AJP - Lung Cellular and Molecular Physiology, Vol 268, Issue 5 862-L867, Copyright © 1995 by American Physiological Society
TGF-beta regulates production of NO in pulmonary artery smooth muscle cells by inhibiting expression of NOS
J. Finder, W. W. Stark Jr, D. K. Nakayama, D. Geller, K. Wasserloos, B. R. Pitt and P. Davies
Division of Pediatric Pulmonology, Children's Hospital of Pittsburgh 15261, USA.
We have previously reported that a mixture of lipopolysaccharide and
cytokines 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). The
current study tests the hypothesis that transforming growth factor-beta
(TGF-beta) modulates this process. Accordingly, RPASM were treated with a
mixture of LPS (10 micrograms/ml) and the cytokines interleukin-1 beta (5
U/ml), tumor necrosis factor-alpha (500 U/ml), and interferon-gamma (100
U/ml). In the absence of TGF-beta 1, NO production (indicated by
colorimetric assay of cumulative nitrite levels at 24 h) was greatly
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 inhibition,
indicating that the effect of TGF-beta 1 was not due to limitation of
enzyme substrate or cofactor tetrahydrobiopterin, respectively. Northern
and Western analyses showed that TGF-beta 1 reduced levels of iNOS mRNA and
protein to baseline at all time points examined up to 24 h. Complete
suppression of iNOS protein expression was evident even when TGF-beta 1 was
added at postinduction time points. One mechanism of action of TGF-beta 1
was demonstrated in experiments in which degradation of iNOS protein was
greatly increased by the addition of TGF-beta 1. These results demonstrate
that TGF-beta 1 regulates production of NO in RPASM by inhibiting iNOS
expression in part by increasing degradation of existing iNOS protein.
