title: Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte-inducible nitric oxide synthase expression.
abstract: BACKGROUND
  Induced nitric oxide synthase (iNOS) is induced in hepatocytes by shock and inflammatory stimuli. Excessive NO from iNOS mediates shock-induced hepatic injury and death, so understanding the regulation of iNOS will help elucidate the pathophysiology of septic shock. In vitro, cytokines induce iNOS expression through activation of signaling pathways including mitogen-activated protein kinases and nuclear factor κB. Cytokines also induce calcium (Ca(2+)) mobilization and activate calcium-mediated intracellular signaling pathways, typically through activation of calmodulin-dependent kinases (CaMK). Calcium regulates NO production in macrophages but the role of calcium and calcium-mediated signaling in hepatocyte iNOS expression has not been defined.
  
  
  METHODS
  Primary rat hepatocytes were isolated, cultured, and induced to produce NO with proinflammatory cytokines. Calcium mobilization and Ca(2+)-mediated signaling were altered with ionophore, Ca(2+) channel blockers, and inhibitors of CaMK.
  
  
  RESULTS
  The Ca(2+) ionophore A23187 suppressed cytokine-stimulated NO production, whereas Ethylene glycol tetraacetic acid and nifedipine increased NO production, iNOS messenger RNA, and iNOS protein expression. Inhibition of CaMK with KN93 and CBD increased NO production but the calcineurin inhibitor FK 506 decreased iNOS expression.
  
  
  CONCLUSIONS
  These data demonstrate that calcium-mediated signaling regulates hepatocyte iNOS expression and does so through a mechanism independent of calcineurin. Changes in intracellular calcium levels may regulate iNOS expression during hepatic inflammation induced by proinflammatory cytokines.
authors.affiliation: The Hiram C. Polk, Jr. MD Department of Surgery, the Price Institute for Surgical Research, University of Louisville, Louisville, Kentucky.
authors.forename: Baochun
authors.initials: B
authors.lastname: Zhang
authors.affiliation: The Hiram C. Polk, Jr. MD Department of Surgery, the Price Institute for Surgical Research, University of Louisville, Louisville, Kentucky.
authors.forename: Will
authors.initials: W
authors.lastname: Crankshaw
authors.affiliation: The Hiram C. Polk, Jr. MD Department of Surgery, the Price Institute for Surgical Research, University of Louisville, Louisville, Kentucky.
authors.forename: Ryan
authors.initials: R
authors.lastname: Nesemeier
authors.affiliation: The Hiram C. Polk, Jr. MD Department of Surgery, the Price Institute for Surgical Research, University of Louisville, Louisville, Kentucky.
authors.forename: Jay
authors.initials: J
authors.lastname: Patel
authors.affiliation: The Hiram C. Polk, Jr. MD Department of Surgery, the Price Institute for Surgical Research, University of Louisville, Louisville, Kentucky.
authors.forename: Ikenna
authors.initials: I
authors.lastname: Nweze
authors.affiliation: The Hiram C. Polk, Jr. MD Department of Surgery, the Price Institute for Surgical Research, University of Louisville, Louisville, Kentucky.
authors.forename: Jaganathan
authors.initials: J
authors.lastname: Lakshmanan
authors.affiliation: The Hiram C. Polk, Jr. MD Department of Surgery, the Price Institute for Surgical Research, University of Louisville, Louisville, Kentucky. Electronic address: briang.harbrecht@louisville.edu.
authors.forename: Brian G
authors.initials: BG
authors.lastname: Harbrecht
mesh_terms: D000818:Animals; D019703:Calcineurin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D061207:Calcium Ionophores; D020013:Calcium Signaling; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008297:Male; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D017207:Rats, Sprague-Dawley
chemical_list: D061207:Calcium Ionophores; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019703:Calcineurin; D002118:Calcium
keywords: Cytokines; Hepatocyte; Liver; NOS2; Nitric oxide synthase; Sepsis; Shock