CALCIUM-ACTIVATED CHLORIDE CHANNELS IN BOVINE PULMONARY-ARTERY ENDOTHELIAL-CELLS

1. We characterized Ca2+-activated Cl- currents in calf pulmonary arte ry endothelial (CPAE) cells by using a combined patch clamp and fura-2 microfluorescence technique to simultaneously measure ionic currents and the intracellular Ca2+ concentration, [Ca2+](i). 2. Various proced ures that increased [Ca2+](i), such as stimulation with ATP or ionomyc in, or loading the cells with Ca2+ via the patch pipette, activated a strongly outwardly rectifying current with a reversal potential close to the Cl- equilibrium potential. Changing the extracellular Cl- conce ntration shifted this reversal potential as predicted for a Cl- curren t. Buffering Ca2+ rises with BAPTA prevented ATP from activating the c urrent. 3. Ca2+-activated Cl- currents could be distinguished from vol ume-activated Cl- currents, which were sometimes coactivated in the sa me cell. The latter showed much less outward rectification, their acti vation was voltage independent, and they could be inhibited by exposin g the cells to hypertonic solutions. 4. The permeability ratio for the Ca2+-activated conductance of the anions iodide:chloride:gluconate wa s 1.71 +/- 0.06:1:0.39 +/- 0.03 (n = 12). 5. This Ca2+-activated Cl- c urrent, I-Cl,I-Ca, inactivated rapidly at negative potentials and acti vated slowly at positive potentials. Outward tail currents were slowly decaying, while inward tail currents decayed much faster. 6. 4,4'Diis othiocyanatostilbene-2,2'-disulphonic acid (DIDS) and niflumic acid in hibited I-Cl,I-Ca in a voltage-dependent manner, i.e. they exerted a m ore potent block at positive potentials. The block by N-phenylanthraci lic acid (NPA), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and ta moxifen was voltage independent. Niflumic acid and tamoxifen were the most potent blockers. 7. The single-channel conductance was 7.9 +/- 0. 7 pS (n = 15) at 300 mM extracellular Cl-. The channel open probabilit y was high at positive potentials, but very small at negative potentia ls. 8. It is concluded that [Ca2+](i) activates small-conductance Cl- channels in endothelial cells, which coexist with the volume-activated Cl- channels described previously.