Melatonin binding sites in the ovine brain and pituitary: characterization during the oestrous cycle

The distribution of putative melatonin receptors in the sheep has been investigated using in vitro autoradiography and the high affinity, high specific activity ligand 2-[(125) l]iodomelatonin. A wide distribution of specific labelling was found in both the ovine brain and pituitary gland as previously reported. Several novel areas of binding were also identified in the present study, including a fine layer of labelling at the medial edge of the diagonal band of Broca, the trigeminal nucleus, laminae II and III of the substantia gelatinosa, the molecular layer of the cerebellum as well as a scattered labelling in the pars distalis of the pituitary. There was no evidence of specific labelling in any of the peripheral tissues examined. Characterization studies performed on both neuronal and pituitary melatonin binding sites revealed that binding was time- and temperature-dependent and reversible on addition of 1 μM melatonin. The binding of 2-[(125) l]iodomelatonin was also competitively inhibited by increasing concentrations of 2-iodomelatonin and melatonin. The inhibition constants (K(i) ) estimated for each of these substances were similar for both neuronal and pituitary sites. Saturation studies also revealed similarities between neuronal and pituitary tissues with 2-[(125) l]iodomelatonin binding specifically to a single class of high affinity binding sites. Values for equilibrium constants (K(d) ) were within a range of 28 to 48 pM, and values were found to be not significantly different amongst the four regions of the brain investigated and the pars tuberalis of the pituitary. In contrast, the concentration of 2-[(125) l]iodomelatonin binding sites (B(max) ) ranged from 3 to 218fmol/mg protein and were maximal for the pars tuberalis. Saturation studies on brain and pituitary tissues taken from ewes killed either on the day of oestrus or during the luteal phase of the oestrous cycle, indicated that no differences exist in the affinity or concentration of 2-[(125) l]iodomelatonin binding in any region between the two times of the cycle investigated.