Abstract-Although recent investigations have suggested that a Rho-kinase-mediated Ca 2ϩ sensitization of vascular smooth muscle contraction plays a critical role in the pathogenesis of cerebral and coronary vasospasm, the upstream of this signal transduction has not been elucidated. In addition, the involvement of protein kinase C (PKC) may also be related to cerebral vasospasm. We recently reported that sphingosylphosphorylcholine (SPC), a sphingolipid, induces Rho-kinase-mediated Ca 2ϩ sensitization in pig coronary arteries. The purpose of this present study was to examine the possible mediation of SPC in Ca 2ϩ sensitization of the bovine middle cerebral artery (MCA) and the relation to signal transduction pathways mediated by Rho-kinase and PKC. In intact MCA, SPC induced a concentration-dependent (EC 50 ϭ3.0 mol/L) contraction, without [Ca 2ϩ ] i elevation. In membrane-permeabilized MCA, SPC induced Ca 2ϩ sensitization even in the absence of added GTP, which is required for activation of G-proteins coupled to membrane receptors. The SPC-induced Ca 2ϩ sensitization was blocked by a Rho-kinase inhibitor (Y-27632) and a dominantnegative Rho-kinase, but not by a pseudosubstrate peptide for conventional PKC, which abolished the Ca 2ϩ -independent contraction induced by phorbol ester. In contrast, phorbol ester-induced Ca 2ϩ sensitization was resistant to a Rho-kinase inhibitor and a dominant-negative Rho-kinase. In primary cultured vascular smooth muscle cells, SPC induced the translocation of cytosolic Rho-kinase to the cell membrane. We propose that SPC is a novel messenger for Rho-kinase-mediated Ca 2ϩ sensitization of cerebral arterial smooth muscle and, therefore, may play a pivotal role in the pathogenesis of abnormal contraction of the cerebral artery such as vasospasm. The SPC/Rho-kinase pathway functions independently of the PKC pathway.

Eicosapentaenoic acid (EPA), but not its metabolites (docosapentaenoic acid and docosahexaenoic acid), stimulated nitric oxide (NO) production in endothelial cells in situ and induced endothelium-dependent relaxation of bovine coronary arteries precontracted with U46619. EPA induced a greater production of NO, but a much smaller and more transient elevation of intracellular Ca 2+ concentration ([Ca 2+ ]i), than did a Ca 2+ ionophore (ionomycin). EPA stimulated NO production even in endothelial cells in situ loaded with a cytosolic Ca 2+ chelator 1,2-bis-o-aminophenoxythamine-NP P,NP P,NP P-tetraacetic acid, which abolished the [Ca 2+ ]i elevations induced by ATP and EPA. The EPA-induced vasorelaxation was inhibited by N gnitro-L-arginine methyl ester. Immunostaining analysis of endothelial NO synthase (eNOS) and caveolin-1 in cultured endothelial cells revealed eNOS to be colocalized with caveolin in the cell membrane at a resting state, while EPA stimulated the translocation of eNOS to the cytosol and its dissociation from caveolin, to an extent comparable to that of the eNOS translocation induced by a [Ca 2+ ]i-elevating agonist (10 W WM bradykinin). Thus, EPA induces Ca 2+ -independent activation and translocation of eNOS and endothelium-dependent vasorelaxation. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Abstract-We recently reported that sphingosylphosphorylcholine (SPC) is a novel messenger for Rho-kinase-mediated Ca 2ϩ 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 2ϩ 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 2ϩ ] i levels and the Ca 2ϩ -dependent contraction induced by high K ϩ 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