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Store-Operated Calcium Influx and Stimulation of Steroidogenesis in Rat Leydig Cells: Role of Ca²⁺-Activated K⁺ Channels

University of Padova, Clinica Medica 3, 35128 Padova, Italy

Address all correspondence and requests for reprints to: Prof. Carlo Foresta, University of Padova, Clinica Medica 3, Via Ospedale 105, 35128 Padova, Italy. E-mail: forestac{at}protec.it

This study evaluates the role of internal calcium store depletion in the activation of ionic fluxes and steroidogenesis in adult rat Leydig cells. Thapsigargin and cyclopiazonic acid, two inhibitors of Ca²⁺-adenosine triphosphatase of internal Ca²⁺ stores induced a dose-dependent rise in intracellular Ca²⁺ concentrations following kinetics that would not be expected if the calcium rise was dependent only on internal calcium store depletion, but it was in keeping with the presence of calcium influx from the external medium. In fact, chelation of external calcium with EGTA during the plateau phase reduced the intracellular calcium concentration to basal levels. When added in calcium-free medium, thapsigargin and cyclopiazonic acid still induced a rise in the intracellular calcium concentration that was transient, and when calcium was added back to the medium, a rapid and sustained intracellular calcium increase was observed. Thapsigargin and cyclopiazonic acid induced a dose-dependent rise in testosterone secretion in the presence and absence of calcium in the external medium, although in calcium-free medium this stimulatory effect was lower. Leydig cell plasma membrane potential monitoring demonstrated that thapsigargin and cyclopiazonic acid induced first a rapid hyperpolarization, followed by a sustained depolarization phase that was reversed by the addition of the calcium-chelating agent EGTA. In the absence of calcium in the external medium the first phase of hyperpolarization was still present, but it was not followed by plasma membrane depolarization but by the slow return of plasma membrane potential to resting levels. The readdition of calcium to the external medium induced the rapid plasma membrane depolarization. Plasma membrane hyperpolarization was completely abolished by Leydig cell preincubation with the K⁺ channel blockers tetraethylammonium and charybdotoxin. Leydig cell preincubation with K⁺ channel inhibitors 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.

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