Store-operated calcium influx and stimulation of steroidogenesis in rat leydig cells: Role of Ca2+ -activated K+ channels

This study evaluates the role of internal calcium store depletion in the ac tivation of ionic fluxes and steroidogenesis in adult rat Leydig cells. Tha psigargin and cyclopiazonic acid, two inhibitors of Ca2+-adenosine triphosp hatase of internal Ca2+ stores induced a dose-dependent rise in intracellul ar Ca2+ concentrations following kinetics that would not be expected if the calcium rise was dependent only on internal calcium store depletion, but i t was in keeping with the presence of calcium influx from the external medi um. In fact, chelation of external calcium with EGTA during the plateau pha se reduced the intracellular calcium concentration to basal levels. When ad ded in calcium-free medium, thapsigargin and cyclopiazonic acid still induc ed a rise in the intracellular calcium concentration that was transient, an d when calcium was added back to the medium, a rapid and sustained intracel lular calcium increase was observed. Thapsigargin and cyclopiazonic acid in duced a dose-dependent rise in testosterone secretion in the presence and a bsence of calcium in the external medium, although in calcium-free medium t his stimulatory effect was lower. Leydig cell plasma membrane potential mon itoring demonstrated that thapsigargin and cyclopiazonic acid induced first a rapid hyperpolarization, followed by a sustained depolarization phase th at was reversed by the addition of the calcium-chelating agent EGTA. In the absence of calcium in the external medium the first phase of hyperpolariza tion was still present, but it was not followed by plasma membrane depolari zation but by the slow return of plasma membrane potential to resting level s. The readdition of calcium to the external medium induced the rapid plasm a membrane depolarization. Plasma membrane hyperpolarization was completely abolished by Leydig cell preincubation with the K+ channel blockers tetrae thylammonium and charybdotoxin. Leydig cell preincubation with K+ channel i nhibitors reduced the thapsigargin-stimulated Ca2+ influx from the external medium and testosterone secretion. These results suggest that internal Ca2 + stores depletion in rat Leydig cells induces a rise in intracellular Ca2, determining important plasma membrane potential variations that influence testosterone secretion.