Biomedical subjects

Sei Kobayashi

Publications and source records attributed to Sei Kobayashi.

10 recordsLinked to original sources

PubMed2006

Cell membrane-derived lysophosphatidylcholine activates cardiac ryanodine receptor channels.

Lysophosphatidylcholine (LPC) is metabolized from a membrane phospholipid and modulates a variety of channels in the plasma membrane (PM). We examined LPC modulation of cardiac ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR) using the planar lipid bilayer method to measure the single-channel currents. Micromolar concentrations of LPC increased the open probability of the reconstituted RyR channels irrespective of whether LPC was added to the cis or trans chamber. LPC also increased the membrane capacitance of the bilayer. The effects of LPC contrasted well with those of sphingosylphosphorylcholine (SPC). Taken together, these results suggest that amphipathic lipid LPC does not bind directly to the RyR channel protein, but rather, is incorporated into the bilayer membrane and activates the channel. Thus, we consider cell membrane-derived LPC to be a putative endogenous mediator that activates not only plasma membrane channels but also RyR channels and induces arrhythmogenic Ca(2+) mobilization in cardiomyocytes.

Animals↗

PubMed2006

Cholesterol primes vascular smooth muscle to induce Ca2 sensitization mediated by a sphingosylphosphorylcholine-Rho-kinase pathway: possible role for membrane raft.

Hypercholesterolemia is a major risk factor involved in abnormal cardiovascular events. Rho-kinase-mediated Ca(2+) sensitization of vascular smooth muscle (VSM) plays a critical role in vasospasm and hypertension. We recently identified sphingosylphosphorylcholine (SPC) and Src family tyrosine kinase (Src-TK) as upstream mediators for the Rho-kinase-mediated Ca(2+) sensitization. Here we report the strong linkage between cholesterol and the Ca(2+) sensitization of VSM mediated by a novel SPC/Src-TK/Rho-kinase pathway in both humans and rabbits. The extent of the sensitization correlated well with the total cholesterol or low-density lipoprotein cholesterol levels in serum. However, an inverse correlation with the serum level of high-density lipoprotein cholesterol was observed, and a correlation with other cardiovascular risk factors was nil. When cholesterol-lowering therapy was given to patients and rabbits with hypercholesterolemia, the SPC-induced contractions diminished. Depletion of VSM cholesterol by beta-cyclodextrin resulted in a loss of membrane caveolin-1, a marker of cholesterol-enriched lipid raft, and inhibited the SPC-induced Ca(2+) sensitization and translocation of Rho-kinase from cytosol to the cell membrane. Vasocontractions induced by membrane depolarization and by an adrenergic agonist were cholesterol-independent. Our data support the previously unreported concept that cholesterol potentiates the Ca(2+) sensitization of VSM mediated by a SPC/Src-TK/Rho-kinase pathway, and are also compatible with a role for cholesterol-enriched membrane microdomain, a lipid raft. This process may play an important role in the development of abnormal vascular contractions in patients with hypercholesterolemia.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

[Clinical phase trial of concurrent chemoradiotherapy combined TS-1 and nedaplatin].

We have treated head and neck carcinoma by concurrent chemoradiotherapy combined with 5-fluorouracil (5-FU) and cisplatin (CDDP). However,this chemoradiotherapy could not show an enormous effect in the advanced carcinoma of Stage III and IV. Therefore,we changed the contents of the chemotherapy, i.e., we replaced 5-FU, one of the agents with time dependency, to continuous administration of TS-1 for 2 weeks,also replacing CDDP, one of the agents with dose dependency, to nedaplatin (CDGP) in order to reduce kidney dysfunction. In this concurrent chemoradiotherapy, oral TS-1 was continued for 2 weeks and CDGP was administered on the 4 th day from the start of TS-1. In addition, radiotherapy was performed concurrently. In this way,we performed a phase I clinical trial of concurrent chemoradiotherapy combining TS-1 and nedaplatin (CDGP). As for the incidence of adverse events,grade 3 mucositis due to radiation was observed in two patients. As a result of the phase I clinical trial,we decided the maximum-tolerated dose (MTD) of TS-1 to be 80 mg/m2 (maximum 120 mg/body) and 100 mg/m2 for CDGP, and then determined the recommended dose(RD) of TS-1 as 80 mg/m2 (maximum 120 mg/ body) TS-1 and of CDGP as 9 0 mg/m2 CDGP.

Adult↗

PubMed2004

ERK1/2 regulates intracellular ATP levels through alpha-enolase expression in cardiomyocytes exposed to ischemic hypoxia and reoxygenation.

Extracellular signal-regulated kinase 1/2 (ERK1/2) is known to function in cell survival in response to various stresses; however, the mechanism of cell survival by ERK1/2 remains poorly elucidated in ischemic heart. Here we applied functional proteomics by two-dimensional electrophoresis to identify a cellular target of ERK1/2 in response to ischemic hypoxia. Approximately 1500 spots were detected by Coomassie Brilliant Blue staining of a sample from unstimulated cells. The staining intensities of at least 50 spots increased at 6-h reoxygenation after 2-h ischemic hypoxia. Of the 50 spots that increased, at least 4 spots were inhibited in the presence of PD98059, a MEK inhibitor. A protein with a molecular mass of 52 kDa that is strongly induced by ERK1/2 activation in response to ischemic hypoxia and reoxygenation was identified as alpha-enolase, a rate-limiting enzyme in the glycolytic pathway, by liquid chromatography-mass spectrometry and amino acid sequencing. The expressions of the alpha-enolase mRNA and protein are inhibited during reoxygenation after ischemic hypoxia in the cells containing a dominant negative mutant of MEK1 and treated with a MEK inhibitor, PD98059, leading to a decrease in ATP levels. alpha-Enolase expression is also observed in rat heart subjected to ischemia-reperfusion. The induction of alpha-enolase by ERK1/2 appears to be mediated by c-Myc. The introduction of the alpha-enolase protein into the cells restores ATP levels and prevents cell death during ischemic hypoxia and reoxygenation in these cells. These results show that alpha-enolase expression by ERK1/2 participates in the production of ATP during reoxygenation after ischemic hypoxia, and a decrease in ATP induces apoptotic cell death. Furthermore, alpha-enolase improves the contractility of cardiomyocytes impaired by ischemic hypoxia. Our results reveal that ERK1/2 plays a role in the contractility of cardiomyocytes and cell survival through alpha-enolase expression during ischemic hypoxia and reoxygenation.

Adenosine Triphosphate↗

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.

Animals↗

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.

Animals↗

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.

Animals↗

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.

Animals↗