Inhibition of tumor necrosis factor-α (tnf-α) release from vascular smooth muscle cells smc bv elevated camp

L. M. Zhang, S. K. Leeper-Woodford, S. K. Erceg, M. R. Castresana, W. H. Newman

Research output: Contribution to journalArticle

Abstract

We recently reported that vascular tissue and SMC are capable of releasing TNF-α when stimulated by bacterial lipopolysacharide (LPS). Here, we tested the hypothesis that increases in intracellular cAMP would inhibit TNF-α release. SMC were cultured from pig coronary artery. Cells were incubated in the presence of 20 μg/ml LPS alone or with the addition of forskolin (FSK). At zero time and 1, 3 and 6 hr, the medium was removed and analyzed for TNF-α by the L929 cell cytotoxicity assay. Intracellular cAMP was extracted with 0.1N HCl and analyzed by radioimmunoassay. LPS alone stimulated the release of TNF-α from SMC all, 3 and 6 hr. For instance at 1 hr, TNF-α in the medium from LPS stimulated cells was 9.4 ± 2.0 units/mg/cell protein vs 1.1 ± 0.4 units/mg/cell protein in control (p < 0.05, 7 cell isolations). FSK inhibited LPS stimulated TNF-α release in a concentration-dependent manner. In the presence of LPS and 1 or 10 μM FSK, TNF-α release at 1 hr was 3.3 ± 1.1 and 0.8 ± 0.2 units/mg cell protein, respectively (p < 0.05 vs. LPS alone). A similar supression of TNF-α by FSK was seen at 3 and 6 hr. Intracellular cAMP increased from 10.1 ± 0.7 pmol/mg cell protein to 31.8 ± 3.3 within 5 min in the presence of 1 μM FSK and remained elevated at 18.2 ± 2.9 pmol/mg protein at 6 hr. Similar inhibition of LPS stimulated TNF-α release from intact coronary vessel was seen with FSK or 8-Br-cAMP. For instance at 6 hr, TNF-α in the medium was 485 ± 23 units/g tissue in the presence of LPS alone and was reduced to 125 ± 24 by 10 μM FSK and 72 ± 22 by 1 mM 8-Br-cAMP. These results indicate that in vascular smooth muscle cells, LPS stimulated TNF-α release is inhibited by elevated intracellular cAMP.

Original languageEnglish (US)
Pages (from-to)A70
JournalFASEB Journal
Volume10
Issue number3
StatePublished - Dec 1 1996

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Colforsin
Vascular Smooth Muscle
Smooth Muscle Myocytes
Muscle
Tumor Necrosis Factor-alpha
Cells
Proteins
Coronary Vessels
Tissue
Cell Separation
Cytotoxicity
Radioimmunoassay
Blood Vessels
Assays
Swine

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Inhibition of tumor necrosis factor-α (tnf-α) release from vascular smooth muscle cells smc bv elevated camp. / Zhang, L. M.; Leeper-Woodford, S. K.; Erceg, S. K.; Castresana, M. R.; Newman, W. H.

In: FASEB Journal, Vol. 10, No. 3, 01.12.1996, p. A70.

Research output: Contribution to journalArticle

Zhang, L. M. ; Leeper-Woodford, S. K. ; Erceg, S. K. ; Castresana, M. R. ; Newman, W. H. / Inhibition of tumor necrosis factor-α (tnf-α) release from vascular smooth muscle cells smc bv elevated camp. In: FASEB Journal. 1996 ; Vol. 10, No. 3. pp. A70.
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abstract = "We recently reported that vascular tissue and SMC are capable of releasing TNF-α when stimulated by bacterial lipopolysacharide (LPS). Here, we tested the hypothesis that increases in intracellular cAMP would inhibit TNF-α release. SMC were cultured from pig coronary artery. Cells were incubated in the presence of 20 μg/ml LPS alone or with the addition of forskolin (FSK). At zero time and 1, 3 and 6 hr, the medium was removed and analyzed for TNF-α by the L929 cell cytotoxicity assay. Intracellular cAMP was extracted with 0.1N HCl and analyzed by radioimmunoassay. LPS alone stimulated the release of TNF-α from SMC all, 3 and 6 hr. For instance at 1 hr, TNF-α in the medium from LPS stimulated cells was 9.4 ± 2.0 units/mg/cell protein vs 1.1 ± 0.4 units/mg/cell protein in control (p < 0.05, 7 cell isolations). FSK inhibited LPS stimulated TNF-α release in a concentration-dependent manner. In the presence of LPS and 1 or 10 μM FSK, TNF-α release at 1 hr was 3.3 ± 1.1 and 0.8 ± 0.2 units/mg cell protein, respectively (p < 0.05 vs. LPS alone). A similar supression of TNF-α by FSK was seen at 3 and 6 hr. Intracellular cAMP increased from 10.1 ± 0.7 pmol/mg cell protein to 31.8 ± 3.3 within 5 min in the presence of 1 μM FSK and remained elevated at 18.2 ± 2.9 pmol/mg protein at 6 hr. Similar inhibition of LPS stimulated TNF-α release from intact coronary vessel was seen with FSK or 8-Br-cAMP. For instance at 6 hr, TNF-α in the medium was 485 ± 23 units/g tissue in the presence of LPS alone and was reduced to 125 ± 24 by 10 μM FSK and 72 ± 22 by 1 mM 8-Br-cAMP. These results indicate that in vascular smooth muscle cells, LPS stimulated TNF-α release is inhibited by elevated intracellular cAMP.",
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