title: Deletion of the sphingosine kinase-1 gene influences cell fate during hypoxia and glucose deprivation in adult mouse cardiomyocytes.
abstract: OBJECTIVE
  Activation of sphingosine kinase (SphK), which has two known isoforms, is responsible for the synthesis of sphingosine 1-phosphate (S1P), a cell survival factor. We tested the following hypotheses: 1] cardiac myocytes null for the SphK1 gene are more vulnerable to the stress of hypoxia+glucose deprivation; 2] the monoganglioside GM-1, which activates SphK via protein kinase C epsilon, is ineffective in SphK1-null myocytes; 3] S1P generated by SphK activation requires cellular export to be cardioprotective.
  
  
  METHODS
  We cultured adult mouse cardiac myocytes from wildtype and SphK1-null mice (deletion of exons 3-6) and measured cell viability by trypan blue exclusion.
  
  
  RESULTS
  In wildtype adult mouse cardiomyocytes subjected to 4 h of hypoxic stress+glucose deprivation, cell viability was significantly higher than in SphK1-null cardiomyocytes. SphK1-null cells also displayed more mitochondrial cytochrome C release. Cell death induced by hypoxia+glucose deprivation was substantially prevented by pretreatment with exogenous S1P in both wildtype and SphK1-null myocytes, but S1P was effective at a lower concentration in wildtype cells. Hence, the absence of the Sphk1 gene did not affect receptor coupling or downstream signal transduction. Pretreatment for 1 h with 1 microM of the monoganglioside GM-1 increased survival in wildtype cells, but not in SphK1-null myocytes. Thus, activation of SphK1 by GM-1 leads to cell survival. In wildtype cells, enhanced survival produced by GM-1 was abrogated by pretreatment either with 300 nM of the S1P(1) receptor-selective antagonist VPC23019 or with 100 ng/ml of pertussis toxin for 16 h before exposure to hypoxia+glucose deprivation.
  
  
  CONCLUSIONS
  As the effect of GM-1 is blocked both at the receptor and the G-protein (Gi) levels, we conclude that S1P generated by GM-1 treatment must be exported from the cell and acts in a paracrine or autocrine manner to couple with its cognate receptor.
authors.affiliation: Cardiology Section, Veterans Affairs Medical Center, University of California, San Francisco, CA 94121, USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Tao
authors.forename: Jianqing
authors.initials: J
authors.lastname: Zhang
authors.forename: Donald A
authors.initials: DA
authors.lastname: Vessey
authors.forename: Norman
authors.initials: N
authors.lastname: Honbo
authors.forename: Joel S
authors.initials: JS
authors.lastname: Karliner
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western / Q000379:methods; D015687:Cell Hypoxia; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D045304:Cytochromes c / Q000032:analysis / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D036145:Ginsenosides / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000494:pharmacology; D008246:Lysophospholipids / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008929:Mitochondria, Heart / Q000378:metabolism; D017202:Myocardial Ischemia / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000201:enzymology* / Q000473:pathology; D037342:Pertussis Toxin / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D000099157:Sphingosine Kinase
chemical_list: D015415:Biomarkers; D045304:Cytochromes c; D004791:Enzyme Inhibitors; D036145:Ginsenosides; D005947:Glucose; D007004:Hypoglycemic Agents; D008246:Lysophospholipids; D037342:Pertussis Toxin; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate; C112772:ginsenoside compound K