Kimiko Mogami

Eicosapentaenoic acid (EPA) induces Ca2+-independent activation and translocation of endothelial nitric oxide synthase and endothelium-dependent vasorelaxation

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 Ca2+ concentration ([Ca2+]i), than did a Ca2+ ionophore (ionomycin). EPA stimulated NO production even in endothelial cells in situ loaded with a cytosolic Ca2+ chelator 1,2‐bis‐o‐aminophenoxythamine‐N′,N′,N′‐tetraacetic acid, which abolished the [Ca2+]i elevations induced by ATP and EPA. The EPA‐induced vasorelaxation was inhibited by N ω ‐nitro‐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 [Ca2+]i‐elevating agonist (10 μM bradykinin). Thus, EPA induces Ca2+‐independent activation and translocation of eNOS and endothelium‐dependent vasorelaxation.

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 &mgr;mol/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.

Involvement of Src Family Protein Tyrosine Kinases in Ca2+ Sensitization of Coronary Artery Contraction Mediated by a Sphingosylphosphorylcholine-Rho-Kinase Pathway

Abstract— We recently reported that sphingosylphosphorylcholine (SPC) is a novel messenger for Rho-kinase–mediated Ca2+ 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 Ca2+ 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 [Ca2+]i levels and the Ca2+-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 Ca2+ sensitization of VSM contractions mediated by a SPC-Rho-kinase pathway.

Sphingosylphosphorylcholine induces Ca2+-sensitization of vascular smooth muscle contraction: possible involvement of Rho-kinase

Sphingosylphosphorylcholine (SPC), a sphingolipid, concentration‐dependently (1–50 μM) induced contraction and slight elevation of the cytosolic Ca2+ concentration ([Ca2+]i) in smooth muscle of the pig coronary artery, the result being a marked increase in the force/[Ca2+]i ratio. In α‐toxin‐ or β‐escin‐permeabilized, but not Triton X‐100‐permeabilized, vascular strips, SPC induced contraction at constant [Ca2+]i (pCa 6.3) in the absence of GTP, whereas a G‐protein‐coupled receptor agonist, histamine, required the presence of GTP to induce the contraction. The Rho‐kinase blocker, Y‐27632 (10 μM) abolished the SPC‐induced Ca2+‐sensitization, without affecting the Ca2+‐induced contraction. These results suggest that SPC induces Ca2+‐sensitization of force in vascular smooth muscle, presumably through the activation of Rho‐kinase (or a related kinase).

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

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. An active moiety (UR‐4501) was isolated by high‐performance liquid chromatography after large‐scale preparation of metabolites. 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. UR‐4501 produced a concentration‐dependent inhibition (3–100 μM) of ADP (10 μM)‐induced human platelet aggregation, whereas 2‐oxo‐ticlopidine (3–100 μM) did not elicit inhibitory responses. UR‐4501 (10–100 μM) strongly inhibited ADP‐ and collagen‐induced aggregation and slightly inhibited thrombin‐induced aggregation. The inhibition of rat washed platelet aggregation by UR‐4501 (100 μM) persisted, even after the platelets had been washed twice. These results suggest that UR‐4501 is the molecule responsible for the in vivo activities of ticlopidine.

Sphingosylphosphorylcholine induces cytosolic Ca2+ elevation in endothelial cells in situ and causes endothelium‐dependent relaxation through nitric oxide production in bovine coronary artery

Sphingosylphosphorylcholine (SPC) increased intracellular Ca2+ concentration ([Ca2+]i) and nitric oxide (NO) production in endothelial cells in situ on bovine aortic valves, and induced endothelium‐dependent relaxation of bovine coronary arteries precontracted with U‐46619. The SPC‐induced vasorelaxation was inhibited by N ω‐monomethyl‐L‐arginine, an inhibitor of both constitutive and inducible NO synthase (NOS), but not by 1‐(2‐trifluoromethylphenyl) imidazole, an inhibitor of inducible NOS (iNOS). Immunoblotting revealed that endothelial constitutive NOS, but not iNOS, was present in endothelial cells in situ on the bovine aortic valves. We propose that SPC activates [Ca2+]i levels and NO production of endothelial cells in situ, thereby causing an endothelium‐dependent vasorelaxation.

Sphingomyelinase causes endothelium-dependent vasorelaxation through endothelial nitric oxide production without cytosolic Ca2+ elevation

Neutral sphingomyelinase (N‐SMase) elevated nitric oxide (NO) production without affecting intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ on aortic valves, and induced prominent endothelium‐dependent relaxation of coronary arteries, which was blocked by N ω ‐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 (C8‐ceramide) mimicked the responses to N‐SMase. We propose the involvement of N‐SMase and ceramide in Ca2+‐independent eNOS activation and NO production in endothelial cells in situ, linking to endothelium‐dependent vasorelaxation.

Phosphorylation of proteins and apoptosis induced by c-Jun N-terminal kinase1 activation in rat cardiomyocytes by H2O2 stimulation

Cytokines and various cellular stresses are known to activate c-Jun N-terminal kinase-1 (JNK1), which is involved in physiological function. Here, we investigate the activation of JNK1 by oxidative stress in H9c2 cells derived from rat cardiomyocytes. H(2)O(2) (100 microM) significantly induces the tyrosine phosphorylation of JNK1 with a peak 25 min after the stimulation. The amount of JNK1 protein remains almost constant during stimulation. Immunocytochemical observation shows that JNK1 staining in the nucleus is enhanced after H(2)O(2) stimulation. To clarify the physiological role of JNK1 activation under these conditions, we transfected antisense JNK1 DNA into H9c2 cells. The antisense DNA (2 microM) inhibits JNK1 expression by 80% as compared with expression in the presence of the sense DNA, and significantly blocks H(2)O(2)-induced cell death. Consistent with the decrease in cell number, we detected condensation of the nuclei, a hallmark of apoptosis, 3 h after H(2)O(2) stimulation in the presence of the sense DNA for JNK1. The antisense DNA of JNK1 inhibits the condensation of nuclei by H(2)O(2). Under these conditions, the H(2)O(2)-induced phosphorylation of proteins with molecular masses of 55, 72, and 78 kDa is blocked by treatment with the antisense DNA for JNK1 as compared with the sense DNA for JNK1. These findings suggest that JNK1 induces apoptotic cell death in response to H(2)O(2), and that the cell death may be involved in the phosphorylations of 55, 72, and 78 kDa proteins induced by JNK1 activation.

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.

Molecular cloning of rat transmembrane domain protein of 40 kDa regulated in adipocytes and its expression in H9c2 cells exposed to ischemic hypoxia and reoxygenation

We clone a 1230 bp complementary DNA encoding rat transmembrane domain protein of 40 kDa regulated in adipocytes (TPRA40), an orphan receptor, by reverse transcription-polymerase chain reaction using H9c2 cells derived from embryonic rat heart. The deduced amino acid sequence of rat TPRA40 consists of 369 amino acids and has a longer carboxyl terminus than that of the mouse protein. The level of TPRA40 mRNA decreases significantly throughout ischemic hypoxia and reoxygenation.