Vol. 282, Issue 1, 496-504, 1997

Involvement of Phospholipase C-2 in Activation of Mitogen-Activated Protein Kinase and Phospholipase A₂ by Zooxanthellatoxin-A in Rabbit Platelets¹

Mun-Chual Rho, Norimichi Nakahata, Hideshi Nakamura, Akio Murai and Yasushi Ohizumi

Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aobaku, Sendai 980 (M.-C.R., N.N., Y.O.) and Department of Chemistry, Faculty of Sciences, Hokkaido University, Sapporo 060, Japan (H.N., A.M.)

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 µM) completely inhibited ZT-A-induced TXB₂ release, it partially inhibited the MAPK activation. The remained MAPK activation was completely inhibited by genistein (50 µM). Genistein (50 µM), by itself, abolished TXB₂ release induced by ZT-A. ZT-A (2 µM) 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)2. The phosphorylation of PLC-2 and the phosphoinositide hydrolysis were also partially inhibited by indomethacin (10 µM), and were abolished by a combined treatment of indomethacin (10 µM) and genistein (50 µM). ZT-A- (2 µM) induced MAPK activation in the presence of indomethacin (10 µM) was concentration-dependently inhibited by staurosporine and calphostin C, protein kinase C inhibitors. PD98059 (50 µM), a MAPK kinase inhibitor, also inhibited ZT-A-induced TXB₂ release. Depletion of external Ca⁺⁺ abolished ZT-A- (2 µM) 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-2 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.