Neylon C B, Little P J, Cragoe E J, Bobik A
Alfred Baker Medical Unit, Alfred Hospital, Prahran, Australia.
Circ Res. 1990 Oct;67(4):814-25. doi: 10.1161/01.res.67.4.814.
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.
我们使用pH敏感染料2',7'-双（2-羧乙基）-5（6）-羧基荧光素（BCECF）和22Na+流入速率，在含有HCO3-的生理盐溶液（PSS）中研究了从人乳内动脉培养的平滑肌细胞中的细胞内pH（pHi）调节机制。当细胞在CO2/HCO3(-)缓冲的PSS中孵育时，从等效的细胞内酸中毒中恢复pHi的速度比在HEPES缓冲的PSS中更快。pHi的恢复依赖于细胞外Na+（Km，13.1 mM）；然而，它并未被4-乙酰氨基-4'-异硫氰酸根合芪-2,2'-二磺酸（SITS）减弱，这表明不存在SITS敏感的HCO3(-)依赖机制。相反，恢复似乎主要依赖于对5-（N-乙基-N-异丙基）氨氯吡脒（EIPA）敏感的过程，这表明涉及Na+/H+交换和一种先前未描述的EIPA敏感的Na(+)和HCO3(-)依赖机制。在细胞ATP耗尽后，实现了这种HCO3(-)依赖机制与Na+/H+交换之间的区分。在这些条件下，在HEPES缓冲的PSS中，细胞内酸中毒刺激的NH4Cl诱导的22Na+流入速率明显减弱，但在CO2/HCO3(-)缓冲的PSS中则没有。EIPA似乎也对这两种机制有不同的抑制作用。在含有20 mM Na+的HEPES缓冲的PSS中，细胞内酸中毒诱导的22Na+流入的EIPA抑制曲线是单相的（IC50，39 nM），而在相同的CO2/HCO3(-)缓冲的PSS中，抑制曲线呈现双相特征（IC50，37.3 nM和312 microM）。综上所述，结果表明Na+/H+交换和一种先前未描述的EIPA敏感Na(+)和HCO3(-)依赖机制在调节人血管平滑肌的pHi中起重要作用。后一种机制的参与程度取决于细胞内酸中毒的严重程度，从严重细胞内酸中毒时的约25%到较轻、更接近生理水平的诱导酸中毒时的50%不等。
