Nitric oxide regulates actin reorganization through cGMP and Ca2+/calmodulin in RAW 264.7 cells

Nitric oxide (NO) has been reported to be involved in the regulation of pse udopodia formation, phagocytosis and adhesion in macrophages through the re organization of actin. In the present study, we directly separated the glob ular (G) and filamentous (F) actin from quiescent or NO-stimulated macropha ge-like cell line RAW 264.7 cells in order to investigate the dynamic redis tribution of actin pools. We also focused on the regulatory mechanisms of a ctin assembly, induced by NO and its possible subsequent signaling pathway. We showed that predominant G-actin coexisted with Triton X-100-insoluble f ilamentous (TIF) and Triton X-100-soluble filamentous actin in resting RAW 264.7 cells. The exogenous NO produced by (+/-)-(E)-2-[(E)-hydroxyimino]-6- methoxy-4-methyl-5-nitro-3-hexenamide (NOR1), the endogenous NO induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN gamma), and dibutyryl-c GMP increased the contents of TIF-actin in dose- and time-dependent manners and altered its morphology. The increase in the TIF-actin contents induced by NOR1 or LPS plus IFN gamma was efficiently blocked by the radical scave nger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxal in-1-one or the arginine analogue N-G-monomethyl-L-arginine acetate, respec tively. Preincubation with the calmodulin antagonist W-7 almost completely blocked the NO-induced TIF-actin increase and morphological change. On the other hand, preincubation with C3 transferase, an inhibitor of Rho protein, efficiently prevented the change in cell morphology, but had no effect on the TIF-actin increase. We postulate that cGMP and subsequent Ca2+/calmodul in may be key regulators of actin reorganization in NO-stimulated RAW 264.7 cells. (C) 2001 Elsevier Science B.V. All rights reserved.