Chloride (Cl-) channels were characterized in vascular smooth muscle cells (VSMC) using radioisotope flux and patch-clamp electrophysiological techniques. Transmembrane 125iodine (125I) efflux from subcultured (Passage 1-5) rat aortic VSMCs was used as an indicator of Cl- movements to study the relationship between intracellular calcium concentration ([Ca2+]i) and Cl- channel activity. Angiotensin II (Ang II) (10(-7) M) and adenosine 5'-triphosphate (ATP) (10(-4) M) induced rapid increases (9.7- and 14.9-fold, respectively) in 125I efflux rates. We found that both Ang II- and ATP-stimulated 125I efflux and [Ca2+]i increases were completely abolished after brief incubation (20 microM, 20 min) with the acetoxymethyl ester of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), a membrane-permeable Ca2+ chelator. However, when external EGTA was used to blunt agonist-stimulated Ca2+ influx, 125I efflux was still increased in response to Ang II and ATP. These data suggest that Ca2+ release from intracellular sites is sufficient to activate Cl- channels in response to Ang II and ATP. Using standard patch-clamp electrophysiological techniques, we found that Ang II, a Ca(2+)-mobilizing agonist, stimulated outward Cl- currents (gCl = 75 pS) in cell-attached (C/A) patches of primary and subcultured VSMCs. Collectively, these data suggest that Ang II and other vasoconstrictor agents stimulate Cl- channel activity via increases in [Ca2+]i. Cl- channel activation may help to depolarize the VSMC membrane leading to increased Ca2+ influx during agonist stimulation.
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