Novel 5-(N-Ethyl-N-isopropyl)amiloride-Sensitive Na+- and HCO3 -Dependent Mechanism

We investigated in a physiological salt solution (PSS) containing HCO3the intracellular pH (pHi) regulating mechanisms in smooth muscle cells cultured from human internal mammary arteries, using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and 22Na' influx rates. The recovery of pH; from an equivalent intracellular acidosis was more rapid when the cells were incubated in COJHCO3--buffered PSS than in HEPES-buffered PSS. Recovery of pH; was dependent on extracellular Na+ (K., 13.1 mM); however, it was not attenuated by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), indicating the absence of SITS-sensitive HCO3 -dependent mechanisms. Recovery instead appeared mostly dependent on processes sensitive to 5-(N-ethyl-N-isopropyl)amiloride (EIPA), indicating the involvement of Na+/H+ exchange and a previously undescribed EIPA-sensitive Na+and HCO3 -dependent mechanism. Differentiation between this HCO3 -dependent mechanism and Na+/H+ exchange was achieved after depletion of cellular ATP. Under these conditions, the NH4CI-induced 22Na' influx rate stimulated by intracellular acidosis was markedly attenuated in HEPES-buffered PSS but not in C021HC03-buffered PSS. EIPA also appeared to inhibit the two mechanisms differentially. In HEPES-buffered PSS containing 20 mM Na+, the EIPA inhibition curve for the intracellular acidosis-induced 22Na' influx was monophasic (IC , 39 nM), whereas in an identical C02IHCO3--buffered PSS, the inhibition curve exhibited biphasic characteristics (IC50, 37.3 nM and 312 ,uM). Taken together, the results indicate that Na+/H+ exchange and a previously undescribed EIPA-sensitive Na+and HCO3 -dependent mechanism play an important role in regulating the pH; of human vascular smooth muscle. The involvement of the latter mechanism depends on the severity of the intracellular acidosis, varying from approximately 25% in severe intracellular acidosis up to 50%o at lesser, more physiological, levels of induced acidosis. (Circulation Research 1990;67:814-825)