Kaplan D L, Boron W F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Biol Chem. 1994 Feb 11;269(6):4116-24.
In a study of intracellular pH (pHi) regulation, we compared c-H-ras-transformed fibroblasts (FT9s) with parental NIH 3T3 cells. Cells were grown on a coverslip and placed in a flow-through cuvette; pHi was monitored using a fluorescent dye. Whether cells were in a CO2-free or CO2-containing solution, steady-state pHi was approximately 0.5 higher in FT9s than in 3T3s. Rates of pHi recovery from acid loads in the presence of CO2 indicate that total net acid extrusion (Jtotal) was far greater in FT9s than 3T3s in the pHi range 6.3-7.4. In both FT9s and 3T3s, pHi recovery was blocked by removing Na+. In FT9s, most of Jtotal was blocked by ethyl isopropyl amiloride (EIPA) and was probably due to Na-H exchange. A smaller component was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and required Cl- and was probably due to Na(+)-dependent Cl-HCO3 exchange. In 3T3s, the DIDS-sensitive and Cl(-)-dependent components of Jtotal were very small. The dominant acid-extruder in 3T3s appears to be a Na-H exchanger insensitive to 50 microM EIPA. We determined the flux-pHi relationships for both the Na-H and Na(+)-dependent Cl-HCO3 exchangers in both FT9s and 3T3s. p21ras alkaline shifts the pHi profile of each transporter by approximately 0.7, without affecting the Vmax. These shifts in the flux-pHi relationships provide a mechanism for the large increase in steady-state pHi produced by long-term expression of p21ras.
在一项关于细胞内pH值（pHi）调节的研究中，我们将c-H-ras转化的成纤维细胞（FT9s）与亲本NIH 3T3细胞进行了比较。细胞生长在盖玻片上，并置于流通式比色皿中；使用荧光染料监测pHi。无论细胞处于无CO₂还是含CO₂的溶液中，FT9s中的稳态pHi均比3T3s中的高约0.5。在有CO₂存在的情况下，从酸负荷中恢复pHi的速率表明，在6.3 - 7.4的pHi范围内，FT9s中的总净酸排出量（Jtotal）远大于3T3s。在FT9s和3T3s中，去除Na⁺均会阻止pHi的恢复。在FT9s中，大部分Jtotal被乙基异丙基氨氯吡咪（EIPA）阻断，可能是由于Na⁺-H⁺交换。一个较小的部分被4,4'-二异硫氰基芪-2,2'-二磺酸盐（DIDS）抑制，且需要Cl⁻，可能是由于Na⁺依赖的Cl⁻-HCO₃交换。在3T3s中，Jtotal的DIDS敏感且Cl⁻依赖的部分非常小。3T3s中主要的酸排出器似乎是一种对50 μM EIPA不敏感的Na⁺-H⁺交换器。我们确定了FT9s和3T3s中Na⁺-H⁺交换器以及Na⁺依赖的Cl⁻-HCO₃交换器的通量-pHi关系。p21ras使每个转运体的pHi曲线碱性偏移约0.7，而不影响Vmax。通量-pHi关系的这些偏移为p21ras长期表达导致的稳态pHi大幅升高提供了一种机制。
