Trapping of nitric oxide produced during denitrification by extracellular hemoglobin.

426

186

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.

Section: Resultsmentioning

confidence: 99%

Activated murine macrophages secrete a metabolite of arginine with the bioactivity of endothelium-derived relaxing factor and the chemical reactivity of nitric oxide.

Stuehr

Gross

Sakuma

et al. 1989

426

186

“…M-0 cytostasis was blocked 97% in the presence of reduced myoglobin and 44% in the presence ofan Fe-catalyzed superoxide generating system. These systems scavenge NO-and have been used to prevent its biological effects (8,20,24,25). In addition to scavenging NO., FeS04 and myoglobin may have helped injured cells to recover faster from cytostasis by furnishing Fe to replenish intracellular pools and rebuild FeS clusters (10,31).…”

Section: Discussionmentioning

confidence: 99%

“…are not yet well understood. For example, the flux of NO-reaching a target will depend on the concentrations of species that can scavenge it, such as oxygen (9), superoxide (20), reduced hemes (23,24), transition metals (37), and thiols (38), as well as on the activity of species that can protect NO-, such as superoxide dismutase (39). NO.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells.

Stuehr

Nathan

1989

1,622

771

Once activated by agents such as IFN-y and bacterial LPS, macrophages (MO)t can inhibit the growth of a wide variety of tumor and microbial targets (1, 2) . Although MO products such as hydrogen peroxide, TNFa, and IL-1 cause cytostasis and/or cytotoxicity (3-5), in many cases these mediators do not appear to be involved . With some targets, Mo-mediated cytostasis and injury to the mitochondria) electron transport chain (METC) require a process associated with MO oxidation of the guanido nitrogens of L-arginine to N02 -/NO3 -(6). However, it is unknown if a metabolite of L-arginine causes these injuries, and if so, which metabolite. Activated Mo have recently been shown to release a compound similar to or identical with the reactive radical nitric oxide (NO .) during metabolism of L-arginine to N02 -/NO3 -(7). This report identifies NO. (or a closely related product) as a mediator of MO-induced cytostasis and mitochondria) respiratory inhibition in lymphoma cells . Materials and MethodsReagents. Cells were cultured in minimum Eagle's medium, a modification (aMEM) or RPMI 1640 (RPMI ; KC Biological Inc ., Lenexa, KS), both supplemented with 8% bovine calf serum (CS; HyClone Systems, Logan, UT), L-glutamine (584 mg/liter), penicillin (50 U/ml), and streptomycin (50 pg/ml) . Catalase and N°-monomethyl-L-arginine (NMA) were from Calbiochem-Behring Corp. La Jolla, CA. NO . gas (99% pure) and N2 gas (45 ppm 02) were from Matheson Gas Products, East Rutherford, NJ . [Methyl-3 H]TdR (2 Ci/mmol) was from New England Nuclear, Boston, MA. Pure IFN-y was generously provided by Genentech, South San Francisco, CA . LPS (Escherichia coli serotype 0127 : B8) and all other reagents were from Sigma Chemical Co., St . Louis, MO. Concentrated stock solutions were prepared in culture medium (for myoglobin, ascorbate, catalase, NMA, LPS, and IFN-y) or saline (for NaN02, NaN03, FeS04) and sterile filtered (0 .22 pm, Millipore, Danvers, MA) .

“…A second nitric oxide scavenging system, with a different mode of action, was also tested. When kept in the ferrous state, heine proteins such as hemoglobin and myoglobin avidly bind and inactivate nitric oxide (23,40,41). Fig.…”

Section: Nitric Oxide Mediates the Z-arginine-dependent Inhibition Of Phi Recovery Since Me Metabolism Of I-arginine Leads Tomentioning

confidence: 99%

Nitric oxide derived from L-arginine impairs cytoplasmic pH regulation by vacuolar-type H+ ATPases in peritoneal macrophages.

Swallow

Grinstein

Sudsbury

et al. 1991

Snmm~ar.y The ability of macrophages (Mos) to function within an acidic environment has been shown to depend on cytoplasmic pH (pHi) regulation by vacuolar-type H + ATPases. Mcs metabolize t-arginine via an oxidative pathway that generates nitric oxide, nitrate, and nitrite. Since each of these products could potentially inhibit vacuolar-type H + ATPases, we investigated the effect of t-arginine metabolism on Me pHi regulation in thioglycolate-elicited murine peritoneal Mcs. H + ATPase-mediated pHi recovery from an imposed cytoplasmic acid load was measured fluorometrically. When Mcs were incubated with t-arginine (0.25-2.0 mM), their rate of pHi recovery declined progressively from 2 to 6 h of incubation. By contrast, the recovery rate of cells incubated in arginine-free medium remained stable over the same period. The impairment of pHi recovery was specific for I.-arginine, and was blocked competitively by NG-monomethylr-arginine, demonstrating its dependence on t-arginine metabolism. In addition, the inhibition of pHi recovery was enhanced by lipopolysaccharide, an agent known to stimulate t-arginine metabolism by Mos. Scavenging the t-arginine metabolite nitric oxide with either ferrous sulphate or ferrous myoglobin prevented the inhibition of phi recovery, implying that t-argininederived nitric oxide was the species responsible for the inhibition. This concept was supported by the finding of elevated nitrite levels in the supernatant ofceUs incubated in I.-arginine. Furthermore, incubation of Mcs with sodium nitroprusside mimicked the t-arginine-dependent inhibition of H + ATPase activity. Treatment with the cyclic GMP analogue, 8-bromoguanosine 3':5'-cyclic monophosphate, similarly impaired Me phi recovery, suggesting that a nitric oxide-stimulated elevation of cyclic GMP may contribute to the r-arginine-dependent inhibition of pHi regulation.