6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2h-pyran-2-one and thiazolyl-blue

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC). To understand its mitogenic signaling events, we have studied the role of calcium-independent phospholipase A2 (iPLA2). Without affecting its levels, thrombin increased iPLA2 activity in a time-dependent manner in VSMC. Thrombin also induced arachidonic acid release and DNA synthesis by about 2-fold as compared with control. Down-regulation of iPLA2 activity by its specific inhibitor, bromoenol lactone, or its expression by antisense oligonucleotides, significantly reduced thrombin-induced arachidonic acid release and DNA synthesis in VSMC. To learn the mechanism of thrombin-stimulated iPLA2 activity, we next tested the role of p38 MAPK. Thrombin stimulated p38 MAPK phosphorylation and activity in a time-dependent manner in VSMC. Inhibition of p38 MAPK activity by SB203580 and SB202190 resulted in decreased iPLA2 activity, arachidonic acid release, and DNA synthesis induced by thrombin in VSMC. Together, these results for the first time demonstrate that iPLA2 plays a role in thrombin-induced arachidonic acid release and growth in VSMC and that these responses are mediated by p38 MAPK.

Topics: Animals; Arachidonic Acid; Blotting, Western; Coloring Agents; DNA; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Group VI Phospholipases A2; Imidazoles; Male; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Naphthalenes; Oleic Acid; Oligonucleotides, Antisense; p38 Mitogen-Activated Protein Kinases; Phospholipases A; Phospholipases A2; Pyridines; Pyrones; Rats; Rats, Sprague-Dawley; Signal Transduction; Tetrazolium Salts; Thiazoles; Thrombin; Time Factors