PROLONGED EXPOSURE TO MELATONIN LEADS TO TIME-DEPENDENT SENSITIZATION OF ADENYLATE-CYCLASE AND DOWN-REGULATES MELATONIN RECEPTORS IN PARS TUBERALIS CELLS FROM OVINE PITUITARY

In photoperiodic mammals, seasonal cycles of growth and reproduction are cued by changes in the duration of the nocturnal profile of secretion of the pineal hormone melatonin. To investigate the likely mode of action of this hormone on target tissues, the effect of prolonged treatment with melatonin on the sensitivity of the adenylate cyclase (AC) system was examined in primary cultures of ovine pars tuberalis (PT) cells. When cells were exposed to melatonin (100 pm or 1 mum) for 16 h, and the hormone was then removed by a series of washes, basal production of cAMP was elevated over that observed in cells not treated with melatonin. Moreover, the rate of accumulation of cAMP after stimulation with forskolin (1 muM) was markedly enhanced in cells previously treated with melatonin compared to that in untreated controls. This sensitization by melatonin of the basal and forskolin-stimulated responses developed gradually and was half-maximal after approximately 8 h of exposure. There was no significant difference between the sensitizing effects of the two melatonin concentrations used. Treatment with melatonin for 24 h reduced the total amount of specific [I-125]iodomelatonin binding in PT cell membranes by 30-50%. However, over the same period there was no reduction in the ability of a maximal (1 muM) concentration of melatonin to inhibit forskolin-stimulated cAMP production, indicating the presence of an excess capacity of melatonin receptors in cultured PT cells. Nevertheless, treatment with melatonin for 16 h did result in a 10-fold increase in the IC50 for the inhibition by melatonin of forskolin-stimulated cAMP production. The enhancement of cAMP production after prolonged treatment with melatonin was not masked by the inclusion of isobutylmethylxanthine (1 mm) during the subsequent challenge with forskolin, suggesting that sensitization was not due to a reduction in the activity of cAMP-phosphodiesterase. In control cells, aluminium fluoride caused an inhibition of forskolin-stimulated cAMP production. Prolonged treatment with melatonin abolished the inhibitory effect of aluminium fluoride, suggesting that treatment with melatonin caused a shift in the net balance between the G-protein-mediated stimulatory and inhibitory influences on the AC system. The sensitization of AC was not blocked by the inclusion of cycloheximide (10 mug/ml) during prolonged exposure to melatonin, suggesting that de novo protein synthesis is not a requirement for this effect of the hormone. These results constitute the first demonstration of an independent action of melatonin on ovine PT cells that is dependent upon the duration of the endocrine stimulus. As such, their significance is discussed in the context of photoperiodic time measurement.