Effects of a nitric oxide donor, the substratum of NO-synthase, and an inhibitor of NO-synthase on the ROS-generation activity of phagocytes in the course of ascites tumor growth

Experiments in vitro were performed to investigate the effects of the nitric oxide donor (SNP), the substratum of NO-synthase (L-arginine), and the inhibitor of NO-synthase (nitroarginine) on the ROS-generating activity of blood plasma polymorphonuclear leucocytes and ascitic fluid macrophages isolated at different times of tumor (Zaidel hepatoma) growth in animal organism. It was found that in the initial period of tumor growth the nitric oxide donor at a concentration of 8 x 10(-5) M reduced the potential ROS-generating activity of macrophages by 38.5 +/- 9.0% and that of polymorphic-nuclear leucocytes of plasma by 27.6 +/- 7.0 %. However, the dynamics of this process during tumor growth was conservative: variations in the production of ROS by phagocytes were 10 +/- 3.0%. L-arginine induced a decrease in the ROS-generating activity of granulocytes and mononucleares by 25-30%. This fact points to an inducible inhibiting effect of NO-synthase on the ROS-generating activity of NADPH-oxidase in the course of tumor growth. The inhibitor of NO-synthase, nitroarginine, produced a monotonous increase in the ROS-generating activity of phagocytes isolated from the tumor at different periods of its growth. The use NO-synthase inhibitors for increasing ROS levels in the region of tumor growth may favor the suppression of tumor cell growth in vivo.     

Induction of nitric oxide synthase is a necessary precondition for expression of tumor necrosis factor-independent tumoricidal activity by activated macrophages.

Various bacteria and bacterial products induce in pure, lymphocyte-free bone marrow-derived mononuclear phagocytes (BMM?) the generation of tumor necrosis factor, nitric oxide (NO) synthase, NO and nitrite (NO2-), the flow of L-arginine to citrulline, and tumoricidal activity. The flow of L-arginine to citrulline and formation of NO/NO2- on the one hand and expression of tumoricidal activity were not always closely related; however, these parameters were suppressed in a dose-dependent manner by the flavoprotein inhibitor, diphenyleneiodonium (DPI) and the L-arginine analogue, NG-monomethyl-L-arginine (NMMA). The findings support the concept of a central role of the NO synthase pathway in the generation of tumor necrosis factor-independent tumoricidal activity by activated macrophages but the exact conditions which enable the transfer of the lytic principle from the effector to the target cell remain to be elucidated.     

Modulation of nitric oxide (NO) biosynthesis in lactobacilli

We characterized effects of nitric oxide synthase (NOS) substrate L-arginine and classical inhibitors of mammalian NOS on nitric oxide (NO) biosynthesis in probiotic bacteria Lactobacillus plantarum 8P-A3. NO-synthase origin of nitric oxide detected by fluorescent NO indicator 1,2-diaminoanthraquinone (DAA) was confirmed by induction of NO production by exogenous L-arginine. None of the used inhibitors of three isoforms of mammalian NOSs (L-NAME, L-NIL, nNOS inhibitor I) showed significant inhibitory effect of lactobacillar NO-synthase activity.     

Effect of nitric oxide on phagocytic activity of lipopolysaccharide-induced macrophages: possible role of exogenous L-arginine

Among the antimicrobial mechanisms associated with macrophages, NO produced by iNOS plays a major role in intracellular killing, but the relationship between NO and phagocytic activity after injection of inflammatory agents into the peritoneal cavity is not clear. The aim of the present study was to investigate the effect of nitric oxide (NO) on macrophage function after treatment with intraperitoneal lipopolysaccharide (LPS) and the role of exogenous L-arginine administration in this event. Six experimental groups and one control group, each consisting of seven Wistar rats were used: Group I: Control; Group II: LPS; Group III: LPS+L-arginine; Group IV: LPS+L-arginine+Aminoguanidine; Group V: LPS+Aminoguanidine; Group VI: L-arginine; Group VII: Aminoguanidine. Macrophage phagocytic activity and total plasma nitrite levels were increased in the LPS group. In the LPS+L-arginine group, both the phagocytic activity and total plasma nitrite levels showed large increases. Administration of aminoguanidine (AG), a specific iNOS inhibitor, abolished macrophage phagocytic activity and total plasma nitrite levels in the LPS and LPS+L-arginine groups. As a result, we showed that NO produced by macrophages has a role not only in intracellular killing, but also in phagocytic activity.     

Reduction of radiation-induced nitrative stress in leucocytes and kidney cells of rats upon administration of polyphenolic complex concentrates from red wine

The research has shown that exposure to ionizing radiation at the dose of 30 cGy leads to the activation of NO-synthase way of nitrogen oxide synthesis, as well as to the accumulation of its stable metabolites and 3'-nitrotyrosine modified proteins in rat peripheral blood leucocytes and the renal cortical layer. NO-synthase activity was preserved at the control value through the consumption of red wine naturalpolyphenolic complex concentrates by the irradiated animals. The content of proteins modified by tyrosine nitration decreased in the early period of post-radiation exposure due to the influence of the investigated concentrate. Thus the ability of red wine natural polyphenolic complex concentrates to prevent adverse changes in L-arginine/NO system and, therefore, inhibit the development of nitrative stress induced by low doses of ionizing radiation has been proved experimentally.     

Macrophage cytotoxicity against Entamoeba histolytica trophozoites is mediated by nitric oxide from L-arginine.

The killing of Entamoeba histolytica trophozoites by phagocytes involves oxidative and nonoxidative mediators. In this study, we determine whether L-arginine-derived nitric oxide (NO) is involved in the killing of E. histolytica trophozoites by activated murine macrophages in vitro. Elicited peritoneal and bone marrow-derived macrophages activated with IFN-gamma alone or with IFN-gamma and LPS killed 62 to 73% of amebae, concomitant with increased levels of nitrate (NO2). Depletion of L-arginine by addition of arginase to culture medium abrogated macrophage amebicidal activity. NG-monomethyl L-arginine, an L-arginine analog, competitively inhibited NO2 release and amebicidal activity in a dose-dependent fashion, without affecting H2O2 production; however, the addition of excess L-arginine competitively restored macrophage amebicidal effects. In culture, sodium nitrite and sodium nitroprusside were cytotoxic to E. histolytica and this was reversed by the addition of myoglobin. Exogenously added FeSO4 prevented macrophage cytotoxicity. Addition of superoxide dismutase, a scavenger of O2-, partially inhibited amebicidal activity, without influencing NO2 production. Untreated and LPS-exposed macrophages produced high levels of H2O2 independent from NO2 production and amebicidal effects. However, the addition of catalase, a scavenger of H2O2, inhibited both amebicidal activity and NO2 production by activated macrophages. Our results demonstrate that NO is the major cytotoxic molecule released by activated macrophages for the in vitro cytotoxicity of E. histolytica and that O2- and H2O2 may be cofactors for the NO effector molecule.     

Involvement of nitric oxide-induced NADPH oxidase in adventitious root growth and antioxidant defense in Panax ginseng

Nitric oxide (NO) affects the growth and development of plants and also affects plant responses to various stresses. Because NO induces root differentiation, we examined whether or not it is involved in increased ROS generation. Treatments with sodium nitroprusside (SNP), an NO donor, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a specific NO scavenger, and Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), an NO synthase (NOS) inhibitor, revealed that NO is involved in the adventitious root growth of mountain ginseng. Supply of an NO donor, SNP, activates NADPH oxidase activity, resulting in increased generation of O₂ ^·−, which subsequently induces growth of adventitious roots. Moreover, treatment with diphenyliodonium chloride (DPI), an NADPH oxidase inhibitor, individually or with SNP, inhibited root growth, NADPH oxidase activity, and O₂ ^·− anion generation. Supply of the NO donor, SNP, did not induce any notable isoforms of enzymes; it did, however, increase the activity of pre-existing bands of NADPH oxidase, superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, and glutathione reductase. Enhanced activity of antioxidant enzymes induced by SNP supply seems to be responsible for a low level of H₂O₂ in the adventitious roots of mountain ginseng. It was therefore concluded that NO-induced generation of O₂ ^·− by NADPH oxidase seems to have a role in adventitious root growth of mountain ginseng. The possible mechanism of NO involvement in O₂ ^·− generation through NADPH oxidase and subsequent root growth is discussed.     

Regulation of the rate of synthesis of nitric oxide by Mg(2+) and hypoxia. Studies in rat heart mitochondria

Summary.  In isolated rat heart mitochondria, L-arginine is oxidized by a nitric oxide synthase (mtNOS) achieving maximal rates at 1 mM L-arginine. The NOS inhibitor N^G-nitro-L-arginine methyl ester (NAME) inhibits the increase in NO production. Extramitochondrial free magnesium inhibited NOS production by 59% at 3.2 mM. The mitochondrial free Mg²⁺ concentration increased to different extents in the presence of L-arginine (29%), the NO donor (S-nitroso-N-acetylpenicillamine) (105%) or the NOS inhibitors L-NAME (48%) or N^G-nitro-L-arginine methyl ester, N^G-monomethyl-L-arginine (L-NMMA) (53%). Under hypoxic conditions, mtNOS activity was inhibited by Mg²⁺ by up to 50% after 30 min of incubation. Reoxygenation restored the activity of the mtNOS to pre-hypoxia levels. The results suggest that in heart mitochondria there is an interaction between Mg²⁺ levels and mtNOS activity which in turn is modified by hypoxia and reoxygenation. Received April 2, 2001 Accepted September 21, 2001     

Gastroprotection by pentoxyfilline against stress-induced gastric damage. Role of lipid peroxidation, antioxidizing enzymes and proinflammatory cytokines.

Impairment of blood perfusion in gastric mucosa results in the formation of erosions and ulcers. Nitric oxide (NO), produced via activity of NO-synthase (NOS), appears to be a one of major factors, involved in the regulation of the gastric blood flow (GBF). Inhibition of this enzyme by N-nitro-L-arginine (L-NNA) results in local decrease of NO production, reduces GBF and impairs gastric mucosal integrity, the effects that can be reversed by the pretreatment with L-arginine, the NOS substrate. However, little information is available regarding the contribution of reactive oxygen species (ROS)-induced lipid peroxidation and NO to the mechanism of gastric mucosal integrity. Therefore, the aim of our present study was to determine the action of pentoxyfilline (PTX), an inhibitor of tumor necrosis factor alpha (TNFalpha) with or without NOS inhibition by L-NNA administration in rats with water immersion and restraint stress (WRS)-induced gastric lesions. Experiments were carried out on 100 male Wistar rats. The gastric blood flow (GBF) was measured using laser Doppler flowmeter. The area of gastric lesions was determined by planimetry and the levels of proinflammatory cytokines (IL-1beta and TNFalpha) were measured by ELISA. Colorimetric assays were employed to determine gastric mucosal levels of lipid peroxidation products, such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) and antioxidant enzymes including superoxide dismutase (SOD) activity, as well as tissue concentration of reduced glutathione (GSH). Administration of PTX significantly attenuated the gastric lesions, induced by 3.5 h of WRS and this was accompanied by the rise in the GBF and a significant decrease in plasma proinflammatory cytokines (IL-1beta and TNFalpha) levels, as well as the reduction of lipid peroxidation. Exposure of rats to WRS suppressed the SOD and GSH activities and these effects were reversed by PTX. The protective and hyperemic effects of PTX, as well as an increase in mucosal SOD activity and GSH concentration were counteracted by pretreatment with L-NNA, but restored by the pretreatment with L-arginine, a NOS substrate. We conclude that PTX exerts beneficial, gastroprotective effect against WRS-induced gastric lesions due to enhancement in gastric microcirculation, possibly mediated by the enhanced NOS activity as well as local action of NO and by the attenuation of oxidative metabolism and generation proinflammatory cytokines.     

Generation of reactive oxygen species by polymorphonuclear leukocytes and its modulation by calcium ions during tumor growth

The generation of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNL) and the role of Ca2+ in regulating their activity during Zajdela hepatoma growth in the animal peritoneal cavity were studied. We found a marked increase in the ROS-generating activity of PMNL in circulating blood, the result of increases in both the specific activity of leukocytes and total number of PMNL in circulating blood. The ROS-generating activity of PMNL was substantially activated by Ca2+ ions and a calcium ionophore (ionomycin), but this effect virtually completely disappeared during tumor growth. Perhaps the high ROS-generating activity of PMNL and the lack of the sensitivity to extracellular Ca2+ during tumor growth in the organism are due to an accumulation of intracellular Ca2+ ions.     

5,6-Dihydroxyindole-2-carboxylic acid,a diffusible melanin precursor,is a potent stimulator of lipopolysaccharide-induced production of nitric oxide by J774 macrophages

Pre-incubation of J774 murine macrophages with 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a diffusible intermediate in the biosynthesis of eumelanins, leads to a marked increase in the levels of nitric oxide (NO) produced by lipopolysaccharide (LPS)-induced NO-synthase (iNOS). The effect varies with DHICA concentratior being maximum at a concentration of 1 × 10⁻⁶M, and is suppressed by the NOS inhibitor N^G-monomethyl-L-arginine (L-NMMA). No stimulation is observed when macrophages are exposed to DHICA after activation with LPS, indicating that the indole does not affect the catalytic activity of iNOS. These results point to a hitherto unrecognized role of DHICA as a chemical messenger mediating interaction between active melanocytes and macrophages in epidermal inflammatory and immune responses.     

Generation of EPR-detectable nitrosyl-iron complexes in tumor target cells cocultured with activated macrophages.

After immunostimulation, murine macrophages oxidize L-arginine into nitric oxide (NO) which acts as an effector molecule. In this study, we attempted to establish whether activated macrophage-derived NO forms paramagnetic complexes in tumor target cells which do not express by themselves the L-arginine:NO pathway. Accordingly, murine L1210 leukemia cells were cocultivated with activated peritoneal macrophages from Bacillus-Calmette-Guérin-infected mice, or activated in vitro with interferon-gamma. In control experiments, macrophages were prevented from producing nitrogen oxides by incubation with NG-monomethyl-L-arginine, a specific inhibitor of the L-arginine:NO pathway. After coculture, L1210 cells were removed from adherent macrophage monolayers and analyzed by electron paramagnetic resonance at 77 K. In the L1210 cells cultured with activated macrophages, we detected a signal typical of nitrosyl-iron-sulfur complexes, with g values of 2.041 and 2.015. This signal was not present when L1210 cells were either cultured alone or cocultured with activated macrophages in the presence of NG-monomethyl-L-arginine. Mitochondria from activated macrophage-injured L1210 cells also exhibited the signal with g values of 2.041 and 2.015. These results show that when tumor target cells undergo cell-to-cell contact with activated macrophages during culture, the macrophages promote target cell nitrosylation in compartments like mitochondria.     

Effects of Nitrate and L-Arginine on Content of Nitric Oxide and Activities of Antioxidant Enzymes in Roots of Wheat Seedlings and Their Heat Resistance

Separate and combined effects of nitrate (NaNO₃) and L-arginine as potential sources of nitric oxide (NO) on the content of endogenous NO in roots of wheat (Triticum aestivum L.) seedlings and on their heat resistance were studied. Both agents increased the seedling resistance to the damaging heating; the effect was maximal at 20 mM NaNO₃ or 5 mM L-arginine. The treatment with L-arginine elevated the NO content in the roots within the first 2 h of the treatment. Nitrate caused a stronger and longer rise in nitric oxide. Activity of nitrate reductase considerably (2–3 times) increased in the roots exposed to nitrate. The augmentation in the nitric oxide level caused by nitrate or L-arginine was prevented by the root pretreatment with an inhibitor of nitrate reductase (sodium tungstate) or an inhibitor of animal NO-synthase—N^G-nitro-L-arginine methyl ester (L-NAME). Upon the combined treatment with NaNO₃ and L-arginine, the nitrateinduced stimulation of the nitrate reductase activity, NO level in the roots, and seedling heat resistance were less pronounced than after separate application. In the presence of L-NAME, the negative influence of L-arginine on nitrate effects was markedly attenuated. The plant exposure to nitrate or L-arginine increased the activities of antioxidant enzymes (superoxide dismutase, catalase, and guaiacol peroxidase). A mixture of NaNO₃, and L-arginine caused weaker effects. It was suggested that nitrate-dependent and arginine-dependent pathways of NO formation are antagonistic to each other in wheat roots.     

Effect of sodium nitroprusside on heat resistance of wheat coleoptiles: Dependence on the formation and scavenging of reactive oxygen species

The effect of nitric oxide donor sodium nitroprusside (SNP) on resistance of coleoptiles of 4-day-old etiolated seedlings of wheat (Triticum aestivum L., cv. Elegiya) to damaging heating (10 min at 43°C) and possible dependence of this effect on changes in the activities of enzymes producing and scavenging reactive oxygen species (ROS) were studied. Treatment of coleoptiles with 500 μM SNP considerably boosted generation of superoxide anion radical therein. This effect was substantially suppressed by blocker of calcium channels (lanthanum chloride), calmodulin antagonist (chlorpromazine), and inhibitor of NADPH-oxidase (imidazole) but not by peroxidase inhibitor (salicylhydroxamic acid). NO donor activated antioxidant enzymes (superoxide dismutase, catalase, and soluble peroxidase) and elevated heat resistance of wheat coleoptiles. NO scavenger methylene blue, antioxidant agent ionol, calcium antagonists, and NADPH-oxidase inhibitor imidazole substantially reduced the elevation of heat resistance of wheat coleoptiles induced by NO donor. It was concluded that SNP-induced heat resistance of coleoptiles depended on calcium and ROS, whose production is probably boosted by activation of NADPH-oxidase.     

Interaction between reactive oxygen species and nitric oxide in the microvascular response to systemic hypoxia.

Systemic hypoxia results in oxidative stress due to a change in the reactive oxygen species (ROS)-nitric oxide (NO) balance. These experiments explored two mechanisms for the altered ROS-NO balance: 1) decreased NO synthesis by NO synthase due to limited O(2) substrate availability and 2) increased superoxide generation. ROS levels and leukocyte adherence in mesenteric venules of rats during hypoxia were studied in the absence and presence of an NO donor [spermine NONOate (SNO)] and of the NO precursor L-arginine. We hypothesized that if the lower NO levels during hypoxia were due to O(2) substrate limitation, L-arginine would not prevent hypoxia-induced microvascular responses. Graded hypoxia (produced by breathing 15, 10, and 7.5% O(2)) increased both ROS (123 +/- 6, 148 +/- 11, and 167 +/- 3% of control) and leukocyte adherence. ROS levels during breathing of 10 and 7.5% O(2) were significantly attenuated by SNO (105 +/- 6 and 108 +/- 3%, respectively) and L-arginine (117 +/- 5 and 115 +/- 2%, respectively). Both interventions reduced leukocyte adherence by similar degrees. The fact that the effects of L-arginine were similar to those of SNO does not support the idea that NO generation is impaired in hypoxia and suggests that tissue NO levels are depleted by the increased ROS during hypoxia.     

Arginase activity in frog urinary bladder epithelial cells and its involvement in regulation of nitric oxide production

The activity of arginase converting arginine into ornithine and urea is of particular interest among many factors regulating NO production in the cells. It is known that by competing with NO-synthase for common substrate (arginine), arginase can affect NO synthesis. In the present work, properties of arginase from the common frog Rana temporaria L. urinary bladder epithelial cells containing the NO-synthase were characterized, and possible contribution of arginase to regulation of NO production by epithelial cells was studied. It has been shown that the enzyme has temperature optimum in the range of 55–60°C, K _M for arginine 23 mM, and V _max about 10 nmole urea/mg of protein/min, and its activity was efficiently inhibited by (S)-(2-boronoethyl)-L-cysteine (BEC), an inhibitor of arginase, at concentrations from 10^?6 to 10^?4 M. The comparison of arginase activity in various frog tissues revealed the following pattern: liver > kidney > brain > urinary bladder (epithelium) > heart > testis. The arginase activity in isolated urinary bladder epithelial cells was 3 times higher that in the intact urinary bladder wall. To evaluate the role of arginase in regulation of NO production, the epithelial cells were cultivated in the media L-15 or 199 containing different amounts of arginine; the concentration of NO₂ ^?, the stable NO metabolites, was de-termined in the cultural fluid after 18–20 h of cell incubation. The vast majority of the produced nitrites are associated with NOS activity, as L-NAME, the NO inhibitor, decreased their accumulation by 77.1% in the L-15 medium and by 80% in the 199 medium. BEC (10^?4 M) increased nitrite production by 18.0% ± 2.7% in the L-15 medium and by 24.4% ± 3.5% in the 199 medium (p < 0.05). The obtained data indicate a relatively high activity of arginase in the frog urinary bladder epithelium and its involvement in regulation of NO production.     

A dual mechanism for impairment of GABAA receptor activity by NMDA receptor activation in rat cerebellum granule cells

The function of the GABA_A receptor has been studied using the whole cell voltage clamp recording technique in rat cerebellum granule cells in culture. Activation of NMDA-type glutamate receptors causes a reduction in the effect of GABA. Full GABA_A receptor activity was recovered after washing out NMDA and NMDA action was prevented in a Mg⁺⁺ containing medium. The NMDA effect was also absent when extracellular Ca⁺⁺ was replaced by Ba⁺⁺ and when 10 mM Bapta was present in the intracellular solution. Charge accumulations via voltage activated Ca⁺⁺ channels greater than the ones via NMDA receptors do not cause any reduction in GABA_A receptor function, suggesting that Ca⁺⁺ influx through NMDA receptor channels is critical for the effect. The NMDA effect was reduced by including adenosine-5′-O-3-thiophosphate (ATP-γ-S) in the internal solution and there was a reduction in the NMDA effect caused by deltamethrin, a calcineurin inhibitor. Part of the NMDA induced GABA_A receptor impairment was prevented by prior treatment with L-arginine. Analogously, part of the NMDA effect was prevented by blockage of NO-synthase activity by N _ω -nitro-L-arginine. A combination of NO-synthase and calcineurin inhibitors completely eliminated the NMDA action. An analogous result was obtained by combining the NO-synthase inhibitor with the addition of ATP-γ-S to the pipette medium. The additivity of the prevention of the NMDA impairment of GABA_A receptor by blocking the L-arginine/NO pathway and inhibiting calcineurin activity suggests an independent involvement of these two pathways in the interaction between NMDA and the GABA_A receptor. On the one hand Ca⁺⁺ influx across NMDA channels activates calcineurin and dephosphorylates the GABA_A receptor complex directly or dephosphorylates proteins critical for the function of the receptor. On the other hand, Ca⁺⁺ influx activates NO-synthase and induces nitric oxide production, which regulates such receptors via protein kinase G activity. Received: 22 July 1996 / Accepted: 29 October 1996     

An inhibitor of macrophage arginine transport and nitric oxide production (CNI-1493) prevents acute inflammation and endotoxin lethality.

BACKGROUND: Nitric oxide (NO), a small effector molecule produced enzymatically from L-arginine by nitric oxide synthase (NOS), is a mediator not only of important homeostatic mechanisms (e.g., blood vessel tone and tissue perfusion), but also of key aspects of local and systemic inflammatory responses. Previous efforts to develop inhibitors of NOS to protect against NO-mediated tissue damage in endotoxin shock have been unsuccessful, largely because such competitive NOS antagonists interfere with critical vasoregulatory NO production in blood vessels and decrease survival in endotoxemic animals. Accordingly, we sought to develop a pharmaceutical approach to selectively inhibit NO production in macrophages while sparing NO responses in blood vessels. MATERIALS AND METHODS: The process of cytokine-inducible L-arginine transport and NO production were studied in the murine macrophage-like cell line (RAW 264.7). A series of multivalent guanylhydrazones were synthesized to inhibit cytokine-inducible L-arginine transport. One such compound (CNI-1493) was studied further in animal models of endothelial-derived relaxing factor (EDRF) activity, carrageenan inflammation, and lethal lipopolysaccharide (LPS) challenge. RESULTS: Upon activation with cytokines, macrophages increase transport of L-arginine to support the production of NO by NOS. Since endothelial cells do not require this additional arginine transport to produce NO, we reasoned that a competitive inhibitor of cytokine-inducible L-arginine transport would not inhibit EDRF activity in blood vessels, and thus might be effectively employed against endotoxic shock. CNI-1493, a tetravalent guanylhydrazone, proved to be a selective inhibitor of cytokine-inducible arginine transport and NO production, but did not inhibit EDRF activity. In mice, CNI-1493 prevented the development of carrageenan-induced footpad inflammation, and conferred protection against lethal LPS challenge. CONCLUSIONS: A selective inhibitor of cytokine-inducible L-arginine transport that does not inhibit vascular EDRF responses is effective against endotoxin lethality and significantly reduces inflammatory responses.     

Metabolic Activity of Macrophages Infected with Hantavirus,an Agent of Hemorrhagic Fever with Renal Syndrome

Monocytes/macrophages are thought to play an important role in pathogenesis of viral infections. These cells are involved in distribution and persistence of viruses in the organism and also influence the regulation of immune reactions. The functional and enzymatic activities of macrophages infected with an agent of hemorrhagic fever with renal syndrome were analyzed for the first time. This disease is caused by a virus of the Hantavirus genus, the Bunyaviridae family. Activities of ectoenzymes 5 -nucleotidase and ATPase of the plasma membrane of the hantavirus-infected macrophages decreased along with the antigen accumulation in the infected cells. The contact of phagocytes with hantavirus resulted in activation in the cells of the oxygen-dependent metabolism and NO-synthase. The NO-synthase-dependent system of the infected macrophages was activated earlier than their oxygen-dependent system. The intracellular contents of acid and alkaline phosphatases increased within the first hours after the infection. The bactericidal activity of the hantavirus-infected macrophages relatively to Staphylococcus aureus increased during the specific antigen accumulation in the phagocytes. Thus, the infection of macrophages with hantavirus was associated with intracellular metabolic changes.     

Hemodynamic responses induced by modulation of the nitric oxide system and mitochondrial permeability in the medullary cardiovascular nuclei of rats

In acute experiments on normotensive rats and those with genetically determined hypertension (urethane anesthesia), we studied hemodynamic effects resulting from modulation of the activities of neuronal NO synthase (NOS-1), arginase II, and superoxide dismutase, and also of the mitochondrial permeability in medullary cardiovascular neurons. Unilateral microinjections of either a nitric oxide (NO) donor, sodium nitroprusside, or a substrate for endogenous NO synthesis, L-arginine, into the medullary cardiovascular nuclei (nucl. tractus solitarius, NTS, nucl. ambiguous, AMB, paramedian nucleus, PMn, and lateral reticular nucleus LRN) were shown to induce hemodynamic responses with rather similar dynamics in both normotensive and spontaneously hypertensive rats, although in the latter the reactions were more intense. Injections of an antagonist of NOS-1, N^G nitro-L-arginine (L-NNA), into the medullary nuclei under study in spontaneously hypertensive rats resulted in shifts of the systemic arterial pressure (SAP), which did not differ dramatically from those observed in normotensive animals. The data obtained serve as the background for the suggestion that the functional activity of NOS-1 is not fundamentally impaired under hypertension conditions, but, probably, the amount of the substrate for adequate synthesis of NO via the NO-synthase pathway of metabolism of L-arginine is insufficient. Considering this, we examined the functional activity of arginase, an enzyme that also, similarly to NOS, uses L-arginine for metabolic transformation. Injections of antagonists of arginase, norvaline or α-difluoromethylornithine hydrochloride (DFMO), into populations of the medullary neurons under study induced similar shifts of the SAP in normotensive and spontaneously hypertensive rats, and those responses did not differ significantly from the effects of inhibition of the NOS-1 activity. Thus, both the above-mentioned enzymes are potentially active in normotensive and spontaneously hypertensive rats; so, a possibility for their competition for L-arginine in certain situations does exist. Modulation of the mitochondrial permeability in medullary cardiovascular neurons in normotensive and spontaneously hypertensive rats induced significant hemodynamic effects. In particular, an increase in the mitochondrial permeability in the medullary cardiovascular nuclei by injections of an inductor of mitochondrial permeability transition pore (mPTP) opening, phenylarsine oxide (PAO), was accompanied by SAP drops in both normotensive and spontaneously hypertensive rats; the effects were dose-dependent and, in some cases, irreversible. A decrease in the mitochondrial permeability in the neurons under study by injections of an inhibitor of mPTP, melatonin, induced mostly hypertensive responses, although in some experiments we observed hypotensive and two-phase responses. Neirofiziologiya/Neurophysiology, Vol. 39, No. 3, pp. 232–244, May–June, 2007.