TNF-alpha inhibits cAMP-mediated phosphorylation of eNOS at serine-633 in endothelial cells

Introduction

Inflammatory mechanisms are recognized as players in the pathophysiology of cardiovascular diseases. High levels of tumor necrosis factor-α (TNF-α) occur in heart failure leading to pleiotropic effects. In blood vessels, the second messenger cyclic-adenosine monophosphate (cAMP) mediates vasodilation, inhibits proliferation and improves barrier function. Also, cAMP participates in nitric oxide (NO) synthesis by promoting protein kinase A (PKA)-mediated phosphorylation of endothelial NO synthase (eNOS) at serine-633 (ser633-eNOS). Vascular dysfunction is a hallmark of heart failure, but how inflammatory cytokines influence vascular cAMP pathways is unclear.

Objective

The aim of this study is to examine the influence of TNF-α exposure on the cAMP pathway in endothelial cells.

Method

Human coronary artery endothelial cells (HCAEC) were cultured between passages 5 and 7. Sub-confluent HCAEC were exposed to TNF-α (10 ng/mL) or vehicle. After 24 h treatment, cells were stimulated for 30 min with isoprenaline (1 μM) or combination of forskolin analogue (L-858051, 30 μM) and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 300 μM) in order to stimulate cAMP production, or vehicle. Cells were then harvested in ice-cold lysis buffer and frozen. Cell lysates were processed for immunoblot studies. Signals obtained on phosphorylated sites in eNOS and vasodilator-stimulated phosphoprotein (VASP) were normalized to the respective signal obtained for total protein. Where relevant, protein level was normalized to GAPDH level.

Results

In control condition, L-858051 with IBMX stimulated the phosphorylation of ser633-eNOS and ser157-VASP significantly (n = 4, P < 0.05). Cell exposure to TNF-α induced expression of intercellular adhesion molecule ICAM-1. TNF-α significantly decreased total eNOS expression compared to control condition (P < 0.05). Interestingly, TNF-α abolished the effect of L-858051 with IBMX on ser633-eNOS phosphorylation, but did not alter that of ser157-VASP (P < 0.05). Isoprenaline had no significant effect on any phosphorylation site.

Conclusion

Exposure of HCAEC to TNF-α decreased cAMP-related phosphorylation of eNOS while leaving intact the phosphorylation of VASP, a standard surrogate for PKA activation in vascular cells. Future experiments will investigate whether TNF-α diruspts specific cAMP signaling compartments in endothelial cells, which may participate to vascular dysfunction.