Sei Kobayashi

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.

Angiogenesis induced by the implantation of self-bone marrow cells: a new material for therapeutic angiogenesis.

Bone marrow contains various primitive cells that are thought to secrete several angiogenic growth factors and may also differentiate into endothelial cells. The present study was conducted to investigate the possibility that bone marrow cells could be a novel material to induce angiogenesis. The expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in rat bone marrow cells was examined by immunohistochemistry. The production of VEGF was compared in tissue culture supernatant under the conditions of normoxia and hypoxia. The process of angiogenesis that occurred following the implantation of bone marrow cells was determined using a rat cornea model. VEGF- and bFGF-positive cells were found in rat bone marrow. The production of VEGF from bone marrow cells was significantly more enhanced by hypoxic conditions than by normoxic conditions. The rat cornea model showed that bone marrow cell implantation created new vessels. The implantation of self-bone marrow cells is a novel and simple method of inducing angiogenesis.

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 α-enolase, a rate-limiting enzyme in the glycolytic pathway, by liquid chromatography-mass spectrometry and amino acid sequencing. The expressions of the α-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. α-Enolase expression is also observed in rat heart subjected to ischemia-reperfusion. The induction of α-enolase by ERK1/2 appears to be mediated by c-Myc. The introduction of the α-enolase protein into the cells restores ATP levels and prevents cell death during ischemic hypoxia and reoxygenation in these cells. These results show that α-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, α-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 α-enolase expression during ischemic hypoxia and reoxygenation.

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.

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 Ca2+ 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 Ca2+ sensitization. Here we report the strong linkage between cholesterol and the Ca2+ 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 &bgr;-cyclodextrin resulted in a loss of membrane caveolin-1, a marker of cholesterol-enriched lipid raft, and inhibited the SPC-induced Ca2+ 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 Ca2+ 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.

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.

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.

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.