Functional coupling of Na(+)-K(+)-2Cl- cotransport and Ca(2+)-dependent K+ channels in vascular endothelial cells.
To determine whether the activation of Na(+)-K(+)-2Cl- cotransport by Ca(2+)-mobilizing agonists is a direct effect of Ca2+ or is secondary to activation of Ca(2+)-dependent K+ channels [via cell shrinkage or decreased intracellular Cl- concentration ([Cl-]), we measured K+ fluxes in aortic endothelial cells in response to ATP and bradykinin. With either agonist there was an immediate bumetanide-insensitive efflux inhibitable by the K+ channel blockers tetrabutylammonium (TBA, 23 mM) and quinidine (1 mM), followed several minutes later by increased bumetanide-sensitive efflux or influx (Na(+)-K(+)-2Cl- cotransport). ATP induced a loss of cell K+ that was prevented by TBA and augmented by bumetanide. Both TBA and quinidine prevented the stimulation of cotransport by agonists but not by hypertonic shrinkage. Raising medium [K+] to prevent K+ loss also blocked activation of cotransport by agonists. The results indicate that the stimulation of Na(+)-K(+)-2Cl- cotransport by Ca2+ is not direct but instead is indirect via activation of Ca(2+)-dependent K+ channels and a resulting decrease in cell volume and intracellular [Cl-]. This suggests that at least one role of Na(+)-K(+)-2Cl- cotransport in endothelial cells is to maintain cell volume and intracellular [Cl-] during agonist stimulation.
O'Neill
W C
WC
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Steinberg
D F
DF
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D009994:Osmolar Concentration; D015221:Potassium Channels / Q000502:physiology*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters
D002352:Carrier Proteins; D015221:Potassium Channels; D000644:Quaternary Ammonium Compounds; D028021:Sodium-Potassium-Chloride Symporters; D000255:Adenosine Triphosphate; C009405:tetrabutylammonium; D002118:Calcium