Agwu D E, McCall C E, McPhail L C
Department of Medicine, Wake Forest University Medical Center, Winston-Salem, NC 27103.
J Immunol. 1991 Jun 1;146(11):3895-903.
In human neutrophils, the chemotactic tripeptide FMLP and the protein kinase C activator PMA stimulate the breakdown of 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine ([3H]EAPC) and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid ([3H]-EAPA) and 1-O-[3H]alkyl-2-acylglycerol ([3H]EAG) via mechanism(s) that may involve the activation of phospholipase D. We have investigated the regulation of phospholipase D by determining the effects of elevation of intracellular levels of cAMP on receptor-mediated and PMA-induced breakdown of [3H]-EAPC and formation of products. Pretreatment of neutrophils with either 10(-3) M dibutyryl-cAMP or 10(-5) M PGE2, in the presence of 4 x 10(-4) M isobutylmethylxanthine (IBMX), inhibited FMLP- and leukotriene B4-induced breakdown of [3H]EAPC and formation of [3H]EAPA and [3H]EAG. Inhibition was apparent at all time points of stimulation examined (15-120 s). In addition, the mass of diradyl-phosphatidic acid was decreased in FMLP-stimulated neutrophils pretreated with PGE2 and IBMX. In contrast, pretreatment of cells with PGE2 and IBMX did not inhibit PMA-induced breakdown of [3H]EAPC and the formation of products at 3 and 10 min after stimulation. Furthermore, formation of 1-O-[3H]alkyl-2-acyl-phosphatidylethanol, produced by phospholipase D in the presence of ethanol by a transphosphatidylation reaction, was significantly inhibited by pretreatment of cells with PGE2 and IBMX in FMLP- but not PMA-stimulated neutrophils. This differential modulation by cAMP of receptor-mediated and PMA-induced activation of phospholipase D suggests agonist-dependent activation of separate pathways and/or activation of separate phospholipase D enzymes. Thus, cAMP elevation may exert inhibitory effects directly on the phospholipase D activated by FMLP and/or on sites proximal to the enzyme that are involved in signal transmission.
在人类中性粒细胞中，趋化三肽FMLP和蛋白激酶C激活剂PMA通过可能涉及磷脂酶D激活的机制，刺激1-O-[3H]烷基-2-酰基-sn-甘油-3-磷酸胆碱（[3H]EAPC）的分解以及1-O-[3H]烷基-2-酰基磷脂酸（[3H]-EAPA）和1-O-[3H]烷基-2-酰基甘油（[3H]EAG）的形成。我们通过测定细胞内cAMP水平升高对受体介导的以及PMA诱导的[3H]-EAPC分解和产物形成的影响，研究了磷脂酶D的调节作用。在存在4×10(-4) M异丁基甲基黄嘌呤（IBMX）的情况下，用10(-3) M二丁酰-cAMP或10(-5) M前列腺素E2对中性粒细胞进行预处理，可抑制FMLP和白三烯B4诱导的[3H]EAPC分解以及[3H]EAPA和[3H]EAG的形成。在所检测的刺激的所有时间点（15 - 120秒），抑制作用均很明显。此外，在用前列腺素E2和IBMX预处理的FMLP刺激的中性粒细胞中，二酰基磷脂酸的量减少。相比之下，用前列腺素E2和IBMX对细胞进行预处理，在刺激后3分钟和10分钟时，并不抑制PMA诱导的[3H]EAPC分解和产物形成。此外，在乙醇存在下由磷脂酶D通过转磷脂酰基反应产生的1-O-[3H]烷基-2-酰基磷脂酰乙醇的形成，在FMLP刺激而非PMA刺激的中性粒细胞中，被用前列腺素E2和IBMX对细胞进行预处理显著抑制。cAMP对受体介导的和PMA诱导的磷脂酶D激活的这种差异调节表明，不同激动剂依赖于不同途径的激活和/或不同磷脂酶D酶的激活。因此，cAMP升高可能直接对由FMLP激活的磷脂酶D和/或对参与信号传递的该酶近端位点发挥抑制作用。
