Inhibitton of prolactin release by stimulation of presynaptic serotonin autoreceptors

Life Sciences, Vol. 31, pp. 2641-2646 Printed in the U.S.A.

Pergamon Press

INHIBITION OF PROLACTIN RELEASE BY STIMULATION OF PRESYNAPTIC SEROTONIN AUTORECEPTORS James A. Clemens and Michael E. Roush Department of CNS/Endocrine Research The L i l l y Research Laboratories Eli L i l l y and Company, Indianapolis, Indiana 46285 (Received in final form August 26, 1982)

Summary The effects of5-methoxy-N,N-dimethyltryptamine (5-MeODMT), a serotonin agonist with a preferential action on presynaptic autoreceptors, on prolactin release in male rats was determined. Basal serum prolactin levels were not altered after administration of 1.0, 2.0, 5.0, lO.O or 20.0 mg/kg of 5-MeODMT. Pretreatment with 5-MeODMT reduced prolactin release by agents that depend on serotonergic neurotransmission for part of t h e i r prolactin release stimulation. Prolactin release in response to L-5hydroxytryptophan (5-HTP) or morphine was s i g n i f i c a n t l y reduced by pretreatment of the rats with 5-MeODMT. The results of this experiment indicate that 5-MeODMT acts as a presynaptic serotonin autoreceptor stimulant and not as a postsynaptic serotonin agonist on the neuronal systems that control prolactin release. Serotonin has been reported to have a stimulatory influence on prolactin release ( I - 3 ) . Serotonin agonists have been shown to stimulate prolactin release, and their action can be antagonized with serotonin antagonists (3-6). The stimulatory influence of serotonin on prolactin release is believed to be mediated through the release of a prolactin releasing factor (7). The major serotonergic input to the hypothalamus is from the midbrain raphe nuclei (8). Elimination of the serotonergic input to the hypothalamus by the process of surgical deafferentation blocks the a b i l i t y of fluoxetine, a serotonin uptake i n h i b i t o r , to potentiate prolactin release by L-5-hydroxytryptophan (5-HTP) (9). After deafferentation, the serotonin nerve terminals degenerate thereby removing the sites in which uptake occurs. Lesions of the dorsal raphe reduce prolactin release, while electrical stimulation of the dorsal raphe increases prolactin release (lO). Thus, various manipulations of the serotonergic neuronal systems have been reported to a l t e r prolactin release in a consistent manner. One way that serotonergic neuronal a c t i v i t y can be altered is by stimulation or inhibition of autoreceptors located on serotonin neurons. Presynaptic autoreceptors are involved in the modulation of the release of several neurotransmitters in the central nervous system ( l l , 1 2 ) . 5-MethoxyN,N-dimethyltryptamine (5-MeODMT) decreases serotonin turnover in the CNS (13), and i t exerts a powerful inhibitory influence on serotonin neurons of the midbrain raphe nuclei when given systemically (14). After microionto~horetic application of 5-MeODMT to serotonergic neurons of the midbrain raphe and to neurons of the lateral geniculate body and the amygdala which a l l receive dense serotonergic innervation, i t was found that 5-MeODMT possessed a 0024-3205/82/232641-06503.00/0 Copyright (c) 1982 Pergamon Press Ltd.

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four-fold greater a b i l i t y to stimulate serotonin autoreceptors than to stimulate postsynaptic serotonin receptors (15). 5-MeODMT was reported to increase serum prolactin levels in normal male rats (16) and in male rats pretreated with the serotonin neurotoxin, 5,7dihydroxytryptamine (17). In both of the above studies 5-MeODMT was assumed to be a postsynaptic serotonin agonist. Recently, repeated administration of 5-MeODMT every three hours for a total of four injections was shown to potentiate the prolactin-releasing properties of serotonin agonists including 5-MeODMT (18). The potentiation of prolactin release was thought to be due to the formation of supersensitive postsynaptic serotonin receptors. I f 5-MeODMT p r e f e r e n t i a l l y stimulates presynaptic autoreceptors on serotonin neurons, then i t would be expected to interfere with the action of substances, the effects of which, are mediated through serotonergic neurotransmission. One such substance is 5-HTP. Small doses of 5-HTP are taken up into serotonin neurons, decarboxylated and released as serotonin. Another example is opiates. Opiates are believed to stimulate prolactin release, at least in part, through serotonin neurons (19). Therefore, a presynaptic action of 5MeODMT would be expected to diminish the effectiveness of both 5-HTP and opiates as prolactin releasers. The present study was performed to test the above hypothesis, and to compare the effects of 5-MeODMT with those of known serotonin agonists. Methods

Adult male Sprague-Dawley derived rats 3 months of age were purchased from Harlan Industries, Cumberland, Indiana. The rats were housed under controlled temperature (24 • l ° C) and lighting (Vita Lights, 14 hrs. l i g h t ) . All rats were allowed to adapt to our animal room conditions for about lO days before the experiments were performed. The effect of 5-MeODMT on prolactin release was determined by injecting various doses of 5-MeODMT, i . p . , and collecting blood by decapitation 30 min l a t e r . Each treatment group contained lO rats. The effect of 5-MeODMT on the L-5-hydroxytryptophan (5-HTP) and morphine induced prolactin release was determined by pretreating the rats with either 5 or lO mg/kg of 5-Me-ODMT, i . p . , followed by either 5-HTP (20 mg/kg, i . p . ) or morphine sulfate (lO mg/kg, i . p . ) 15 min l a t e r . The rats were decapitated 25 min after 5-HTP or morphine sulfate. To demonstrate the effect of postsynaptic serotonin agonists on prolactin release male rats received single i . p . injections of l-(m-trifluoromethylphenyl)-piperazine (TMPP), m-chlorophenylpiperazine (CPP) or quipazine. The rats were decapitated 30 minutes after treatment. After decapitation trunk blood was collected and allowed to clot at 4° C, after which i t was centrifuged. After centrifugation the serum was collected and assayed for prolactin by radioimmunoassay with reagents supplied by Dr. A. Parlow, Harbor General Hospital. Serum levels of prolactin were expressed as ng NIAMDD-prolactin-RP-I per ml. Quipazine was synthesized in the L i l l y Research Laboratories, and the other chemicals were purchased from Aldrich Chemical Co. Results The results of the f i r s t experiment indicate that 5-MeODMT did not alter serum prolactin levels at any dose tested (Table l ) .

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Table 2 shows the e f f e c t of pretreatment with 5-MeODMT on the a b i l i t y of 5-HTP and morphine to release p r o l a c t i n . 5-MeODMTadministration at a dose of 5 mg/kg c l e a r l y antagonized the release of p r o l a c t i n by morphine, and a dose of I0 mg/kg was required to antagonize the release of p r o l a c t i n by 5-HTP. All of the serotonin agonists (TMPP, CPP and quipazine) stimulated prolact i n release (Table 3). TMPP and CPP were more potent p r o l a c t i n releasers than quipazine. TABLE 1 Effects of 5-Methoxy-N,N-Dimethyltryptamine (5-MeODMT) on Prolactin Release in Male Rats a Dose of (5-MeODMT~ (mg/kg) 0 1.0 2.0 5.0 I0.0 20.0

Serum Prolactin Levels (ng/ml) Exp. 1 Exp. 2 14.4 ± 1.6 b 12.1 * 2.0 12.6,1.4 14.3"1.1 16.8 * 3.4 23.0*6.7

11.0 ± 2.5 7.5 ± 1.4 10.2,1.4 12.6"2.7 17.4 * 4.9 14.5,2.5

aTen rats were used on each dose. bMean * standard e r r o r . Discussion The f a i l u r e of 5-MeODMT to stimulate p r o l a c t i n release, even when administered at high doses, indicates that t h i s compound does not act as a postsynaptic serotonin agonist on the neuronal system that controls p r o l a c t i n r e lease. This study does not confirm some of the e a r l i e r reports on the prolact i n releasing e f f e c t s of 5-MeODMT (16-18), although in two of these studies (16,18) the rats were decapitated 15 min a f t e r 5-MeODMT treatment. In the present study, i f p r o l a c t i n levels were elevated at 15 min a f t e r 5-MeODMT treatment, they must have returned to baseline by 30 min. I n t e r e s t i n g l y , the behavioral syndrome believed to be due to a c t i v a t i o n of postsynaptic receptors subsided by 30 min a f t e r treatment. Thus, by 30 min a f t e r treatment with 5-MeODMT, the primary e f f e c t of the compound may be that of stimulation of presynaptic autoreceptors, and the period of postsynaptic a c t i v a t i o n may be very b r i e f . While the action of 5-MeODMT on p r o l a c t i n release c e r t a i n l y does not p a r a l l e l that of the postsynaptic serotonin agonists reported here, i t s a c t i v i t y is consistent with that expected from a presynaptic autoreceptor stimulant. Evidence for t h i s compound being a p r e f e r e n t i a l stimulant at serotonin presynaptic autoreceptors has been reported (14,15). I t is known that administration of 5-HTP can displace v e s i c u l a r catecholamines at the same time that i t increases serotonin neuronal function (20) thereby making i t d i f f i c u l t to i n t e r p r e t the mechanism of 5-HTP action. Howe v e r , the dose of 5-HTP used in t h i s study is below the minimal e f f e c t i v e dose f o r producing a change in dopamine turnover (21). The antagonism by 5-MeODMT of the release of p r o l a c t i n in response to 5-HTP indicates t h a t , at the dose of 5-HTP used in t h i s study (20 mg/kg), the formation of serotonin from 5-HTP must have been from uptake and decarboxylation in serotonin neurons. This is

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TABLE 2 E f f e c t s o f 5-Methoxy-N,N-Dimethyltryptamine (5-MeODMT) Pretreatment on P r o l a c t i n Release in Response to L-5-Hydroxytryptophan (5-HTP) and Morphine Group and Treatment Schedule Time 0 15 min I. 2. 3. 4. 5. 6. 7. 8.

Saline 5-MeODMT (5 mg/kg) 5-MeODMT (5 mg/kg) 5-MeODMT (5 mg/kg) 5-MeODMT ( I 0 mg/kg) 5-MeODMT (lO mg/kg) Saline Saline

Saline Saline 5-HTP Morphine Saline 5-HTP 5-HTP Morphine

Serum P r o l a c t i n Levels (nglml) 17.9 28.6 47.6 71.2 25.0 22.3 68.7 130.6

• 1.8 a ± 6.3 ± II.6 b ± 16.3b, e ± 9.0 • 3.5 d * 16.1 b ± 1.8 c

aMean ± standard e r r o r . vs group 1 Cp<.O01 vs group l vs group 7 ep<.05 vs group 8

~p<.05 p<.05

TABLE 3 E f f e c t s of Serotonin Agonists on P r o l a c t i n Release Serum P r o l a c t i n Level (ng/ml)

Treatment Saline Quipazine Quipazine TMPPb TMPP CPPc CPP

(I0 (40 ( 5 (I0 ( 5 (I0

12.1 25.9 50.3 24.7 53.2 30.9 89.3

mglkg) mg/kg) mg/kg) mg/kg) mg/kg) mg/kg)

i 2.5 a * 4.3 d ± 9.2 e ± 4.1 d * 8.5 e ± 5.0 d ± I0.5 e

aMean m standard e r r o r . bTMPP = l - ( m - t r i f l u o r o m e t h y l p h e n y l ) - p i p e r a z i n e . cCpP : m - c h l o r o p h e n y l p i p e r a z i n e . dp<.05 vs s a l i n e c o n t r o l . ep<.Ol vs s a l i n e c o n t r o l .

in agreement w i t h e a r l i e r work showing t h a t raphe l e s i o n s blocked the e f f e c t of 5-HTP on p r o l a c t i n release (22). The r e d u c t i o n by also is in agreement of p r o l a c t i n release through s e r o t o n e r g i c

5-MeODMT o f the morphine-induced release of p r o l a c t i n w i t h a p r e s y n a p t i c a c t i o n f o r 5-MeODMT. The s t i m u l a t i o n by morphine has been reported to be mediated in part neurons (19).

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5-MeODMT at the doses used in this study, produces a behavioral syndrome similar to that produced by 5-HTP administration to rats pretreated with monoamine oxidase inhibitors (23). The syndrome is characterized by the presence of tremor, r i g i d i t y , s t i f f t a i l , forepaw treading and head weaving. This a c t i v i t y has been attributed to stimulation of postsynaptic serotonin receptors. However, more than one postsynaptic serotonin receptor has been reported to exist in the brain (24), and not all central serotonin agonists produce the behavioral syndrome. Thus, i t is feasible that 5-MeODMTmay have stimulated the postsynaptic receptors involved in the behavioral syndrome but had no influence on the postsynaptic serotonin receptors involved in the stimulation of prolactin release. Perhaps other CNS postsynaptic serotonin receptors exist which are incapable of being stimulated by 5-MeODMT, or that 5-MeODMT stimulates only a particular subset of serotonin postsynaptic receptors. On the other hand, the waning of the behavioral syndrome at 30 min may indicate that the postsynaptic effects are terminated by that time. I f this were the case, our results indicate that the presynaptic effects of 5-MeODMT are of a longer duration. Because of the doubt that exists regarding the a b i l i t y of the peripheral serotonin antagonists to block central serotonin receptors (25), th~ presynaptic autoreceptor stimulating a c t i v i t y of 5-MeODMTmay be of use in investigating the role of serotonin in several of the physiological conditions where prolactin levels are elevated. References 1. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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