REGULATION OF INTRACELLULAR PH IN J774 MURINE MACROPHAGE CELLS - H+ EXTRUSION PROCESSES
REGULATION OF INTRACELLULAR PH IN J774 MURINE MACROPHAGE CELLS - H+ EXTRUSION PROCESSES
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DOI：
10.1152/ajpcell.1995.268.1.c210
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发表时间：
1995-01-01
影响因子：
5.5
通讯作者：
MORAN, A
中科院分区：
文献类型：
--
作者：
MCKINNEY, LC;MORAN, A
在鼠巨噬细胞系J774.1中表征了细胞内pH（pH（i））调节的机制，使用2 '，7'-双（羧乙基）-5（6）-羧基荧光素测量pH（i）。在标称无HCO 3条件下，非粘附J774.1细胞的3静息pH（i）为7.53 +/- 0.02（n = 86），粘附细胞的为7.59 +/- 0.02（n = 97）。在HCO 3-/CO2存在下，pH（i）值分别降低至7.41 +/- 0.02（n = 12）和7.40 +/- 0.01（n = 28）。阿米洛利是Na+/H+交换的抑制剂，不影响静息pH（i）。液泡型H+-ATP酶抑制剂[巴弗洛霉素A（1）、N-乙基马来酰亚胺（NEM）、7-氯-4-硝基苯-2-氧杂-1，3-二叠氮（NBD）和对氯汞苯磺酸（pCMBS）]使pH（i）降低至少0.2个pH单位。其他类型的H+-ATP酶（寡霉素，叠氮化物，钒酸盐和哇巴因）的抑制剂没有效果。通过弱缓冲细胞悬液的细胞外pH值变化测量的H+外排抑制遵循相同的药理学特征，表明pH值（i）的降低是由于H+外排抑制。还研究了从施加的细胞内酸负荷中恢复的机制。在NaCl-Hanks溶液中，pH（i）在2分钟内以指数方式恢复正常。阿米洛利或N-甲基-葡糖胺替代细胞外Na+浓度可抑制初始恢复率> 90%。液泡H+-ATP酶的抑制剂也抑制恢复。NEM和NBD非特异性地抑制所有恢复。巴弗洛霉素A（1）和pCMBS不抑制阿米洛利敏感的初始恢复部分，但当pH（i）高于7.0时，它们确实抑制了恢复的晚期成分。我们得出结论，Na+/H+交换器主要负责从酸负荷中恢复，但不调节静息pH（i）。相反，液泡H+-ATP酶调节J774细胞的静息pH（i），但对从酸化中恢复几乎没有贡献。
Mechanisms of intracellular pH (pH(i)) regulation were characterized in the murine macrophage cell line J774.1, using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to measure pH(i). Under nominally HCO3--free conditions, 3 resting pH(i) of nonadherent J774.1 cells was 7.53 +/- 0.02 (n = 86), and of adherent cells was 7.59 +/- 0.02 (n = 97). In the presence of HCO3-/CO2, pH(i) values were reduced to 7.41 +/- 0.02 (n = 12) and 7.40 +/- 0.01 (n = 28), respectively. Amiloride, an inhibitor of Na+/H+ exchange, did not affect resting pH(i). Inhibitors of a vacuolar type H+-ATPase [bafilomycin A(1), N-ethylmaleimide (NEM), 7-chloro-4-nitrobenz-2-oxa-1,3-diazide (NBD), and p-chloromercuriphenylsulfonic acid (pCMBS)] reduced pH(i) by at least 0.2 pH units. Inhibitors of other classes of H+-ATPases (oligomycin, azide, vanadate, and ouabain) were without effect. Inhibition of H+ efflux, measured by the change in extracellular pH of a weakly buffered cell suspension, followed the same pharmacological profile, indicating that the reduction of pH(i) was due to inhibition of H+ extrusion. Mechanisms of recovery from an imposed intracellular acid load were also investigated. In NaCl-Hanks' solution, pH(i) recovered exponentially to normal within 2 min. The initial rate of recovery was inhibited > 90% by amiloride or by replacement of extracellular Na+ concentration by N-methyl-glucamine. Inhibitors of the vacuolar H+-ATPase also inhibited recovery. NEM and NBD nonspecifically inhibited all recovery. Bafilomycin A(1) and pCMBS did not inhibit the initial amiloride-sensitive portion of recovery, but they did inhibit a late component of recovery when pH(i) was above 7.0. We conclude that the Na+/H+ exchanger is primarily responsible for recovery from an acid load but does not regulate resting pH(i). Conversely, a vacuolar H+-ATPase regulates the resting pH(i) of J774 cells but contributes little to recovery from acidification.