King G G, Lohrmann W E, Ickes J W, Feldman G M
Department of Medicine, McGuire Veterans Affairs Medical Center, Richmond 23249.
Am J Physiol. 1994 Jul;267(1 Pt 1):G119-28. doi: 10.1152/ajpgi.1994.267.1.G119.
Colonocytes must regulate intracellular pH (pHi) while they transport H+ and HCO3-. To investigate the membrane transport processes involved in pHi regulation, colonocyte pHi was measured with 2,'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in intact segments of rat distal colon mounted on a holder that fits into a standard fluorometer cuvette and allows independent superfusion of mucosal and serosal surfaces. When NCECF-acetoxymethyl ester was in the mucosal solution only, BCECF loaded surface colonocytes with a high degree of selectivity. In HEPES-buffered solutions, basal pHi was 7.31 +/- 0.01 (n = 68), and pHi was dependent on extracellular Na+. Cells acidified in Na(+)-free solution, and pHi rapidly corrected when Na+ was returned. pHi recovered at 0.22 +/- 0.01 pH/min (n = 6) when Na+ was introduced into the mucosal solution and at 0.02 +/- 0.01 pH/min (n = 7) when Na+ was absent from the mucosal solution. The presence or absence of Na+ in the serosal solution did not affect pHi. This indicated that the Na(+)-dependent pHi recovery process is located in the apical cell membrane, but not in the basolateral membrane. Because amiloride (1 mM) inhibited Na(+)-dependent pHi recovery by 75%, Na+/H+ exchange appears to be present in the apical membrane. Because Na(+)-independent pHi recovery was not affected by K(+)-free media, 50 microM SCH-28080, 100 nM bafilomycin A1, or Cl(-)-free media, this transport mechanism does not involve a gastriclike H(+)-K(+)-ATPase, a vacuolar H(+)-ATPase, or a Cl-/base exchanger. In summary, pHi was selectively measured in surface colonocytes by this technique. In these cells, the Na+/H+ exchange activity involved in pHi regulation was detected in the apical membrane, but not in the basolateral membrane.
结肠上皮细胞在转运H⁺和HCO₃⁻时必须调节细胞内pH（pHi）。为了研究参与pHi调节的膜转运过程，使用2,'7'-双（2-羧乙基）-5（6）-羧基荧光素（BCECF）测量大鼠远端结肠完整节段中的结肠上皮细胞pHi，该节段安装在一个适合标准荧光计比色皿的固定器中，并允许对黏膜和浆膜表面进行独立的灌流。当NCECF-乙酰氧基甲酯仅存在于黏膜溶液中时，BCECF以高度的选择性加载表面结肠上皮细胞。在HEPES缓冲溶液中，基础pHi为7.31±0.01（n = 68），且pHi依赖于细胞外Na⁺。细胞在无Na⁺溶液中酸化，当恢复Na⁺时pHi迅速校正。当将Na⁺引入黏膜溶液时，pHi以0.22±0.01 pH/分钟（n = 6）的速度恢复，而当黏膜溶液中不存在Na⁺时，pHi以0.02±0.01 pH/分钟（n = 7）的速度恢复。浆膜溶液中Na⁺的存在与否不影响pHi。这表明依赖Na⁺的pHi恢复过程位于顶端细胞膜而非基底外侧膜。因为氨氯地平（1 mM）抑制依赖Na⁺的pHi恢复达75%，所以顶端膜中似乎存在Na⁺/H⁺交换。因为不依赖Na⁺的pHi恢复不受无K⁺培养基、50 μM SCH-28080、100 nM巴弗洛霉素A1或无Cl⁻培养基的影响，所以这种转运机制不涉及胃型H⁺-K⁺-ATP酶、液泡型H⁺-ATP酶或Cl⁻/碱基交换体。总之，通过该技术在表面结肠上皮细胞中选择性地测量了pHi。在这些细胞中，在顶端膜而非基底外侧膜中检测到了参与pHi调节的Na⁺/H⁺交换活性。
