Bevensee M O, Bashi E, Schlue W R, Boyarsky G, Boron W F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Am J Physiol. 1999 Mar;276(3):C674-83. doi: 10.1152/ajpcell.1999.276.3.C674.
Using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we examined the effect of hyperosmolar solutions, which presumably caused cell shrinkage, on intracellular pH (pHi) regulation in mesangial cells (single cells or populations) cultured from the rat kidney. The calibration of BCECF is identical in shrunken and unshrunken mesangial cells if the extracellular K+ concentration ([K+]) is adjusted to match the predicted intracellular [K+]. For pHi values between approximately 6.7 and approximately 7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH2O) is threefold larger than in unshrunken cells (approximately 300 mosmol/kgH2O). In the nominal absence of CO2/HCO-3, exposing cell populations to a HEPES-buffered solution supplemented with approximately 300 mM mannitol (600 mosmol/kgH2O) causes steady-state pHi to increase by approximately 0.4. The pHi increase is due to activation of Na+/H+ exchange because, in single cells, it is blocked in the absence of external Na+ or in the presence of 50 microM ethylisopropylamiloride (EIPA). Preincubating cells in a Cl--free solution for at least 14 min inhibits the shrinkage-induced pHi increase by 80%. We calculated the pHi dependence of the Na+/H+ exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) the pHi dependence of the total acid-extrusion rate and 2) the pHi dependence of the EIPA-insensitive acid-loading rate. Shrinkage alkali shifts the pHi dependence of Na+/H+ exchange by approximately 0.7 pH units.
我们使用对pH敏感的染料2',7'-双(2-羧乙基)-5(6)-羧基荧光素（BCECF），研究了可能导致细胞皱缩的高渗溶液对从大鼠肾脏培养的系膜细胞（单细胞或细胞群体）细胞内pH（pHi）调节的影响。如果将细胞外K+浓度（[K+]）调整为与预测的细胞内[K+]相匹配，BCECF在皱缩和未皱缩的系膜细胞中的校准是相同的。对于大约6.7至大约7.4之间的pHi值，皱缩细胞（600 mosmol/kgH2O）的内在缓冲能力比未皱缩细胞（大约300 mosmol/kgH2O）大三倍。在名义上不存在CO2/HCO-3的情况下，将细胞群体暴露于补充有大约300 mM甘露醇（600 mosmol/kgH2O）的HEPES缓冲溶液中会导致稳态pHi增加约0.4。pHi的增加是由于Na+/H+交换的激活，因为在单细胞中，在没有外部Na+或存在50 microM乙基异丙基氨氯地平（EIPA）的情况下，该交换被阻断。将细胞在无Cl-的溶液中预孵育至少14分钟可使皱缩诱导的pHi增加抑制80%。我们通过将1）总酸分泌速率的pHi依赖性和2）EIPA不敏感的酸加载速率的pHi依赖性相加，计算了等渗和高渗条件下细胞群体中Na+/H+交换速率的pHi依赖性。皱缩碱使Na+/H+交换的pHi依赖性移动约0.7个pH单位。
