Luteotropic Hormone (LH) effects on T‐type calcium currents and intracellular calcium transients are mediated by PKA in mice Leydig cells

Abstract

Previous results from our laboratory have shown that LH is able to increase the intracellular Ca2+ concentration ([Ca2+]i) in Leydig cells, a process triggered by calcium entry through a T‐type Ca2+ channel. Here we describe the effects of modulators of PKC and PKA on the Ca2+ currents, measured with the patch clamp technique, and on the overall [Ca2+]i transients with fluorescence detection. Treatment of the cells with LH (1 μg/ml) increased the peak current at −20 mV from −2.4 ± 0.7 to −3.8 ± 0.8 pA/pF (n = 22, p = 0.0381). PMA (1 μM) increased the peak current to −3.6 ± 0.4 pA/pF (n = 6, p = 0.00033). Only minor changes were observed in the voltage dependence of activation, inactivation or deactivation of the currents. Experiments with the calcium sensitive dye Fluo‐3, showed that inhibition of both PKC and PKA with 400 nM staurosporine blocks the [Ca2+]i changes induced by LH (n = 37 cells). The specific inhibitor of PKA, H89 (10 μM) abolished the LH induced rise in [Ca2+]i (n = 48 cells). PMA slowly increases the fluorescence, but the subsequent addition of 1 μg/ml LH still triggers the typical [Ca2+]i transient showing that block of PKC alone does not inhibit the effect of LH. Chelerythrine, a PKC inhibitor, also does not avoid the Ca2+ transients. Taken together, our results support the conclusion that PKA plays a leading role in mediating the LH induced intracellular Ca2+ changes.Financial Support: FAPESP, CNPq