The functional significance of the pars tuberalis (PT) of the mammalian adenohypophysis has remained an enigma (1, 2). One view of its function is that it acts as an auxiliary gland to support the endocrine role of the pars distalis (PD) (2), as it has been shown to contain immunocytochemically identifiable thyrotrophs and gonadotrophs (1). Many of the cells of the PT are, however, ultrastructurally unique suggesting an independent function for this tissue. Our recent demonstration that the PT of the rat is a major binding site for the ligand iodomelatonin lends further support to this idea (3). We have utilized the highly specific ligand ['251]melatonin, and have demonstrated that it binds exclusively, with very high affinity, to the PT but not the PD of the adult sheep adenohypophysis. These findings support the conclusion that the PT has a distinct role in relation to melatonin action and seasonal reproduction.The tissue specificity of ['2sII]melatonin binding to the hypothalamic-pituitary complex of the sheep brain has been revealed by in vitro autoradiography. Fig. IA and I B shows the images produced on X-ray film by sagittal sections (20 pm thick) through the sheep brain and pituitary, after incubation with either 50 pM ['251]melatonin alone (Fig. 1~) or in the presence of 1 pM melatonin (Fig. 16). The tissues underlying these images are shown in a section after staining with toluidine blue (Fig. Ic). These results reveal [I 2sI]melatonin labelling is discretely localized on the PT of the adenohypophysis, and that the specific labelling can be completely displaced by excess cold melatonin. No labelling was found in either the P D or the median eminence of the hypothalamus. This result confirms our conclusions reached for the rat (3), and provides further support for the view, that reports which ascribe ['ZSI]melatonin labelling to the median eminence (4, 5) and the anterior lobe of the pituitary (5) may have mistaken these sites for the PT.The binding characteristics of the PT for ['2sI]melatonin were examined using homogenates of ovine PT obtained from sheep slaughtered at an abattoir during their anoestrus period. In excess of 500 PT were used in these experiments. The binding of ['2sI]melatonin was found to be dependent upon membrane concentration, time and temperature of incubation (Fig. 2~, 26). The specific binding of [1251]melatonin was shown to be linearly related to membrane concentration (Fig. 2~) .The time-course of binding was found to differ considerably from those reported for other species (4, 6, 7). At 37 "C binding increases rapidly over the first 2 h before reaching equilibrium between 2 to 4 h, remaining stable for up to 6 h. Binding is also reversible, as seen by the dissociation of [' 2sI]melatonin from PT membrane in the presence of excess cold melatonin (1 pM) (Fig. 26). The dissociation rate constant (k-1) is estimated to be 0.015 min-', and the association FIG. 1 (A) and (B). Images produced on X-ray film by 20 prn thick sagittal sections through the hypothalamic-pituitary...

Primary cultures of ovine pars tuberalis (PT) cells of the pituitary were established to investigate the mode of action of melatonin. The heterogeneous population of cells was shown to bind the radioligand 2-[125I]-melatonin over 72 h in culture, although there was a progressive decline in specific binding with time. In cells cultured for 24 h, forskolin (1 μmol/l) was found to stimulate a 12-fold increase in cyclic AMP accumulation. This response could be inhibited by melatonin in a dose-dependent manner, with an IC50 of approx. 6 pmol/l. However, melatonin did not inhibit basal levels of cyclic AMP. In homogenates of ovine PT, forskolin stimulated a dose-dependent increase in cyclic AMP, although the magnitude of this response was found to be lower than that observed in cells. This response was not inhibited by either 10 nmol or 1 μmol melatonin/l, and was also unaffected by GTP. These results provide the first evidence that the melatonin-binding site on ovine PT, recently characterized using the radioligand 2-[125I]-melatonin, functions as a physiological receptor.

A high-affinity, discretely localized melatonin receptor has been characterized and mapped within the brain and pituitary of the Syrian hamster using the high specific activity ligand [ '251] In mammals, the pineal hormone melatonin is synthesized and secreted into the circulation with a precise nocturnal rhythm (I). The melatonin signal thus acts as an endocrine representation of the dark-phase (2). Melatonin has been shown to influence a variety of processes, including circadian cycles of activity in the rat (3) and seasonal reproductive cyclicity in photoperiodic species such as the sheep and the hamster (1). The mode of action of melatonin is, however, obscure due to the fact that the sites of action of the hormone have remained unidentified. A number of studies have documented relatively lowaffinity melatonin receptors (K, within the nanomolar range) using membrane homogenate receptor assays. The localization of these low-affinity receptors within the brain, pituitary and other tissues is remarkably ubiquitous and, as yet, their physiological significance is unknown (4-9). The widespread distribution found for the low-affinity melatonin receptor contrasts with reports that the photoperiodic effects of melatonin can only be induced by very localized intracerebral administration of the compound (10-12).The use of in vitro receptor-site autoradiography in conjunction with membrane homogenate receptor assays, utilizing the high specific activity ligand [1251]iodomelatonin, has led to the identification of discretely localized melatonin receptors in the brain and pituitary of the foetal and adult hamster (13,14), the adult rat (15-19) and the ovine pituitary (20). Using the same combined approach, we have investigated the distribution and characteristics of a high-affinity melatonin receptor in the brain and pituitary of the adult Syrian hamster, an animal used extensively to study the influence of melatonin on circadian rhythmicity and seasonal reproduction.

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 microM) 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 microM) 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 [125I]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 microM) 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 micrograms/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.(ABSTRACT TRUNCATED AT 400 WORDS)

Bordetella pertussis toxin (islet activating protein, IAP) has been used to investigate the G-proteins involved in mediating the action of the melatonin receptor. Melatonin inhibits iorskolin-stimulated cyclic AMP production in ovine pars tuberalis (PT) cells. In cells treated with IAP for 16 h this response is attenuated in a dose-dependent manner, but not abolished. IAP catalyses the incorporation of [(32) P-ADP]ribose into a 41 kd protein present in PT membranes, but this labelling can be reduced if PT cells are preincubated with IAP for 16 h. Treatment of crude membrane preparations with IAP (20 /ig/ml) suppresses the binding of 2-[(125) l]iodomelatonin by 20%, whereas 1 mM GTP alone reduces binding by 40%, and in combination with IAP its effect is additive (60% inhibition). Therefore, these results indicate that the melatonin receptor acts via two G-proteins, one pertussis toxin-sensitive and the other pertussis toxin-insensitive.