Intracellular pH in human arterial smooth muscle. Regulation by Na+/H+ exchange and a novel 5-(N-ethyl-N-isopropyl)amiloride-sensitive Na(+)- and HCO3(-)-dependent mechanism
- Alfred Hospital, Prahran (Australia)
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.
- OSTI ID:
- 6284142
- Journal Information:
- Circulation Research (An Official Journal of the American Heart Association); (USA), Vol. 67:4; ISSN 0009-7330
- Country of Publication:
- United States
- Language:
- English
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550201* - Biochemistry- Tracer Techniques