Increased Nitrite Production Research Articles

The inhibition by dantrolene of L-arginine transport and nitric oxide synthase in rat alveolar macrophages.

Dantrolene decreases the free cytosolic Ca2+ level via inhibition of calcium release from the sacroplasmic reticulum. However, the effect of dantrolene on L-arginine transport and nitric oxide synthase (NOS) activity is still unknown. In this study, we examined the effects of dantrolene on L-arginine transport and activity of inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) in rat alveolar macrophages. Incubation of cells with LPS (1 microg/mL) and IFN-gamma (100 u/mL) for 24 h resulted in significant increases in nitrite production and L-arginine transport. In the presence of dantrolene (100 microM) or inhibitors of NOS, such as aminoguanidine (100 microM), N(G)-monomethyl-L-arginine (100 microM), the nitrite production and L-arginine transport were significantly inhibited compared with that in the LPS + IFN-gamma group. Furthermore, the results of kinetic analysis indicate that the suppression of L-arginine transport by dantrolene was caused by selective decrease of the velocity of transport (Vmax) without affecting the affinity (Km) for L-arginine. In addition, dantrolene also attenuated the activity of iNOS in a dose-dependent manner. We conclude that the mechanisms by which dantrolene attenuated NO synthesis may be associated with the inhibition of availability of L-arginine by reducing the affinity for L-arginine, accompanied by a parallel decrease of the activity of iNOS. In this study, we demonstrated that dantrolene, a drug that reduces the intracellular Ca2+ level, can inhibit L-arginine availability and inducible nitric oxide synthase activity in macrophages. Our finding may provide a novel therapeutic approach using dantrolene to prevent hypotension associated with an activation of inducible nitric oxide synthase in endotoxemia.

Neutral endopeptidase and angiotensin-converting enzyme inhibitors increase nitric oxide production in isolated canine coronary microvessels by a kinin-dependent mechanism.

Bradykinin is a substrate for both neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE). Our previous studies showed that ACE inhibitors can stimulate nitric oxide production in coronary microvessels, which is mediated by local kinins. Whether inhibition of NEP also can affect local vascular NO production has not been established. To determine the role of NEP in the control of NO production, coronary microvessels were isolated from seven mongrel dogs. Two NEP inhibitors, phosphoramidon and thiorphan, and an ACE inhibitor, ramiprilat, were used. Nitrite, the metabolite of NO in aqueous solution, was measured by using the Griess reaction. Phosphoramidon and thiorphan (10(-6) M) increased nitrite production from 80 +/- 6 to 136 +/- 6 and 144 +/- 7 pmol/mg, respectively. Ramiprilat (10(-8) M) increased nitrite production from 78 +/- 6 to 155 +/- 7 pmol/mg wet weight. The effect of these agents on nitrite release was blocked by L-NAME, which inhibits NO synthase, HOE-140, which blocks bradykinin B2-receptor, and dichloroisocoumarin, which blocks kinin-forming enzymes. These results clearly indicate that inhibition of kinin metabolism by using neutral endopeptidase inhibitors increases NO production from coronary microvessels. Thus neutral endopeptidase plays an important role in local kinin-modulated NO production in the coronary microcirculation and NEP inhibitors may be useful clinical tools in treatment of cardiovascular disease.

Effects of vesnarinone on nitric oxide synthesis in rat cardiac myocytes.

The purpose of this study was to investigate the effects of vesnarinone on nitric oxide (NO) synthesis in cardiac myocytes. We measured the accumulation of nitrite, a stable oxidation product of NO synthase (iNOS) protein in cultured neonatal rat cardiac myocytes. Incubation of the cultures with interleukin-1 beta (IL-1 beta; 10 ng/ml) and tumor necrosis factor alpha (TNF alpha; 10 ng/ml) caused a marked increase in nitrite production. Although vesnarinone by itself showed no effect on nitrite accumulation, it enhanced cytokine-induced nitrite production by cardiac myocytes in a dose-dependent manner. The effect of vesnarinone was completely abolished in the presence of NG-monomethyl-L-arginine or actinomycin D. The vesnarinone-induced nitrite production was accompanied by increased iNOS protein expression. In the presence of dibutyryl-cAMP, cytokine-induced nitrite accumulation was further increased, but the stimulatory effect on vesnarinone on nitrite accumulation was diminished. The effect of vesnarinone was also inhibited by Rp-8-Br-cAMPS, a competitive inhibitor of protein kinase A, in a dose-dependent manner. These findings indicate that vesnarinone increases NO synthesis in cytokine-stimulated cardiac myocytes, at least partially through a cAMP-dependent pathway.

Monocyte-vascular smooth muscle cell interaction enhances nitric oxide production.

The adhesive interaction of monocytes and vascular smooth muscle cells (VSMCs) has been suggested to be a regulatory signal in the cellular activation that is involved in the pathogenesis of atherosclerosis. We investigated the effects of monocyte-VSMC interaction on inducible nitric oxide (NO) synthase expression. NO production by the cultured cells was determined by measuring the nitrite content of the culture media using the Griess reagent. The expression of inducible NO synthase protein was assayed by Western blotting. Interleukin-1 beta (IL-1 beta) induced nitrite production by VSMCs in a time-dependent manner. The addition of the mouse monocyte cell line J774 to IL-1 beta-stimulated VSMCs further increased nitrite production in a monocyte number-dependent manner. Enhanced nitrite production by coculture was accompanied by increased inducible NO synthase protein accumulation. Addition of tumor necrosis factor-alpha (TNF-alpha) also enhanced IL-1 beta-induced nitrite production by VSMCs, but TNF-alpha showed no effect in the presence of monocytes. Coculture of monocytes and VSMCs in the presence of IL-1 beta secreted substantial amounts of TNF-alpha. The production of nitrite by coculture was markedly inhibited by an anti-TNF-alpha antibody. The present study revealed that direct cell-to-cell interaction between monocytes and VSMCs enhances NO production, suggesting an important role for their interaction in the pathogenesis of atherosclerosis.

Role of endothelial kinins in control of coronary nitric oxide production.

The purpose of the present study was to determine whether interventions that promote kinin production or decrease kinin inactivation affect nitric oxide production in isolated canine coronary microvessels. Accordingly, bradykinin (10[-8] to 10[-5] mol/L), ramiprilat (10[-10] to 10[-8] mol/L), A23187 (10[-8] to 10[-6] mol/L), kallikrein (1 to 20 U/mL), and kininogen (0.5 to 10 microg/mL) were used to stimulate endothelium-dependent nitric oxide production. Receptor antagonists, serine protease inhibitors, and a kinin antibody were used to inactivate local kallikrein-kinin activity. Nitrite, the metabolite of nitric oxide in aqueous solution, was measured using the Griess reaction. All the agonists significantly increased nitrite release. For instance, the highest dose of bradykinin, ramiprilat, A23187, kallikrein, and kininogen markedly increased nitrite production, from 60+/-10 to 156+/-12, 153+/-11, 161+/-15, 176+/-15, and 168+/-16 pmol/mg (all P<.05), respectively. The increased nitrite production caused by these agents was not only blocked by N omega-nitro-L-arginine methyl ester (L-NAME) and HOE 140 (which blocks B2 kinin receptor) but by the kinin antibody also. For instance, nitrite production elicited by bradykinin, ramiprilat, A23187, and kininogen was reduced to 95+/-8, 87+/-8, 94+/-11, and 85+/-11 pmol/mg (all P<.05), respectively, by the kinin antibody. Carbachol-induced nitrite production (from 66+/-8 to 144+/-13) was blocked by L-NAME but not by HOE 140 or the kinin antibody. These results suggest that either increasing kininogen to promote endogenous kinin formation or inhibiting angiotensin-converting enzyme to decrease kinin breakdown, increases nitric oxide production in isolated coronary microvessels. These data indicate that a microvessel kallikrein-kinin system has an important role in the control of nitric oxide production in coronary microvessels.

Nitric oxide synthase activity and mRNA expression in chicken macrophages

The activity of inducible nitric oxide synthase (iNOS) enzyme was quantified in chicken macrophages. Macrophages from Cornell K-strain (B15B15), GB1 (B13B13), and GB2 (B6B6) chickens and a transformed cell line (MQ-NCSU) were incubated with or without varying concentrations of bacterial lipopolysaccharide (LPS). The culture supernatants were tested for the presence of nitrite. Macrophages from either source produced minimal nitrite (< 4.4 microM/1 x 10(6) cells) levels without LPS stimulation. However, nitrite levels produced by K-strain (42 microM) and MQ-NCSU (41 microM) macrophages were higher (P < 0.05) than those produced by the GB1 (14 microM) and GB2 (14 microM) per 1 x 10(6) macrophages with optimum LPS concentration range of 50 ng to 1 microgram/mL. The addition of an L-arginine analog, NGMMLA, at a concentration of 200 microM completely abolished nitrite production. The addition of 10% vol/vol lymphokines exhibited an additive effect on nitrite production in conjunction with LPS. The increased nitrite production by the K-strain and MQ-NCSU macrophages corresponded to an increased expression of iNOS mRNA as compared to the mRNA produced by GB1 and GB2 macrophages. The iNOS mRNA kinetics study revealed that mRNA levels peaked between 6 to 12 h. The cells from avian lymphoid lineage failed to produce any detectable iNOS activity. These studies showed that macrophages from varying sources differ in NOS activity and implied that genetic background may dictate the extent of arginine-mediated contribution in various biological and immunological functions.

Serotonin inhibits nitric oxide synthesis in rat vascular smooth muscle cells stimulated with interleukin-1

We investigated the effects of serotonin (5-hydroxytryptamine; 5-HT) on nitric oxide (NO) synthesis in vascular smooth muscle cells. We measured the production of nitrite, a stable metabolite of NO, and the expression of inducible NO synthase protein in cultured rat vascular smooth muscle cells. Incubation of the cultures with interleukin-1 β (10 ng/ml) caused a significant increase in nitrite production. 5-HT inhibited nitrite production by interleukin-1 β-stimulated vascular smooth muscle cells in a concentration-dependent manner (10 −8–10 −5 M). 5-HT-induced inhibition of nitrite production was accompanied by decreased inducible NO synthase protein accumulation in vascular smooth muscle cells. Addition of the 5-HT 2 receptor antagonist ketanserin, but not the 5-HT 1A receptor antagonist spiroxatrine, inhibited the effect of 5-HT. On the other hand, the 5-HT 2 receptor agonist α-methyl-5-HT, but not the 5-HT 1A receptor agonist (±)-8-hydroxy-2-(di- n-propylamino) tetralin, decreased interleukin-1 β-induced nitrite production by vascular smooth muscle cells. 5-HT significantly increased protein kinase C activity in vascular smooth muscle cells, and the protein kinase C inhibitor calphostin C dose-dependently abolished the effect of 5-HT on nitrite production. After protein kinase C activity was functionally depleted by treatment of cells with phorbol 12-myristate 13-acetate for 24 h, the effect of 5-HT was abolished. These results indicate that 5-HT acts on 5-HT 2 receptors and inhibits NO synthesis in interleukin-1 β-stimulated vascular smooth muscle cells at least partially through a protein kinase C-dependent pathway.

Nitric oxide mediates down regulation of lipoprotein lipase activity induced by tumor necrosis factor- α in brown adipocytes

We previously reported that tumor necrosis factor- α (TNF- α)/cachectin suppresses lipoprotein lipase activity and its gene expression in brown adipocytes differentiated in culture. Recent evidence suggests that the effect of TNF- α over various cells is related to the enhanced production of nitric oxide (NO). The present study examined whether the suppressive effect of TNF- α on lipoprotein lipase activity is mediated by production of NO in the brown adipocytes. A reverse transcription-polymerase chain reaction (RT-PCR) assay revealed that TNF- α caused a concentration- and time-dependent expression of inducible NO synthase in brown adipocytes. Increasing concentrations of TNF- α (0.5–50 ng/ml) for 24 h resulted in a concentration-dependent decrease in lipoprotein lipase activity with reciprocal increase in nitrite production in the medium. The suppressive effect of TNF- α on lipoprotein lipase activity was significantly prevented by NO synthase inhibitors, N G-nitro- l-arginine methyl ester ( l-NAME) and aminoguanidine, but not by d-NAME, an inactive isomer. Furthermore, 8-bromoguanosine 3′,5′-cyclic monophosphate, cell permeant cGMP, suppressed lipoprotein lipase activity and 1 H-[1,2,4] oxadiazolo[4,3- a]quinoxalin-1-one, a selective inhibitor for soluble guanylate cyclase, restored the TNF- α-suppressed lipoprotein lipase activity. These results suggest that TNF- α stimulates brown adipocytes to express inducible NO synthase, followed by production of NO, which in turn mediates the suppressive effect of TNF- α on lipoprotein lipase activity. The effect of NO is mediated, at least partly, through production of cGMP.

ANG II stimulates endothelial nitric oxide synthase expression in bovine pulmonary artery endothelium.

Although angiotensin II (ANG II) is a known pulmonary vasoconstrictor, the purpose of this study was to examine the effect of ANG II on pulmonary artery endothelial cell nitric oxide synthase (ecNOS) mRNA and protein expression. Cultured bovine pulmonary artery endothelial (BPAE; passages 5-8) cells were incubated for 0-12 h with 10(-6) M ANG II. Total RNA was extracted, and ecNOS expression was assessed by Northern blot analysis. In BPAE cells, ecNOS mRNA was significantly increased 2.4 +/- 0.3-fold (P < 0.05 vs. basal; n = 5) 6 h after the addition of ANG II over basal levels. In & similar time course, it was found that ecNOS protein concentrations are increased 247 +/- 62% (P < 0.05 vs. basal; n = 8) over basal levels 4 h after ANG II addition. There is a second protein peak 8 h after ANG II addition in which ecNOS was increased 333 +/- 145% over basal (P < 0.05, n = 3). These data suggest that ANG II stimulates ecNOS mRNA expression and are followed by increased levels of ecNOS protein in cultured BPAE cells, consistent with an observed increase in nitrite production. Both the increase in ecNOS protein and mRNA expression could be inhibited with the ANG II receptor antagonist saralasin. Additionally, actinomycin D, an inhibitor of transcription, prevented the rise in mRNA at 6 h while cycloheximide inhibited the initial protein peak. The effects of ANG II on ecNOS were specific for the pulmonary artery endothelium. Addition of ANG II did not increase ecNOS protein or mRNA expression in parallel studies in bovine coronary artery endothelium. The stimulation of ecNOS by ANG II may act to protect the lung and maintain low pulmonary artery pressures in the renin-angiotensin model of systemic hypertension.

Adenosine stimulates nitric oxide synthesis in vascular smooth muscle cells.

The aim was to investigate the effects of adenosine on nitric oxide (NO) synthesis in vascular smooth muscle cells. NO and cAMP synthesis was measured in confluent rat vascular smooth muscle cells in culture at passage 5-10, using Griess reagent and an enzyme immunoassay kit, respectively. The expression of inducible NO synthase mRNA was assayed by Northern blotting. Incubation of cultures with interleukin-1 beta (10 ng/ml) for 24 h caused a significant increase in nitrite production. The interleukin-1 beta-induced nitrite production by vascular smooth muscle cells was significantly increased by adenosine or its stable analogue, 2-chloroadenosine, in a dose-dependent manner. The adenosine A2a receptor antagonist, KF17837, but not the A1 receptor antagonist, DPCPX, significantly inhibited 2-chloroadenosine-mediated nitrite production. The 2-chloroadenosine-mediated nitrite production by interleukin-1 beta-stimulated cells was accompanied by increased inducible NO synthase mRNA accumulation. In the presence of dibutyryl-cAMP (1 mM), interleukin-1 beta-induced nitrite accumulation was further increased, but the effect of 2-chloroadenosine was not additive or synergistic. Addition of 2-chloroadenosine dose-dependently increased intracellular cAMP levels of vascular smooth muscle cells. These results indicate that adenosine acts on A2 receptors and augments NO synthesis in interleukin-1 beta-stimulated vascular smooth muscle cells, at least partially through a cAMP-dependent pathway.

Adenosine stimulates nitric oxide synthesis in rat cardiac myocytes.

We investigated the effects of adenosine on nitric oxide (NO) synthesis by measuring the production of nitrite, a stable metabolite of NO, in cultured neonatal rat cardiac myocytes. Incubation of cultures with interleukin-1 beta (10 ng/ml) for 24 h caused a significant increase in nitrite production. The interleukin-1 beta-induced nitrite production by cardiac myocytes was significantly increased by adenosine or its stable analog 2-chloroadenosine in a dose-dependent manner (10(-7)-10(-4) M). The adenosine A2-receptor antagonist KF-17837 (10(-6) M), but not the A1-receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (10(-6) M), significantly inhibited 2-chloroadenosine-mediated nitrite production. The 2-chloroadenosine-induced nitrite production by interleukin-1 beta-stimulated cells was accompanied by inducible NO synthase mRNA and protein accumulation. In the presence of N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) (10(-3) M) or isoproterenol (10(-5) M), interleukin-1 beta-induced nitrite accumulation was further increased, but the effect of 2-chloroadenosine was not additive or synergistic. The protein kinase C inhibitor calphostin C did not inhibit the effect of 2-chloroadenosine. These results indicate that adenosine acts on A2 receptors and augments NO synthesis in interleukin-1 beta-stimulated cardiac myocytes, at least partially through a cAMP-dependent pathway.

Enhancement of humoral and cellular immunity by vitamin E after embryonic exposure

In the present study, the amnion of turkey and chicken embryos were injected 3 d prior to hatch with different levels of vitamin E (VE). In Experiments 1 and 2, turkey embryos received 10, 20, and 30 IU of VE. In Experiment 3, broiler embryos received 10 IU VE. In all three experiments, sham-injected control embryos (0 IU VE) received 300 microL of saline. In Experiments 1 and 2 (turkey embryos), 20 and 30 IU of VE reduced (P < or = 0.05) percentage hatchability below that of controls. At hatch, poults exhibited a dose related increase (P < 0.05) in plasma VE levels. Mean BW gain up to 35 d and relative bursa of Fabricius and spleen weights were not different among treatment groups. When challenged at 7 d posthatch, total (P < 0.05) and IgM (P < 0.08) anti-SRBC antibodies were higher in 10 IU VE poults than in controls. Immunoglobulin G levels did not differ among the treatment groups. Poults in the 10 IU VE group had higher (P < 0.002) numbers of Sephadex-elicited inflammatory exudate cells, as well as a greater percentage of phagocytic macrophages (P < 0.0001). Additionally, the numbers of SRBC per phagocytic macrophage were greater (P < 0.001), than in control poults at 4 wk of age. In Experiment 3, chick embryos exposed to 10 IU VE, exhibited no differences in hatchability, BW gain, or bursal and splenic weights from the sham-exposed group. However, total and IgM antibody responses against SRBC were greater (P < 0.01) in the 10 IU VE group at 7 d postinjection. A secondary SRBC challenge given at 14 d after primary injection resulted in higher total (P < 0.07) and IgG (P < 0.04) antibody responses in the 10 IU VE chicks than in the controls. Similarly, broiler chicks (10 IU VE) had more Sephadex-elicited abdominal exudate cells (P < 0.07), and greater macrophage phagocytic potential (P < 0.0001). In ovo VE exposure (10 IU) also increased nitrite production (P < 0.04) by chick macrophages. The results from this study demonstrated an enhanced antibody and macrophage response and suggest that in ovo exposure with VE may improve posthatch poult and broiler quality.

Modulation of Myocardial Oxygen Consumption Through ACE Inhibitors

In this issue of Circulation ,1 Zhang and coworkers provide convincing evidence that ACE inhibitors reduce myocardial oxygen consumption by preventing the degradation of bradykinin, which subsequently stimulates the release of nitric oxide (NO). They propose that NO inhibits mitochondrial respiration, thus reducing myocyte oxygen use. This is an exciting observation because it provides a new mechanism whereby ACE inhibitors might benefit ischemia beyond simply inhibiting the formation of angiotensin II, thus reducing the vasoconstrictor effect of this octapeptide. These results are further evidence for a role of bradykinin in the response of tissues to ACE inhibitors. The mechanism enabling these interactions to occur is coming to light on the basis of studies such as the current one from Zhang et al. Interest in bradykinin and related kinins as cardiovascular mediators has grown tremendously in the past several years, largely because it has been recognized that they potently stimulate release of NO and prostacyclin from endothelial cells. Bradykinin and Lys-bradykinin, respectively, are formed when one of a family of enzymes, known as kallikreins, act on either heavy- or low-molecular-weight kininogens.2 This action involves cleavage of amino acids from both the C- and N-terminal ends of the kininogen peptides and takes place both in plasma (for heavy-molecular-weight kininogen) and tissues (for low-molecular-weight kininogen). The kininogens were previously thought to be expressed primarily by hepatocytes; however, it is now clear that many tissues, including vascular cells3 4 and the heart,5 also express kininogens. The kallikreins as well are made by many cell types and are produced by vascular cells in a continuous fashion.6 Thus, many tissues, including the heart and vascular cells, have an intact kallikrein/kininogen system capable of producing bradykinin. The mechanisms whereby bradykinin production is regulated remain unclear; however, there is some evidence that flow …

ACE inhibitors promote nitric oxide accumulation to modulate myocardial oxygen consumption.

ACE inhibitors potentiate kinin-nitric oxide (NO)-dependent coronary vascular dilation, and NO can modulate myocardial oxygen consumption. Whether ACE inhibitors also affect myocardial O2 consumption has not been established. Production of nitrite, a metabolite of NO in aqueous solution, in coronary microvessels and O2 consumption in myocardium were quantified with the use of in vitro tissue preparations, the Greiss reaction, and a Clark-type O2 electrode. In coronary microvessels, kininogen (the precursor of kinin; 10 micrograms/mL) and three ACE inhibitors (captopril, enalaprilat, or ramiprilat; 10(-8) mol/L) increased nitrite production from 76 +/- 6 to 173 +/- 15, 123 +/- 12, 125 +/- 12, and 153 +/- 12 pmol/mg, respectively (all P < .05). In myocardium, kininogen (10 micrograms/mL) and captopril, enalaprilat, or ramiprilat (10(-4) mol/L) reduced cardiac O2 consumption by 41 +/- 2%, 19 +/- 3%, 25 +/- 2%, and 35 +/- 2%, respectively. The changes in both nitrite release and O2 consumption in vitro were blocked by N omega-nitro-L-arginine methyl ester or N omega-nitro-L-arginine, inhibitors of endogenous NO formation. The effects were also blocked by HOE 140, which blocks the bradykinin B2-kinin receptor, and serine protease inhibitors, which inhibit local kinin formation. Our data indicate that stimulation of local kinin formation by use of a precursor for kinin formation or inhibition of kinin degradation by use of ACE inhibitors increases NO formation and is important in the control of cardiac O2 consumption. Vasodilation and control of myocardial O2 consumption by NO may contribute importantly to the therapeutic actions of ACE inhibitors in cardiac disease states.

Endothelin-1 inhibits nitric oxide synthesis in vascular smooth muscle cells.

We investigated the effects of endothelin-1 on nitric oxide synthesis in vascular smooth muscle cells. We measured the production of nitrite, a stable metabolite of nitric oxide, and the expression of inducible nitric oxide synthase mRNA and protein in cultured rat vascular smooth muscle cells. Incubation of the cultures with interleukin-1 beta (10 ng/mL) for 24 hours caused a significant increase in nitrite production. Endothelin-1 significantly decreased the interleukin-1 beta-induced nitrite production by vascular smooth muscle cells in a dose-dependent manner (10(-11) to 10(-8) mol/L). Incubation with interleukin-1 beta for 24 hours induced expression of inducible nitric oxide synthase mRNA and protein in vascular smooth muscle cells, whereas endothelin-1 showed a suppressive effect on their expressions. Addition of the endothelin type A receptor antagonist BQ-485, but not the endothelin type B receptor antagonist BQ-788, dose-dependently inhibited the effect of endothelin-1. After protein kinase C activity was functionally depleted by treatment of cells with phorbol 12-myristate 13-acetate for 24 hours, the effect of endothelin-1 was abolished. These results indicate that endothelin-1 acts on endothelin type A receptors and inhibits nitric oxide synthesis in interleukin-1 beta-stimulated vascular smooth muscle cells at least partially through a protein kinase C-dependent pathway.

Glutathione regulates nitric oxide synthase in cultured hepatocytes.

The authors determine the relationship between glutathione and nitric oxide (NO) synthesis in cultured hepatocytes. Glutathione is a cofactor for a number of enzymes, and its presence is essential for maximal enzyme activity by the inducible macrophage nitric oxide synthase (iNOS), which produces the reactive nitric oxide radical. Hepatocytes contain substantial quantities of glutathione, and this important tripeptide is decreased in hepatocytes stressed by ischemia/reperfusion or endotoxemia. Endotoxemia also induces the synthesis of inflammatory cytokines that result in the production of nitric oxide from hepatocytes by iNOS, suggesting that hepatocytes may be attempting to synthesize nitric oxide at times when intracellular glutathione is reduced. Hepatocytes were cultured with buthionine sulfoximine and 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) to inhibit glutathione. After exposure to cytokines, NO synthesis was assessed by supernatant nitrite levels, cytosolic iNOS enzyme activity, and iNOS mRNA levels. Inhibition of glutathione synthesis with buthionine sulfoximine or inhibition of glutathione reductase activity with BCNU inhibited nitrite synthesis. Both buthionine sulfoximine and BCNU inhibited the induction of iNOS mRNA, as detected by Northern blot analysis. Exogenous glutathione increased cytokine-stimulated iNOS induction, overcame the inhibitory effects of BCNU, and increased nitrite production by intact hepatocytes, induced hepatocyte cytosol, and partially purified hepatocyte iNOS. In cultured hepatocytes, adequate glutathione levels are required for optimal nitric oxide synthesis. This finding is predominantly due to an effect on iNOS mRNA levels, although glutathione also participates in the regulation of iNOS enzyme activity.

Ebselen and cytokine-induced nitric oxide synthase expression in insulin-producing cells

Interleukin-1 (IL-1) may be a mediator of β-cell damage in insulin-dependent diabetes mellitus (IDDM). The IL-1 mechanism of action on insulin-producing cells probably includes activation of the transcription nuclear factor ϰB (NF-ϰB), increased transcription of the inducible form of nitric oxide synthase (iNOS) and the subsequent production of nitric oxide (NO). Reactive oxygen intermediates, particularly H 2O 2, have been proposed as second messengers for NF-κB activation. In the present study, we tested whether ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a glutathione peroxidase mimicking compound, could counteract the effects of IL-1β, H 2O 2 and alloxan in rat pancreatic islets and in the rat insulinoma cell line RINm5F (RIN cells). Some of these experiments were also reproduced in human pancreatic islets. Ebselen (20 μM) prevented the increase in nitrite production by rat islets exposed to IL-1β for 6 hr and induced significant protection against the acute inhibitory effects of alloxan or H 2O 2 exposure, as judged by the preserved glucose oxidation rates. However, ebselen failed to prevent the increase in nitrite production and the decrease in glucose oxidation and insulin release by rat islets exposed to IL-1β for 24 hr. Ebselen prevented the increase in nitrite production by human islets exposed for 14 hr to a combination of cytokines (IL-1β, tumor necrosis factor-α and interferon-γ). In RIN cells, ebselen counteracted both the expression of iNOS mRNA and the increase in nitrite production induced by 6 hr exposure to IL-β but failed to block IL-1β-induced iNOS expression following 24 hr exposure to the cytokine. Moreover, ebselen did not prevent IL-1β-induced NF-κB activation. As a whole, these data indicate that ebselen partially counteracts cytokine-induced NOS activation in pancreatic β-cells, an effect not associated with inhibition of NF-κB activation.

Investigation of partial and full nitrification characteristics of fertilizer wastewaters in a submerged biofilm reactor

Nitrification characteristics of a high-strength fertilizer wastewater were studied in an upflow submerged biofilm reactor. The aim was the investigation of nitrogen removal rates with regard to ammonium removal and the determination of NOx-N production rates in terms of nitrile build-up and nitrate production. For this purpose the biofilm reactor was operated at various loading rates and DO levels. The system could reach high ammonium removal rates up to about 0.6 kgNH4-Nm−3d−1 (13.2 gNH4-Nm−2d−1). In a number of runs nitrification was incomplete and nitrite reached a considerable level. One of the most important factors for nitrite build-up was the relative ratio of bulk oxygen to bulk ammonium. More important than this was the ratio of bulk oxygen to bulk free ammonia. When this ratio was lower than 10, nitrite was the dominant species in the produced NOx-N. Also the increase in volumetric loading rate generally led to an increase in nitrite production. Thus, in order to achieve partial nitrification, free ammonia inhibition can be enhanced by operating at relatively low DO, high bulk ammonium and high bulk pH values. The main inhibition in the treatment of these wastes was substrate inhibition at indicated operating conditions. Since the concentration of free nitrous acid was very low, product inhibition could be disregarded.

Ethanol potentiates lipopolysaccharide- or interleukin-1 β-induced nitric oxide generation in RBE4 cells

Our present study investigated the effects of ethanol treatment on inducible nitric oxide (NO) synthase pathway from lipopolysaccharide- or interleukin-1β-treated cultured rat blood-brain barrier cell line (rat brain endothelial 4 cells: RBE4 cells). Cells were lipopolysaccharide- or interleukin-1β-treated with or without ethanol (50, 100 or 200 mM) for 16 or 24 h. Inducible NO synthase activity and mRNA expression were measured using Griess reaction and reverse transcription-polymerase chain reaction (RT-PCR) respectively. In the absence of lipopolysaccharide or interleukin-1β, ethanol treatments failed to stimulate inducible NO synthase gene expression. Lipopolysaccharide or interleukin-1β increased nitrite production and inducible NO synthase mRNA levels, and ethanol potentiated this effect. We concluded that ethanol could aggravate the consequences of NO generation by RBE4 cells after inducible NO synthase induction following inflammation or sepsis. This ethanol action on NO generation could contribute to circulatory failure associated with shock due to sepsis or hemorrhage, and alter blood-brain barrier permeability.

Influence of Pneumocystis carinii on nitrite production by rat alveolar macrophages.

Nitrite production by rat alveolar macrophages was studied to determine the role of L-arginine oxidation in the interaction between these cells and Pneumocystis carinii. Alveolar macrophages from rats obtained from two different breeders were used: rats from Janvier breeder had latent P. carinii infection, while those from Charles River breeder were bred in a germ-free environment. Pneumocystis carinii increased in vitro nitrite generation by unstimulated alveolar macrophages from Janvier rats only, and this was blocked by NG-monomethyl-L-arginine. Incubation of cells from Janvier and Charles River rats with lipopolysaccharide and/or interferon-gamma increased nitrite production to a similar extent. Pneumocystis carinii partially decreased nitrite release by activated alveolar macrophages, and this was still inhibited by NG-monomethyl-L-arginine. In the presence of P. carinii, superoxide dismutase used as a superoxide anion scavenger had no effect on nitrite production by activated cells. These results show that prior exposure to P. carinii leads to nitric oxide production by rat alveolar macrophages. Although the magnitude of this production seems to be moderate, it is of biological significance since cells of P. carinii-naive rats do not generate nitrite whereas those of latently infected rats do.