title: Role of neuronal nitric oxide synthase in lipopolysaccharide-induced tumor necrosis factor-alpha expression in neonatal mouse cardiomyocytes.
abstract: OBJECTIVE
  Neuronal nitric oxide synthase (nNOS) has been shown to regulate intracellular calcium in cardiomyocytes. Calcium in turn modulates extracellular signal-related kinase (ERK) signaling, which is important in tumor necrosis factor-alpha (TNF-alpha) expression during lipopolysaccharide (LPS) stimulation. However, the role of nNOS in LPS-induced TNF-alpha expression is not known. We hypothesized that nNOS suppresses LPS-induced TNF-alpha expression by inhibiting the calcium/ERK signaling pathway.
  
  
  RESULTS
  Cultured neonatal mouse cardiomyocytes were challenged with LPS for 4 h. While there was no change in the basal Ca(2+) concentration, LPS increased peak Ca(2+) levels. LPS stimulation increased TNF-alpha mRNA and protein levels in wild-type cells however, the responses were enhanced in nNOS(-/-) cardiomyocytes. Treatment with an antisense oligonucleotide against nNOS also significantly enhanced TNF-alpha expression during LPS stimulation. Furthermore, LPS-induced ERK phosphorylation was significantly increased in the nNOS(-/-) compared to wild-type cardiomyocytes. The enhanced TNF-alpha expression in nNOS(-/-) cardiomyocytes was abrogated by an L-type calcium channel blocker verapamil or ERK1 siRNA. Finally, myocardial ERK phosphorylation and TNF-alpha expression were increased while cardiac function was decreased in endotoxemia in nNOS(-/-) compared to wild-type mice.
  
  
  CONCLUSIONS
  nNOS inhibits LPS-induced TNF-alpha expression in cardiomyocytes and improves cardiac function in endotoxemia. The inhibitory role of nNOS is mediated by a reduction in L-type calcium channel-dependent ERK signaling in cardiomyocytes.
authors.affiliation: Cardiology Research Laboratory, Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada.
authors.forename: Nicola
authors.initials: N
authors.lastname: Geoghegan-Morphet
authors.forename: Dylan
authors.initials: D
authors.lastname: Burger
authors.forename: Xiangru
authors.initials: X
authors.lastname: Lu
authors.forename: Venkatachalem
authors.initials: V
authors.lastname: Sathish
authors.forename: Tianqing
authors.initials: T
authors.lastname: Peng
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Sims
authors.forename: Qingping
authors.initials: Q
authors.lastname: Feng
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D019446:Endotoxemia / Q000276:immunology* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000235:genetics; D032383:Myocytes, Cardiac / Q000276:immunology* / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010477:Perfusion; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000378:metabolism*
chemical_list: D008070:Lipopolysaccharides; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D052248:Nitric Oxide Synthase Type I; D020929:Mitogen-Activated Protein Kinase Kinases; D002118:Calcium