endocrinology 100: 722, 1977. MacLeod, R. M., Regulation of prolactin secretion, In Martini, L., and W. F. Ganong (eds.), Frontiers in Neuroendocrinology, vol. 4, Raven Press, New York, 1976, p. 169. Lu, K. H., and J. Meites, Effects of L-dopa on serum prolactin and PIF in intact and hypophysectomized, pituitary-grafted rats, Endocrinology 91: 868, 1972. Chen, H. J., and J. Meites, Effects of biogenic amines and TRH on release of prolactin and TSH in the rat, Endocrinology 96: 10, 1975. Martin, J. B., Neural regulation of growth hormone secretion, Medical Progress Report, N EnglJMed 288: 1384, 1973. Willoughby, J. O., P. Brazeau, and J. B. Martin, Pulsatile growth hormone and prolactin: effects of (+) butaclamol, a dopaminergic receptor blocking agent, Endocrinology 101: 1298, 1977. Collu, R., F. Fraschini, P. Visconti, and L. Martini, Adrenergic and serotoninergic control of growth hormone secretion in adult male rats, Endocrinology 90: 1231, 1972. Wirz-Justice, A., I. Lancranjan, and W. Piihringer, The effect of a new soluble ester of L-5-hydroxytryptophan on mood, growth hormone and prolactin release in healthy subjects, Proc Vth Int Congr Endocrinol, Hamburg, p. 43 (Abstract), 1976. Chambers, J. W., and G. M. Brown, Neurotransmitter regulation of growth hormone and ACTH in the rhesus monkey: effects of biogenic amines, Endocrinology 98: 420, 1976. Smythe, G. A., J. F. Brandstater, and L. Lazarus, Serotoninergic control of rat growth hormone secretion, Neuroendocrinology 17: 245, 1975. Koe, W. B., and A. Weissman, P-chlorophenylalanine: a specific depletor of brain serotonin, J Pharm Exp Ther 154: 499, 1966. Lawson, D. M., and R. R. Gala, The influences of adrenergic, dopaminergic, cholinergic and serotoninergic drugs on plasma prolactin levels in ovariectomized, estrogen-treated rats, Endocrinology 96: 313, 1975. Davis, S. L., and M. L. Borger, Hypothalamic catecholamine effects on plasma levels of prolactin and growth hormone in the sheep, Endocrinology 92: 303, 1973. Fuxe, K., and T. Hokfelt, Central monoaminergic systems and hypothalamic function, In Martini, L, M. Motta, and F. Fraschini (eds.), The Hypothalamus, Academic Press, New York, 1970, p. 136. Grandison, L., M. Gelato, and J. Meites, Inhibition of prolactin secretion by cholinergic drugs, Proc Soc Exp Biol Med 145: 1236, 1974. Kamberi, I. A, R. S. Mical, and J. C. Porter, Effects of melatonin and serotonin on the release of FSH and prolactin, Endocrinology 88: 1288, 1971. Lu, K.-H., and J. Meites, Effects of serotonin precursors and melatonin on serum prolactin release in rats, Endocrinology 93: 152, 1973. Gil-Ad, I., F. Zambotti, M. O. Carruba, L. Vicentini, and E. E. Miiller, Stimulatory role for brain serotoninergic system on prolactin secretion in the male rat, Proc Soc Exp Biol Med 151: 512, 1976. Kordon, C, C. A. Blake, and C. H. Sawyer, Participation of serotoninergic-containing neurons in the suckling-induced rise in plasma prolactin levels in lactating rats, Neuroendocrinology 13: 213, 1973/74. Caligaris, L. L, and S. Taleisnik, Involvement of neurons containing 5-hydroxytryptamine in the mechanism of prolactin release induced by estrogen, J Endocrinol 62: 25, 1974. Marchlewska-Koj, A., and L. Krulich, The role of central
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PHARMACOLOGY OF GH AND PRL SECRETION monoamines in the stress-induced prolactin release in the rat, Fed Proc 34: 252, 1975. 32. Terry, L. C, J. 0. Willoughby, P. Brazeau, J. B. Martin, and Y. Patel, Antiserum to somatostatin prevents stress-induced inhibition of growth hormone secretion in the rat, Science 192: 565, 1976. 33. Terry, L. C, A. Saunders, J. Audet, J. O. Willoughby, P. Brazeau, and J. B. Martin, Physiologic secretion of growth hormone and prolactin in male and female rats, Clin EndocrinolB: 19S, 1977.
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34. Martin, J. B., The role of hypothalamic and extrahypothalamic structures in the control of GH secretion, In Raiti, S. (ed.), Proceedings of the NIH Symposium on Growth Hormone, NIH Publication no. 74-612,1974, p. 223. 35. Martin, J. B., J. Kontor, and P. Mead, Plasma GH responses to hypothalamic, hippocampal and amygdaloid electrical stimulation: effects of variation in stimulus parameters and treatment with a-methyl-p-tyrosine (aMT), Endocrinology 92: 1354, 1973.
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Vol. 102, No. 1 Printed in U.S.A.
Neuropharmacological Regulation of Episodic Growth Hormone and Prolactin Secretion in the Rat JOSEPH B. MARTIN,* DOMINIQUE DURAND,f WENDY GURD, GAIL FAILLE, JUDY AUDET, AND PAUL BRAZEAU^ Division of Neurology, Department of Medicine, The Montreal General Hospital and Department of Neurology and Neurosurgery, The Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada ABSTRACT. The effects on GH and PRL secretion of several pharmacological agents known to modify central neurotransmitter action were determined in unanesthetized male rats. Phenoxybenzamine, an a-adrenergic blocker (5 mg/kg iv), abolished episodic GH secretion and caused elevation of serum PRL levels. Propranolol, a /?-adrenergic blocker (5 mg/kg iv), had no effect on GH secretion and caused a small but significant depression in PRL levels. Parachlorophenylalanine methyl ester, an inhibitor of tryptophan hydroxylase (300-350 mg/kg ip), resulted in significant inhibition of GH pulsatile secretion and suppressed PRL levels. Methysergide hydrogen maleinate (25 mg/kg iv), a ser-
otonin receptor antagonist, also inhibited GH secretion, but produced a transient stimulation in PRL levels. Atropine sulfate (2 mg/kg iv) caused significant suppression in GH secretion, but had no effect on PRL. Picrotoxin, a y-aminobutyric acid antagonist, in a subconvulsive dose of 1-3 mg/kg iv, also depressed GH episodic secretion but did not affect PRL levels. These results indicate that several neurotransmitters, i.e., norepinephrine, serotonin, acetylcholine, and Y-aminobutyric acid, found in high concentration in the hypothalamus, influence GH and PRL secretion. (Endocrinology 102: 106,1978)
T
HERE is considerable evidence that certain central nervous system neurotransmitters, notably dopamine, norepinephrine, and serotonin exert an influence on GH secretion via effects on the brain (see References 1 and 2 for review). Most studies directed toward clarification of the role of these substances have depended upon pharmacological stimulation of GH secretion or block of such stimulation by other pharmacological agents. For example, L-dopa, apomorphine, clonidine, and 5-hydroxytryptophan are reported to stimulate GH secretion in states of low basal secretion in several species (2). On the other hand, GH release in man induced by insulin hypoglycemia, arginine, L-dopa, exercise, and
vasopressin is blocked by prior administration of phentolamine, an a-adrenergic blocker (2), and insulin-induced GH secretion is also abolished by serotonin antagonists (3, 4). In contrast, physiological GH secretion in man, as exemplified by sleep-associated GH release, is not affected by chlorpromazine or a-adrenergic blockade (5), and is variably reported to be unaffected (6) or enhanced (7) by serotonin antagonists. The objective of the present experiments was to evaluate systematically the influence of dopamine, norepinephrine, serotonin, and other central nervous system neurotransmitters in regulation of physiological episodic GH secretion in the unanesthetized male rat. Previous studies from our laboratory have documented that GH secretion in undisturbed male rats is characterized by an ultradian Received April 27, 1977. * Research Associate, Medical Research Council of rhythm with GH surges occurring at 3.0- to Canada. To whom requests for reprints should be ad3.5-h intervals (8-10). Individual bursts of GH dressed. f Present address: Laboratoire de Physiologie, U.E.R. secretion result in rapid increases in plasma des Sciences Pharmaceutiques, 4 avenue de l'Observatoire, levels from undetectable (
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FIG. 2. Effect of phenoxybenzamine on GH secretion in four individual rats. Control injection, given on one sample day, had no effect on episodic GH secretion (O O). Arrows and numbers at top of figure show GH values greater than 300 ng/ml. Phenoxybenzamine resulted in inhibition of GH ( • • ) . Arrows below each abscissa indicate time of injection.
levels returned to basal low values before the second dose. This stimulatory effect on PRL was significant when quantitated by planimetry (Table 1).
GH and PRL data from seven animals given 1 or 3 mg/kg who showed no convulsive effects are presented here. GH secretion was significantly inhibited by picrotoxin, whereas PRL secretion was unaffected (Table 1).
Effects ofatropine on GH and PRL secretion Atropine produced significant inhibition of GH secretion (Fig. 3, bottom, and Table 1), but had no effect on PRL. The suppression in GH was evident immediately after administration of the first dose. Effect ofpicrotoxin on GH and PRL secretion The subconvulsive dose of picrotoxin was determined in preliminary experiments by iv administration of the drug in doses ranging from 0.5-5 mg/kg. A dose of 1 mg/kg had no observable effect, whereas 3 mg/kg induced convulsions in three of seven animals. The
Discussion These studies suggest that several CNS neurotransmitters are involved in the regulation of GH and PRL secretion in the rat. The importance of the present experiments is that they provide data on the role of such substances in physiological hormone secretion as opposed to results obtained from pharmacologically induced responses. The results confirm our earlier hypothesis of the importance of a-adrenergic mechanisms in the regulation of episodic GH secretion (11). a-Adrenergic blockade with phenoxybenzamine was effective in most rats in totally
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MARTIN ET AL.
110 200 r
PCPA (9)
rGH
• rPRL ATROPINE
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FIG. 3. Effects of PCPA, MES, and atropine on GH and PRL secretion. PCPA significantly suppressed secretion of both hormones (cf. Table 1). MES inhibited GH and stimulated PRL. Atropine suppressed GH but had no effect on PRL levels.
abolishing GH secretion, whereas /^-blockade with propranolol had no effect. The suppressive effect of phenoxybenzamine was greater than that induced by any other drug. These findings implicate norepinephrine as a neurotransmitter in hypothalamic control of GH secretion and permit speculation that this substance stimulates growth hormone-releasing factor secretion in species as divergent as rat and primate (15). The present studies do not indicate that ^-blockade enhances GH secretion as has been shown to occur in man (2). The role of dopamine in episodic GH secretion appears to be much less important. Willoughby et al. (16) reported that (+)-butaclamol, a specific, potent dopamine antagonist, resulted in only a minor suppression of GH episodic secretion. Pimozide, another dopamine antagonist, has similar effects (Martin,
Endo ' > 1978 Vol 102 i N o l
unpublished data). In both cases, the effects were much less than those noted with phenoxybenzamine in the present experiments. Serotonin has been* frequently implicated in the regulation of GH secretion. Collu et al (17) reported that intraventricular serotonin caused GH secretion in urethane-anesthetized rats. 5-Hydroxy-tryptophan, the precursor of serotonin, is effective in causing GH release when administered systemically to man (18), monkey (19), and rat (20). Cyproheptadine and MES, both serotonin antagonists, inhibit insulin-induced GH secretion in man (3, 4) and block the rise in GH that follows administration of 5-hydroxy-tryptophan in the rat (20). In the present experiments, administration of PCPA, which causes depletion of serotonin in brain (21), and MES, a serotonin receptor antagonist, both had a marked suppressive effect on episodic GH secretion. These data are consistent with a potentiating role of serotonin in physiological GH secretion and are in agreement with previous reports that postulate a role of serotonin in GH stimulation (17, 18). The present data also suggest that acetylcholine and GABA have stimulatory effects on GH secretion. Atropine, a muscarinic cholinergic blocker, and picrotoxin, a GABA antagonist, both resulted in significant GH suppression. These effects were less than that observed with phenoxybenzamine or PCPA. The role of acetylcholine and GABA, both of which are present in considerable concentration in the hypothalamus, in the regulation of physiological GH secretion has not been previously investigated. The effects of the drugs used in the present experiments on PRL secretion are more difficult to interpret. Phenoxybenzamine and MES both caused highly significant stimulation of PRL secretion, whereas PCPA, propranolol, atropine, and picrotoxin had no effect or produced slight suppression of secretion. The rise in PRL after phenoxybenzamine was remarkably large and sustained, an effect greater than that observed after administration of aMT (11), which results in depletion
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PHARMACOLOGY OF GH AND PRL SECRETION
of both norepinephrine and dopamine, or after administration of dopamine receptor blockers such as butaclamol (16) or pimozide (Martin, unpublished results). Several other studies have shown stimulation of PRL secretion after a-adrenergic blockade. Lawson and Gala (22) reported elevation in plasma PRL in female ovariectomized rats after intra-arterial administration of phenoxybenzamine in a dose of 25 mg/kg but not after 5 or 10 mg/kg. Phenoxybenzamine also elevates PRL in sheep (23). However, there is also evidence that a-adrenergic stimulation enhances PRL secretion. We reported previously that clonidine further augmented elevated PRL levels in male rats treated with aMT (11). Fuxe and Hokfelt (24) have shown that activation of noradrenergic receptors by clonidine decreases activity of tuberoinfundibular dopaminergic neurons. It is possible that phenoxybenzamine may have acted by stimulation of a-adrenergic receptors or by blocking other biogenic amine receptors as suggested by Lawson and Gala (22). Propranolol had a small inhibitory effect on PRL in the present studies. This is in agreement with the report of Lawson and Gala (22), who found only minor effects of /?-adrenergic blockade on PRL secretion. In their studies, high doses (50 mg/kg) elicited a modest rise in PRL. These same workers found no effect after atropine administration in doses as high as 50 mg/kg. Grandison et al. (25) similarly found no effect of atropine on PRL in male rats. Serotonin is generally regarded to act via the CNS to stimulate PRL secretion (13). Intraventricular administration of serotonin (26) or systemic administration of the serotonin precursors, tryptophan or 5-OH tryptophan, is reported to cause elevation of PRL in the rat (27). Conversely, inhibition of 5hydroxytryptophan synthesis by PCPA is reported to reduce baseline PRL levels in the male rat (28), a finding confirmed in the present experiments. PCPA also blocks sucklinginduced PRL release in the female rat (29) and the serotonin blocker MES inhibits the rise in PRL that accompanies estrogen injection (30) or stress in the rat (31). Our finding that MES caused transient elevation in PRL
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is similar to the results reported by Lawson and Gala (22) using the serotonin receptor antagonist methiothepin. Thus, the observed stimulatory effect on PRL might be attributed to initial stimulation of serotonin receptors or to other effects such as inhibition of dopamine receptors. There is no evidence available to either confirm or negate these possibilities. Inhibition of dopamine receptors would seem unlikely since dopamine blockade has minimal inhibitory effects on episodic GH secretion (16), whereas in the present experiments, MES produced marked suppression. The fact that depletion of serotonin with PCPA failed to increase PRL secretion argues against an inhibitory role of serotonin in PRL control and provides additional evidence that the MES effect may have been due to initial serotonin receptor activation. Since GH secretion is inhibited and PRL is released by a variety of stresses in the rat (2, 12, 32), consideration of this possibility is important in interpretation of the results with phenoxybenzamine and MES. Against this possibility are the facts that the rats showed no observable behavioral effects from the drugs, the time course of the effects of MES and phenoxybenzamine is different, and most stress effects on PRL secretion are brief, rather than sustained (33). The effect of the injection technique, per se, can be excluded as a factor since none of the control rats showed a PRL response. Although it is not possible to exclude the role of stress entirely, it is likely that the results observed are due to a direct pharmacological effect. Systemic administration of drugs as used in the present experiments provides no evidence with respect to exact site of action of these agents. Dopamine receptors have been postulated to exist on PRL-secreting cells in pituitary (see Reference 12 for review), but no evidence is available to implicate a direct effect of biogenic amines on pituitary GHsecreting cells. The amines have no effect on GH secretion in pituitary monolayer cultures (W. Vale, personal communication; P. Brazeau, unpublished data). Evidence for a neural site of action in GH regulation was provided in earlier experiments that used electrical
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MARTIN ET AL.
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stimulation of various brain sites in pentobarbital-anesthetized rats. In those experiments, GH release after hypothalamic stimulation was not affected by administration of aMT, whereas GH release induced by stimulation of the hippocampus and basolateral amygdala was completely blocked by prior administration of the drug (34, 35). In further studies, it was shown that phenoxybenzamine, an a-adrenergic blocker, but not propranolol, a (3 blocker, also suppressed amygdaloid-induced GH release. PCPA was shown to prevent amygdaloid-induced GH release but had no effect on hypothalamic-mediated GH stimulation. The interpretation given to these results was that relay of electrically induced GH release from extrahypothalamic to hypothalamic regions was dependent upon a-adrenergic and serotonergic mechanisms, whereas hypothalamic-induced GH release was unaffected by these drugs.
Acknowledgments We are grateful to the Medical Research Council of Canada for financial support. The generous provision of radioimmunoassay materials by the NIAMDD is acknowledged. We thank Tina Crossfield for technical assistance and Miss Debbie Davis for secretarial assistance.
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12. 13. 14. 15. 16.
17. 18.
19. 20. 21. 22.
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correlation to sleep phases in the rat, Endocrinology 98: 593, 1976. Tannenbaum, G. S., J. B. Martin, and E. Colle, Ultradian growth hormone rhythm in the rat: effects of feeding, hyperglycemia and insulin-induced hypoglycemia, Endocrinology 99: 270, 1976. Durand, D., J. B. Martin, and P. Brazeau, Evidence for a role of a-adrenergic mechanisms in regulation of episodic growth hormone secretion in the rat, Endocrinology 100: 722, 1977. MacLeod, R. M., Regulation of prolactin secretion, In Martini, L., and W. F. Ganong (eds.), Frontiers in Neuroendocrinology, vol. 4, Raven Press, New York, 1976, p. 169. Lu, K. H., and J. Meites, Effects of L-dopa on serum prolactin and PIF in intact and hypophysectomized, pituitary-grafted rats, Endocrinology 91: 868, 1972. Chen, H. J., and J. Meites, Effects of biogenic amines and TRH on release of prolactin and TSH in the rat, Endocrinology 96: 10, 1975. Martin, J. B., Neural regulation of growth hormone secretion, Medical Progress Report, N EnglJMed 288: 1384, 1973. Willoughby, J. O., P. Brazeau, and J. B. Martin, Pulsatile growth hormone and prolactin: effects of (+) butaclamol, a dopaminergic receptor blocking agent, Endocrinology 101: 1298, 1977. Collu, R., F. Fraschini, P. Visconti, and L. Martini, Adrenergic and serotoninergic control of growth hormone secretion in adult male rats, Endocrinology 90: 1231, 1972. Wirz-Justice, A., I. Lancranjan, and W. Piihringer, The effect of a new soluble ester of L-5-hydroxytryptophan on mood, growth hormone and prolactin release in healthy subjects, Proc Vth Int Congr Endocrinol, Hamburg, p. 43 (Abstract), 1976. Chambers, J. W., and G. M. Brown, Neurotransmitter regulation of growth hormone and ACTH in the rhesus monkey: effects of biogenic amines, Endocrinology 98: 420, 1976. Smythe, G. A., J. F. Brandstater, and L. Lazarus, Serotoninergic control of rat growth hormone secretion, Neuroendocrinology 17: 245, 1975. Koe, W. B., and A. Weissman, P-chlorophenylalanine: a specific depletor of brain serotonin, J Pharm Exp Ther 154: 499, 1966. Lawson, D. M., and R. R. Gala, The influences of adrenergic, dopaminergic, cholinergic and serotoninergic drugs on plasma prolactin levels in ovariectomized, estrogen-treated rats, Endocrinology 96: 313, 1975. Davis, S. L., and M. L. Borger, Hypothalamic catecholamine effects on plasma levels of prolactin and growth hormone in the sheep, Endocrinology 92: 303, 1973. Fuxe, K., and T. Hokfelt, Central monoaminergic systems and hypothalamic function, In Martini, L, M. Motta, and F. Fraschini (eds.), The Hypothalamus, Academic Press, New York, 1970, p. 136. Grandison, L., M. Gelato, and J. Meites, Inhibition of prolactin secretion by cholinergic drugs, Proc Soc Exp Biol Med 145: 1236, 1974. Kamberi, I. A, R. S. Mical, and J. C. Porter, Effects of melatonin and serotonin on the release of FSH and prolactin, Endocrinology 88: 1288, 1971. Lu, K.-H., and J. Meites, Effects of serotonin precursors and melatonin on serum prolactin release in rats, Endocrinology 93: 152, 1973. Gil-Ad, I., F. Zambotti, M. O. Carruba, L. Vicentini, and E. E. Miiller, Stimulatory role for brain serotoninergic system on prolactin secretion in the male rat, Proc Soc Exp Biol Med 151: 512, 1976. Kordon, C, C. A. Blake, and C. H. Sawyer, Participation of serotoninergic-containing neurons in the suckling-induced rise in plasma prolactin levels in lactating rats, Neuroendocrinology 13: 213, 1973/74. Caligaris, L. L, and S. Taleisnik, Involvement of neurons containing 5-hydroxytryptamine in the mechanism of prolactin release induced by estrogen, J Endocrinol 62: 25, 1974. Marchlewska-Koj, A., and L. Krulich, The role of central
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PHARMACOLOGY OF GH AND PRL SECRETION monoamines in the stress-induced prolactin release in the rat, Fed Proc 34: 252, 1975. 32. Terry, L. C, J. 0. Willoughby, P. Brazeau, J. B. Martin, and Y. Patel, Antiserum to somatostatin prevents stress-induced inhibition of growth hormone secretion in the rat, Science 192: 565, 1976. 33. Terry, L. C, A. Saunders, J. Audet, J. O. Willoughby, P. Brazeau, and J. B. Martin, Physiologic secretion of growth hormone and prolactin in male and female rats, Clin EndocrinolB: 19S, 1977.
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34. Martin, J. B., The role of hypothalamic and extrahypothalamic structures in the control of GH secretion, In Raiti, S. (ed.), Proceedings of the NIH Symposium on Growth Hormone, NIH Publication no. 74-612,1974, p. 223. 35. Martin, J. B., J. Kontor, and P. Mead, Plasma GH responses to hypothalamic, hippocampal and amygdaloid electrical stimulation: effects of variation in stimulus parameters and treatment with a-methyl-p-tyrosine (aMT), Endocrinology 92: 1354, 1973.
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