Synergistic regulation of NOS II expression by IL-1β and TNF-α in cultured rat colonic smooth muscle cells

Interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and lipopolysaccharide (LPS) were examined for their ability to regulate the activity and protein levels of inducible nitric oxide synthase (NOS II) in cultured rat colonic smooth muscle cells. Treatment with these agents resulted in a time-dependent increase in NOS II activity. After 48 h, NOS II activity, measured as L-[H-3]citrulline production, was increased 24.3 +/- 6.9 pmol.min(-1).mg protein(-1) by 1 nM IL-1 beta and 3.2 +/- 1.1 pmol.min(-1).mg protein(-1) by 1 nM TNF-alpha, and increased synergistically by a combination of the two (51.8 +/- 7.3 pmol.min(-1).mg protein(-1)). Measurement of NOS II activity as nitrite production yielded similar results: IL-1 beta, 27.2 +/- 1.2; TNF-alpha, 1.6 +/- 0.1; and IL-1 beta + TNF-alpha, 46.8 +/- 3.2 pmol.min(-1).mg protein(-1) above basal. LPS (10 mu g/ml) had a small but significant effect at 48 h that was only additive with that of IL-1 beta. The increase in NOS II activity induced by IL-1 beta and TNF-alpha was inhibited 73-86% by transforming growth factor-beta 1 (TGF-beta 1). The NOS isoform induced by IL-1 beta and TNF-alpha was identified as NOS II by Western immunoblot analysis and confirmed by its 66-97% inhibition by 100 mu M S-methylisothiourea, a selective NOS II inhibitor, and its Ca2+-independent activity. We conclude that the cytokines IL-1 beta and TNF-alpha act independently and synergistically to stimulate NOS II expression and enzymatic activity in rat colonic smooth muscle through a mechanism sensitive to inhibition by TGF-beta 1.