Release of Tumor Necrosis Factor-α from Coronary Smooth Muscle: Activation of NF-κB and Inhibition by Elevated Cyclic AMP

Abstract

Background.Evidence suggests that tumor necrosis factor-α (TNF-α) 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-α. The aims were to determine if bacterial lipopolysaccharide (LPS) could stimulate production; if activation of the nuclear regulatory factor, NF-κB, was associated with production; and if intracellular cAMP regulates TNF-α in coronary vasculature through a mechanism involving NF-κB.Material and methods.LPS was used to stimulate TNF-α production. Forskolin (FSK) and 8-Br-cAMP were added to tissue and cells in order to elevate intracellular cAMP. TNF-α release into the bathing medium was measured by the L929 cell cytotoxicity assay. Intracellular cAMP was determined by radioimmunoassay. NF-κB activation was determined in whole cell extracts by electrophoretic mobility shift assay.Results.In segments of coronary arteries, LPS stimulated TNF-α 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-α 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-α release. For instance at 6 h in the presence of LPS and FSK or 8-Br-cAMP, TNF-α 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-α release which was inhibited in a concentration-dependent manner by a rise in intracellular cAMP induced by FSK. In cultured cells release of TNF-α stimulated by LPS was associated with activation of NF-κB. Neither FSK nor 8-Br cAMP inhibited activation of NF-κB by LPS.Conclusions.Porcine coronary arteries produce TNF-α from a smooth muscle cell source. Production stimulated by LPS was inhibited by elevated intracellular cAMP and was associated with activation of NF-κB. However, activation of NF-κB was not inhibited by elevated cAMP, suggesting that the regulatory action of this cyclic nucleotide could lie downstream from activation of the TNF-α gene. These results support the view that coronary vessels can be a source of TNF-α possibly involved in heart disease.