Hiura M, Ozawa M, Ohtsuka T, Takesue H, Yamaguchi M, Okamura N, Ishibashi S
Department of Physiological Chemistry, Hiroshima University School of Medicine, Japan.
Arch Biochem Biophys. 1991 Nov 15;291(1):31-7. doi: 10.1016/0003-9861(91)90101-n.
Conditions for superoxide anion (O2-) production were examined in guinea pig polymorphonuclear leukocytes (PMNL). When PMNL were suspended in the hypotonic medium, O2- production was significantly enhanced by concurrent treatment with low concentrations of 1-oleoyl-2-acetylglycerol (OAG), a cell-permeable protein kinase C activator. Such hypotonicity or OAG alone had little effect on the production. Other protein kinase C activators also markedly enhanced O2- production in combination with hypotonicity, but not in the isotonic medium. Protein kinase C inhibitors, H-7 and staurosporine, dose-dependently inhibited the production. These observations indicate that protein kinase C participates in such synergistic O2- production with hypotonicity. Phosphorylation of 46-kDa protein(s), which was commonly enhanced in paralleled with an activation of NADPH oxidase in guinea pig PMNL, was increased by treatment with 10 microM OAG, but the phosphorylation was little altered by hypotonic treatment. Intracellular calcium concentration, arachidonate release, and 1,2-diacylglycerol and phosphoinositide concentrations were slightly altered by hypotonic treatment. A change in phosphatidate (PA) production in PMNL was induced by hypotonic treatment either by itself or in combination with OAG treatment. These results suggest that the combination of cell membrane changes by hypotonic treatment accompanied by the increase in PA and 46-kDa protein phosphorylation by protein kinase C provides the conditions required for a marked increase in O2- production. Hypotonicity may be a good tool for studying the mechanism of priming in the activation of NADPH oxidase.
在豚鼠多形核白细胞（PMNL）中检测了超氧阴离子（O2-）产生的条件。当PMNL悬浮于低渗培养基中时，同时用低浓度的1-油酰基-2-乙酰甘油（OAG，一种可透过细胞的蛋白激酶C激活剂）处理，O2-的产生显著增强。单独的这种低渗或OAG对产生的影响很小。其他蛋白激酶C激活剂与低渗联合时也显著增强O2-的产生，但在等渗培养基中则不然。蛋白激酶C抑制剂H-7和星形孢菌素剂量依赖性地抑制产生。这些观察结果表明蛋白激酶C参与了这种与低渗协同的O2-产生。在豚鼠PMNL中，通常与NADPH氧化酶激活同时增强的46 kDa蛋白的磷酸化，通过用10 microM OAG处理而增加，但低渗处理对磷酸化几乎没有改变。低渗处理轻微改变了细胞内钙浓度、花生四烯酸释放以及1,2-二酰甘油和磷酸肌醇浓度。低渗处理单独或与OAG处理联合诱导了PMNL中磷脂酸（PA）产生的变化。这些结果表明，低渗处理引起的细胞膜变化与PA增加以及蛋白激酶C介导的46 kDa蛋白磷酸化相结合，为O2-产生的显著增加提供了所需条件。低渗可能是研究NADPH氧化酶激活中启动机制的良好工具。
