ENDOTHELIN-INDUCED CHANGES IN INTRACELLULAR PH AND CA2(-MUSCLE - ROLEOF NA+-H+ EXCHANGE() IN CORONARY SMOOTH)

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. Intra cellular pH (pH(i)) and [Ca2+](i) were measured using 2,7-bis-carboxye thyl-5(6)-carboxyfluorescein and the acetoxymethyl ester of fura-2 res pectively. In HCO3-- free buffer (pH = 7.4), ET-1 (0.1-50 nM) induced a sustained, dose-dependent increase in pH(i). ET-1 (10 nM) increased pH(i) from 6.83 +/- 0.01 to 6.93 +/- 0.02 (P < 0.01). The alkalinizati on was blocked by the Na+-H+ exchange inhibitor, 5-(N-ethyl-N-isopropy l)amiloride (EIPA, 3 mu M) or by substitution of Na+ with N-methylgluc amine or choline chloride (P < 0.05). Recovery of pH(i) in response to acidification, induced by washout of a 20 mM NH4Cl prepulse, was > 90 % inhibited by EIPA (3 mu M), confirming the presence of an ET-1-resp onsive 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 n ot inhibited by removal of extracellular Ca2+ ([Ca2+](i)). However, co mplete blockade of the ET-1-mediated [Ca2+](i) response using the intr acellular Ca2+-chelator, (2-amino-5-methylphenoxy)ethane-NNN'N'-tetraa cetic acid tetraacetoxymethyl ester] (MAPTAM), in [Ca2+](o)-free media , demonstrated that an increment in [Ca2+](i) is required for activati on of the Na+-H+ exchanger by ET-1. The ET-1-induced rise in [Ca2+](i) was not associated with a rise in pH(i) 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 chang es in [Ca2+](i).