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.
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.
Jun
C D
CD
Department of Microbiology and Immunology, Wonkwang University School of Medicine, Chonbuk, Korea.
Han
M K
MK
Kim
U H
UH
Chung
H T
HT
D000199:Actins / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology; D002448:Cell Adhesion; D002478:Cells, Cultured; D007371:Interferon-gamma / Q000494:pharmacology; D007797:Laminin; D008070:Lipopolysaccharides / Q000494:pharmacology; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008809:Mice, Inbred C3H; D009569:Nitric Oxide / Q000502:physiology*; D009599:Nitroprusside / Q000494:pharmacology; D010587:Phagocytosis / Q000187:drug effects; D011554:Pseudopodia / Q000502:physiology*; D011994:Recombinant Proteins; D019323:omega-N-Methylarginine / Q000494:pharmacology
D000199:Actins; D007797:Laminin; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D009599:Nitroprusside; D000246:Adenosine Diphosphate Ribose; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D007371:Interferon-gamma; D001905:Botulinum Toxins; C096324:botulinum toxin type C