Feedback inhibition of nitric oxide synthase activity by nitric oxide

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Abstract
1 A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon-gamma (IFN-γ, 10 u ml−1) plus lipopolysaccharide (LPS, 10 ng ml−1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2 The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3 The decline in the NO synthase activity was significantly slowed down in cells cultured in a low l-arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4 The addition of NO generators, S-nitroso-acetyl-penicillamine (SNAP) or S-nitroso-glutathione (GSNO) markedly inhibited the NO synthase activity in a dose-dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5 SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6 The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly.