Regulation of membrane-associated iPLA2 activity by a novel PKC isoform in ventricular myocytes.

Thrombin stimulation of rabbit ventricular myocytes increases membrane-associated, Ca2+-independent phospholipase A2 (iPLA2) activity, resulting in accelerated hydrolysis of membrane plasmalogen phospholipids and increased production of arachidonic acid and lysoplasmenylcholine. This study was designed to investigate the signal transduction pathways involved in activation of membrane-associated iPLA2. Incubation of isolated membrane fractions suspended in Ca2+-free buffer with thrombin or phorbol 12-myristate 13-acetate resulted in a two- to threefold increase in iPLA2 activity. Prior treatment with the PKC inhibitor GF-109203X blocked iPLA2 activation by thrombin. These data suggest that a novel PKC isoform present in the membrane fraction modulates iPLA2 activity. Immunoblot analysis revealed a significant portion of PKC-epsilon present in the membrane fraction, but no other membrane-associated novel PKC isoform was detected by this method. These data indicate that activation of membrane-associated iPLA2 is mediated by a membrane-associated novel PKC isoform in thrombin-stimulated rabbit ventricular myocytes.

Inhibition of platelet-activating factor (PAF) acetylhydrolase by methyl arachidonyl fluorophosphonate potentiates PAF synthesis in thrombin-stimulated human coronary artery endothelial cells.

We have previously demonstrated that thrombin stimulation of endothelial cells results in increased membrane-associated, Ca(2+)-independent phospholipase A2 (iPLA2) activity, accelerated hydrolysis of membrane plasmalogen phospholipids, and production of several biologically active phospholipid metabolites, including prostacyclin and platelet-activating factor (PAF) that is abolished by pretreatment with the iPLA2-selective inhibitor bromoenol lactone. This study was designed to further investigate the role of alternative PLA2 inhibitors, including methyl arachidonyl fluorophosphonate (MAFP, an inhibitor of cytosolic PLA2 isoforms), on phospholipid turnover and PAF production from thrombin-stimulated human coronary artery endothelial cells (HCAECs). Paradoxically, pretreatment of HCAEC with MAFP (5-25 microM) resulted in a significant increase in PAF production in both unstimulated and thrombin-stimulated cells that was found to be a direct result of inhibition of PAF acetylhydrolase (PAF-AH) activity. Pretreatment with MAFP did not significantly inhibit HCAEC PLA2 activity, possibly due to the localization of PLA2 activity in the membrane fraction rather than the cytosol. Bromoenol lactone did not inhibit PAF-AH activity, even at concentrations as high as 20 microM. We conclude that MAFP augments thrombin-stimulated PAF production by inhibition of PAF catabolism without affecting membrane-associated iPLA2 activity.