Biomedical subjects

Sei Kobayashi

Publications and source records attributed to Sei Kobayashi.

5 recordsLinked to original sources

PubMed2004

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.

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PubMed2003

Proteasome-dependent decrease in Akt by growth factors in vascular smooth muscle cells.

Akt is activated by growth factors to regulate various aspects of vascular smooth muscle cell function. Platelet-derived growth factor (PDGF) and insulin-like growth factor-1 activated Akt in vascular smooth muscle cells with a rapid reduction of total Akt protein that lasted for several hours. The downregulation of Akt required phosphatidylinositol 3-kinase activity, but not intrinsic Akt activity. The downregulation of Akt was abrogated by MG-132, a proteasome inhibitor, but not by inhibitors of calpain or cathepsins. Akt was found in ubiquitin immune complex after PDGF treatment. Proteasome-dependent degradation of Akt may provide a counter-regulatory mechanism against overactivation of Akt.

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PubMed2003

Ca2+ and voltage dependence of cardiac ryanodine receptor channel block by sphingosylphosphorylcholine.

The effect of sphingosylphosphorylcholine (SPC) on the cytoplasmic Ca(2+) and voltage dependence of channel gating by cardiac ryanodine receptors (RyR) was examined in lipid bilayer experiments. Micromolar concentrations of the lysosphingolipid SPC added to cis solutions rapidly and reversibly decreased the single-channel open probability (P(o)) of reconstituted RyR channels. The SPC-induced decrease in P(o) was marked by an increase in mean closed time and burst-like channel gating. Gating kinetics during intraburst periods were unchanged from those observed in the absence of the sphingolipid, although SPC induced a long-lived closed state that appeared to explain the observed decrease in channel P(o). SPC effects were observed over a broad range of cis [Ca(2+)] but were not competitive with Ca(2+). Interestingly, the sphingolipid-induced, long-lived closed state displayed voltage-dependent kinetics, even though other channel gating kinetics were not sensitive to voltage. Assuming SPC effects represent channel blockade, these results suggest that the blocking rate is independent of voltage whereas the unblocking rate is voltage dependent. Together, these results suggest that SPC binds directly to the cytoplasmic side of the RyR protein in a location in or near the membrane dielectric, but distinct from cytoplasmic Ca(2+) binding sites on the protein.

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PubMed2002

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.

Acylation↗

PubMed2002

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.

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