Proceedings of The Physiological Society

University of Cambridge (2008) Proc Physiol Soc 11, C113

Oral Communications

LH-induced depolarization of membrane potential in mice Leydig cells can function as a trigger for the subsequent intracellular calcium increase

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

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

Testosterone synthesis in Leydig cells is regulated by the Luteinizing Hormone (LH) and external Ca2+ is required for this process. Using fluo3 confocal microscopy we show that cAMP causes intracellular Ca2+ transients that depend crucially on the Ca2+ influx via a T-type calcium channel located at the plasma membrane of mice isolated Leydig cells. While the involvement of Ca2+ in LH induced steridogenesis is well known, the actual mechanisms that determine the intracellular Ca2+ signaling processes are poorly understood. Similarly to cAMP, the LH dependent [Ca2+]i increase is inhibited by removal of the extracellular Ca2+ or by addition of 400 µM NiSO4. The kinetic properties of the [Ca2+]i transients are, however, different. The mean rise time (tp) for LH (1 µg/ml) is 64 ± 4.2 s (n = 60 cells), whereas for 400 µM dibutyril-cAMP it is 38.2 ± 5.3 s (n = 44 cells), and the amplitude of the fluorescence signal (ΔF) is 2.9 ± 0.4 times the baseline fluorescence for cAMP and 3.3 ± 0.2 for LH. Ryanodine receptors are involved in the rise of [Ca2+]i since 100 µM ryanodine blocks the [Ca2+]i increase subsequent to the addition of 1 µg/ml of LH to the bath (4 experiments). Interestingly, removal of ryanodine under these conditions leads to [Ca2+]i oscillations. Three isoforms of IP3 receptors were also identified by immunocytochemistry with a highly heterogeneous spatial distribution. Despite the presence of IP3 receptors, addition of 100 µM 2-APB had no effect on the LH induced [Ca2+]i changes (tp= 87.2 ± 5.8 s, ΔF= 0.8 ± 0.1; n = 12 cells, 4 experiments). The LH induced [Ca2+]i increase is also evident inside the mitochondria as revealed by experiments with mitotracker green (Invitrogen Corporation). Measurements of the membrane potential of Leydig cells with Di-4-ANEPPS (Invitrogen Corporation) show that treatment with 1 µg/ml LH causes a depolarization around 30 mV (n=3 experiments). We argue that this change in membrane potential may function as the driving force for the Ca2+ influx trough the T-type channels, constituting therefore the initial signal for subsequent Ca2+ release from the ER via the ryanodine and the IP3 receptors. The details on how the structure and the spatial distribution of the above mentioned molecules determines the observed Ca2+ dynamics still remains to be elucidated.

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