Martinez-Zaguilan R, Lynch R M, Martinez G M, Gillies R J
Department of Biochemistry, University of Arizona College of Medicine, Tuscon 85724.
Am J Physiol. 1993 Oct;265(4 Pt 1):C1015-29. doi: 10.1152/ajpcell.1993.265.4.C1015.
Mammalian cells generally regulate their intracellular pH (pHi) via collaboration between Na(+)-H+ exchanger and HCO3- transport. In addition, a number of normal mammalian cells have been identified that express H(+)-adenosinetriphosphatases (ATPases) in their plasma membranes. Because tumor cells often maintain a high pHi, we hypothesized that they might functionally express H(+)-ATPases in their plasma membranes. In the first phase of the present study, we screened 19 normal and tumorigenic human cell lines for the presence of plasmalemmal H(+)-ATPase activity using bafilomycin A1 to inhibit V-type H(+)-ATPase and Sch-28080 to inhibit P-type H(+)-K(+)-ATPase. Bafilomycin A1 decreased pHi in the six tumor cell lines with the highest resting pHi in the absence of HCO3-. Sch-28080 did not affect pHi in any of the human cells. Simultaneous measurement of pH in the cytoplasm and in the endosomes/lysosomes localized the activity of bafilomycin to the plasma membrane in three cell lines. In the second phase of this study, these three cell lines were shown to recover from NH4(+)-induced acid loads in the absence of Na+. This recovery was inhibited by N-ethylmaleimide, bafilomycin A1, and ATP depletion and was not significantly affected by vanadate, Sch-28080, or hexamethyl amiloride. These results indicate that a vacuolar type H(+)-ATPase is expressed in the plasma membrane of some tumor cells.
哺乳动物细胞通常通过钠氢交换体和碳酸氢根转运之间的协同作用来调节细胞内pH值（pHi）。此外，已鉴定出许多正常哺乳动物细胞在其质膜中表达氢-三磷酸腺苷酶（H(+)-ATPases）。由于肿瘤细胞通常维持较高的pHi，我们推测它们可能在质膜中功能性地表达H(+)-ATPases。在本研究的第一阶段，我们使用巴弗洛霉素A1抑制V型H(+)-ATPase和Sch-28080抑制P型H(+)-K(+)-ATPase，筛选了19种正常和致瘤性人类细胞系，以检测质膜H(+)-ATPase活性的存在。在没有碳酸氢根的情况下，巴弗洛霉素A1降低了六种静息pHi最高的肿瘤细胞系的pHi。Sch-28080对任何人类细胞的pHi均无影响。同时测量细胞质和内体/溶酶体中的pH值将巴弗洛霉素的活性定位到三个细胞系的质膜上。在本研究的第二阶段，这三个细胞系在没有钠离子的情况下能够从铵离子诱导的酸负荷中恢复。这种恢复受到N-乙基马来酰胺、巴弗洛霉素A1和ATP耗竭的抑制，而钒酸盐、Sch-28080或六甲铵对其没有显著影响。这些结果表明，某些肿瘤细胞的质膜中表达了液泡型H(+)-ATPase。
