INTRACELLULAR PH IN HUMAN ARTERIAL SMOOTH-MUSCLE - REGULATION BY NA+/H+ EXCHANGE AND A NOVEL 5-(N-ETHYL-N-ISOPROPYL)AMILORIDE-SENSITIVE NA+-DEPENDENT AND HCO3--DEPENDENT MECHANISM

被引:84
作者
NEYLON, CB
LITTLE, PJ
CRAGOE, EJ
BOBIK, A
机构
[1] BAKER MED RES INST,COMMERCIAL RD,PRAHRAN,VIC 3181,AUSTRALIA
[2] ALFRED HOSP,ALFRED BAKER MED UNIT,PRAHRAN,VIC 3181,AUSTRALIA
[3] MERCK SHARP & DOHME LTD,W POINT,PA 19486
关键词
Amiloride derivatives; ATP; Cell culture; Intracellular pH; Vascular smooth muscle;
D O I
10.1161/01.RES.67.4.814
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
We investigated in a physiological salt solution (PSS) containing HCO3- the intracellular pH (pH(i)) 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(i) 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 pH(i) was dependent on extracellular Na+ (K(m), 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 apparent 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 μM). 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(i) 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.
引用
收藏
页码:814 / 825
页数:12
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