Activated murine macrophages secrete a metabolite of arginine with the bioactivity of endothelium-derived relaxing factor and the chemical reactivity of nitric oxide.
L-arginine-dependent synthesis of nitrite (NO2-) and nitrate (NO3-) by macrophages correlates with and is required for their execution of nonspecific cytotoxicity toward some tumor cells and microbes. However, the bioactive L-arginine metabolites responsible for cytotoxicity are unknown. Mammalian endothelial cells have recently been shown to release nitric oxide (NO.); we therefore determined if this reactive metabolite was synthesized by activated murine macrophages. Macrophage-derived NO. was detected by two independent methods: a bioassay for NO.-mediated relaxation of preconstricted rings of rabbit aorta; and a spectroscopic measurement of the reaction of NO. with clostridial ferredoxin, an Fe-S protein. After activation with IFN-gamma and LPS, macrophages continuously secreted a substance that relaxed rabbit aortic rings denuded of endothelium. Production of the vasorelaxant was enhanced by 0.5 mM L-arginine and inhibited reversibly by NG-methylated L-arginine analogs that block macrophage NO2-/NO3- synthesis. The vasorelaxant was scavenged by ferrous myoglobin, was labile, and was neither NO2- nor a cyclooxygenase metabolite. Activated M phi also secreted a substance that bleached Fd, a reaction carried out by NO. and NO2, but not NO2-. Macrophage bleaching of Fd correlated directly with time, cell number, and concomitant NO2-/NO3- production, required L-arginine, and was independent of reactive oxygen intermediates. Thus, activated murine M phi release NO. and/or a closely related, highly reactive nitrogen oxide such as NO2, during their conversion of L-arginine to NO2-/NO3-. NO. and NO2 may mediate L-arginine-dependent pathologic effects of M phi, as well as physiologic effects not previously considered for this widely distributed cell type.
Stuehr
D J
DJ
Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College, New York, New York 10021.
Gross
S S
SS
Sakuma
I
I
Levi
R
R
Nathan
C F
CF
D000818:Animals; D001011:Aorta; D001120:Arginine / Q000378:metabolism* / Q000494:pharmacology; D001681:Biological Assay; D001685:Biological Factors / Q000494:pharmacology*; D055598:Chemical Phenomena; D002621:Chemistry; D004730:Endothelium, Vascular; D005288:Ferredoxins / Q000378:metabolism; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D009126:Muscle Relaxation / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000502:physiology; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D009573:Nitrites / Q000378:metabolism; D009585:Nitrogen Dioxide / Q000032:analysis / Q000378:metabolism; D011817:Rabbits; D013053:Spectrophotometry
D001685:Biological Factors; D005288:Ferredoxins; D009566:Nitrates; D009573:Nitrites; D009569:Nitric Oxide; D001120:Arginine; D009585:Nitrogen Dioxide