Voltage-sensitive chloride channels of large conductance in the membrane of pig aortic endothelial cells.
Single, large-conductance chloride-selective channels were studied in the membrane of pig aortic endothelial cells. These channels were usually inactive in cell-attached recordings and activated spontaneously upon formation of inside-out patches or amphotericin B-perforated vesicles. Channel activity was voltage dependent, with a maximum open probability within the range of -20 mV to + 20 mV. Addition of 1 mM Zn2+ to either the cytoplasmic or extracellular side blocked channel activity reversibly. Extracellular 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) blocked the channels; the concentration necessary for half-maximum blockade was 100 mumol/l. The frequency of observing channels in cell-attached patches increased from less than 5% to 27% when cells were treated for several minutes with 1 mumol/l bradykinin and to 80% in the presence of the calcium ionophore A23187 (1 mumol/l). Both agents increase the cytoplasmic Ca2+ concentration, thereby stimulating nitric oxide (NO) synthesis and cGMP formation in endothelial cells. Sodium nitroprusside (100 mumol/l), which spontaneously releases NO, did not increase Cl- channel activity in intact cells. Polymyxin B (100 mumol/l), an inhibitor of protein kinase C, clearly enhanced Cl- channel activity in intact cells, resulting in the observation of Cl- channels in 70% of cell-attached patches. Our results demonstrate the existence of a large-conductance (LC-type) Cl- channel in vascular endothelium which is subject to a complex cellular regulation, possibly involving inhibition via phosphorylation by protein kinase C, and activation by a Ca2(+)-dependent process which is different from the NO/cGMP pathway.
Groschner
K
K
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Austria.
Kukovetz
W R
WR
D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004594:Electrophysiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D066298:In Vitro Techniques; D007473:Ion Channels / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D008566:Membranes / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D013552:Swine; D015032:Zinc / Q000494:pharmacology
D002712:Chlorides; D007473:Ion Channels; D000666:Amphotericin B; D011493:Protein Kinase C; D015032:Zinc; D002118:Calcium