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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 585/2/339    most recent jphysiol.2007.137950v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Costa, R. R. Articles by Varanda, W. A. Search for Related Content PubMed PubMed Citation Articles by Costa, R. R. Articles by Varanda, W. A. Related Collections Cellular

¹ Department of Physiology, School of Medicine of Ribeirão Preto/University of São Paulo, 14049–900 Ribeirão Preto/São Paulo, Brazil

Luteinizing hormone (LH) regulates testosterone synthesis in Leydig cells by inducing an intracellular increase in cAMP concentration. LH also increases the intracellular calcium concentration ([Ca²⁺]_i), dependent on the presence of Ca²⁺ in the extracellular medium ([Ca²⁺]_e) for its effect. Despite these evidences, the identity of a pathway for calcium entry has remained elusive and the relationship between cAMP and [Ca²⁺]_i has been questioned. Here we show that mice Leydig cells do have an inward Ca²⁺ current carried by T-type Ca²⁺ channels. In 10 mM [Ca²⁺]_e, the currents start to be activated at –60 mV, reaching maximal amplitude of 1.8 ± 0.3 pA pF^–1 at –20 mV. Currents were not modified by Ba²⁺ or Sr²⁺, were suppressed in Ca²⁺-free external solution, and were blocked by 100 µM nickel or 100 µM cadmium. The K_i for Ni²⁺ is 2.6 µM and concentrations of Cd²⁺ smaller than 50 µM have a very small effect on the currents. The calcium currents displayed a window centred at –40 mV. The half-voltage (V_0.5) of activation is –30.3 mV, whereas the half-voltage steady-state inactivation is –51.1 mV. The deactivation time constant (_deactivation) is around 3 ms at –35 mV. Confocal microscopy experiments with Fluo-3 loaded cells reveal that both LH and dibutyryl-cAMP (db-cAMP) increase [Ca²⁺]_i. The db-cAMP induced calcium increase was dependent on Ca²⁺ influx since it was abolished by removal of extracellular Ca²⁺ and by 400 µM Ni²⁺. [Ca²⁺]_i increases in regions close to the plasma membrane and in the cell nucleus. Similar effects are seen when Leydig cells are depolarized by withdrawing K⁺ from the extracellular solution. Altogether, our studies show that Ca²⁺ influx through T-type Ca²⁺ channels in the plasma membrane of Leydig cells plays a crucial role in the intracellular calcium concentration changes that follow binding of LH to its receptor.

(Received 6 June 2007; accepted after revision 5 October 2007; first published online 11 October 2007)
Corresponding author W. A. Varanda: Department of Physiology, School of Medicine of Ribeirão Preto/University of São Paulo, Av. Bandeirantes, 3900, 14049–900 Ribeirão Preto/São Paulo Brazil. Email: wvaranda{at}fmrp.usp.br

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