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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 series of N-naphthylethyl amide derivatives were synthesized and evaluated as melatonin receptor ligands. The affinity of each compound for the melatonin receptor was determined by binding studies using [2-125I]iodomelatonin on ovine pars tuberalis membrane homogenates. Structure-activity relationships led to the conclusion that naphthalene is a bioisostere of the indole moiety of melatonin. Moreover it appears that the affinity is strongly affected by the size of the substituent of the nitrogen of the amidic function. Many of these ligands give biphasic dose-response curves which suggests that there may be two melatonin receptor subtypes within the ovine pars tuberalis cells. The replacement of naphthalene by benzofuran or benzothiophene did not strongly alter the affinity for the melatonin receptor. In contrast, the benzimidazole analogue was a poor ligand. Compound 7, the naphthalenic analogue of melatonin, a selective ligand of the melatonin receptor and an agonist derivative, has been selected for clinical development.

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.