Fineman I, Hart D, Nord E P
Department of Medicine, School of Medicine, State University of New York, Stony Brook 11794.
Am J Physiol. 1990 Apr;258(4 Pt 2):F883-92. doi: 10.1152/ajprenal.1990.258.4.F883.
The role of an anion exchange pathway in the regulation of intracellular pH (pHi) under alkaline load and steady-state conditions and the modulation of this transporter by pHi was investigated in confluent monolayers of cloned JTC-12 cells, derived from monkey kidney proximal tubule. Regulation of pHi was fluorometrically monitored using the pH-sensitive probe, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Monolayers in which pHi was rapidly elevated by removal of HCO3(-)-CO2 from the bathing medium demonstrated an absolute requirement for Cl- to recover toward base-line pHi. The recovery process proceeded in the absence of Na+, was inhibited 80% by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and was unaffected by 10(-4) M amiloride. When extracellular pH (pHo) was serially lowered from 7.4 to 6.7, recovery from an alkaline load induced by removal of HCO3(-)-CO2 from the medium occurred only when pHi was elevated above approximately 7.25. Below pHi approximately 7.25 no recovery toward initial pHi was observed. When pHi was elevated above approximately 7.25 with pHo maintained at 6.7, the recovery process ceased at pHi approximately 7.25 despite favorably oriented Cl- and OH- chemical gradients. Consistent with these observations, removal of Cl- from the medium of cells buffered with 25 mM HCO3(-)-5% CO2 at pHo 7.4 (in the absence of Na+) resulted in reversible elevation of pHi, whereas in a solution buffered to pHo 6.7 with 5 mM HCO3(-)-5% CO2, removal of Cl- failed to elevate pHi. Under steady-state conditions in the presence of 25 mM HCO3(-)-5% CO2 at pHo 7.4, pHi was 7.40 +/- 0.02 and reversibly decreased to 7.23 +/- 0.01 on removal of Na+ (in the presence of amiloride) from the bathing medium, indicating that the Cl(-)-base exchanger is operative under basal conditions and functions as a base extruder. In summary the JTC-12 cell possesses a Na(+)-independent Cl(-)-base exchange mechanism that is operative under alkaline load and steady-state conditions. pHi but not pHo modulates the activity of this transport pathway, and below pHi approximately 7.25 the exchanger is quiescent.
在源自猴肾近端小管的克隆JTC - 12细胞的汇合单层中，研究了阴离子交换途径在碱性负荷和稳态条件下对细胞内pH（pHi）的调节作用以及pHi对该转运体的调节。使用pH敏感探针2',7'-双（2 - 羧乙基）-5（6）-羧基荧光素（BCECF）通过荧光法监测pHi的调节。通过从浴液中去除HCO3(-)-CO2使pHi迅速升高的单层细胞显示，恢复至基线pHi绝对需要Cl-。恢复过程在无Na+的情况下进行，4,4'-二异硫氰基芪-2,2'-二磺酸可抑制80%，而10(-4) M氨氯吡咪对其无影响。当细胞外pH（pHo）从7.4连续降低至6.7时，仅当pHi升高至约7.25以上时，才会从因从培养基中去除HCO3(-)-CO2诱导的碱性负荷中恢复。在pHi约7.25以下，未观察到恢复至初始pHi。当pHo维持在6.7且pHi升高至约7.25以上时，尽管Cl-和OH-化学梯度有利，但恢复过程在pHi约7.25时停止。与这些观察结果一致，在pHo 7.4（无Na+）时，从用25 mM HCO3(-)-5% CO2缓冲的细胞培养基中去除Cl-导致pHi可逆性升高，而在用5 mM HCO3(-)-5% CO2缓冲至pHo 6.7的溶液中，去除Cl-未能升高pHi。在pHo 7.4存在25 mM HCO3(-)-5% CO2的稳态条件下，pHi为7.40±0.02，从浴液中去除Na+（存在氨氯吡咪）后可逆性降至7.23±0.01，表明Cl(-)-碱交换体在基础条件下起作用并作为碱外排体发挥功能。总之，JTC - 12细胞具有一种不依赖Na+的Cl(-)-碱交换机制，该机制在碱性负荷和稳态条件下起作用。pHi而非pHo调节该转运途径的活性，且在pHi约7.25以下，交换体处于静止状态。
