Hays S R, Alpern R J
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.
J Gen Physiol. 1991 Oct;98(4):791-813. doi: 10.1085/jgp.98.4.791.
Apical membrane H+ extrusion in the renal outer medullary collecting duct, inner stripe, is mediated by a Na(+)-independent H+ pump. To examine the regulation of this transporter, cell pH and cell Ca2+ were measured microfluorometrically in in vitro perfused tubules using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2, respectively. Apical membrane H+ pump activity, assayed as cell pH recovery from a series of acid loads (NH3/NH+4 prepulse) in the total absence of ambient Na+, initially occurred at a slow rate (0.06 +/- 0.02 pH units/min), which was not sufficient to account for physiologic rates of H+ extrusion. Over 15-20 min after the initial acid load, the rate of Na(+)-independent cell pH recovery increased to 0.63 +/- 0.09 pH units/min, associated with a steady-state cell pH greater than the initial pre-acid load cell pH. This pattern suggested an initial suppression followed by a delayed activation of the apical membrane H+ pump. Replacement of peritubular Na+ with choline or N-methyl-D-glucosamine resulted in an initial spike increase in cell Ca2+ followed by a sustained increase in cell Ca2+. The initial rate of Na(+)-independent cell pH recovery could be increased by elimination of the Na+ removal-induced sustained cell Ca2+ elevation by: (a) performing studies in the presence of 135 mM peritubular Na+ (1 mM peritubular amiloride used to inhibit basolateral membrane Na+/H+ antiport); (b) clamping cell Ca2+ low with dimethyl-BAPTA, an intracellular Ca2+ chelating agent; or (c) removal of extracellular Ca2+. Cell acidification induced a spike increase in cell Ca2+. The late acceleration of Na(+)-independent cell pH recovery was independent of Na+ removal and of the method used to acidify the cell, but was eliminated by prevention of the cell Ca2+ spike and markedly delayed by the microfilament-disrupting agent, cytochalasin B. This study demonstrates that peritubular Na+ removal results in a sustained elevation in cell Ca2+, which inhibits the apical membrane H+ pump. In addition, rapid cell acidification associated with a spike increase in cell Ca2+ leads to a delayed activation of the H+ pump. Thus, cell Ca2+ per se, or a Ca(2+)-activated pathway, can modulate H+ pump activity.
肾外髓集合管内带顶端膜的氢离子排出由一种不依赖钠离子的氢离子泵介导。为了研究这种转运体的调节机制，分别使用2',7'-双（羧乙基）-5（6）-羧基荧光素和fura-2，通过微荧光法在体外灌注的肾小管中测量细胞pH值和细胞钙离子浓度。顶端膜氢离子泵活性通过在完全没有细胞外钠离子的情况下，对一系列酸负荷（氨/铵预脉冲）后细胞pH值的恢复情况进行测定。最初，恢复速率较慢（0.06±0.02 pH单位/分钟），这不足以解释生理状态下的氢离子排出速率。在最初的酸负荷后15 - 20分钟内，不依赖钠离子的细胞pH值恢复速率增加到0.63±0.09 pH单位/分钟，同时稳态细胞pH值高于酸负荷前的初始细胞pH值。这种模式表明顶端膜氢离子泵最初受到抑制，随后延迟激活。用胆碱或N - 甲基 - D - 葡萄糖胺替代肾小管周围的钠离子会导致细胞钙离子浓度先出现一个尖峰增加，随后持续升高。不依赖钠离子的细胞pH值恢复的初始速率可以通过以下方法提高：消除因去除钠离子而导致的细胞钙离子持续升高，方法包括：（a）在135 mM肾小管周围钠离子存在的情况下进行实验（使用1 mM肾小管周围氨氯吡咪抑制基底外侧膜钠离子/氢离子反向转运体）；（b）用细胞内钙离子螯合剂二甲基 - BAPTA将细胞钙离子钳制在低水平；或（c）去除细胞外钙离子。细胞酸化会导致细胞钙离子浓度出现尖峰增加。不依赖钠离子的细胞pH值恢复的后期加速与钠离子去除以及细胞酸化的方法无关，但通过防止细胞钙离子尖峰可消除这种加速，并且微丝破坏剂细胞松弛素B会使其明显延迟。这项研究表明，去除肾小管周围的钠离子会导致细胞钙离子持续升高，从而抑制顶端膜氢离子泵。此外，与细胞钙离子尖峰增加相关的快速细胞酸化会导致氢离子泵延迟激活。因此，细胞钙离子本身或钙离子激活的途径可以调节氢离子泵的活性。
