sphingosine-1-phosphate and chelerythrine

Sphingosine-1-phosphate (S1P) protects neonatal rat cardiac myocytes from hypoxic damage through unknown signaling pathways. We tested the hypothesis that S1P-induced cardioprotection requires activation by the epsilon-isoform of protein kinase C (PKC epsilon) by subjecting hearts isolated from PKC epsilon knockout mice and wild-type mice to 20 min of global ischemia and 30 min of reperfusion. Pretreatment with a 2-min infusion of 10 nM S1P improved recovery of left ventricular developed pressure (LVDP) in both wild-type and PKC epsilon knockout hearts and reduced the rise in LV end-diastolic pressure (LVEDP) and creatine kinase (CK) release. Pretreatment for 2 min with 10 nM of the ganglioside GM-1 also improved recovery of LVDP and suppressed CK release in wild-type hearts but not in PKC epsilon knockout hearts. Importantly, GM-1 but not S1P, increased the proportion of PKC epsilon localized to particulate fractions. Our results suggest that GM-1, which enhances endogenous S1P production, reduces cardiac injury through PKC epsilon-dependent intracellular pathways. In contrast, extracellular S1P induces equivalent cardioprotection through PKC epsilon-independent signaling pathways.

Topics: Alkaloids; Animals; Animals, Newborn; Benzophenanthridines; Blotting, Western; Cells, Cultured; Creatine Kinase; Enzyme Inhibitors; G(M1) Ganglioside; Isoenzymes; Lysophospholipids; Mice; Mice, Knockout; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Phenanthridines; Protein Kinase C; Signal Transduction; Sphingosine; Ventricular Function, Left

The lysophospholipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) stimulate cellular proliferation and affect numerous cellular functions by signaling through G protein-coupled endothelial differentiation gene-encoded (Edg) receptors. S1P and LPA also act as survival factors in many cell types, but have not previously been studied in cardiac myocytes. We incubated neonatal rat cardiac myocytes either in room air/1% CO2 (normoxia) or in an atmosphere of 99% N2/1%CO2 (hypoxia) at 37 degrees C for 18-20 h in the absence of glucose. Cell viability was measured using a calcein ester green fluorescence assay. Under normoxic conditions 88.7+/-1.0% of the cells were viable after 18-20 h. Severe hypoxia reduced viability to 61.3+/-4.3% (n=6, P<0.05). In myocytes preincubated with either 10 microM S1P or 1 microM LPA for 2 h, the effects of severe hypoxia on cell viability were prevented resulting in survival equivalent to normoxia. Neither the protein kinase C inhibitor chelethyrine (1 microM) nor the mitochondrial K(ATP) channel antagonist 5-hydroxydecanoic acid, (5-HD, 100 microM) had any effect on myocyte survival during severe hypoxia, but both agents completely abolished the ability of S1P to rescue cardiac myocytes from hypoxic cell death. We also tested the effects of dimethylsphingosine (DMS), which inhibits sphingosine kinase synthesis of S1P. Incubation of neonatal rat cardiac myocytes with 10 microM DMS for 2 h in the presence of serum resulted in 25-30% cell death during 18-20 h of normoxia. DMS-induced cell death was prevented by concurrent preincubation with either S1P or GM-1, a ganglioside that activates sphingosine kinase to increase intracellular levels of S1P. We conclude that both S1P and LPA are cardioprotective for hypoxic neonatal rat ventricular myocytes. S1P acts through cellular membrane receptors by signaling mechanisms involving protein kinase C and mitochondrial K(ATP) channels. Both endogenous and exogenously applied S1P are effective in preventing cell death induced by inhibition of sphingosine kinase.

Topics: Alkaloids; Animals; Animals, Newborn; Anti-Arrhythmia Agents; Benzophenanthridines; Cardiotonic Agents; Cell Hypoxia; Cell Survival; Cells, Cultured; Culture Media, Serum-Free; Decanoic Acids; Enzyme Inhibitors; Gelsolin; Heart; Hydroxy Acids; Lysophospholipids; Myocardium; Phenanthridines; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Sphingosine

Sphingolipid breakdown products [ceramide, sphingosine, and sphingosine-1-phosphate (SPP)] are emerging as a new class of bioactive molecules. In agreement with previous studies, treatment of human promyelocytic leukemia HL-60 cells with 1-alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induced a transient increase of ceramide levels within 2 h, which then returned to basal levels within 8 h. In contrast, sphingosine kinase activity increased more slowly and reached maximal levels only after 20 h of exposure, leading to a concomitant increase in SPP level. Unlike treatments with cell-permeable ceramide analogues or sphingomyelinase, which induce apoptosis, 1,25-(OH)2D3 did not induce apoptosis, despite the early formation of ceramide. Moreover, prolonged treatment of HL-60 cells with 1,25-(OH)2D3 suppressed ceramide-induced apoptosis. There was a correlation between the time course and dose response of the activation of sphingosine kinase by 1,25-(OH)2D3 and the protection against apoptosis. In contrast, treatment with all-trans-retinoic acid neither stimulated sphingosine kinase activity nor protected cells from ceramide-induced apoptosis. Treatment with SPP protected HL-60 cells from ceramide-induced apoptosis, and N,N-dimethylsphingosine (DMS), a competitive inhibitor of sphingosine kinase, prevented the survival effect of 1,25-(OH)2D3. The effect of DMS was counteracted by SPP, suggesting that SPP is a critical component of the cytoprotective effect of 1,25-(OH)2D3. Chelerythrine chloride, an inhibitor of protein kinase C, markedly reduced sphingosine kinase activity and the apoptosis-sparing effect of 1,25-(OH)2D3, and conversely, the tumor promoter 12-O-tetradecanoylphorhol-13-acetate not only suppressed ceramide-induced apoptosis but also stimulated sphingosine kinase activity. Moreover, the protective effect of 12-O-tetradecanoylphorbol-13-acetate was blocked by DMS. Collectively, our observations indicate that the cytoprotective effect of 1,25-(OH)2D3 is mediated by SPP, which is formed as a consequence of activation of sphingosine kinase.

Topics: Alkaloids; Apoptosis; Benzophenanthridines; Calcitriol; Carcinogens; Cell Survival; Ceramides; Dose-Response Relationship, Drug; Enzyme Inhibitors; HL-60 Cells; Humans; Lysophospholipids; Phenanthridines; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Tetradecanoylphorbol Acetate; Time Factors