Nitric oxide inhibits superoxide production by inflammatory polymorphonuclear leukocytes

Nitric oxide(NO ·) has a complex role in the inflammatory response. Inthis study, we modified the levels of endogenous NO · in vivoin an acute model of inflammation and evaluated the interactionsbetween NO · and superoxide anion() produced bypolymorphonuclear leukocytes (PMNs) accumulated in the inflamed area.We injected phosphate-buffered saline (control group), 6 µmol ofL-N⁵-(1-iminoethyl)ornithine(L-NIO group), or 6 µmol ofL-arginine (L-arginine group) into thegranuloma pouch induced by carrageenan in rats. plus (indicative of NO · generation) was 188 nmol in the exudate of the control group, but itdecreased in the L-NIO group(P < 0.05) and increased in theL-arginine group(P < 0.05). When PMNsfrom treated rats were incubated in vitro, the productionof superoxide anion () decreased by ~46% in theL-arginine group. Furthermore, was inhibited in PMNs whenL-arginine was addedto the incubation medium before phorbol 12-myristate 13-acetatestimulation but not when added simultaneously. Our results suggest aprotective role for NO · in inflammation, through theinactivation of NADPH oxidase and the consequent impairment of production for cell-mediatedinjury.

     

Neuregulin stimulates myogenic differentiation in an autocrine manner.

During myogenesis, mononucleated myoblasts form multinucleated myotubes by membrane fusion. Efficiency of this intercellular process can be maximized by a simultaneous progress, with a time window, of other neighboring myoblasts in the differentiation program. This phenomenon has been described as the community effect. It proposes the existence of a molecule that acts as a differentiation-inducing signal to a group of identical cells. Here, we show that neuregulin is a strong candidate for this molecule in myoblast differentiation. The expression of neuregulin increased rapidly but transiently at early stage of differentiation of rat L6 cells. Neuregulin showed a potent differentiation-promoting activity in membrane fusion and expression of myosin heavy chain. The antibodies raised against neuregulin and its cognate receptor ErbB3, which were capable of neutralizing the signal pathway, inhibited myotube formation and expression of myosin heavy chain in both L6 cells and primary rat myoblasts. The progress of differentiation was mostly halted after the expression of myogenin and cell cycle arrest. These results suggest that the activation of an autocrine signaling of neuregulin may provide a basic mechanism for the community effect observed in the differentiation of the embryonic muscle cells.     

Phosphorylase kinase from human polymorphonuclear leukocytes.

Phosphorylase kinase from human polymorphonuclear leukocytes was investigated in a gel filtered crude preparation (17,000 x g supernatant). It was found to exist in two forms, one (the phosphorylated form) more active than the other (the dephosphorylated form). Interconversion between the two forms was carried out by a cyclic AMP dependent protein kinase and phosphoprotein phosphatase, respectively. The ratio of activity measured at pH 8.0 and 6.0 was 0.36 for the non-activated and 0.83 for the activated form, which is in contrast to the behaviour of phosphorylase kinase from muscle. Km app for the substrate phosphorylase b was 650 U/ml and 85 U/ml for the non-activated and activated form, respectively, whereas Km app for ATP was 0.03 mM and identical for the two forms. The non-activated form of phosphorylase kinase was activated by Ca2+ in the range 10(-7)--5 . 10(-6) M, which may have physiological importance, whereas the activated form was insensitive to variations in Ca2+ concentration between 10(-9) and 10(-3) M.     

Nitric oxide production by rat bronchoalveolar macrophages or polymorphonuclear leukocytes following intratracheal instillation of lipopolysaccharide or silica

Exposure of the lung to lipopolysaccharide (LPS) or silica results in an activation of alveolar macrophages (AMs), recruitment of polymorphonuclear leukocytes (PMNs) into bronchoalveolar spaces, and the production of free radicals. Nitric oxide (NO) is one of the free radicals generated by bronchoalveolar lavage (BAL) cell populations following either LPS or silica exposure. The purpose of the present study was to assess the relative contributions of AMs and PMNs to the amounts of NO produced by BAL cells following intratracheal (IT) instillation of either LPS or silica. Male Sprague Dawley rats (265-340 g body wt.) were given LPS (10 mg/100 g body wt.) or silica (5 mg/100 g body wt.). BAL cells were harvested 18-24 h post-IT and enriched for AMs or PMNs using density gradient centrifugation. Media levels of nitrate and nitrite (NOx; the stable decomposition products of NO) were then measured 18 h after ex vivo culture of these cells. Following IT exposure to either LPS or silica, BAL cell populations were approximately 20% AMs and approximately 80% PMNs. After density gradient centrifugation of BAL cells from LPS- or silica-treated rats, cell fractions were obtained which were relatively enriched for AMs (approximately 60%) or PMNs (approximately 90%). The amounts of NOx produced by the AM-enriched fractions from LPS- or silica-treated rats were approximately 2-4-fold greater than that produced by the PMN-enriched fractions. Estimations of the relative contribution of AMs or PMNs to the NOx produced indicated that: (i) following LPS treatment, 75%-89% of the NOx was derived from AMs and 11%-25% from PMNs; and (ii) following silica treatment, 76%-100% of the NOx was derived from AMs and 0-24% from PMNs. Immunohistochemistry for inducible NO synthase on lung tissue sections supported these findings. We conclude that AMs are the major source of the NO produced by BAL cells during acute pulmonary inflammatory responses to LPS or silica.     

Monokines released during short-term Fc gamma receptor phagocytosis up-regulate polymorphonuclear leukocytes and monocyte-phagocytic function.

Freshly explanted monocytes phagocytosing IgG antibody-coated erythrocyte targets (EIgG) release a factor(s) that stimulates phagocytosis by neighboring monocytes and polymorphonuclear leukocytes (PMN). Culture supernatants obtained after 30-min incubation of adherent monocytes with EIgG, but not unopsonized sheep erythrocytes, markedly up-regulated the extent of PMN phagocytosis and enhanced the rate at which monocytes ingested EIgG. The presence of this factor(s) was first evident in phagocytic studies in which monocytes were prepared by a colloidal silica-based continuous gradient technique (Sepracell-Mn). After introduction of erythrocyte targets, there was a 20- to 30-min delay before initiation of phagocytosis that was not observed with monocytes prepared by the standard Percoll-gradient technique. Experiments suggest that, when compared with monocytes prepared by the Percoll-gradient method, Sepracell-Mn monocytes are closer to a base line state of activation with regard to the expression of Fc gamma RI and the ability to ingest EIgG. The mechanism of PMN upregulation by the monocyte factor(s) was explored. Monocyte supernatants did not induce an increase in the surface expression of PMN Fc gamma RI, II, or III. Neither anti-TNF, anti-IL-2, nor anti-GM-CSF had any significant effect on monocyte supernatant activity. Neutrophil activating protein-1 was not detected by ELISA. In contrast, anti-IL-1 completely blocked the effect of the supernatant on subsequent monocyte phagocytosis, and partially inhibited its effect on PMN phagocytosis. Furthermore, it was shown that RIL-1 as well as TNF markedly enhanced monocyte and PMN ingestion of EIgG. These results suggest that monocytes, after Fc gamma R-mediated phagocytosis, release monokines, including at least IL-1, which enhance the phagocytic function of neighboring PMN and monocytes to augment the host defense process.     

Nitric oxide as an antioxidant.

Benzoate monohydroxy compounds, and in particular salicylate, were produced during interaction of ferrous complexes with hydrogen peroxide (Fenton reaction) in a N2 environment. These reactions were inhibited when Fe complexes were flushed, prior to the addition in the model system, by nitric oxide. Methionine oxidation to ethylene by Fenton reagents was also inhibited by nitric oxide. Myoglobin in several forms such as metmyoglobin, oxymyoglobin, and nitric oxide-myoglobin were interacted with an equimolar concentration of hydrogen peroxide. Spectra changes in the visible region and the changes in membrane (microsomes) lipid peroxidation by the accumulation of thiobarbituric acid-reactive substances (TBA-RS) were determined. The results showed that metmyoglobin and oxymyoglobin were activated by H2O2 to ferryl myoglobin, which initiates membrane lipid peroxidation; but not nitric oxide-myoglobin, which, during interaction with H2O2, did not form ferryl but metmyoglobin which only poorly affected lipid peroxidation. It is assumed that nitric oxide, liganded to ferrous complexes, acts to prevent the prooxidative reaction of these complexes with H2O2.     

Interferon-beta from melanoma cells suppresses the proliferations of melanoma cells in an autocrine manner

Interferon (IFN)-alpha and IFN-beta have been utilized in the treatment of melanoma as a form of cytokine therapy. While previous studies have demonstrated that melanocytes and melanoma cells produce a number of cytokines, it remains unclear whether or not melanocytes and melanoma cells per se produce IFN-alpha or IFN-beta. In the present study, we investigated the expression of IFN-alpha or IFN-beta in human melanocytes and five melanoma cell lines: G-361, C32TG, MMAc, MEWO and VMRC-MELG at both mRNA and protein levels. Both IFN-alpha and IFN-beta mRNA were detected in normal human melanocytes. Likewise, IFN-alpha mRNA was detected in all five melanoma cell lines. However, IFN-beta mRNA was only detected in one melanoma cell line, VMRC-MELG. When melanocytes and melanoma cells were treated with a potent IFN inducer, polyinosinic:polycytidylic acid (poly I:C), the mRNA expression of both IFN-alpha and IFN-beta was significantly upregulated. Poly I:C was not able to induce melanocytes or melanoma cells to produce detectable amounts of IFN-alpha protein, but able to induce a significant amount of IFN-beta in melanocytes and two of the melanoma cell lines: MMAc and VMRC-MELG. Moreover, similar to exogenous IFN-alpha and IFN-beta, poly I:C significantly inhibited the proliferation of all five melanoma cell lines. This suppressive effect was partially blocked by anti-IFN-beta antibody treatment in the IFN-beta-producing melanoma cell lines: MMAc and VMRC-MELG, but not in the non-IFN-beta-producing cell lines: G-361, C32TG and MEWO. Collectively, these studies have demonstrated for the first time that human melanocytes and melanoma cells produce IFN-beta. Furthermore, melanoma cells are capable of suppressing their own proliferation via secretion of endogenous IFN-beta. This finding may have important implications for melanoma therapy.     

Nitric oxide modulates arterial baroreflex control of heart rate in conscious lambs in an age-dependent manner

Experiments were carried out in conscious chronically instrumented lambs aged 1 (n = 6) and 6 wk (n = 5) to evaluate the arterial baroreflex control of heart rate (HR) during postnatal maturation and to investigate any modulatory role of endogenously produced nitric oxide (NO). Before and after intravenous administration of 20 mg/kg of the L-arginine analog N(G)-nitro-L-arginine methyl ester (L-NAME), the arterial baroreflex was assessed by measuring HR responses to increases and decreases in systolic arterial pressure achieved by intravenous administration of phenylephrine and sodium nitroprusside. The HR range over which the baroreflex operates and minimum HR as well as maximum gain were greater at 1 than at 6 wk of age. These age differences were abolished in the presence of L-NAME, which decreased the HR range and gain of the arterial baroreflex control of HR at 1 but not at 6 wk of age. These data provide new information that age-dependent effects of the arterial baroreflex appear to result from effects of endogenously produced NO.     

The actin released from profilin--actin complexes is insufficient to account for the increase in F-actin in chemoattractant-stimulated polymorphonuclear leukocytes

Chemoattractant stimulation of polymorphonuclear leukocytes is associated with a nearly two-fold rise in actin filament content. We examined the role of the actin monomer sequestering protein, profilin, in the regulation of PMN actin filament assembly during chemoattractant stimulation using a Triton extraction method. Poly-L-proline-conjugated Sepharose beads were used to assess the relative concentration of actin bound to profilin with high enough affinity to withstand dilution (profilin-actin complex) and DNase I-conjugated beads to measure the relative concentration of actin in the Triton-soluble fraction not bound to profilin. Actin associated with the Triton-insoluble fraction (F-actin) was also measured. In unstimulated PMN, the relative concentration of actin bound to profilin was maximum. After FMLP stimulation, profilin released actin monomers within 10 s, with the profilin-actin complex concentration reaching a nadir by 40 s and remaining low as long as the cells were exposed to chemoattractant (up to 30 min). If FMLP was dissociated from PMN membrane receptors using t-BOC, actin reassociated with profilin within 20 s. Quantitative analysis of these reactions, however, revealed that profilin release of and rebinding to actin could account for only a small percentage of the total change in F-actin content. Determination of the total profilin and actin concentrations in PMN revealed that the molar ratio of profilin to actin was 1 to 5.2. When purified actin was polymerized in PMN Triton extract containing EGTA, removal of profilin from the extract minimally affected (12% reduction) the high apparent critical concentration at which actin began to assemble. Although profilin released actin at the appropriate time to stimulate actin assembly during exposure to chemoattractants, the concentration of profilin in PMN was insufficient to explain the high unpolymerized actin content in unstimulated PMN and the quantity of actin released from profilin too small to account for the large shifts from unpolymerized to polymerized actin associated with maximal chemoattractant stimulation.     

Nitric oxide activates Rap1 and Ral in a Ras-independent manner

Rap1 and Ral, the small GTPases belonging to the Ras superfamily, have recently attracted much attention; Ral because of Ral-specific guanine nucleotide exchange factors which are regulated by direct binding to Ras and Rap1 because of its proposed role as an antagonist of Ras signaling. We have previously demonstrated that nitric oxide (NO) activates Ras and proposed the structural basis of interaction between NO and Ras. In the present study we have shown that NO activates Rap1 and Ral in a time- and concentration-dependent manner. Using activation-specific probes for Rap1 and Ral, it was found that the NO-generating compounds SNP and SNAP could activate both Rap1 and Ral in Jurkat and PC12 cell lines. To investigate the involvement of Ras in NO mediated activation of Rap1 and Ral, we used PC12 cell lines expressing either the Ras mutant C118S (Cys118 mutated to Ser) or N17 (GDP-locked and inactive). We had previously shown that NO fails to activate Ras in these mutant cell lines. However, here it was found that Rap1 and Ral were activated by NO in these cell lines. The evidence presented in this study unambiguously demonstrates the existence of Ras-independent pathways for NO mediated activation of Rap1 and Ral.     

Characterization of two gangliosides from human leukemic polymorphonuclear leukocytes.

Eight bands of gangliosides, from human polymorphonuclear leukocytes were demonstrated by thin-layer chromatography. Bands 4 and 5 were isolated and purified in sufficient amounts to allow their biochemical identification by thin-layer chromatography, gas chromatography and sequential action of glycosidases and neuraminidase. The major ganglioside was characterised as N-acetylneuraminylgalactosyl-beta-N-acetylglucosaminyl-beta-galactosyl-beta-glucosylceramide. A second ganglioside was tentatively identified as N-acetylneuraminyl-galactosyl-beta-N-acetylglucosaminyl-beta-(N-acetylneuraminyl)galactosyl-beta-glucosylceramide. Both gangliosides isolated were hydrolysed by neuraminidase. However, treatment of the intact cells with neuraminidase did not alter the ganglioside pattern.     

Increased insulin-insensitive glucose transport in polymorphonuclear leukocytes from non-insulin-dependent diabetic patients.

We studied the transport rate of a non-metabolizable hexose analogue, 3-O-methyl-D-glucose, in polymorphonuclear leukocytes (insulin-insensitive cells) from patients with untreated non-insulin-dependent diabetes mellitus. The mean glucose transport rate was significantly elevated in the diabetic patients compared with healthy controls (13.3 +/- 3.7 vs 10.4 +/- 2.5 fl/cell.sec, mean +/- SD, p less than 0.01). In the diabetic subjects, glucose transport rates were positively correlated with HbA1c levels (r = 0.563, p less than 0.01) but had no relations with ambient plasma glucose concentrations. Short-term incubation with 20 mM D-glucose had no effect on glucose transport in those cells. When glucose transport rates, HbA1c and fasting plasma glucose levels were simultaneously measured at weekly intervals over a four-week period in three diabetic subjects, the alterations in transport rates generally paralleled the changes observed in HbA1c levels rather than plasma glucose concentrations. It can be concluded that unlike insulin-sensitive cells such as adipocytes and muscle, glucose transport in human polymorphonuclear leukocytes, which are insulin insensitive cells, is increased in patients with non-insulin-dependent diabetes mellitus. Long-term, not short-term, derangement of glucose metabolism seems to be associated with increased glucose transport rate found in those patients.     

Cytochalasin E-induced oxidative metabolism in polymorphonuclear leukocytes.

The extent of the cyanide-resistent oxidative burst of polymorpho-nuclear leukocytes after stimulation with cytochalasin E was shown to depend markedly on the osmolarity of the cell-suspension medium. With granulocyte concentrations up to 2 X 10(6) cells/ml, optimal oxygen consumption and releases of H2O2 and superoxide anions were reached at 180 mOsmol and 2 X 10(-5) M cytochalasin E. After removal of unbound activator, the cellular oxidative activity remained unaltered and continued to depend on the used osmotic conditions. It is proposed that binding of cytochalasin to the plasma membrane induces an irreversible activation of the oxidative system, whereas the resulting metabolic activity depends on conformational changes in the plasma membrane.     

Nitric oxide and atherosclerosis: an update.

Nitric oxide (NO) is a molecule that has gained recognition as a crucial modulator of vascular disease. NO has a number of intracellular effects that lead to vasorelaxation, endothelial regeneration, inhibition of leukocyte chemotaxis, and platelet adhesion. Endothelium damage induced by atherosclerosis leads to the reduction in bioactivity of endothelial NO synthase (eNOS) with subsequent impaired release of NO together with a local enhanced degradation of NO by increased generation of reactive oxygen species with subsequent cascade of oxidation-sensitive mechanisms in the arterial wall. Many commonly used vasculoprotective agents have their therapeutic actions through the production of NO. L-Arginine, the precursor of NO, has demonstrated beneficial effects in atherosclerosis and disturbed shear stress. Finally, eNOS gene polymorphism might be an additional risk factor that may contribute to predict cardiovascular events. However, further studies are needed to understand the possible clinical implications of these correlations.     

Tetramethylpyrazine scavenges superoxide anion and decreases nitric oxide production in human polymorphonuclear leukocytes

Tetramethylpyrazine is one of the active ingredients of the Chinese herb Ligusticum wallichii Franchat. By electron spin resonance spin trapping methods, effects of tetramethylpyrazine on superoxide anion and nitric oxide generated by human polymorphonuclear leukocytes were studied. During the respiratory burst of polymorphonuclear leukocytes induced by N-formylmethionyl-leucyl-phenylalanine, tetramethylpyrazine scavenges superoxide anion dose-dependently, and decreases the production of nitric oxide significantly, but shows no influence on oxygen consumption. These results suggest that the effective protection of tetramethylpyrazine against ischemic brain injury might be due to its scavenging of reactive oxygen species and regulation on nitric oxide production, and consequent prevention of peroxynitrite formation.