Rho M C, Nakahata N, Nakamura H, Murai A, Ohizumi Y
Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
J Pharmacol Exp Ther. 1997 Jul;282(1):496-504.
Zooxanthellatoxin-A (ZT-A), a polyhydroxypolyene isolated from a symbiotic dinoflagellate Symbiodinium sp., caused thromboxane A2-(TXA2) dependent and genistein-sensitive aggregation in rabbit platelets. Our study was performed to clarify the mechanism of the action of ZT-A. ZT-A caused an increase in tyrosine phosphorylation of 42-kDa protein, which is defined as p42 mitogen-activated protein kinase (MAPK) by immunoprecipitation. Although indomethacin (10 microM) completely inhibited ZT-A-induced TXB2 release, it partially inhibited the MAPK activation. The remained MAPK activation was completely inhibited by genistein (50 microM). Genistein (50 microM), by itself, abolished TXB2 release induced by ZT-A. ZT-A (2 microM) stimulated liberation of arachidonic acid and the subsequent metabolites such as TXB2 and 12-hydroperoxyeicosatetraenoic acid. However, ZT-A-stimulated phosphoinositide hydrolysis which was due to an increase in tyrosine phosphorylation of phospholipase C-(PLC)gamma2. The phosphorylation of PLC-gamma2 and the phosphoinositide hydrolysis were also partially inhibited by indomethacin (10 microM), and were abolished by a combined treatment of indomethacin (10 microM) and genistein (50 microM). ZT-A- (2 microM) induced MAPK activation in the presence of indomethacin (10 microM) was concentration-dependently inhibited by staurosporine and calphostin C, protein kinase C inhibitors. PD98059 (50 microM), a MAPK kinase inhibitor, also inhibited ZT-A-induced TXB2 release. Depletion of external Ca++ abolished ZT-A- (2 microM) induced MAPK activation, phosphoinositide hydrolysis, arachidonic acid liberation and TXB2 release. These results suggest that ZT-A stimulates a protein tyrosine kinase in the presence of external Ca++, resulting in the activation of MAPK probably via PLC-gamma2 and protein kinase C. The MAPK stimulated a liberation of arachidonic acid that is rapidly converted to TXA2. The released TXA2 causes aggregation accompanied with second stimulation of MAPK cascade.
虫黄藻毒素 - A（ZT - A）是从共生甲藻共生藻属中分离出的一种多羟基多烯，可引起兔血小板中血栓素A2 -（TXA2）依赖性和染料木黄酮敏感的聚集。我们进行这项研究以阐明ZT - A的作用机制。ZT - A导致42 kDa蛋白的酪氨酸磷酸化增加，通过免疫沉淀法将其定义为p42丝裂原活化蛋白激酶（MAPK）。虽然吲哚美辛（10 microM）完全抑制ZT - A诱导的TXB2释放，但它仅部分抑制MAPK激活。剩余的MAPK激活被染料木黄酮（50 microM）完全抑制。染料木黄酮（50 microM）自身可消除ZT - A诱导的TXB2释放。ZT - A（2 microM）刺激花生四烯酸的释放以及随后的代谢产物，如TXB2和12 - 氢过氧二十碳四烯酸。然而，ZT - A刺激的磷脂酰肌醇水解是由于磷脂酶C -（PLC）γ2的酪氨酸磷酸化增加所致。PLC - γ2的磷酸化和磷脂酰肌醇水解也被吲哚美辛（10 microM）部分抑制，并被吲哚美辛（10 microM）和染料木黄酮（50 microM）联合处理所消除。在吲哚美辛（10 microM）存在下，ZT - A -（2 microM）诱导的MAPK激活被蛋白激酶C抑制剂星形孢菌素和钙磷蛋白C浓度依赖性抑制。MAPK激酶抑制剂PD98059（50 microM）也抑制ZT - A诱导的TXB2释放。细胞外Ca++的耗尽消除了ZT - A -（2 microM）诱导的MAPK激活。花生四烯酸的释放以及TXB2释放。这些结果表明，ZT - A在细胞外Ca++存在下刺激蛋白酪氨酸激酶，可能通过PLC - γ2和蛋白激酶C导致MAPK激活。被刺激的MAPK促进花生四烯酸的释放，花生四烯酸迅速转化为TXA2。释放的TXA2导致聚集并伴随着MAPK级联的二次刺激。
