Indirect stimulation of Ca(2+)-activated Cl- current by Na+/Ca2+ exchange in rabbit portal vein smooth muscle

Macroscopic currents were recorded in freshly dissociated smooth muscle cells from the rabbit portal vein using the tight seal whole cell recording mode (22 degrees C). In some experiments, the indo 1 fluorescence technique was used to simultaneously monitor the changes in the concentration of free intracellular Ca2+ ([Ca2+]i; indo 1 ratio, 400/500 nm). In cells exposed to tetraethylammonium chloride (TEA) to inhibit K+ channels and 1-10 microM nifedipine or nicardipine to inhibit L-type Ca2+ channels, cell dialysis with 30 mM Na+ increased [Ca2+]i and induced membrane current consistent with the activation of Ca(2+)-activated Cl- channels [ICl(Ca)]. From holding potential (HP) of -60 mV, high intracellular Na+ concentration ([Na+]i)-mediated current was instantaneous in response to 0.5- to 10-s voltage clamp pulses from -80 to +20 mV; steps ranging from +20 to +80 mV evoked slow time-dependent outward current (I(t); superimposed on the instantaneous current) and voltage-dependent Ca2+ transient; on return to HP, slow inward tail current appeared that reflected deactivation of I(t). Both current components 1) exhibited outward rectifying properties, 2) reversed near the predicted equilibrium potential for Cl-, 3) were stimulated by elevation of extracellular Ca2+ concentration, 4) were abolished when the cells were dialyzed with 5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 5) were inhibited by extracellular application of niflumic acid (50 microM). Complete replacement of extracellular Na+ concentration with tetramethylammonium increased both the instantaneous and time-dependent components of ICl(Ca), resting [Ca2+]i at -60 mV and Ca2+ transient at +40 mV. Cell dialysis with Na(+)-free pipette solution prevented these effects. Our results are consistent with an indirect mechanism of stimulation of ICl(Ca), which involves intracellular Ca2+ accumulation via reverse-mode electrogenic Na+/Ca2+ exchange activity.