Kummerow D, Hamann J, Browning J A, Wilkins R, Ellory J C, Bernhardt I
Biophysics Section, Institute of Biology, Humboldt University Berlin, Invalidenstrasse 42, 10115 Berlin, Germany.
J Membr Biol. 2000 Aug 1;176(3):207-16. doi: 10.1007/s00232001089.
The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K(+) efflux and Cl(-) loss. When human erythrocytes were suspended in a physiological NaCl solution (pH(o) = 7.4), the measured pH(i) was 7.19 + or - 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution, an immediate alkalinization increased the pH(i) to 7.70 + or - 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl(-) content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison of the calculated H(+) influx with the measured unidirectional K(+) efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were enhanced by decreasing the ionic strength of the solution resulting in a H(+) influx and a K(+) efflux in LIS solution of 108.2 + or - 20.4 mmol (l(cells) hr)(-1) and 98.7 + or - 19.3 mmol (l(cells) hr)(-1), respectively. For bovine and porcine erythrocytes, in LIS media, H(+) influx and K(+) efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions. Quinacrine, a known inhibitor of the mitochondrial K(+)(Na(+))/H(+) exchanger, inhibited the K(+) efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K(+)(Na(+))/H(+) exchanger in the human erythrocyte membrane.
使用对pH敏感的荧光染料BCECF研究了不同实验条件下红细胞细胞内pH的变化，并将其与（哇巴因+布美他尼+乙二醇双四乙酸）不敏感的K⁺外流和Cl⁻损失相关联。当人红细胞悬浮于生理NaCl溶液（pHₒ = 7.4）中时，测得的pHᵢ为7.19±0.04，并且在30分钟内保持恒定。当红细胞转移至低离子强度（LIS）溶液中时，立即发生碱化，使pHᵢ升高至7.70±0.15，随后细胞酸化速度较慢。LIS培养基中细胞的碱化归因于带3介导的效应，因为观察到细胞内Cl⁻含量迅速损失约80%，这对已知的阴离子转运抑制剂敏感。在细胞酸化的情况下，比较不同细胞外离子强度下计算出的H⁺内流与测得的单向K⁺外流，显示出与接近1:1化学计量比的相关性。通过降低溶液的离子强度，两种通量均增强，导致LIS溶液中的H⁺内流和K⁺外流分别为108.2±20.4 mmol·(l细胞·小时)⁻¹和98.7±19.3 mmol·(l细胞·小时)⁻¹。对于牛和猪的红细胞，在LIS培养基中，H⁺内流和K⁺外流的幅度相当，但仅约为LIS条件下人红细胞中观察到的通量的10%。喹吖因是一种已知的线粒体K⁺(Na⁺)/H⁺交换体抑制剂，可使LIS溶液中的K⁺外流抑制约80%。我们的结果为人类红细胞膜中存在K⁺(Na⁺)/H⁺交换体提供了证据。
