Intracellular pH regulation during spreading of human neutrophils.
Intracellular pH regulation during spreading of human neutrophils.
复制标题
DOI：
10.1083/jcb.133.6.1391
复制
发表时间：
1996-06
期刊：
影响因子：
--
通讯作者：
Grinstein S
中科院分区：
文献类型：
--
作者：
Demaurex N;Downey GP;Waddell TK;Grinstein S
本文用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（pHi）的调节。在粘性基质上铺展引起快速和持续的胞质碱化。这种pHi的增加是通过省略外部Na+来防止的，这表明它是由Na+/H+交换的激活引起的。在选择性阻断Na+H+反向转运蛋白的NHE-1亚型的浓度下，化合物HOE 694也阻止了细胞毒性诱导的碱化。通过任一程序抑制Na+/H+交换揭示了扩散后相当大的细胞溶质酸化，指示细胞内酸产生。过量的酸产生引起的，至少部分地，由呼吸爆发的激活，因为酸化密切相关的超氧化物的产生，测量在单个扩散的中性粒细胞与二氢罗丹明-123，和很少的酸生产中观察到的存在下，二苯碘鎓，NADPH氧化酶的阻断剂。此外，慢性肉芽肿病患者的中性粒细胞不产生超氧化物，不能酸化。当β 2整联蛋白在涂有抗CD 18抗体的表面上铺展期间被选择性激活时，观察到相当的pHi变化。当通过用可溶性抗CD 18抗体预处理排除整合素接合时，与纤维蛋白原上的扩散相关的pHi变化显著降低。抑制微丝装配与细胞松弛素D排除扩散和随之而来的废除超氧化物的产生和相关的pHi的变化，表明细胞骨架重组和/或参与的粘附受体的数量增加所需的响应。当氧化酶被阻断或当pHi被尼日利亚菌素夹在生理值附近时，中性粒细胞正常扩散。然而，当pHi被夹在酸性值时，扩散被强烈抑制。我们的研究结果表明，中性粒细胞释放超氧化物扩散，产生细胞内的酸生产的爆发。伴随的Na+/H+反向转运的激活不仅防止了由NADPH氧化酶释放的酸的有害作用，而且诱导了净细胞溶质碱化。由于中性粒细胞的几种功能在酸性pHi下受到抑制，pHi调节机制与氧化酶的协同激活沿着对于持续的杀微生物活性是必不可少的。
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.