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ABSTRACT

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.

ABSTRACT

A new member of the G protein-coupled receptor superfamily has been isolated from an ovine genomic library with a probe generated by the application of the PCR technique, using cDNA synthesized on a mRNA template isolated from the ovine pars tuberalis. This genomic clone encodes a novel receptor of 325 amino acids with seven transmembrane domains. These domains share homology with other members of this family, but the best homology is with the recently cloned human MC-1 (50% in the transmembrane domains) and MC-3 (69% in the transmembrane domains) MSH receptors and the human ACTH (42% in the transmembrane domains) receptor. When this receptor was expressed in Cos7 cells, it was able to bind a potent analogue of α-MSH, [Nle⁴, d-Phe⁷]-α-MSH (NDP-MSH), with high affinity. This binding could be displaced by pro-opiomelanocortinderived and related peptides, with the order of potency NDP-MSH>α-MSH=ACTH>β-MSH and with no effect of γ-MSH, δ-MSH or β-endorphin. The expressed receptor was demonstrated to be functionally coupled to the adenylate cyclase second messenger pathway, with α-MSH, β-MSH and ACTH stimulating cyclic AMP production. The amount of the mRNA for this receptor was found to be very low. The tissue distribution of this receptor could only be observed using the reverse transcription-PCR technique and the receptor was found to be present in a number of somatic tissues. These data indicate that this is a new and distinct member of the melanocortin receptor family.

ABSTRACT

The effect of aluminium fluoride (AlF₄ ⁻) has been studied on inositol phosphate accumulation, calcium mobilization, cyclic AMP production and [2-¹²⁵I]iodomelatonin binding in ovine pars tuberalis cells. These cells have high-affinity receptors for, and respond to, melatonin through inhibition of forskolin-stimulated adenylate cyclase. In the presence of 10 mm LiCl, AlF₄ ⁻ stimulated the net accumulation of inositol monophosphate and inositol bisphosphate. Consistent with these findings, AlF₄ ⁻ increased intracellular calcium; although this response was attenuated in calcium-depleted medium, indicating that the calcium response comprises both intracellular and extracellular components. Melatonin was ineffective on either basal or AlF₄ ⁻-stimulated turnover of inositol phosphates. In concordance with the inositol phosphate response, melatonin had no effect on either the AlF₄ ⁻-stimulated or the basal calcium levels. AlF₄ ⁻ blocked the increase in cyclic AMP stimulation by l μm forskolin, being as effective as melatonin, achieving approximately 90% inhibition. AlF₄ ⁻ also attenuated the binding of [2-¹²⁵I]iodomelatonin to ovine pars tuberalis membranes by 15%. At the concentration used, these results are consistent with the interpretation that AlF₄ ⁻ activates many G protein-mediated responses, and thus imply that the inhibitory pathway for cyclic AMP predominates over the stimulatory arm, whereas there can only be a stimulatory pathway linked to phosphoinositide metabolism in ovine pars tuberalis cells.