Hou W, Arita Y, Morisset J
Département de Médecine, Faculté de Médecine Université de Sherbrooke, Québec, Canada.
Cell Signal. 1996 Nov;8(7):487-96. doi: 10.1016/s0898-6568(96)00104-0.
This study was performed to evaluate the effect of human recombinant basic fibroblast growth factor on arachidonic acid release from rat pancreatic acini and to determine the cellular mechanism involved. From enzymatic assays, basic fibroblast growth factor did not significantly stimulate phospholipase A2 activity, whereas it significantly increased diacylglycerol lipase activity. Validity of phospholipase A2 or diacylglycerol lipase inhibitors was confirmed by their ability to inhibit phospholipase A2 or diacylglycerol lipase activities. Basic fibroblast growth factor increased intracellular accumulation and extracellular release of arachidonic acid from metabolically labelled acinar cells in a concentration- and time-dependent manner. This effect was maximal with 50 pM basic fibroblast growth factor and became significant after a 5-min incubation period. The protein tyrosine kinase inhibitor, 0.5 mM genistein, inhibited arachidonic acid release in basic fibroblast growth factor-stimulated acini, whereas 100 microM vanadate, a protein tyrosine phosphatase inhibitor, enhanced arachidonic acid release. Two phospholipase A2 inhibitors, mepacrine and aristolochic acid, failed to attenuate basic fibroblast growth factor-stimulated arachidonic acid release. A diacylglycerol lipase inhibitor RHC 80267 at 150 microM and 50 microM completely inhibited 50 pM basic fibroblast growth factor-induced intracellular accumulation and extracellular release of arachidonic acid, respectively. Furthermore, basic fibroblast growth factor stimulated arachidonic acid release was also inhibited by 10 microM U73122 and by 100 nM staurosporine, phospholipase C and protein kinase C respective inhibitors. Wortmannin, an inhibitor of basic fibroblast growth factor-stimulated phospholipase D, did not affect arachidonic acid release. 100 nM 4 beta-phorbol 12-myristate 13-acetate also increased arachidonic acid release, an effect also inhibited by staurosporine. Taken together, these data demonstrate activation of diacylglycerol lipase and arachidonic acid release in pancreatic acini upon stimulation by basic fibroblast growth factor, and strongly indicate that arachidonic acid release in response to basic fibroblast growth factor depends upon the sequential action of tyrosine kinase, phospholipase C, protein kinase C and diacylglycerol lipase but not from phospholipase A2 not phospholipase D activation.
本研究旨在评估重组人碱性成纤维细胞生长因子对大鼠胰腺腺泡细胞花生四烯酸释放的影响，并确定其涉及的细胞机制。酶活性测定结果显示，碱性成纤维细胞生长因子并未显著刺激磷脂酶A2的活性，却能显著提高二酰基甘油脂肪酶的活性。磷脂酶A2或二酰基甘油脂肪酶抑制剂对相应酶活性的抑制能力证实了其有效性。碱性成纤维细胞生长因子能以浓度和时间依赖性方式增加代谢标记腺泡细胞内花生四烯酸的积累及细胞外释放。这种效应在50 pM碱性成纤维细胞生长因子时达到最大，并在孵育5分钟后变得显著。蛋白酪氨酸激酶抑制剂0.5 mM染料木黄酮可抑制碱性成纤维细胞生长因子刺激的腺泡细胞中花生四烯酸的释放，而蛋白酪氨酸磷酸酶抑制剂100 microM钒酸盐则增强了花生四烯酸的释放。两种磷脂酶A2抑制剂，即米帕林和马兜铃酸，未能减弱碱性成纤维细胞生长因子刺激的花生四烯酸释放。150 microM和50 microM的二酰基甘油脂肪酶抑制剂RHC 80267分别完全抑制了50 pM碱性成纤维细胞生长因子诱导的细胞内花生四烯酸积累和细胞外释放。此外，10 microM U73122和100 nM星形孢菌素（分别为磷脂酶C和蛋白激酶C的抑制剂）也抑制了碱性成纤维细胞生长因子刺激的花生四烯酸释放。渥曼青霉素是碱性成纤维细胞生长因子刺激的磷脂酶D的抑制剂，它对花生四烯酸释放没有影响。100 nM 4β-佛波醇12-肉豆蔻酸酯13-乙酸盐也增加了花生四烯酸的释放，这种效应也被星形孢菌素抑制。综上所述，这些数据表明碱性成纤维细胞生长因子刺激后，胰腺腺泡细胞中二酰基甘油脂肪酶被激活且花生四烯酸释放增加，并且强烈表明碱性成纤维细胞生长因子诱导的花生四烯酸释放依赖于酪氨酸激酶、磷脂酶C、蛋白激酶C和二酰基甘油脂肪酶的顺序作用，而非磷脂酶A2或磷脂酶D的激活。
