Differential effects of phorbol 12-myristate 13-acetate and diacylglycerols on thromboxane A2-independent phospholipase A2 activation in collage-stimulated human platelets.
We investigated the priming effects of protein kinase C (PKC) activators such as phorbol 12-myristate 13-acetate (PMA), 1,2-DiC8 and OAG, and 1,3-DiC8 (a poor activator of PKC) on thromboxane A2 (TxA2)-independent phospholipase A2 (PLA2) activation in human platelets using collagen and A23187 as agonists. We measured PLA2 activation in collagen-stimulated platelets in the presence of BW755C, which abolished TxA2 synthesis, rise in cytosolic Ca2+, and aggregation. In the presence of PMA (50 nM), the amount of arachidonic acid (AA) released in platelets stimulated with collagen and A23187 represented 300% (13.85 nmol versus 4.5 nmol) and 400% (28 nmol versus 7 nmol) of controls (without PMA), respectively, while 1,2-DiC8, OAG, and 1,3-DiC8 increased TxA2-independent AA release by 50% in A23187-stimulated platelets and had no effect on the release of AA in collagen-stimulated platelets. Interestingly, 1,3-DiC8, which is a poor activator of PKC, was as effective as the other two DAGs (OAG and 1,2-DiC8) in priming TxA2-independent PLA2 activation, but was less effective than PMA in platelets stimulated with A23187. These results suggest that the TXA2-dependent IP3-mediated rise in cytosolic Ca2+ may not be obligatory for priming PLA2 activation in the presence of PMA in collagen-stimulated platelets. In contrast, 1,2-DiC8, OAG, and 1,3-DiC8 likely enhanced PLA2 activation via intracellular Ca2+ as they selectively affect this enzyme only in A23187-stimulated platelets. We also observed a significant increase in both saturated (palmitic and stearic acids) and unsaturated fatty acids (oleic and linoleic acids) in platelets stimulated by collagen or A23187 in the presence of PMA (50 nM), but not in the presence of DAGs. These findings imply that PMA may also affect the activation of DAG/MAG lipases, PLA1, or nonspecific PLA2. Since both 1,2-DiC8 and OAG exert no significant effect on the release of these fatty acids, the effects observed with PMA on DAG lipase/PLA1 may not involve a PKC-dependent mechanism. We, therefore, conclude that the mechanisms by which PMA and DAGs prime PLA2 activation are different and that the priming mechanism by DAGs may not involve PKC, but may require a rise in intracellular Ca2+.
Reddy
S
S
Department of Pediatrics and Child Health, Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada.
Rao
G H
GH
Murthy
M
M
D015772:4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D003094:Collagen / Q000494:pharmacology*; D004075:Diglycerides / Q000494:pharmacology*; D004789:Enzyme Activation; D006801:Humans; D010741:Phospholipases A / Q000378:metabolism*; D054466:Phospholipases A1; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013928:Thromboxane A2 / Q000502:physiology*
D004075:Diglycerides; D000001:Calcimycin; D013928:Thromboxane A2; D015772:4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; D003094:Collagen; D010741:Phospholipases A; D054466:Phospholipases A1; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium