Increased intracellular cyclic adenosine 3', 5'-monophosphate inhibits release of tumor necrosis factor-alpha from human vascular tissue and cultured smooth muscle cells.

Objectives: We recently reported that bacterial lipopolysaccharide stimulates release of tumor necrosis factor (TNF)-alpha from both human vascular tissue and cultured smooth muscle cells. In the current study, we tested the hypothesis that increased intracellular cyclic adenosine 3',5'-monophosphate (cAMP) could inhibit TNF-alpha release.

Design: Prospective, repeated-measures analysis.

Setting: Academic research laboratory.

Subjects: Segments of internal mammary artery and saphenous vein from patients undergoing coronary artery bypass surgery.

Measurements And Main Results: Segments of saphenous vein and internal mammary artery and confluent smooth muscle cells cultured from these vessels were incubated in the presence of 20 micrograms/mL bacterial lipopolysaccharide, alone or with the addition of forskolin or 8-Br-cAMP. At 0, 1, 3, 6, 18, and 24 hrs, the incubation medium was removed from vessel segments or cells and was analyzed for biologically active TNF-alpha, using the L929 cell cytotoxicity assay. cAMP was extracted from tissue and cells with 0.1 N HCl and was analyzed by radioimmunoassay. Bacterial lipopolysaccharide stimulated the release of TNF-alpha from internal mammary smooth muscle cells at all time points. For example, at 6 hrs, TNF-alpha concentration in the medium from lipopolysaccharide-stimulated cells was 20 +/- 1.6 U/mg of cell protein, compared with 0.9 +/- 0.5 U/mg of cell protein in control cell medium (p < .05). Forskolin-inhibited bacterial lipopolysaccharide stimulated TNF-alpha release. In the presence of lipopolysaccharide and forskolin, TNF-alpha release at 6 hrs was 8.6 +/- 1.5 U/mg of cell protein (p < .05 vs. in the presence of bacterial lipopolysaccharide alone). Bacterial lipopolysaccharide, alone, had no effect on intracellular cAMP. Forskolin increased intracellular cAMP levels to 74.0 +/- 12 pmol/mg of cell protein at 6 hrs from a control level of 7.7 +/- 0.4 pmol/mg (p < .05). The 8-Br-cAMP, an agent that mimics the action of intracellular cAMP, also inhibited TNF-alpha release stimulated by lipopolysaccharide. Similar inhibition by forskolin and 8-Br-cAMP on TNF-alpha release was obtained with smooth muscle cells from saphenous vein. Finally, in tissue segments from either internal mammary artery or saphenous vein, both forskolin and 8-Br-cAMP inhibited lipopolysaccharide-stimulated TNF-alpha release.

Conclusions: These results are consistent with the conclusion that vascular tissue, particularly the smooth muscle cell, is a source of TNF-alpha. Further, bacterial lipopolysaccharide-stimulated tumor TNF-alpha release can be inhibited by increased intracellular cAMP.