Release of tumor necrosis factor-α from coronary smooth muscle

TY - JOUR

T1 - Release of tumor necrosis factor-α from coronary smooth muscle

T2 - Activation of NF-κB and inhibition by elevated cyclic AMP

AU - Newman, Walter H.

AU - Zhang, Li Ming

AU - Lee, Dong H.

AU - Dalton, Martin L.

AU - Warejcka, Debra J.

AU - Castresana, Manuel R

AU - Leeper-Woodford, Sandra K.

PY - 1998/1/1

Y1 - 1998/1/1

N2 - 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.

AB - 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.

KW - CAMP

KW - Coronary artery

KW - NF-κB

KW - Smooth muscle cells

KW - TNF-α

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U2 - 10.1006/jsre.1998.5456

DO - 10.1006/jsre.1998.5456

M3 - Article

VL - 80

SP - 129

EP - 135

JO - Journal of Surgical Research

JF - Journal of Surgical Research

SN - 0022-4804

IS - 2

ER -