Bidani A, Brown S E, Heming T A, Gurich R, Dubose T D
Department of Medicine, University of Texas Medical Branch, Galveston 77550.
Am J Physiol. 1989 Jul;257(1 Pt 1):C65-76. doi: 10.1152/ajpcell.1989.257.1.C65.
The pulmonary macrophage plays a primary role in the immunological defense of the lung. Although many studies have been devoted to elucidation of its phagocytic and secretory functions, little is known of its membrane transport properties or of how it regulates intracellular pH (pHi). The purpose of this study, therefore, was to determine base-line pHi and the mechanism(s) by which the cell recovers pHi when challenged with an intracellular acid load. Through the use of the pH-sensitive fluorescent dye, 2,7-biscarboxyethyl-5(6)-carboxy-fluorescein (BCECF), base-line pHi was estimated to be 7.24 +/- 0.03. Cells were acidified by two methods, nigericin and weak acids, while recovery (dpHi/dt) was monitored. The rate of recovery was found to be independent of external Na+ and K+ and was insensitive to amiloride. Pretreatment with 4,4'-diiso-thiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of Cl- -HCO3- exchange, was also without effect on recovery from an intracellular acid load in these cells, under nominally HCO3- -free conditions. In contrast, N-ethylmaleimide (NEM) and N,N'-dicyclohexylcarbodiimide, nonspecific inhibitors of proton adenosinetriphosphatases (ATPases), virtually abolished pHi recovery. Efflux of H+ equivalents by pulmonary macrophages was measured by techniques involving both pH stat titration and the effect on fluorescence of extracellular BCECF. Basal H+ extrusion was approximately 2.75 +/- 0.64 nmol H+.min-1.10(6) cells-1 and was enhanced to approximately 26.0 +/- 6.95 nmol H+.min-1.10(6) cells-1 in acid-loaded cell suspensions. The basal rate of H+ extrusion was reduced to approximately 0.84 +/- 0.31 nmol H+.min-1.10(6) cells-1 in the presence of 1 mM NEM. These results suggest that recovery of cytoplasmic pH from an intracellular acid load, as well as regulation of pHi, under the conditions examined, is not mediated by a Na+-H+ exchanger in these cells. Rather, the data are consistent with the presence of an H+-ATPase in the plasma membrane of pulmonary macrophages.
肺巨噬细胞在肺部的免疫防御中起主要作用。尽管许多研究致力于阐明其吞噬和分泌功能，但对其膜转运特性或其如何调节细胞内pH（pHi）却知之甚少。因此，本研究的目的是确定基线pHi以及细胞在受到细胞内酸负荷挑战时恢复pHi的机制。通过使用对pH敏感的荧光染料2,7-双（羧乙基）-5（6）-羧基荧光素（BCECF），估计基线pHi为7.24±0.03。细胞通过两种方法酸化，即尼日利亚菌素和弱酸，同时监测恢复情况（dpHi/dt）。发现恢复速率与细胞外Na+和K+无关，并且对氨氯吡脒不敏感。在名义上无HCO3-的条件下，用Cl--HCO3-交换抑制剂4,4'-二异硫氰基芪-2,2'-二磺酸预处理对这些细胞从细胞内酸负荷中的恢复也没有影响。相反，质子腺苷三磷酸酶（ATP酶）的非特异性抑制剂N-乙基马来酰亚胺（NEM）和N,N'-二环己基碳二亚胺几乎完全消除了pHi的恢复。通过涉及pH稳态滴定和细胞外BCECF对荧光影响的技术测量肺巨噬细胞中H+当量的流出。基础H+排出量约为2.75±0.64 nmol H+.min-1.10(6)细胞-1，在酸负荷细胞悬液中增加到约26.0±6.95 nmol H+.min-1.10(6)细胞-1。在1 mM NEM存在下，基础H+排出速率降低到约0.84±0.31 nmol H+.min-1.10(6)细胞-1。这些结果表明，在所研究的条件下，细胞内酸负荷后细胞质pH的恢复以及pHi的调节不是由这些细胞中的Na+-H+交换体介导的。相反，数据与肺巨噬细胞质膜中存在H+-ATP酶一致。
