Signal Transduction in Neutrophil Activation - Search for the real 2nd messenger

我们在中性粒细胞中寻找激活或灭活超氧化物生成酶（NADPH氧化酶）的信号分子，发现磷脂酸（PA）可以激活该酶，精胺可以灭活该酶。PA是由磷脂酰胆碱通过磷脂酶D的作用而产生的，磷脂酶D在细胞激活时被激活。当加入渗透中性粒细胞时，PA立即激发活性。发现活化与Ca^<2+>，二酰基甘油或蛋白激酶c无关。活化是由PA微摩尔浓度引起的。O_2^-的产生速率与生理刺激的产生速率相似。这些结果表明，PA可能作为第二信使激活细胞内NADPH氧化酶。精胺的研究进展[j] .生物化学，生物物理，2003,(1)。]。为了阐明其抑制机制，本研究考察了精胺对NADPH氧化酶无细胞活化的影响。精胺抑制sds诱导的氧化酶活化（IC_<50>=18muM）。这种抑制作用对精胺的前体胺具有特异性。精胺没有改变NADPH的K_m和SDS的最佳活化浓度。发现该胺抑制半重组无细胞系统，并可能干扰酶组分的组装以形成活性复合物，特别是通过与细胞质亚基p67phox结合。这些结果表明，静息中性粒细胞中的精胺抑制超氧化物的产生，磷脂酸抑制超氧化物的产生，磷脂酸在细胞刺激时增加。

We have searched for the signaling molecules which activate or inactivate the superoxide generating enzyme (NADPH oxidase) in neutrophils, and found that hosphatidic acid (PA) may activate the enzyme and spermine inactivate it.PA is derived from phosphatidylcholine by action of phosphlipase D which is known to activate upon cell activation. When added to permeabilised neutrophils, PA elicited the activity immediately. The activation was found independent of Ca^<2+>, diacylglycerol, or protein kinase c. The activation was caused by PA micromolar concentrations. And the rate of O_2^- generation was similar to that by physiological stimuli. These results show that PA may searve as a second messenger to activate NADPH oxidase in the cell.Spermine, a celular plyamine, down-regulates superoxide generation [Ogata, Tamura, Takeshita (1992) Biochem.Biophys.Res.Commun.] . In the present study, to elucidate the mechanism for the inhibition, the effect of spermine on cell-free activation of NADPH oxidase was examined. Spermine suppressed the SDS-induced activation of the oxidase (IC_<50>=18muM). The inhibition was specific for spermine over its precursor amines. Spermine did not alter the K_m for NADPH or the optimal concentration of SDS for the activation. The amine was found to inhibit semi-recombinant cell-free system and may interfere with the assembly of the enzyme components to form active complex especially by binding to p67phox, a cytosolic subunit.These results show that superoxide generation is suppressed by spermine in resting neutrophils and clicited by phosphatidic acid which increases upon the cell stimulation.