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Cell Immunol. 1996 Nov 25;174(1):25-34.

Nitric oxide induces ADP-ribosylation of actin in murine macrophages: association with the inhibition of pseudopodia formation, phagocytic activity, and adherence on a laminin substratum.

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1
Department of Microbiology and Immunology, Wonkwang University School of Medicine, Chonbuk, Korea.

Abstract

A recently recognized property of nitric oxide (NO), which would be expected to alter cell function, is the capacity to induce the ADP-ribosylation of various proteins. In these studies we demonstrate that actin present in murine macrophages is a substrate for NO-dependent ADP-ribosylation and that this modification is associated with the modification of cellular functions in murine peritoneal macrophages. A 42-kDa substrate for NO-dependent ADP-ribosylation was identified as actin by binding to DNAse-I and immunoprecipitation with anti-actin antibodies. The amount of actin ADP-ribosylation was correlated with the concentration of sodium nitroprusside (SNP), a NO generating agent, used in each experiment and with the amount of NO produced by activated macrophages. However, a specific inhibitor for NO synthase, N(G)-monomethyl-L-arginine (N(G)MMA), inhibited the ADP-ribosylation of actin by blocking the NO production in the interferon (IFN)-gamma plus lipopolysaccharide (LPS)-stimulated cells. Because the integrity of cytoskeletal protein is involved in shape change, adhesion, and phagocytosis of cells, we elucidated the role of NO-dependent ADP-ribosylation of actin in murine macrophages. A morphology kinetics assay comparing pseudopodial extension values over a 72-hr period showed that IFN-gamma plus LPS-treated macrophages underwent a wave of morphological changes, returning to a round shape after 32 hr. However, incubation of the cells with IFN-gamma plus LPS in the presence of N(G)MMA resulted in spindle-shaped pseudopodia formation and an altered composition of F-actin in macrophages. Adding either SNP or botulinum C2 toxin also inhibited IFN-gamma plus LPS-induced pseudopodia formation even in the presence of N(G)MMA. Flow cytometry revealed that NO inhibits the phagocytosis of fluorescent particles in a reversible manner. Preincubation of the cells with SNP (2 mM) also diminished LPS- or phorbol 12-myristate 13-acetate-induced macrophage adhesion on a laminin substratum. Collectively, in addition to its better-characterized role as a cytolytic mediator, the data illustrate that NO shows negative regulatory roles in cytoskeletal assembly, pseudopodia formation, phagocytosis, and adherence of murine macrophages in association with the ADP-ribosylation of actin.

PMID:
8929451
DOI:
10.1006/cimm.1996.0290
[Indexed for MEDLINE]

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