Autoradiographic anomaly in 125I-melatonin binding revealed in ovine adrenal

Conventional in vitro autoradiographical techniques have been used to screen a number of ovine peripheral tissues for the presence of specific 2-[125I]iodomelatonin binding sites. Intense specific labelling (defined as that displaced by 1 microM melatonin) was observed in the adrenal cortex, and less intense specific binding in the spleen. Subsequent attempts to characterize these adrenal binding sites using in vitro binding assays were unsuccessful. The level of specific binding to both crude membranes and to dispersed whole cell preparations was negligible, and was not correlated with increasing protein or cell concentration. Using quantitative in vitro autoradiography, melatonin was found to competitively inhibit 2-[125I]iodomelatonin binding to sections of the adrenal cortex, but with a considerably lower affinity (IC50 of 1 microM) than that obtained for the ovine pars tuberalis (IC50 of 150 pM) under identical conditions. These results suggest that under the conditions of in vitro autoradiography, 2-[125I]iodomelatonin binds to a finite number of non-receptor sites which are restricted to the ovine adrenal gland. The ability to visualize these sites in the present study may arise through the use of an inappropriately high excess of unlabelled melatonin (1 microM). Consequently to avoid this potential problem, non-specific labelling should perhaps more appropriately be defined as binding in the presence of a concentration of melatonin at approximately 100-fold the value of the Kd (i.e. 10 nM).