Brisseau G F, Tsai O, Nordström T, Marshall J C, Grinstein S, Rotstein O D
Department of Surgery, University of Toronto, Ontario, Canada.
Surgery. 1994 Aug;116(2):268-74; discussion 274-5.
Maintenance of cytoplasmic pH (pHi) close to the physiologic range is vital to normal cellular homeostasis. We have previously reported that a vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) situated in the plasma membrane of macrophages and poised to extrude protons from the cytoplasmic to the extracellular space is an important pHi regulatory mechanism. Since the inflammatory microenvironment is frequently characterized by the influx of cells known to release reactive oxygen metabolites, we performed studies to examine the effect of oxidant stress on pHi regulation in peritoneal macrophages. Specifically, the effect of hydrogen peroxide on V-ATPase-mediated proton extrusion from acid-loaded macrophages was investigated.
Thioglycollate-elicited murine peritoneal macrophages were exposed to varying concentrations of hydrogen peroxide and examined for their ability to recover from an acid-load. pHi was studied by preloading cells with the pH-sensitive fluorescent dye, bis-carboxyethyl-carboxyfluorescein, and monitoring changes in fluorescence under various conditions using a fluorescence spectrometer.
Hydrogen peroxide caused a time- and dose-dependent decrease in proton pump-mediated pHi recovery in peritoneal macrophages. This effect occurred without cytotoxicity and was a specific effect as evidenced by the ability of catalase to reverse the inhibition. Since hydrogen peroxide is known to deplete intracellular ATP, a substrate for V-ATPase activity, we hypothesized that ATP depletion may underlie the effect. These studies showed that hydrogen peroxide-mediated ATP depletion was both necessary and sufficient for the effect. Finally, depletion of intracellular glutathione in vivo by using diethyl maleate increased the sensitivity of V-ATPase activity to oxidant stress.
Oxidant stress within the inflammatory milieu impairs macrophage pHi regulation. This effect is magnified by depletion of intracellular antioxidants, as occurs during sepsis. This represents another mechanism whereby oxidants may contribute to cellular dysfunction associated with inflammatory states.
维持细胞质pH（pHi）接近生理范围对正常细胞稳态至关重要。我们之前报道过，位于巨噬细胞膜上、准备将质子从细胞质排出到细胞外空间的液泡型H(+) - 三磷酸腺苷酶（V - ATP酶）是一种重要的pHi调节机制。由于炎症微环境的特征通常是已知会释放活性氧代谢产物的细胞流入，我们进行了研究以检查氧化应激对腹膜巨噬细胞pHi调节的影响。具体而言，研究了过氧化氢对酸负荷巨噬细胞中V - ATP酶介导的质子排出的影响。
用巯基乙酸盐诱导的小鼠腹膜巨噬细胞暴露于不同浓度的过氧化氢，并检测其从酸负荷中恢复的能力。通过用pH敏感荧光染料双羧乙基羧基荧光素预加载细胞，并使用荧光光谱仪监测各种条件下的荧光变化来研究pHi。
过氧化氢导致腹膜巨噬细胞中质子泵介导的pHi恢复出现时间和剂量依赖性降低。这种效应在没有细胞毒性的情况下发生，并且是一种特异性效应，过氧化氢酶能够逆转这种抑制作用证明了这一点。由于已知过氧化氢会消耗细胞内ATP，而ATP是V - ATP酶活性的底物，我们推测ATP消耗可能是这种效应的基础。这些研究表明，过氧化氢介导的ATP消耗对于这种效应既是必要的也是充分的。最后，通过使用马来酸二乙酯在体内耗尽细胞内谷胱甘肽增加了V - ATP酶活性对氧化应激的敏感性。
炎症环境中的氧化应激损害巨噬细胞的pHi调节。如败血症期间发生的那样，细胞内抗氧化剂的消耗会放大这种效应。这代表了氧化剂可能导致与炎症状态相关的细胞功能障碍的另一种机制。
