Proceedings of The Physiological Society

University of Cambridge (2008) Proc Physiol Soc 11, C114

Oral Communications

PKA mediates intracellular calcium increase induced by LH in mice Leydig cells by modulating Cav3.2 channels

R. R. Costa1, J. F. Aguiar1, W. A. Varanda1

1. Physiology, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.

Binding of Luteinizing hormone (LH) to its receptor, in mice Leydig cells, leads to the activation of both adenyl cyclase and phospholipase C pathways. Action of the LH is also associated to an increase in the intracellular Ca2+ concentration ([Ca2+]I) mediated by calcium entry through a T-type Ca2+ channel present in the plasma membrane (Costa and Varanda, 2007). In order to study the possible pathway by which LH activates the previously reported Ca2+ channel, we investigated the effects of modulators of the PKC and PKA pathways on the Ca2+ currents stimulated by LH. Using confocal microscopy we also tested the effects of these agents on the overall LH induced [Ca2+]I increase. The amplitude of maximal current at -20 mV increases with the addition of 1 µg/ml LH (from -2.4 ± 0.3 in control to -3.1 ± 0.4 pA/pF with LH; n=8, p< 0.00672, t-test) and 1 µM phorbol 12-myristate 13-acetate (PMA) (from -3.6 ± 0.4 with PMA; n=5, p < 0.00009, t-test). Despite of this, no change in the voltage dependence of the activation, inactivation or deactivation was observed. The half voltage (V0.5) for activation at control conditions and 37 oC were -39.4 ± 0.2 mV and -40.1 ± 0.1 in the presence of LH. The V0.5 of steady state inactivation was -51.6 ± 0.9 mV and -52.2 ± 0.9 mV, respectively for control and LH. The V0.5 of activation with PMA was -37.7 ± 0.6 mV and for PMA+LH it was -39.9 ± 0.1 mV. For steady state inactivation the V0.5 values were -49.8 ± 1.4 mV and -51.6 mV respectively. The deactivation time constant, τdeact, at control conditions was 11.6 ± 1.9 ms. For LH, PMA and PMA+LH, the values of τdeactwere =15.3 ± 0.6 ms, τdeact = 24 ± 6. 5 ms and τdeact = 16.17 ± 3.7 ms, respectively. Experiments carried out with the calcium sensitive dye fluo-3, show that inhibition of PKC and PKA with 400 nM stausporine blocks the increase of [Ca2+]I induced by LH. Treatment of the cells with 10 µM H89, an inhibitor of cAMP-dependent protein kinase, also abolishes the elevation of [Ca2+]I caused by LH (n=5). PMA slowly increases the fluorescence when the cells are incubated at 37 oC. Nevertheless, the subsequent addition of 1 µg/ml LH induces the typical increase in [Ca2+]I showing that PKC does not inhibit the LH induced [Ca2+]I rise. Taken together, these results suggest that PKA plays a leading role in mediating the LH action on the [Ca2+]I, despite the fact that PKC is able to increase the amplitude of CaV3.2.

Where applicable, experiments conform with Society ethical requirements