title: Slow channel calcium inhibition blocks proinflammatory gene signaling and reduces macrophage responsiveness.
abstract: BACKGROUND
  This study investigates the possible intracellular mechanisms responsible for calcium antagonist protection in tissue-fixed macrophages, a central modulator of the proinflammatory phenotype.
  
  
  METHODS
  Rabbit alveolar macrophages were exposed to lipopolysaccharide in the presence of different specific calcium antagonists. Cellular and nuclear protein were extracted and analyzed by Western blot for the phosphorylated forms of PYK2, ERK 1/2, and p38, and nuclear translocation of NF-kappaB and AP-1. Tumor necrosis factor-alpha (TNF-alpha) expression was measured by an L929 bioassay on cellular supernatants. Statistical analysis was performed by unpaired Student's t tests.
  
  
  RESULTS
  Cells pretreated with 100 to 500 micromol/L of diltiazem or 50 to 100 micromol/L of verapamil, both slow channel calcium blockers, led to dose-dependent reductions in lipopolysaccharide-induced PYK2 and ERK 1/2 phosphorylation, and nuclear translocation of AP-1 when compared with controls (p < 0.05). Neither inhibitor had any significant effect on p38 or NF-kappaB translocation. EGTA an extracellular calcium chelator, had no significant effect on any intracellular process studied. A dose-dependent reduction in TNF-alpha production was demonstrated with diltiazem and verapamil (p < 0.05), with no effect induced by EGTA.
  
  
  CONCLUSIONS
  Slow channel calcium influx is essential for optimal intracellular signaling through PYK2 and ERK 1/2. This reduced intracellular signaling correlated with reduced AP-1 translocation and TNF-alpha production. Extracellular calcium chelation had no significant effect on intracellular signaling or TNF-alpha production. This study further elucidates the protective mechanism of action of calcium channel blockade by diltiazem and verapamil by reducing intracellular calcium release and down-regulating the excessive proinflammatory phenotype.
authors.affiliation: Department of Surgery, University of Washington, Seattle, Washington 98104, USA. jcuschie@uwashington.edu
authors.forename: Joseph
authors.initials: J
authors.lastname: Cuschieri
authors.forename: David
authors.initials: D
authors.lastname: Gourlay
authors.forename: Iris
authors.initials: I
authors.lastname: Garcia
authors.forename: Sandra
authors.initials: S
authors.lastname: Jelacic
authors.forename: Ronald V
authors.initials: RV
authors.lastname: Maier
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D004110:Diltiazem / Q000494:pharmacology*; D004306:Dose-Response Relationship, Immunologic; D051418:Focal Adhesion Kinase 2; D008070:Lipopolysaccharides / Q000494:pharmacology; D016676:Macrophages, Alveolar / Q000276:immunology / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011817:Rabbits; D018805:Sepsis / Q000378:metabolism; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D014700:Verapamil / Q000494:pharmacology*
chemical_list: D002121:Calcium Channel Blockers; D008070:Lipopolysaccharides; D016328:NF-kappa B; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D014700:Verapamil; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; D020928:Mitogen-Activated Protein Kinases; D004110:Diltiazem