TGF-BETA REGULATES PRODUCTION OF NO IN PULMONARY-ARTERY SMOOTH-MUSCLECELLS BY INHIBITING EXPRESSION OF NOS

We have previously reported that a mixture of lipopolysaccharide and c ytokines stimulates cultured rat pulmonary artery smooth muscle cells (RPASM) to express elevated levels of mRNA for inducible nitric oxide synthase (iNOS), and to produce large amounts of nitric oxide (NO). Th e current study tests the hypothesis that transforming growth factor-b eta (TGF-beta) modulates this process. Accordingly, RPASM were treated with a mixture of LPS (10 mu g/ml) and the cytokines interleukin-1 be ta (5 U/ml), tumor necrosis factor-alpha (500 U/ml), and interferon-ga mma (100 U/ml). In the absence of TGF-beta 1, NO production (indicated by colorimetric assay of cumulative nitrite levels at 24 h) was great ly increased, as previously observed. Under identical conditions, TGF- beta 1 caused a concentration-dependent decrease in NO production. The addition of neither excess L-arginine nor sepiapterin reversed the in hibition, indicating that the effect of TGF-beta 1 was not due to limi tation of enzyme substrate or cofactor tetrahydrobiopterin, respective ly. Northern and Western analyses showed that TGF-beta 1 reduced level s of iNOS mRNA and protein to baseline at all time points examined up to 24 h. Complete suppression of iNOS protein expression was evident e ven when TGF-beta 1 was added at postinduction time points. One mechan ism of action of TGF-beta 1 was demonstrated in experiments in which d egradation of iNOS protein was greatly increased by the addition of TG F-beta 1. These results demonstrate that TGF-beta 1 regulates producti on of NO in RPASM by inhibiting iNOS expression in part by increasing degradation of existing iNOS protein.