Volume-activated chloride channels in mice Leydig cells.

Production and secretion of testosterone in Leydig cells are mainly controlled by the luteinizing hormone (LH). Biochemical evidences suggest that the activity of Cl(-) ions can modulate the steroidogenic process, but the specific ion channels involved are not known. Here, we extend the characterization of Cl(-) channels in mice Leydig cells (50-60 days old) by describing volume-activated Cl(-) currents (I(Cl,swell)). The amplitude of I(Cl,swell) is dependent on the osmotic gradient across the cell membrane, with an apparent EC(50) of approximately 75 mOsm. These currents display the typical biophysical signature of volume-activated anion channels (VRAC): dependence on intracellular ATP, outward rectification, inactivation at positive potentials, and selectivity sequence (I(- )> Cl(- )> F(-)). Staurosporine (200 nM) did not block the activation of I(Cl,swell). The block induced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 128 microM), SITS (200 microM), ATP (500 microM), pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 100 miccroM), and Suramin (10 microM) were described by the permeant blocker model with apparent dissociation constant at 0 mV K(do) and fractional distance of the binding site (delta) of 334 microM and 47 %, 880 microM and 35 %, 2,100 microM and 49%, 188 microM and 27%, and 66.5 microM and 49%, respectively. These numbers were derived from the peak value of the currents. We conclude that I(Cl,swell) in Leydig cells are activated independently of purinergic stimulation, that Suramin and PPADS block these currents by a direct interaction with VRAC and that ATP is able to permeate this channel.

P2X receptors in mouse Leydig cells.

ATP-activated currents were studied in Leydig cells of mice with the patch-clamp technique. Whole cell currents were rapidly activating and slowly desensitizing (55% decrement from the peak value on exposure to 100 microM ATP for 60 s), requiring 3 min of washout to recover 100% of the response. The concentration-response relationships for ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and 2-methylthio-ATP (2-MeS-ATP) were described by the Hill equation with a concentration evoking 50% of maximal ATP response (K(d)) of 44, 110, and 637 microM, respectively, and a Hill coefficient of 2. The order of efficacy of agonists was ATP >or= ATPgammaS > 2-MeS-ATP > 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). alphabeta-Methylene-ATP (alphabeta-MeATP), GTP, UTP, cAMP, and adenosine were ineffective. Suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) blocked the responses in a concentration-dependent manner. The ATP-activated currents were dependent on extracellular pH, being maximal at pH 6.5 and decreasing with both acidification and alkalinization (apparent dissociation constant (pK(a)) of 5.9 and 7.4, respectively). The whole cell current-voltage relationship showed inward rectification and reversed near 0 mV. Experiments performed in bi-ionic conditions for measurement of reversal potentials showed that this channel is highly permeable to calcium [permeability (P)(Ca)/P(Na) = 5.32], but not to chloride (P(Cl)/P(Na) = 0.03) or N-methyl-D-glucamine (NMDG) (P(NMDG)/P(Na) = 0.09). Unitary currents recorded in outside-out patches had a chord conductance of 27 pS (between -90 and -50 mV) and were inward rectifying. The average current passing through the excised patch decreased with time [time constant (tau) = 13 s], resembling desensitization of the macroscopic current. These findings indicate that the ATP receptor present in Leydig cells shows properties most similar to those of cloned homomeric P2X(2).