Elevated concentrations of sphingosylphosphorylcholine in cerebrospinal fluid after subarachnoid hemorrhage: a possible role as a spasmogen.

This study investigates the role of sphingosylphosphorylcholine (SPC) in the mechanisms underlying cerebral vasospasm after subarachnoid hemorrhage (SAH). The levels of SPC were measured in cerebrospinal fluid (CSF) of patients with SAH and also in an experimental canine model. CSF samples were collected from 11 patients with SAH, and from dogs that had received an injection of SPC into the cisterna magna to examine SPC kinetics in the CSF. SPC was assayed using solid-phase extraction and triple quadrupole mass spectrometry. The SPC concentrations in SAH patients on days 3, 8, and 14 after the onset of SAH were significantly higher than those in normal CSF. In the canine model, rapid dilution of SPC in CSF was observed. In combination with data from previous studies, these results suggest that SPC is involved in the development of cerebral vasospasm. Rapid dilution of SPC in CSF suggests that SPC is released into CSF at higher concentrations than those measured in the present study.

Sphingomyelinase causes endothelium-dependent vasorelaxation through endothelial nitric oxide production without cytosolic Ca(2+) elevation.

Neutral sphingomyelinase (N-SMase) elevated nitric oxide (NO) production without affecting intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells in situ on aortic valves, and induced prominent endothelium-dependent relaxation of coronary arteries, which was blocked by N(omega)-monomethyl-L-arginine, a NO synthase (NOS) inhibitor. N-SMase induced translocation of endothelial NOS (eNOS) from plasma membrane caveolae to intracellular region, eNOS phosphorylation on serine 1179, and an increase of ceramide level in endothelial cells. Membrane-permeable ceramide (C(8)-ceramide) mimicked the responses to N-SMase. We propose the involvement of N-SMase and ceramide in Ca(2+)-independent eNOS activation and NO production in endothelial cells in situ, linking to endothelium-dependent vasorelaxation.

Identification of the active metabolite of ticlopidine from rat in vitro metabolites.

1 Ticlopidine is a well-known anti-platelet agent, but is not active by itself in vitro. We identified a metabolite with anti-platelet activity, which was generated after incubation of 2-oxo-ticlopidine with phenobarbital-induced rat liver homogenate in vitro. 2 An active moiety (UR-4501) was isolated by high-performance liquid chromatography after large-scale preparation of metabolites. 3 The chemical structure of UR-4501 was determined by a combination of liquid chromatography mass/mass spectrometry (LC/MS/MS) and nuclear magnetic resonance (NMR) analysis. 4 UR-4501 produced a concentration-dependent inhibition (3-100 microm) of ADP (10 microm)-induced human platelet aggregation, whereas 2-oxo-ticlopidine (3-100 microm) did not elicit inhibitory responses. 5 UR-4501 (10-100 microm) strongly inhibited ADP- and collagen-induced aggregation and slightly inhibited thrombin-induced aggregation. 6 The inhibition of rat washed platelet aggregation by UR-4501 (100 microm) persisted, even after the platelets had been washed twice. 7 These results suggest that UR-4501 is the molecule responsible for the in vivo activities of ticlopidine.

Involvement of Src family protein tyrosine kinases in Ca(2+) sensitization of coronary artery contraction mediated by a sphingosylphosphorylcholine-Rho-kinase pathway.

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.

Sphingosylphosphorylcholine is a novel messenger for Rho-kinase-mediated Ca2+ sensitization in the bovine cerebral artery: unimportant role for protein kinase C.

Although recent investigations have suggested that a Rho-kinase-mediated Ca2+ 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 Ca2+ sensitization in pig coronary arteries. The purpose of this present study was to examine the possible mediation of SPC in Ca2+ 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 (EC50=3.0 micromol/L) contraction, without [Ca2+]i elevation. In membrane-permeabilized MCA, SPC induced Ca2+ sensitization even in the absence of added GTP, which is required for activation of G-proteins coupled to membrane receptors. The SPC-induced Ca2+ sensitization was blocked by a Rho-kinase inhibitor (Y-27632) and a dominant-negative Rho-kinase, but not by a pseudosubstrate peptide for conventional PKC, which abolished the Ca2+-independent contraction induced by phorbol ester. In contrast, phorbol ester-induced Ca2+ 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 Ca2+ 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.