Bonanno J A, Machen T E
Department of Physiology-Anatomy, University of California, Berkeley 94720.
Exp Eye Res. 1989 Jul;49(1):129-42. doi: 10.1016/0014-4835(89)90081-x.
Intracellular pH (pHi) was measured in basal corneal epithelial cells from fresh corneal explants using the pH sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The overlying superficial and wing cells were removed by mechanical scraping to expose basal cells attached to their basal lamina. Tissue pieces with attached, dye-loaded basal cells were mounted in a microscope-stage-perfusion chamber which allowed rapid changes of Ringer's bathing solutions while measuring BCECF fluorescence. In NaCl-Ringer's (bicarbonate free). pHo 7.40, resting cell pHi was 7.34 +/- 0.03 (+/- S.E.M., n = 31). Buffering capacity measured by NH4Cl treatment was 31 mM pH at pHi 7.34 and increased with decreasing pHi. Recovery from 20 mM NH4Cl-induced acid loads was dependent on the presence of Na and inhibited by 1 mM amiloride. Adding amiloride to resting cells caused a slow, reversible acidification (0.04 pH units min-1). These results indicate the presence of Na:H exchange, its role in responding to acid loads and in maintaining resting cell pHi. Activation of Na:H by Nao showed simple saturation kinetics, with Km = 44 mM. Net proton efflux via Na:H exchange increased with decreasing pHi and was enhanced by depleting cells of Nai, suggesting roles for both pHi and Nai in control of Na:H activation.
使用pH敏感荧光染料2'，7'-双（2-羧乙基）-5（6）-羧基荧光素（BCECF）测量新鲜角膜外植体基底角膜上皮细胞中的细胞内pH（pHi）。通过机械刮除去除覆盖的表层细胞和翼状细胞，以暴露附着于其基底膜的基底细胞。将带有附着的、加载了染料的基底细胞的组织块安装在显微镜载物台灌注室中，该灌注室允许在测量BCECF荧光时快速改变林格氏浴液。在无碳酸氢盐的NaCl-林格氏液中，pHo为7.40，静息细胞的pHi为7.34±0.03（±标准误，n = 31）。通过氯化铵处理测量的缓冲能力在pHi为7.34时为31 mM pH，并随着pHi的降低而增加。从20 mM氯化铵诱导的酸负荷中恢复取决于Na的存在，并受到1 mM氨氯吡脒的抑制。向静息细胞中添加氨氯吡脒会导致缓慢的、可逆的酸化（0.04 pH单位/分钟）。这些结果表明存在Na：H交换，其在应对酸负荷和维持静息细胞pHi中的作用。Nao对Na：H的激活表现出简单的饱和动力学，Km = 44 mM。通过Na：H交换的净质子外流随着pHi的降低而增加，并因细胞内Na+耗尽而增强，表明pHi和细胞内Na+在控制Na：H激活中均起作用。
