Molecular and Cellular Endocrinology, 75 (1991) 49-56 49 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50 MOLCEL 02411 The role of calcium in luteinizing hormone/human chorionic gonadotrophin stimulation of Leydig cell immunoactive inhibin secretion in vitro B.J.B. Simpson, G.P. Risbridger, M.P. Hedger and D.M. de Kretser Department of Anatomy, Monash University, Melbourne, Victoria 3168, Australia (Received 20 July 1990; accepted 1 October 1990) Key words: Leydig cell; Inhibin, luteinizing hormone-stimulated; Cyclic AMP; Calcium; (Adult rat) Summary The mechanism by which luteinizing hormone (LH) stimulates Leydig cell immunoactive inhibin (I-inhibin) secretion was investigated using Percoll-purified adult rat Leydig cells. Using a maximally stimulating dose of LH (16 ng/ml), Leydig cell I-inhibin secretion was non-detectable at 1-2 h of incubation, but subsequently increased at all time points during a 25 h incubation period. LH stimulated both Leydig cell content and release of I-inhibin. Increasing concentrations of LH stimulated both inhibin and testosterone immunoactivity in the incubation media over a similar dose-response range, with a 2- to 4-fold rise in I-inhibin secretion at maximal doses of LH. Dibutyryl cAMP stimulated testosterone secretion in a manner similar to that of LH, but I-inhibin secretion was less sensitive than testosterone and a significant stimulation was observed only at the highest doses (200-1000 Fg/ml). LH-stimulated I-inhibin secretion was significantly decreased when Leydig cells were incubated in calcium-depleted (0.15 mM Ca2++ 1 mM EGTA) or low [Ca 2+ ] media (0.15 mM) as compared to normal (1.15 mM) or high [Ca z+] (2-5 mM) media. In contrast, LH-stimulated testosterone secretion remained unchanged by altering extracellular [Ca 2+], and although decreased in the presence of EGTA, testosterone secretion remained significantly greater than basal levels. Furthermore both diltiazem and verapamil completely blocked the LH and dibutyryl cAMP-stimulated increase in Leydig cell I-inhibin, but did not reduce either LH or dibutyryl cAMP-stimulated testosterone production to basal levels. We conclude that LH stimulates both I-inhibin synthesis and release by adult rat Leydig cells in culture, by mechanisms involving calcium. Introduction Inhibin is a glycoprotein hormone primarily of gonadal origin, consisting of two disulphide-linked Address for correspondence: Dr. B.J.B. Simpson, Depart- ment of Anatomy, Monash University, Melbourne, Victoria 3168, Australia. partially homologous subunits termed a and fl, which selectively inhibits the secretion of follicle- stimulating hormone (FSH) from the anterior pituitary (de Kretser and Robertson, 1989). Several studies have demonstrated that the Sertoli cell is a site of production of bioactive and immunoactive inhibin (I-inhibin) in the immature (Steinberger and Steinberger, 1976; Le Gac and de Kretser, 1982) and adult testis (Gonzales et al., 1988), and