We investigated in a physiological
salt solution (PSS) containing HCO3- the 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 pHi from an equivalent intracellular
acidosis was more rapid when the cells were incubated in CO2/HCO3(-)-buffered PSS than in
HEPES-buffered PSS. Recovery of pHi was dependent on extracellular Na+ (Km, 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 NH4Cl-induced 22Na+ influx rate stimulated by intracellular
acidosis was markedly attenuated in
HEPES-buffered PSS but not in CO2/HCO3(-)-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 (IC50, 39 nM), whereas in an identical CO2/HCO3(-)-buffered PSS, the inhibition curve exhibited biphasic characteristics (IC50, 37.3 nM and 312 microM). 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 pHi 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% at lesser, more physiological, levels of induced
acidosis.