Tumor Necrosis Factor-Alpha Dependent Stimulation of Cyclooxygenase Involves Nitric Oxide
The aim of this study was to establish the interaction of nitric oxide (NO) and cyclooxygenase (COX) isoforms in vascular smooth muscle cells (VSMC) of rat aorta. We hypothesize that increased NO levels derived from inducible nitric oxide synthase (iNOS) after challenge with tumor necrosis factor-alpha (TNF) stimulates COX-2. TNF [1ng/ml] induced a transient increase in VSMC COX-2 and iNOS mRNA accumulation, without affecting COX-1 mRNA levels. The kinetics of COX-2 and iNOS mRNA accumulation were similar after challenge with TNF; mRNA peaked at 2 hr (COX-2: ∼8 fold and iNOS: ∼2 fold) and decreased to control levels by approximately 6 hr. COX-2 and iNOS protein expression also increased after TNF challenge (COX-2: ∼8-fold and iNOS: ∼1.5fold). Stimulation with TNF for 24 hrs increased nitrite levels by 4-fold (control: 5.26±0.37 vs. TNF: 18.33±0.66 pmol/μg protein; p<0.001). Aminogunidine (AG; [1mM]), a specific iNOS inhibitor, abolished this stimulation. TNF-mediated PGI2production increased 4.5-fold (control: 120±14.47 vs. TNF: 541±55.05 pg/μg protein; p<0.001) and 2-fold for TXA2 (control 30.67±4.17 vs. TNF: 59+4.0 pg/μg protein; p<0.01). AG pretreatment attenuated TNF mediated increase in PGI2 by 45% (TNF: 541±55.05 vs. TNF+AG: 296±29 pg/μg protein; p<0.001) and TXA2 by 34% (TNF: 59±4.0 vs. TNF+AG: 39±3 pg/μg protein; p<0.01). Sodium Nitroprusside (SNP), an NO donor increased COX-2 protein expression by ∼3 fold after 1 hr of treatment. PGI2 levels also increased after incubation with SNP (control: 136±4.95 vs. SNP: 188±10.39 pg/μg protein; p<0.01); TXA2 levels were unaffected. The data indicate, that TNF-mediated NO production in VSMC is iNOS dependent. Moreover, increased NO levels after exposure to TNF stimulates prostanoid production, possibly via a COX-2 dependent mechanism. We conclude that stimulation of the COX pathway by TNF may involve iNOS-mediated increases in NO levels. This interaction may be relevant to the regulation of hyperplasia/hypertrophy/apoptosis in diseases such as arteriosclerorsis and hypertension, as recent evidence has shown that TNF-mediated increases in VSMC proliferation are COX-2 dependent.