Bianchini L, Todderud G, Grinstein S
Division of Cell Biology, Hospital for Sick Children, Toronto, Canada.
J Biol Chem. 1993 Feb 15;268(5):3357-63.
Activated phagocytes produce large amounts of reactive oxygen intermediates, including peroxides. In addition to their microbicidal effect, it has recently been suggested that reactive oxygen species play a role as intracellular messengers. The mechanism of action remains unknown, but peroxides have been reported to increase tyrosine phosphorylation, an effect potentiated by vanadate. In this report we studied the effects of a combination of H2O2 and vanadate on Ca2+ homeostasis in granulocytic HL60 cells. The peroxides induced a transient elevation of cytosolic [Ca2+] associated with release from internal stores. Ca2+ mobilization was accompanied by increased generation of inositol 1,4,5-trisphosphate, implicating phospholipase C (PLC). A sizable increase in phosphotyrosine accumulation by several polypeptides in the M(r) 20,000 to 250,000 range preceded the [Ca2+] changes. We therefore considered the possibility that tyrosine phosphorylation of a phospholipase mediates the observed effects. Differentiated (granulocytic) HL60 cells did not have detectable levels of PLC gamma 1 but had substantial PLC gamma 2. Immunoprecipitation and immunoblotting experiments demonstrated that PLC gamma 2 becomes tyrosine-phosphorylated upon treatment of the cells with peroxides of vanadate. If associated with activation, such phosphorylation of PLC gamma 2 can account for the rise in [Ca2+]. Although capable of mobilizing internal Ca2+ stores, the peroxides failed to produce the sustained [Ca2+] increase predicted by the "capacitative" model. Mn2+ influx determinations indicated that this is due to impairment of divalent cation entry by the peroxides, uncoupling the plasma membrane from the internal stores. Changes in [Ca2+] homeostasis could mediate some of the messenger actions of reactive oxygen species.
活化的吞噬细胞会产生大量的活性氧中间体，包括过氧化物。除了其杀菌作用外，最近有研究表明活性氧作为细胞内信使发挥作用。其作用机制尚不清楚，但据报道过氧化物可增加酪氨酸磷酸化，钒酸盐可增强这种作用。在本报告中，我们研究了过氧化氢和钒酸盐组合对粒细胞HL60细胞中钙离子稳态的影响。过氧化物诱导细胞溶质[Ca2+]短暂升高，这与细胞内储存库的释放有关。Ca2+动员伴随着肌醇1,4,5-三磷酸生成的增加，这表明磷脂酶C（PLC）参与其中。在[Ca2+]变化之前，分子量在20,000至250,000范围内的几种多肽的磷酸酪氨酸积累有相当大的增加。因此，我们考虑磷脂酶的酪氨酸磷酸化介导观察到的效应的可能性。分化的（粒细胞）HL60细胞没有可检测到的PLCγ1水平，但有大量的PLCγ2。免疫沉淀和免疫印迹实验表明，在用钒酸盐过氧化物处理细胞后，PLCγ2会发生酪氨酸磷酸化。如果与激活相关，PLCγ2的这种磷酸化可以解释[Ca2+]的升高。尽管过氧化物能够动员细胞内的Ca2+储存库，但未能产生“容量性”模型预测的持续[Ca2+]增加。Mn2+流入测定表明，这是由于过氧化物损害了二价阳离子的进入，使质膜与细胞内储存库解偶联。[Ca2+]稳态的变化可能介导活性氧的一些信使作用。
