Release of tumor necrosis factor-α from coronary smooth muscle:: Activation of NF-κB and inhibition by elevated cyclic AMP

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-kappa B, was associated with production; and if intracellular cAMP regulates TNF-alpha in coronary vasculature through a mechanism involving NF-kappa B. Material and 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-kappa B 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-kappa B. Neither FSK nor 8-Br cAMP inhibited activation of NF-kappa B 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-kappa B. However, activation of NF-kappa B 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. (C) 1998 Academic Press.