Changes in binding of iodomelatonin to membranes of Leydig cells and steroidogenesis after prolonged in vitro exposure to melatonin

The aim of the present study was to investigate the effects of prolonged ex posure to melatonin (MLT) on the binding of iodomelatonin to membranes of r at Leydig cells and the subsequent modulation of testosterone and cyclic ad enocine monophosphate (cAMP) secretion from these cells by MLT itself, Leyd ig cells were Percoll-purified from adult rats and cultured in vitro with M LT (1-100 nmol/L) for 16 h. Binding assays with 2((125)-I)iodomelatonin wer e then performed; moreover, testosterone and cAMP secretion during an acute challenge with lutenizing hormone (LH) (20 mIU/mL for 3 h) was assayed by RIA. As a result of prolonged MLT administration, a decrease in maximum bin ding density (B-max) and equilibrium dissociation constant (K-d) of the bin ding of 2(I-125)iodomelatonin to purified cell membranes was noted. Higher testosterone and cAMP secretion during LH challenge were recorded in cells pre-incubated with MLT; notwithstanding, the inhibitory effect of acutely a dministered MLT on LH-challenged secretions was not only retained but also reinforced, as the IC50 was 30% lower in cells pre-treated with the higher concentration of MLT (100 nM), Cycloheximide administration (10 mug/mL for 16 h) did not prevent hyper-sensitization to LH challenge or to acute MLT a dministration on LH challenge. Pertussis toxin (180 ng/mL for 16 h) prevent ed hyper-sensitization to LH, but not to acutely administered MLT. Forskoli n (10 nmol/L) administration abolished either phenomena. in conclusion, pro longed exposure to MLT modulates the secretion of testosterone by cultured rat Leydig cells. Although MLT receptors were reduced, hyper-sensitization to LH challenge and to acutely administered MLT on LH challenge were observ ed with the higher concentration of MLT. Reduction in intracellular cAMP as a result of prolonged administration of MLT, could be the primary cause of both phenomena. On the one hand, reduced cAMP could start re-arrangement o f the G-proteins and thus LH-dependent adenylate cyclase sensitization. On the other hand, reduced cAMP could render the Leydig cells more responsive to MLT itself through a mechanism which does not involve G-protein re-arran gement.