Na+/K+/Cl- cotransport in cultured vascular smooth muscle cells: stimulation by angiotensin II and calcium ionophores, inhibition by cyclic AMP and calmodulin antagonists.
The specific activity of the Na+/K+/Cl cotransporter was assayed by measuring the initial rates of furosemide-inhibitable 86Rb+ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 microM, respectively. The dependence of furosemide-inhibitable 86Rb+ influx on external Na+ and K+ was hyperbolic with apparent Km values of 46 and 4 mM, respectively. The dependence on Cl was sigmoidal. Assuming a stoichiometry of 1:1:2 for Na+/K+/Cl-, a Km of 78 mM was obtained for Cl. In quiescent smooth muscle cells cotransport activity was approximately equal to Na+ pump activity with each pathway accounting for 30% of total 86Rb+ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na+ pump activity was not significantly different in the growing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores. A23187 and ionomycin. The removal of external Ca2+ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited 86Rb+ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D001013:Aorta, Thoracic / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D004232:Diuretics / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000378:metabolism; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters
D002121:Calcium Channel Blockers; D002352:Carrier Proteins; D002712:Chlorides; D004232:Diuretics; D028021:Sodium-Potassium-Chloride Symporters; D000804:Angiotensin II; D000001:Calcimycin; D004533:Egtazic Acid; D012964:Sodium; D011188:Potassium; D002118:Calcium