Harvey B J, Thomas S R, Ehrenfeld J
Département de Biologie, Laboratoire Jean Maetz, Villefranche-sur-Mer, France.
J Gen Physiol. 1988 Dec;92(6):767-91. doi: 10.1085/jgp.92.6.767.
We determined the effects of intracellular respiratory and metabolic acid or alkali loads, at constant or variable external pH, on the apical membrane Na+-specific conductance (ga) and basolateral membrane conductance (gb), principally due to K+, in the short-circuited isolated frog skin epithelium. Conductances were determined from the current-voltage relations of the amiloride-inhibitable cellular current pathway, and intracellular pH (pHi) was measured using double barreled H+-sensitive microelectrodes. The experimental set up permitted simultaneous recording of conductances and pHi from the same epithelial cell. We found that due to the asymmetric permeability properties of apical and basolateral cell membranes to HCO3- and NH+4, the direction of the variations in pHi was dependent on the side of addition of the acid or alkali load. Specifically, changing from control Ringer, gassed in air without HCO3- (pHo = 7.4), to one containing 25 mmol/liter HCO3- that was gassed in 5% CO2 (pHo = 7.4) on the apical side caused a rapid intracellular acidification whereas when this maneuver was performed from the basolateral side of the epithelium a slight intracellular alkalinization was produced. The addition of 15 mmol/liter NH4Cl to control Ringer on the apical side caused an immediate intracellular alkalinization that lasted up to 30 min; subsequent removal of NH4Cl resulted in a reversible fall in pHi, whereas basolateral addition of NH4Cl produced a prolonged intracellular acidosis. Using these maneouvres to change pHi we found that the transepithelial Na+ transport rate (Isc), and ga, and gb were increased by an intracellular alkalinization and decreased by an acid shift in pHi. These variations in Isc, ga, and gb with changing pHi occurred simultaneously, instantaneously, and in parallel even upon small perturbations of pHi (range, 7.1-7.4). Taken together these results indicate that pHi may act as an intrinsic regulator of epithelial ion transport.
我们研究了在外部pH恒定或变化的情况下，细胞内呼吸和代谢性酸或碱负荷对短路离体蛙皮上皮细胞顶膜钠特异性电导（ga）和主要由钾引起的基底外侧膜电导（gb）的影响。电导由氨氯地平抑制的细胞电流途径的电流-电压关系确定，细胞内pH（pHi）使用双管H+敏感微电极测量。该实验装置允许从同一上皮细胞同时记录电导和pHi。我们发现，由于顶膜和基底外侧细胞膜对HCO3-和NH+4的渗透性不对称，pHi变化的方向取决于酸或碱负荷添加的一侧。具体而言，从不含HCO3-（pHo = 7.4）的空气通气的对照林格液改为顶侧含25 mmol/L HCO3-且用5% CO2通气（pHo = 7.4）的溶液会导致细胞内迅速酸化，而当从上皮细胞的基底外侧进行此操作时，则会产生轻微的细胞内碱化。在顶侧将15 mmol/L NH4Cl添加到对照林格液中会导致立即的细胞内碱化，持续长达30分钟；随后去除NH4Cl会导致pHi可逆下降，而在基底外侧添加NH4Cl会产生长时间的细胞内酸中毒。利用这些方法改变pHi，我们发现跨上皮钠转运速率（Isc）、ga和gb会因细胞内碱化而增加，因pHi的酸移而降低。即使在pHi有小的扰动（范围为7.1 - 7.4）时，Isc、ga和gb随pHi变化的这些变化也是同时、瞬间且平行发生的。综合这些结果表明，pHi可能作为上皮离子转运的内在调节因子。
