Faculty Bibliography

ADP-ribosylation is an important post-translational protein modification; however, endogenous substrates are poorly characterized. In these studies we examined the effects of nitric oxide on the ADP-ribosylation of neutrophil proteins. Purified cytosol and plasma membrane were incubated with 32P-NAD (5 microM, 1 microCi, 30 min) in the presence or absence of nitric oxide. Nitric oxide induced the ADP-ribosylation of the 37 kD substrate present only in cytosol. Nitric oxide treatment of plasma membrane plus cytosol revealed the ADP-ribosylation of an additional 43 kD protein. This 43 kD substrate was identified as actin by both phalloidin precipitation and immunoblot (2-D) gel using specific anti-actin antibodies. The data indicate that nitric oxide stimulates the ADP-ribosylation of two discrete substrates in fractionated PMN, one of which can be identified as actin. NO-induced ADP-ribosylation may contribute to the modulatory effect of nitric oxide on neutrophil functions, including F-actin assembly

OBJECTIVE. To examine the effects of nitric oxide (NO) and its more stable derivative, S-nitrosoglutathione (SNO-GSH), on the response of activated T lymphocytes. METHODS. The effects of NO and SNO-GSH on DNA synthesis, interleukin-2 (IL-2) production, IL-2 receptor expression, and cGMP accumulation were determined in phytohemagglutinin-activated peripheral blood mononuclear cells (PBMC) and spleen T cells. RESULTS. Nitric oxide (half-life [T1/2] < 15 seconds) did not inhibit T cell proliferation. However, the derivative SNO-GSH (25 microM) (T1/2 > 2 hours) inhibited DNA synthesis by a mean +/- SD of 65 +/- 19.6% (P < 0.001) in PBMC and 75 +/- 15% (P < 0.001) in spleen cells. Macrophage depletion of PBMC did not abrogate the inhibition. SNO-GSH had no effect on IL-2 production or IL-2 receptor expression. NO (25 microM) increased the cGMP content of PBMC (0.65 +/- 0.15 pmoles/10(6) cells; P < 0.04), as did SNO-GSH (25 microM) in both PBMC (3.8 +/- 1; P < 0.001) and spleen T cells (5.2 +/- 1.2; P < 0.001). Methylene blue and hemoglobin, which are NO inhibitors, inhibited SNO-GSH-induced cGMP accumulation (P < 0.001). CONCLUSION. SNO-GSH inhibits T cell DNA synthesis independently of IL-2 production and in association with cGMP accumulation via a NO-dependent mechanism. We suggest that NO and its S-nitrosothiol derivatives may act as endogenous inhibitors of T cell-mediated inflammation