Salicylate or Aspirin Inhibits the Induction of the Inducible Nitric Oxide Synthase in Rat Cardiac Fibroblasts

To determine if fibroblasts are a source of NO inflammatory myocardial diseases, we have studied the effect of cytokines on the inducible NO synthase (iNOS) in neonatal cardiac fibroblasts and tested whether nonsteroidal anti-inflammatory drugs can diminish the induction of iNOS. In primary cultures, interferon gamma (IFN), interleukin-1 beta (IL-1), or tumor necrosis factor-alpha (TNF) separately did not stimulate nitrite production, whereas IFN combined with IL-1 or TNF synergistically induced iNOS, both at the level of steady state mRNA and nitrite accumulation. Steady state mRNA levels for iNOS were obvious as early as 3 hours after the addition of IFN + TNF and remained elevated for at least 72 hours. Sodium salicylate inhibited cytokine-induced nitrite accumulation in a time- and dose-dependent manner (IC50, 750 mumol/L). The inhibition was reversible and occurred when salicylate was added either before or after cytokine induction. Aspirin (1 mmol/L) also inhibited nitrite production, whereas indomethacin (25 mumol/L) or acetaminophen (100 mumol/L) did not. TNF, either alone or combined with IFN, significantly stimulated prostaglandin E2, which was inhibited by either salicylate (4 mmol/L) or indomethacin (25 mumol/L). Salicylate, when given either before or after IFN + TNF, reduced mRNA levels of iNOS induced by cytokines. Salicylate did not affect iNOS enzymatic activity when added to the cytosolic lysate, although it was able to reduce enzymatic activity to 32% of induced levels when given to intact cells. These studies implicate cardiac fibroblasts as a source of NO in inflammatory cardiac diseases and suggest a possible therapeutic role for salicylate and aspirin in diminishing the steady state levels of iNOS mRNA.