6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2h-pyran-2-one and thrombin-receptor-peptide-(42-55)

Activation of phospholipase A2 (PLA2) and accumulation of lysophosphatidylcholine contribute importantly to arrhythmogenesis during acute myocardial ischemia. We examined thrombin stimulation of PLA2 activity in isolated ventricular myocytes. Basal and thrombin-stimulated cardiac myocyte PLA2 activity demonstrated a distinct preference for sn-1 ether-linked phospholipids with arachidonate esterified at the sn-2 position. The majority of PLA2 activity was calcium independent and membrane associated. Thrombin stimulation of membrane-associated PLA2 occurs in a time- and concentration-dependent fashion. An increase in PLA2 activity was also observed using the synthetic peptide SFLLRNPNDKYEPF (the tethered ligand generated by thrombin cleavage of its receptor). Bromoenol lactone, a selective inhibitor of calcium-independent PLA2, completely blocked thrombin-stimulated increases in PLA2 activity and arachidonic acid release. No significant inhibition of thrombin-induced PLA2 was observed following pretreatment with mepacrine or dibucaine. These data confirm the presence of high-affinity thrombin receptors on isolated cardiac myocytes and demonstrate the specific activation of a unique membrane-associated, calcium-independent PLA2 following thrombin receptor ligation.

Topics: Animals; Arachidonic Acid; Calcium; Cell Membrane; Dibucaine; Enzyme Activation; Enzyme Inhibitors; Female; Heart Ventricles; In Vitro Techniques; Myocardium; Naphthalenes; Peptide Fragments; Phospholipases A; Phospholipases A2; Pyrones; Quinacrine; Rabbits; Receptors, Thrombin; Thrombin

The present study was performed to characterize thrombin-stimulated phospholipase A2 (PLA2) activity and the resultant release of lysophospholipids from endothelial cells. The majority of PLA2 activity in endothelial cells was membrane associated, Ca2+ independent, and arachidonate selective. Incubation with thrombin increased membrane-associated PLA2 activity using both plasmenylcholine and alkylacyl glycerophosphocholine substrates in the absence of Ca2+, with no increase in activity observed with phosphatidylcholine substrate. The increased PLA2 activity was accompanied by arachidonic acid and lysoplasmenylcholine (LPlasC) release from endothelial cells into the surrounding medium. Thrombin-induced changes were duplicated by stimulation with the thrombin-receptor-directed peptide SFLLRNPNDKYEPF. Pretreatment with the Ca2+-independent PLA2 inhibitor bromoenol lactone blocked thrombin-stimulated increases in PLA2 activity, arachidonic acid, and LPlasC release. Stimulation of protein kinase C (PKC) increased basal PLA2 activity and LPlasC production. Thrombin-stimulated PLA2 activity and LPlasC production were enhanced with PKC activation and completely prevented with PKC downregulation. Thus thrombin treatment of endothelial cells activates a PKC-activated, membrane-associated, Ca2+-independent PLA2 that selectively hydrolyzes arachidonylated, ether-linked phospholipid substrates, resulting in LPlasC and arachidonic acid release.

Topics: Animals; Arachidonic Acid; Cells, Cultured; Endothelium, Vascular; Enzyme Activation; Hydrolysis; Lysophospholipids; Naphthalenes; Peptide Fragments; Phosphodiesterase Inhibitors; Phospholipases A; Phospholipases A2; Plasmalogens; Protein Kinase C; Pyrones; Swine; Thrombin