Buckley B J, Whorton A R
Department of Medicine, Duke University Medical Center, Durham, NC 27710.
Biochem J. 1994 Jun 1;300 ( Pt 2)(Pt 2):449-55. doi: 10.1042/bj3000449.
We investigated the mechanism by which the G-protein activators aluminium fluoride and vanadate stimulate arachidonic acid release in pig aortic endothelial cells. Our previous study demonstrated a novel Ca(2+)-independent pathway of phospholipase A2 (PLA2) activation stimulated by aluminium fluoride in this model. In the present study, we found that sodium metavanadate stimulated a rapid concentration-dependent release of [3H]arachidonic acid from prelabelled cells. A more than 3-fold enhancement of arachidonic acid release was achieved in cells treated with 1 mM vanadate for 20 min. Synthesis of prostaglandin products was similarly enhanced. The release of arachidonic acid was not dependent on the presence of extracellular Ca2+, but did require protein synthesis de novo. Both cycloheximide and actinomycin D completely blocked aluminium fluoride- and vanadate-stimulated arachidonic acid release. Because fluoride and vanadate are known protein tyrosine phosphatase inhibitors, it is possible that PLA2 activation occurred secondarily to changes in protein tyrosine phosphorylation. Both aluminium fluoride and vanadate stimulated the rapid phosphorylation of 58, 93 and 120 kDa tyrosine-containing protein substrates. However, in contrast with arachidonic acid release, this response was found to be sensitive to the presence of extracellular Ca2+ and insensitive to blockers of protein synthesis de novo. Furthermore H2O2 treatment resulted in rapid tyrosine phosphorylation of the same substrates without a concomitant increase in arachidonic acid release. These results suggest that the effects of aluminium fluoride and vanadate on PLA2 are not due to changes in protein tyrosine phosphorylation, but do require rapid protein synthesis de novo.
我们研究了G蛋白激活剂氟化铝和钒酸盐刺激猪主动脉内皮细胞释放花生四烯酸的机制。我们之前的研究表明，在该模型中，氟化铝可通过一种新的不依赖Ca(2+)的途径激活磷脂酶A2（PLA2）。在本研究中，我们发现偏钒酸钠能刺激预先标记的细胞快速释放[3H]花生四烯酸，且呈浓度依赖性。用1 mM钒酸盐处理细胞20分钟后，花生四烯酸释放量增加了3倍多。前列腺素产物的合成也同样增强。花生四烯酸的释放不依赖细胞外Ca2+的存在，但确实需要重新合成蛋白质。环己酰亚胺和放线菌素D均可完全阻断氟化铝和钒酸盐刺激的花生四烯酸释放。由于已知氟化物和钒酸盐是蛋白酪氨酸磷酸酶抑制剂，因此PLA2的激活可能继发于蛋白酪氨酸磷酸化的变化。氟化铝和钒酸盐均可刺激58、93和120 kDa含酪氨酸蛋白底物的快速磷酸化。然而，与花生四烯酸释放不同的是，这种反应对细胞外Ca2+的存在敏感，对重新合成蛋白质的阻断剂不敏感。此外，H2O2处理导致相同底物的快速酪氨酸磷酸化，但花生四烯酸释放量并未随之增加。这些结果表明，氟化铝和钒酸盐对PLA2的作用并非由于蛋白酪氨酸磷酸化的变化，而是确实需要重新快速合成蛋白质。
