sphingosine-phosphorylcholine and 2-bromopalmitate

We recently reported that sphingosylphosphorylcholine (SPC) is a novel messenger for Rho-kinase-mediated Ca(2+) sensitization of vascular smooth muscle (VSM) contraction. Subcellular localization and kinase activity of Src family protein kinases (SrcPTKs), except for c-Src, is controlled by a reversible S-palmitoylation, an event inhibited by eicosapentaenoic acid (EPA). We examined the possible involvement of SrcPTKs in SPC-induced Ca(2+) sensitization and effects of EPA. We used porcine coronary VSM and rat aortic VSM cells (VSMCs) in primary culture. An SrcPTKs inhibitor, PP1, and EPA inhibited SPC-induced contraction, concentration-dependently, without affecting [Ca(2+)](i) levels and the Ca(2+)-dependent contraction induced by high K(+) depolarization. A digitized immunocytochemical analysis in VSMCs revealed that SPC induced translocation of Fyn, but not of c-Src, from the cytosol to the cell membrane, an event abolished by EPA. Translocation of Rho-kinase from the cytosol to the cell membrane by SPC was also inhibited by EPA and PP1. The SPC-induced activation of SrcPTKs was blocked by EPA and PP1, but not by Y27632, an Rho-kinase inhibitor. Rho-kinase-dependent phosphorylation of myosin phosphatase induced by SPC was inhibited by EPA, PP1, and Y27632. Translocation and activation of SrcPTKs, including Fyn, play an important role in Ca(2+) sensitization of VSM contractions mediated by a SPC-Rho-kinase pathway.

Topics: Acylation; Animals; Blotting, Western; Calcium; Coronary Vessels; Enzyme Activation; Enzyme Inhibitors; Fatty Acids, Unsaturated; Immunohistochemistry; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Isometric Contraction; Male; Muscle, Smooth, Vascular; Palmitates; Phosphorylcholine; Protein Serine-Threonine Kinases; Protein Transport; Rats; Rats, Wistar; rho-Associated Kinases; Second Messenger Systems; Signal Transduction; Sphingosine; src-Family Kinases; Swine; Vasoconstriction