Intracellular calcium changes in mice Leydig cells are dependent on calcium entry through T-type calcium channels.
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 microm nickel or 100 microm cadmium. The Ki for Ni2+ is 2.6 microm and concentrations of Cd2+ smaller than 50 microm have a very small effect on the currents. The calcium currents displayed a window centred at -40 mV. The half-voltage (V0.5) of activation is -30.3 mV, whereas the half-voltage steady-state inactivation is -51.1 mV. The deactivation time constant (taudeactivation) 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 microm 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.
Costa
Roberta Ribeiro
RR
Department of Physiology, School of Medicine of Ribeirão Preto/University of São Paulo, Av. Bandeirantes, 3900, 14049-900 Ribeirão Preto/São Paulo Brazil.
Varanda
Wamberto Antonio
WA
D000814:Aniline Compounds / Q000493:pharmacokinetics; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020747:Calcium Channels, T-Type / Q000502:physiology*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D007700:Kinetics; D007985:Leydig Cells / Q000187:drug effects / Q000502:physiology*; D007986:Luteinizing Hormone / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D018408:Patch-Clamp Techniques; D014966:Xanthenes / Q000493:pharmacokinetics
D000814:Aniline Compounds; D020747:Calcium Channels, T-Type; D005456:Fluorescent Dyes; D014966:Xanthenes; C059715:Fluo-3; D003994:Bucladesine; D007986:Luteinizing Hormone; D002118:Calcium