Chloride efflux in unstimulated Leydig cells causes autonomous cAMP production and stimulatory/inhibitory steroidogenesis with an efflux inhibitor

Gonadotropins cause immediate chloride secretion in Leydig cells, which still have an unknown physiological. Thyroid and Leydig cells produce more cAMP and steroids, respectively, under low or chloride-free conditions. Here, we show that chloride efflux mediated by incubation of Mouse Leydig Tumor cells (MLTC-1) in hypotonic or chloride-free isotonic buffers results in more cAMP production without any gonadotropic stimulation. MLTC-1 cells incubated with 0.5 mM diphenylamine-2-carboxylate (DPC), a chloride efflux inhibitor, produced increased amounts of testosterone as chloride ions were substituted by gluconate or sulphate ions under isotonic conditions. There was also an up-regulation of steroidogenic acute regulatory protein and 3β-hydroxysteroid dehydrogenase/isomerase type I mRNAs, without human chorionic gonadotropin. With the exception of cAMP production, 2 mM DPC inhibited all of the above, including the transcription of constitutively expressed cholesterol side-chain cleavage enzyme and the L19 house-keeping gene. Although it was speculated that gonadotropin mediated chloride secretion could aid steroid hormone release, we conclude that the real reason for chloride secretion in Leydig cells may be to “kick start” cAMP production.