Neuroscience Letters, 110 (1990) 199 203

199

Elsevier Scientific Publishers Ireland Ltd. NSL 06687

Melatonin modulates diacylglycerol and arachidonic acid metabolism in the anterior pituitary of immature rats Jifi Van~6ek and Lutz Vollrath Department of Anatomy, Johannes Gutenberg University, Mainz ( F.R.G. ) (Received 10 August 1989; Revised version received 24 October 1989; Accepted 26 October 1989)

Key words." Melatonin; Luteinizing hormone-releasing hormone; Diacylglycerol; Arachidonic acid; Anterior pituitary; Organ culture In pituitary glands of immature rats prelabeled in vitro with [3H]arachidonic acid, melatonin diminished the luteinizing hormone-releasing hormone (LHRH)-induced increase in [3H]diacylglycerol accumulation as well as [3H]arachidonic acid release from the tissue. Melatonin reduced also LHRH-stimulated incorporation of [3H]glycerol into pituitary [3H]diacylglycerol. The effect was day-time dependent: in the evening experiment melatonin was effective at 0.1 nM concentration while in the morning it had no effect even at 10 nM concentration. The effect of melatonin was also abolished by pretreatment with pertussis toxin. Diacylglycerol and/or arachidonic acid might serve as 2nd messengers transducing the effect of melatonin at the cellular level.

In photoperiodic rodents, the pineal hormone melatonin transduces the effect of photoperiod on seasonal rhythms [3, 8, 9]. Although adult rats are only marginally photoperiodic, melatonin inhibits the reproductive functions and decreases the levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the immature animals [4]. Recently we have described the distribution of high-affinity melatonin binding sites in the rat brain [10, 12]. Anterior pituitary (AP) of neonatal rats had the highest concentration of the binding sites [11]. Since melatonin inhibits the LHreleasing hormone (LHRH)-induced LH release from neonatal rat pituitary cultured in vitro [5, 6], AP could thus be one of melatonin's targets for regulation of reproduction. Melatonin interaction with its receptors has to induce changes in the intracellular messenger system to affect the cell's function. The nature of the second messenger system transducing the melatonin effect in mammals is unknown. Recently, we have found an inhibitory effect of melatonin on cyclic AMP accumulation in anterior pituitary [! 3]. However, it is generally believed that diacylglycerol (DG), arachidonic Correspondence: J. Vanecek. Present address: Institute of Physiology, Czechoslovak Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czechoslovakia. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

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Fig. I. Effect of melatonin on LHRH-induced increase of [3H]DG and [3H]AA accumulation. Rats were killed at 09.00 h. Hemipituitaries were labeled with [3H]AA (22 nM) for 44 h, washed for 2 h and then stimulated with L H R H (1 /~M) for 3 rain. Melatonin (MEL, 1 /~M) was added L0 rain prior to L H R H . Drugs were added as 100 x concentrated solutions. C, controls. Data are expressed as percentage of total radioactivity incorporated in the tissue (dpm incorporated: C, 753986 + 74094; L H R H , 645980 + 51702: L H R H + MEL, 607683 +46864). Values are given as mean + S.E.M. from 3 samples. *Significantly higher than C ( P < 0.05); ~significantly lower than L H R H ( P < 0.05).

acid (AA) and Ca 24 rather than cyclic nucleotides are the 2nd messengers transducing the effect of L H R H [1, 7, 15]. In this study we therefore examined the effect of melatonin on pituitary D G and AA. [3H]Glycerol (spec. act. 40 Ci/mmol) and [3H]arachidonic acid (spec. act. 225 Ci/ mmol) were supplied from NEN Research Products, Du Pont de Nemours GmbH, F.R.G. All other chemicals were from Sigma. Female Sprague-Dawley rats kept on LD 12:12 (light on from 06.00 to 18.00 h) were decapitated at 10-15 days of age. Their anterior pituitaries were dissected, cut in halves and hemipituitaries were cultured (3T~C, 95% 02-5% CO2) in BGJb containing 1% bovine serum albumin. Tissue was placed on a nylon disc to enhance its transfer. Hemipituitaries from the same rat were never in the same treatment group. Following the indicated treatment the glands were frozen on liquid nitrogen and the lipids were extracted to 50/21 of chloroform:methanol (1:2) containing unlabeled standards. Five/tl were counted directly giving the total lipid radioactivity, 10/21 were subjected to thin layer chromatography (TLC) using Merck silica get plates developed in the upper phase of a mixture ofethylacetate/isooctane/acetic acid/water (140:80:25:120). The Rr values for triglycerides, diacylglycerol and arachidonic acid were 0.94, 0.86 and 0.77, respectively. The spots were visualized by exposure to iodine vapor, cut off and counted using instant scintillation gel. One-way analysis of variance (Newman-Keuls test) was used for statistical evaluation.

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Fig. 2. Effect of melatonin on LHRH-induced stimulation of [3H]AA release. Rats were killed at 08.30 h. Pituitaries prelabeled with [3H]AA (2.6 nM) were transferred through a series of 6-rain incubations in medium (200/A) with or without drug. LHRH (1 /IM) was added at time 0, melatonin (I0 nM) 6 min earlier. Aliquots of the media were collected, extracted and radioactive products were separated by TLC. The rate of [3H]AA release is expressed as percent of individual gland release during the 6 min period preceding LHRH addition (time 0; basal release [DPM]: C, 331+22; LHRH, 409__+37; LHRH + MEL, 308_+ 19). *Significantly higher than C ( P < 0.05); asignificantly lower than LHRH (P < 0.05). See the legend to Fig. 1 for details.

In cultured anterior pituitaries prelabeled to an isotopic equilibrium with [3H]AA, addition of LHRH to the incubation medium induced the increase of [3H]diacylglycerot ([3H]DG) and [3H]AA accumulation within 3 min (Fig. 1). Melatonin significantly diminished the increase in [3H]DG accumulation, while [3H]AA accumulation was not significantly affected. However, melatonin significantly decreased [3H]AA release induced by LHRH (Fig. 2). In order to reach isotopic equilibrium [1] the hemipituitaries in the above described experiments were labeled for 2 days. Under these conditions, the changes in the radioactivity reflect mass changes within DG and AA pools. However, the long period of in vitro labeling abolishes all rhythmic inputs thus rendering impossible the study of the daily changes in melatonin effectiveness. In the following experiments we therefore studied the short term incorporation of [3H]glycerol into pituitary DG. LHRH markedly stimulated incorporation of [3H]glycerol into DG (Fig. 3). In the evening experiment, melatonin significantly diminished LHRH-induced increase in DG labeling already at 0.1 nM concentration (Fig. 3B). In the morning experiment, however, melatonin had no significant effect even at 100-fold higher concentration (Fig. 3A). Melatonin's efficacy is thus markedly decreased in the morning which correlates with insensitivity of reproductive system to morning melatonin injections [2, 9]. The daily differences in melatonin efficacy may be due to desensitization induced by endogenous melatonin, which is synthetized during night.

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Fig. 3. Daily changes in the effect of melatonin on [3H]glycerolincorporation into diacytglycerol.A: melatonin 09.10 11.10 h; B: melatonin 15.10-17.10 h. Hemipituitaries were incubated for 2 h in the presence of [3H]glycerol (2.5/tM) and bacitracin (0.5 mg/ml), with or without LHRH and/or melatonin. Values represent the mean + S.E.M. from 4 samples. *Significantlyhigher than controls (P < 0.05); ,1significantly lower than LHRH only (P