Demaurex N, Downey G P, Waddell T K, Grinstein S
Division of Cell Biology, Hospital for Sick Children, Toronto, Canada.
J Cell Biol. 1996 Jun;133(6):1391-402. doi: 10.1083/jcb.133.6.1391.
The regulation of the intracelluar pH (pHi) during spreading of human neutrophils was studied by a combination of fluorescence imaging and video microscopy. Spreading on adhesive substrates caused a rapid and sustained cytosolic alkalinization. This pHi increase was prevented by the omission of external Na+, suggesting that it results from the activation of Na+/H+ exchange. Spreading-induced alkalinization was also precluded by the compound HOE 694 at concentrations that selectively block the NHE-1 isoform of the Na+H+ antiporter. Inhibition of Na+/H+ exchange by either procedure unmasked a sizable cytosolic acidification upon spreading, indicative of intracellular acid production. The excess acid generation was caused, at least in part, by the activation of the respiratory burst, since the acidification closely correlated with superoxide production, measured in single spreading neutrophils with dihydrorhodamine-123, and little acid production was observed in the presence of diphenylene iodonium, a blocker of the NADPH oxidase. Moreover, neutrophils from chronic granulomatous disease patients, which do not produce superoxide, failed to acidify. Comparable pHi changes were observed when beta 2 integrins were selectively activated during spreading on surfaces coated with anti-CD18 antibodies. When integrin engagement was precluded by pretreatment with soluble anti-CD18 antibody, the pHi changes associated with spreading on fibrinogen were markedly reduced. Inhibition of microfilament assembly with cytochalasin D precluded spreading and concomitantly abolished superoxide production and the associated pHi changes, indicating that cytoskeletal reorganization and/or an increase in the number of adherence receptors engaged are required for the responses. Neutrophils spread normally when the oxidase was blocked or when pHi was clamped near physiological values with nigericin. Spreading, however, was strongly inhibited when pHi was clamped at acidic values. Our results indicate that neutrophils release superoxide upon spreading, generating a burst of intracellular acid production. The concomitant activation of the Na+/H+ antiport not only prevents the deleterious effects of the acid released by the NADPH oxidase, but induces a net cytosolic alkalinization. Since several functions of neutrophils are inhibited at an acidic pHi, the coordinated activation of pHi regulatory mechanisms along with the oxidase is essential for sustained microbicidal activity.
通过荧光成像和视频显微镜相结合的方法，研究了人中性粒细胞铺展过程中细胞内pH值（pHi）的调节。在黏附底物上的铺展导致胞质迅速且持续地碱化。外部Na⁺的缺失可阻止这种pHi升高，表明其源于Na⁺/H⁺交换的激活。化合物HOE 694在选择性阻断Na⁺/H⁺反向转运体的NHE-1亚型的浓度下，也能阻止铺展诱导的碱化。通过上述任何一种方法抑制Na⁺/H⁺交换，都会在铺展时暴露出相当程度的胞质酸化，这表明细胞内有酸产生。过量的酸产生至少部分是由呼吸爆发的激活引起的，因为酸化与超氧化物的产生密切相关，在单个铺展的中性粒细胞中用二氢罗丹明-123测量超氧化物的产生，并且在NADPH氧化酶的抑制剂二苯碘鎓存在的情况下几乎没有酸产生。此外，慢性肉芽肿病患者的中性粒细胞不产生超氧化物，也不会酸化。当在包被抗CD18抗体的表面铺展过程中β2整合素被选择性激活时，观察到了类似的pHi变化。当用可溶性抗CD18抗体预处理阻止整合素结合时，与在纤维蛋白原上铺展相关的pHi变化明显减少。用细胞松弛素D抑制微丝组装可阻止铺展，并同时消除超氧化物的产生和相关的pHi变化，表明细胞骨架重组和/或参与的黏附受体数量增加是这些反应所必需的。当氧化酶被阻断或用尼日利亚菌素将pHi钳制在生理值附近时，中性粒细胞能正常铺展。然而，当pHi被钳制在酸性值时，铺展受到强烈抑制。我们的结果表明，中性粒细胞在铺展时释放超氧化物，产生一阵细胞内酸的产生。同时激活的Na⁺/H⁺反向转运不仅能防止NADPH氧化酶释放的酸的有害影响，还能诱导胞质净碱化。由于中性粒细胞的几种功能在酸性pHi时会受到抑制，pHi调节机制与氧化酶的协同激活对于持续的杀菌活性至关重要。
