Melatonin Receptors on Ovine Pars Tuberalis: Characterization and Autoradiographicai Localization

Abstract

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...

Test of ML23 as an antagonist to the effects of melatonin in the ram

“…In a recent competitive binding study it has been shown that ML23 has a very low potency in inhibiting the binding of 125I-labelled melatonin (0062%) in crude membrane preparations from the hypothalamus of male rats (English & Arendt, 1988). This result has also been confirmed in preparations from the pars tuberalis of sheep (Morgan et al, 1989).…”

Monitoring of Ligand-independent Dimerization and Ligand-induced Conformational Changes of Melatonin Receptors in Living Cells by Bioluminescence Resonance Energy Transfer

“…At equimolar concentrations, the level of BRET detected was independent of the receptor expression levels (data not shown). BRET was observed at receptor levels as low as 20 fmol/mg of protein, a concentration similar to those observed in native tissues such as the retina (70 fmol/mg) (18) and the hypophysal pars tuberalis (40 fmol/mg) (19) indicating that the constitutive homo-and hetero-dimerization detected by BRET did not result from over-expression and could reflect a physiological reality.…”

Melatonin Receptor Sites in the Syrian Hamster Brain and Pituitary. Localization and Characterization Using [125|]lodomelatonin*

“…The pars tuberalis of the pituitary binds ['251]iodomelatonin in the Syrian hamster, the rat (18) and the sheep (20) and is the most intensely labelled region found in all three species (19) (present study and unpublished observation). The influence of photoperiod on the pars tuberalis has previously been shown in the Djungarian hamster where cells in this region exhibit distinct morphological and immunocytochemical changes in animals kept under different light regimes (32, 33).…”

“…(43) and separated by HPLC (18,20) to provide a ligand with close to the maximal specific activity of ca. 2,000 Ci/mmol.…”

“…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)(16)(17)(18)(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.…”