Chao W, Liu H, Hanahan D J, Olson M S
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
J Biol Chem. 1992 Apr 5;267(10):6725-35.
The lipid mediator platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC) has been shown to elicit several important biochemical signaling responses in mammalian cells, including polyphosphoinositide hydrolysis, arachidonic acid release/eicosanoid production, and protein tyrosine phosphorylation. In the present study, the roles of Ca2+ and protein kinase C (PKC), two signaling components of the phospholipase C pathway, in AGEPC-stimulated eicosanoid production and protein tyrosine phosphorylation, were investigated in cultured rat Kupffer cells. AGEPC at nanomolar concentrations induced an increase in intracellular calcium concentration ([Ca2+]i), stimulated membrane PKC activity, and resulted in protein tyrosine phosphorylation. The maximal increase in [Ca2+]i and membrane PKC activity in response to AGEPC were observed within 30-50 s, whereas the AGEPC-induced protein tyrosine phosphorylation reached maximal levels within 2-5 min. [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) but not 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of calcium release from intracellular compartments, nearly abolished the AGEPC-induced increase in [Ca2+]i suggesting involvement of extracellular calcium influx in this event. Both EGTA and TMB-8 abolished or inhibited AGEPC-stimulated protein tyrosine phosphorylation and eicosanoid formation, respectively. The calcium ionophore A23187 alone stimulated eicosanoid production and protein tyrosine phosphorylation with an identical pattern to that of AGEPC. Phorbol myristate acetate (PMA), an activator of PKC, which did not affect [Ca2+]i, mimicked the actions of AGEPC, stimulating eicosanoid production and promoting tyrosine phosphorylation of a set of proteins similar to those phosphorylated following AGEPC stimulation. AGEPC-enhanced tyrosine phosphorylation of some of the protein substrates and eicosanoid production were inhibited in cells "down-regulated" for PKC. Furthermore, both PMA- and AGEPC-stimulated eicosanoid production and protein tyrosine phosphorylation were attenuated or abolished by at least one of the PKC inhibitors, staurosporine, and calphostin C. Taken together, these results are consistent with the conclusions that: (a) AGEPC stimulates the phospholipase-mediated arachidonic acid release/eicosanoid synthesis cascade and protein tyrosine phosphorylation through extracellular Ca(2+)-dependent and PKC-dependent and -independent mechanism(s) and (b) the Ca(2+)-PKC interaction determines the efficacy of the AGEPC-stimulated cellular events.
脂质介质血小板活化因子（1-O-烷基-2-乙酰基-sn-甘油-3-磷酸胆碱，AGEPC）已被证明能在哺乳动物细胞中引发多种重要的生化信号反应，包括多磷酸肌醇水解、花生四烯酸释放/类花生酸生成以及蛋白酪氨酸磷酸化。在本研究中，我们在培养的大鼠库普弗细胞中研究了磷脂酶C途径的两个信号成分钙离子（Ca2+）和蛋白激酶C（PKC）在AGEPC刺激的类花生酸生成和蛋白酪氨酸磷酸化中的作用。纳摩尔浓度的AGEPC可诱导细胞内钙浓度（[Ca2+]i）升高，刺激膜PKC活性，并导致蛋白酪氨酸磷酸化。对AGEPC反应时，[Ca2+]i和膜PKC活性的最大增加在30 - 50秒内观察到，而AGEPC诱导的蛋白酪氨酸磷酸化在2 - 5分钟内达到最大水平。细胞内钙释放抑制剂[乙二胺双（氧乙烯腈）]四乙酸（EGTA）而非8-（N，N-二乙氨基）-辛基-3,4,5-三甲氧基苯甲酸盐酸盐（TMB-8）几乎完全消除了AGEPC诱导的[Ca2+]i升高，表明细胞外钙内流参与了这一过程。EGTA和TMB-8分别消除或抑制了AGEPC刺激的蛋白酪氨酸磷酸化和类花生酸形成。单独的钙离子载体A23187刺激类花生酸生成和蛋白酪氨酸磷酸化的模式与AGEPC相同。佛波酯肉豆蔻酸乙酸酯（PMA）是PKC的激活剂，它不影响[Ca2+]i，模拟了AGEPC的作用，刺激类花生酸生成并促进一组与AGEPC刺激后磷酸化的蛋白质相似的蛋白质的酪氨酸磷酸化。在PKC“下调”的细胞中，AGEPC增强的一些蛋白底物的酪氨酸磷酸化和类花生酸生成受到抑制。此外，PKC抑制剂星形孢菌素和钙泊三醇中的至少一种可减弱或消除PMA和AGEPC刺激的类花生酸生成和蛋白酪氨酸磷酸化。综上所述，这些结果与以下结论一致：（a）AGEPC通过细胞外Ca(2+)依赖性和PKC依赖性及非依赖性机制刺激磷脂酶介导的花生四烯酸释放/类花生酸合成级联反应和蛋白酪氨酸磷酸化；（b）Ca(2+)-PKC相互作用决定了AGEPC刺激的细胞事件的效力。
