Endothelin-induced changes in intracellular pH and Ca2+ in coronary smooth muscle: role of Na(+)-H+ exchange.
The relationship between endothelin-1 (ET-1)-induced stimulation of Na(+)-H+ exchange and intracellular free Ca2+ ([Ca2+]i) was examined in primary cultures of porcine coronary artery smooth muscle cells. Intracellular pH (pHi) and [Ca2+]i were measured using 2,7-bis-carboxyethyl-5(6)-carboxyfluorescein and the acetoxymethyl ester of fura-2 respectively. In HCO3(-)-free buffer (pH = 7.4), ET-1 (0.1-50 nM) induced a sustained, dose-dependent increase in pHi. ET-1 (10 nM) increased pHi from 6.83 +/- 0.01 to 6.93 +/- 0.02 (P < 0.01). The alkalinization was blocked by the Na(+)-H+ exchange inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 3 microM) or by substitution of Na+ with N-methylglucamine or choline chloride (P < 0.05). Recovery of pHi in response to acidification, induced by washout of a 20 mM NH4Cl prepulse, was > 90% inhibited by EIPA (3 microM), confirming the presence of an ET-1-responsive Na(+)-H+ exchanger. Coronary smooth muscle cells responded to ET-1 with a dose-dependent, biphasic increase in [Ca2+]i which was not inhibited by manipulations (EIPA pretreatment or Na(+)-free media) shown to block the Na(+)-H+ exchanger. The ET-1-mediated alkalinization was not inhibited by removal of extracellular Ca2+ ([Ca2+]o). However, complete blockade of the ET-1-mediated [Ca2+]i response using the intracellular Ca(2+)-chelator, [bis-(2-amino-5-methylphenoxy)ethane-NNN'N'-tetraacetic acid tetraacetoxymethyl ester] (MAPTAM), in [Ca2+]o-free media, demonstrated that an increment in [Ca2+]i is required for activation of the Na(+)-H+ exchanger by ET-1. The ET-1-induced rise in [Ca2+]i was not associated with a rise in pHi in the presence of CO2/HCO3-. We conclude that: (1) activation of Na(+)-H+ exchange by ET-1 requires an increment in [Ca2+]i; and (2) ET-1 stimulates EIPA-sensitive Na(+)-H+ exchange, but this stimulation does not modulate ET-1-induced changes in [Ca2+]i.
Hubel
C A
CA
Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, OH 45267-0576, USA.
Highsmith
R F
RF
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002794:Choline / Q000494:pharmacology; D003331:Coronary Vessels / Q000187:drug effects / Q000378:metabolism*; D016232:Endothelins / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008536:Meglumine / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects / Q000378:metabolism*; D013552:Swine
D000889:Anti-Arrhythmia Agents; D016232:Endothelins; D017923:Sodium-Hydrogen Exchangers; D008536:Meglumine; D000584:Amiloride; D012964:Sodium; D002794:Choline; D002118:Calcium; C039614:ethylisopropylamiloride