title: Release of tumor necrosis factor-alpha from coronary smooth muscle: activation of NF-kappaB and inhibition by elevated cyclic AMP.
abstract: BACKGROUND
  Evidence suggests that tumor necrosis factor-alpha (TNF-alpha) is involved in heart diseases such as atherosclerosis. We used porcine coronary arteries and smooth muscle cells cultured from these vessels to study the regulation of production of TNF-alpha. The aims were to determine if bacterial lipopolysaccharide (LPS) could stimulate production; if activation of the nuclear regulatory factor, NF-kappaB, was associated with production; and if intracellular cAMP regulates TNF-alpha in coronary vasculature through a mechanism involving NF-kappaB.
  
  
  METHODS
  LPS was used to stimulate TNF-alpha production. Forskolin (FSK) and 8-Br-cAMP were added to tissue and cells in order to elevate intracellular cAMP. TNF-alpha release into the bathing medium was measured by the L929 cell cytotoxicity assay. Intracellular cAMP was determined by radioimmunoassay. NF-kappaB activation was determined in whole cell extracts by electrophoretic mobility shift assay.
  
  
  RESULTS
  In segments of coronary arteries, LPS stimulated TNF-alpha release which increased with time to a maximum at 6 h (485 +/- 19 units/g tissue) and remained elevated at this level for 24 h. In contrast, the level of TNF-alpha measured at 24 h in medium from coronary tissue not exposed to LPS was 11.1 +/- 4.1 units/g tissue. In the presence of LPS, both FSK and 8-Br-cAMP significantly reduced TNF-alpha release. For instance at 6 h in the presence of LPS and FSK or 8-Br-cAMP, TNF-alpha was 126 +/- 24 and 71.6 +/- 22 units/g tissue, respectively (P < 0.05 vs LPS alone). Tissue levels of cAMP were significantly elevated in the presence of FSK. Similar results were obtained with smooth muscle cells cultured from the coronary arteries; i.e., LPS stimulated TNF-alpha release which was inhibited in a concentration-dependent manner by a rise in intracellular cAMP induced by FSK. In cultured cells release of TNF-alpha stimulated by LPS was associated with activation of NF-kappaB. Neither FSK nor 8-Br cAMP inhibited activation of NF-kappaB by LPS.
  
  
  CONCLUSIONS
  Porcine coronary arteries produce TNF-alpha from a smooth muscle cell source. Production stimulated by LPS was inhibited by elevated intracellular cAMP and was associated with activation of NF-kappaB. However, activation of NF-kappaB was not inhibited by elevated cAMP, suggesting that the regulatory action of this cyclic nucleotide could lie downstream from activation of the TNF-alpha gene. These results support the view that coronary vessels can be a source of TNF-alpha possibly involved in heart disease.
authors.affiliation: Department of Surgery, Mercer University School of Medicine and Medical Center of Central Georgia, Macon, Georgia, 31201, USA.
authors.forename: W H
authors.initials: WH
authors.lastname: Newman
authors.forename: L M
authors.initials: LM
authors.lastname: Zhang
authors.forename: D H
authors.initials: DH
authors.lastname: Lee
authors.forename: M L
authors.initials: ML
authors.lastname: Dalton
authors.forename: D J
authors.initials: DJ
authors.lastname: Warejcka
authors.forename: M R
authors.initials: MR
authors.lastname: Castresana
authors.forename: S K
authors.initials: SK
authors.lastname: Leeper-Woodford
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D005576:Colforsin / Q000494:pharmacology; D003331:Coronary Vessels / Q000187:drug effects / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism*; D006331:Heart Diseases / Q000209:etiology; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000235:genetics; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000378:metabolism*
chemical_list: D008070:Lipopolysaccharides; D016328:NF-kappa B; D015345:Oligonucleotide Probes; D014409:Tumor Necrosis Factor-alpha; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP