Involvement of phospholipase C-gamma2 in activation of mitogen-activated protein kinase and phospholipase A2 by zooxanthellatoxin-A in rabbit platelets.
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.
Rho
M C
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Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Nakahata
N
N
Nakamura
H
H
Murai
A
A
Ohizumi
Y
Y
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D019833:Genistein; D007213:Indomethacin / Q000494:pharmacology; D007529:Isoflavones / Q000494:pharmacology; D008297:Male; D009281:Naphthalenes / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D011090:Polyenes / Q000494:pharmacology*; D011817:Rabbits; D019311:Staurosporine / Q000494:pharmacology; D010738:Type C Phospholipases / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism
D007529:Isoflavones; D009281:Naphthalenes; D010716:Phosphatidylinositols; D011090:Polyenes; C081400:zooxanthellatoxin A; D016718:Arachidonic Acid; D014443:Tyrosine; D019833:Genistein; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D010738:Type C Phospholipases; D019311:Staurosporine; C058819:calphostin C; D007213:Indomethacin