Endothelial cell sodium-potassium-chloride cotransport. Evidence of regulation by Ca2+ and protein kinase C.
Previous studies have shown that vascular endothelial cells exhibit a highly active Na-K-Cl cotransport system that is regulated by a variety of vasoactive hormones and neurotransmitters, suggesting that the cotransporter may play an important role in endothelial cell function. In this study, the regulation of endothelial cell Na-K-Cl cotransport was further investigated by probing the stimulus-transfer pathway by which vasoactive agents stimulate the cotransporter. Specifically, three peptides previously shown to stimulate cotransport activity (angiotensin II, vasopressin, and bradykinin) were evaluated. Na-K-Cl cotransport was assessed in cultured bovine aortic endothelial cells as bumetanide-sensitive K+ influx. Stimulation of Na-K-Cl cotransport by angiotensin II, vasopressin, or bradykinin was found to be reduced either by removal of extracellular Ca2+ or by treatment of the cells with 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate or 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In addition, the calmodulin antagonist W-7 was found to prevent stimulation of endothelial cell Na-K-Cl cotransport by the three peptides. These findings suggest that regulation of endothelial cell cotransport by these vasoactive peptides may be both Ca(2+)- and calmodulin-dependent. Angiotensin II, vasopressin, and bradykinin were also found to elevate phosphatidylinositol hydrolysis in the cultured endothelial cells. Thus, the possibility that regulation of endothelial Na-K-Cl cotransport by these vasoactive peptides also involves diacylglycerol activation of protein kinase C was investigated. A 10-min exposure of the endothelial cells to low doses of phorbol 12-myristate 13-acetate was found to reduce Na-K-Cl cotransport whether in the presence or absence of angiotensin II, vasopressin, or bradykinin. However, down-regulation of protein kinase C by a 40-h exposure to higher doses of the phorbol ester was found to elevate Na-K-Cl cotransport activity under both control and agonist-stimulated conditions, indicating that activation of protein kinase C results in inhibition of endothelial cell Na-K-Cl cotransport. Thus, protein kinase C activation may serve as negative feedback in the stimulus-transfer pathway by which these agonists regulate endothelial cell Na-K-Cl cotransport.
O'Donnell
M E
ME
Department of Human Physiology, School of Medicine, University of California, Davis 95616.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000804:Angiotensin II / Q000502:physiology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001920:Bradykinin / Q000502:physiology; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002417:Cattle; D002712:Chlorides / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology*; D012964:Sodium / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014667:Vasopressins / Q000502:physiology
D002147:Calmodulin; D002712:Chlorides; D004075:Diglycerides; D007295:Inositol Phosphates; D014667:Vasopressins; D000804:Angiotensin II; D000255:Adenosine Triphosphate; D012964:Sodium; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium; D001920:Bradykinin; D002118:Calcium