Nitric Oxide Synthesis In Response Research Articles

Argon-stimulated nitric oxide production and its function in alfalfa cadmium tolerance

Recent results showed that argon may have great potential in both medicines (especially) and agriculture. However, how argon positively influences crop physiology remains elusive. Here, we observed that the stimulation of nitric oxide (NO) production upon cadmium (Cd) stress in hydroponic alfalfa root tissues was strengthened by argon-rich water and/or a NO-releasing compound. The pharmacological results further indicated that above potential source of NO stimulation achieved by argon might be attributed to NO synthase (NOS) and nitrate reductase (NR). Under hydroponic and pot conditions, the improvement of Cd tolerance elicited by argon, confirmed by the alleviation in the plant growth inhibition, oxidative damage, and Cd accumulation, was sensitive to the scavenger of NO. These results suggested a crucial role of argon-induced NO synthesis in response to Cd stress. Subsequent evidence showed that the improved iron homeostasis and increased S-nitrosylation were also dependent on argon-stimulated NO. Above results were matched with the transcriptional profiles of representative target genes involved in heavy metal detoxification, antioxidant defence, and iron homeostasis. Taken together, our results clearly indicated that argon-stimulated NO production contributes to Cd tolerance by favoring important defense strategies against heavy metal exposure.

α-Klotho expression determines nitric oxide synthesis in response to FGF-23 in human aortic endothelial cells.

Endothelial cells (ECs) express fibroblast growth factor (FGF) receptors and are metabolically active after treatment with FGF-23. It is not known if this effect is α-Klotho independent or mediated by humoral or endogenous endothelial α-Klotho. In the present study, we aimed to characterize EC α-Klotho expression within the human vascular tree and to investigate the potential role of α-Klotho in determining FGF-23 mediated EC regulation. Human tissue and ECs from various organs were used for immunohistochemistry and Western blot. Primary cultures of human aortic endothelial cells (HAECs) and human brain microvascular endothelial cells (HBMECs) were used to generate in vitro cell models. We found endogenous α-Klotho expression in ECs from various organs except in microvascular ECs from human brain. Furthermore, FGF-23 stimulated endothelial nitric oxide synthase (eNOS) expression, nitric oxide (NO) production, and cell proliferation in HAECs. Interestingly, these effects were not observed in our HBMEC model in vitro. High phosphate treatment and endothelial α-Klotho knockdown mitigated FGF-23 mediated eNOS induction, NO production, and cell proliferation in HAECs. Rescue treatment with soluble α-Klotho did not reverse endothelial FGF-23 resistance caused by reduced or absent α-Klotho expression in HAECs. These novel observations provide evidence for differential α-Klotho functional expression in the human endothelium and its presence may play a role in determining the response to FGF-23 in the vascular tree. α-Klotho was not detected in cerebral microvascular ECs and its absence may render these cells nonresponsive to FGF-23.

Molecular characterization of argininosuccinate synthase and argininosuccinate lyase from the liver of the African lungfish Protopterus annectens, and their mRNA expression levels in the liver, kidney, brain and skeletal muscle during aestivation.

Argininosuccinate synthase (Ass) and argininosuccinate lyase (Asl) are involved in arginine synthesis for various purposes. The complete cDNA coding sequences of ass and asl from the liver of Protopterus annectens consisted of 1,296 and 1,398bp, respectively. Phylogenetic analyses revealed that the deduced Ass and Asl of P. annectens had close relationship with that of the cartilaginous fish Callorhinchus milii. Besides being strongly expressed in the liver, ass and asl expression were detectable in many tissues/organs. In the liver, mRNA expression levels of ass and asl increased significantly during the induction phase of aestivation, probably to increase arginine production to support increased urea synthesis. The increases in ass and asl mRNA expression levels during the prolonged maintenance phase and early arousal phase of aestivation could reflect increased demand on arginine for nitric oxide (NO) production in the liver. In the kidney, there was a significant decrease in ass mRNA expression level after 6months of aestivation, indicating possible decreases in the synthesis and supply of arginine to other tissues/organs. In the brain, changes in ass and asl mRNA expression levels during the three phases of aestivation could be related to the supply of arginine for NO synthesis in response to conditions that resemble ischaemia and ischaemia-reperfusion during the maintenance and arousal phase of aestivation, respectively. The decrease in ass mRNA expression level, accompanied with decreases in the concentrations of arginine and NO, in the skeletal muscle of aestivating P. annectens might ameliorate the potential of disuse muscle atrophy.

Nutrient timing with low carbohydrate diets

Low carbohydrate diets Various low carbohydrate diets have been shown to provide beneficial changes in body mass, lipid profiles and other health risk factors. Recent evidence indicates that diets with lower glycemic index carbohydrates and increased protein provide greater weight loss and maintenance of the reduced weight as compared with high glycemic and low protein diets. Insulin release is lower with lower blood glucose levels, thereby reducing fatty acid metabolism and storage. High intake of high glycemic response carbohydrates is associated with increased levels of insulin resistance which appears to be the common link between obesity and metabolic syndrome. Nutrient timing Nutrient timing is generally regarded as a nutritional strategy in which precise amounts of particular nutrients are delivered at precise time points, relative to exercise, in order to enhance performance or training effects. This somewhat general definition has been operationally limited by many to diets that utilize high glycemic carbohydrates prior to, during, and/or following exercise. These carbohydrates are considered vital as they provide an energy source as well as inducing increased insulin levels. As insulin directly influences the production of nitric oxide, vascular musculature is relaxed and circulation into the capillary beds of exercising muscles is increased. Carbohydrates, in particular higher glycemic carbohydrates, supply these critical benefits. Low carbohydrate nutrient timing The basic model of low carbohydrate nutrient timing applies specific proven micronutrients for enhanced exercise performance rather than relying on the ingestion of sugar and the subsequent insulin responses. First, reduced carbohydrate intake produces reduced insulin responses which shifts the metabolism to fatty acid utilization. Secondly, various nutritional components can provide additional energy sources and/or produce increased nitric oxide production with subsequent vasodilation. Items such as creatine and beta alanine can influence energy levels by affecting energy replenishment and acting as an anaerobic buffer. Branched chain amino acids provide a third energy source without which muscle tissue may be consumed with intense exercise. Various micronutrients can increase muscle blood flow to some degree. In particular, glycine proprionyl l-carnitine (GPLC) has been shown to dramatically increase nitric oxide synthesis in response to exercise stresses and to significantly increase exercise performance with reduced production of lactate. Conclusions The limited research in the area suggests that some athletes can train and compete in certain settings successfully with relatively low intake of dietary carbohydrates. It has been shown that pre-workout supplements containing common ingredients such as creatine, beta alanine, branched chain amino acids can substantially enhance exercise performance without ingestion of additional carbohydrates. Controlled clinical trials are needed to examine the effectiveness of nutrient timing with a low carbohydrate diet in various sports settings.

Hydrogen peroxide-mediated activation of MAP kinase 6 modulates nitric oxide biosynthesis and signal transduction in Arabidopsis.

Nitric oxide (NO) is a bioactive molecule that functions in numerous physiological and developmental processes in plants, including lateral root development. In this study, we used biochemical and genetic approaches to analyze the function of Arabidopsis thaliana mitogen-activated protein kinase 6 (MPK6) in the regulation of NO synthesis in response to hydrogen peroxide (H₂O₂) during lateral root development. In both mpk6 mutants studied, H₂O₂-induced NO synthesis and nitrate reductase (NR) activity were decreased dramatically. Furthermore, one NR isoform, NIA2, was required for the MPK6-mediated production of NO induced by H₂O₂. Notably, NIA2 interacted physically with MPK6 in vitro and in vivo and also served as a substrate of MPK6. Phosphorylation of NIA2 by MPK6 led to an increase in NR activity, and Ser-627 was identified as the putative phosphorylation site on NIA2. Phenotypical analysis revealed that mpk6-2 and mpk6-3 seedlings produce more and longer lateral roots than wild-type plants did after application of the NO donor sodium nitroprusside or H₂O₂. These data support strongly a function of MPK6 in modulating NO production and signal transduction in response to H₂O₂ during Arabidopsis root development.

Lymphocytes and not IFN-γ mediate expression of iNOS by intestinal epithelium in murine cryptosporidiosis

We hypothesized that unrecognized differences in epithelial expression of inducible nitric oxide synthase (iNOS), resulting from engineered immunodeficiency, could explain the contradictory findings of prior studies regarding the importance of nitric oxide (NO) in murine models of Cryptosporidium parvum infection. Severe combined immunodeficient mice (SCID) failed to constitutively or inducibly express epithelial iNOS or increase NO synthesis in response to C. parvum infection. In contrast, mice lacking IFN-gamma alone induced both epithelial iNOS expression and NO synthesis in response to infection. Accordingly, lymphocytes mediate epithelial expression of iNOS and NO synthesis independent of IFN-gamma in response to C. parvum infection. These findings in large part explain the contradictory conclusions of prior studies regarding the role of iNOS in C. parvum infection.

Roles of ciliary polycystin and survivin in cardiovascular abnormality in PKD

BackgroundPolycystic kidney disease (PKD) is characterized by the development of fluid‐filled cysts in the kidney. However, cardiovascular hypertension and aneurysm represent the most serious life‐threatening complications in PKD patients. We have recently shown that polycystins are localized to the endothelial cilia and function in regulating intracellular calcium and nitric oxide synthesis in response to fluid‐shear stress.Methods and ResultsWe show here that two different PKD endothelial cells, Pkd1 (29%) and Tg737 (32%), are characterized by abnormal cell division (p<0.05, compared to wild type cells). We also observe a significant centrosomal over‐duplication, multipolar spindle formation, nuclear fragmentation and polyploidism leading to chromosome instability and mitotic catastrophe in Pkd1 (16%) and Tg737 (20%). These mitotic abnormalities are associated with the down‐regulation of survivin, an inhibitor of apoptosis and a chromosomal passenger protein involved in mitotic spindle checkpoint and regulation of chromosomal segregation.ConclusionTaken together, our findings provide a novel evidence of the roles for ciliary polycystins in regulating cell division through survivin expression and confirm the proposed involvement of cilia in cell division, parallel to their roles as fluid mechanosensors.

Preoptic neuronal nitric oxide synthase induction by testosterone is consistent with a role in gating male copulatory behavior

Copulatory behaviors are generally dependent on testicular androgens in male vertebrates, being eliminated by castration and reinstated by testosterone administration. It is postulated that a critical factor in this hormonal gating is up-regulation of neuronal nitric oxide synthase (nNOS) in the preoptic area, and consequent enhanced nitric oxide synthesis in response to stimuli associated with a receptive female. Previous studies have suggested that nNOS protein is more abundant in behaviorally relevant preoptic regions of testosterone-exposed animals than in hormone-deprived controls. This study sought to elucidate the molecular events underlying this apparent up-regulation by examining preoptic nNOS mRNA abundance at several time points following testosterone administration in a castration and replacement paradigm. Castrated male whiptails (Cnemidophorus inornatus) were implanted with testosterone, and at four time points over the subsequent 18 days their sexual behavior was tested. A rostral periventricular area previously implicated in hormonal gating of male-typical copulatory behavior was then excised by laser microdissection, and nNOS transcript abundance was assessed by quantitative PCR. As neither this technique nor nNOS mRNA measurements have previously been performed in this area of the brain, expression was concomitantly assayed on adjacent sections by in situ hybridization or NADPH diaphorase histochemistry. Results are consistent with transcriptional up-regulation of nNOS by testosterone and a central role for the enzyme in mediating hormonal gating of copulatory behavior.

Synthesis of Nitric Oxide in Human Osteoblasts in Response to Physiologic Stimulation of Electrotherapy

Electrotherapy for bone healing, remodeling and wound healing may be mediated by modulation of nitric oxide (NO). Using NO-specific fluorophore (DAF-2), we report here that application of non-invasive, physiologic electrical stimulation induces NO synthesis in human osteoblasts, and that such NO generation is comparable to that induced by estrogen treatment. For example, application of a sinusoidal 1 Hz, 2 V/cm (peak to peak) electrical stimulation (ES) increases NO-bound DAF-2 fluorescence intensity by a 2-fold within 60 min exposure by activating nitric oxide synthase (NOS). Increase in the NO level is found to depend critically on the frequency and strength of ES. While the frequency of 1 Hz ES seems optimal, the ES strength >0.5 V/cm is required to induce significant NO increase, however. Nitric oxide synthesis in response to ES is completely prevented by blocking estrogen receptors using a competitive inhibitor, suggesting that NO generation is likely initiated by activation of estrogen receptors at the cell surface. Based on these findings, physiologic stimulation of electrotherapy appears to represent a potential non-invasive, non-genomic, and novel physical technique that could be used to regulate NO-mediated bone density and facilitate bone remodeling without adverse effects associated with hormone therapy.

Blood cell NO synthesis in response to exercise

Nitric oxide (NO) is important for the maintenance of cardiovascular homeostasis and is also involved in immunity and inflammation. The aim of our work was to determine the effects of intense exercise on plasma and blood cell NO handling. Nine voluntary male professional cyclists participated in the study. Blood samples were taken in basal conditions and 3h after finishing a mountain cycling stage. Exercise-induced neutrophilia, lymphopenia, and hemolysis. Plasma and erythrocytes maintained basal nitrite levels, whereas neutrophils and lymphocytes decreased nitrite concentration after intense exercise. Basal iNOS levels and SOD activity were similar in neutrophils and lymphocytes. iNOS levels and SOD activity dropped in neutrophils and rose in lymphocytes after exercise. Arginase activity rose only in lymphocytes. Neutrophil nitrite was correlated with SOD activity and iNOS levels, but not in lymphocytes. iNOS levels were correlated with SOD in both neutrophils and lymphocytes. Intense exercise maintained plasma basal arginine and ornithine concentration, and decreased citrulline concentration. Intense exercise induced important changes in NO handling in neutrophils and lymphocytes, yet the basal picture was maintained in erythrocytes.

Regulation of platelet guanylyl cyclase by collagen: evidence that Glycoprotein VI mediates platelet nitric oxide synthesis in response to collagen

The molecular regulation of nitric oxide synthase (NOS) in blood platelets is an uncharacterised area of platelet biology. We investigated the mechanism of collagen-stimulated NO synthesis in platelets. Our aim was to identify the key collagen receptor and downstream signalling mechanisms linking collagen to NOS activation. Collagen and the GpVI-specific platelet activator collagen-related peptide (CRP-XL) stimulated NO synthesis, as evidenced by increased [(3)H]L-citrulline production, and cyclic GMP (cGMP) formation. After platelet activation by collagen and CRP-XL was normalised, we found no differences in NOS activation or cGMP formation in response to these agonists. Blocking the interaction of collagen with integrin alpha(2)beta(1), a second collagen receptor, failed to affect NOS activation by collagen. These data indicate that collagen-induced NO synthesis is linked to GpVI activation. cGMP formation in response to collagen and CRP-XL required increased intracellular Ca(2+), Src family kinases, phosphatidylinositol 3-kinase (PI3-K) and protein kinase C. By comparison, Gp VI-independent cGMP formation induced by thrombin was Src kinase-dependent, but was independent of PI3-K and PKC. Thus the mechanisms of collagen- and CRP-XL-induced NOS activation were identical, but distinct from that of thrombin. Platelet activation in response to collagen leads to secretion of adenosine diphosphate (ADP) and thromboxane A(2) (TxA(2)). Our results demonstrate that collagen-stimulated cGMP synthesis was enhanced significantly by platelet-derived ADP and TxA(2). These results reveal that collagen stimulates platelet NOS activation through a specific Ca(2+)-dependent GpVI receptor signalling cascade, and demonstrate that collagen-induced cGMP accrual requires the release of secondary platelet agonists.

Low endotoxemia prevents the reduction of gastric blood flow induced by NSAIDs: role of nitric oxide.

1 The role of nitric oxide (NO) in the effects of low endotoxemia on gastric damage and blood flow has been evaluated in indomethacin-treated rats. 2 Pretreatment (-1 h) with endotoxin (40 micro g kg(-1)) reduced gastric damage induced by indomethacin (20 mg kg(-1)) in conscious rats. 3 Endotoxin prevented the reduction in gastric blood flow (laser Doppler flowmetry) induced by indomethacin in pentobarbital-anaesthetised rats. 4 Pretreatment with an NO-synthase (NOS) inhibitor (L-NAME, 1 mg kg(-1)) reversed the protective effect of endotoxin on gastric blood perfusion. 5 Endotoxin did not modify the expression of mRNA for endothelial NOS or inducible NOS in the gastric corpus when evaluated 1 h postinjection. However, a 3.8-fold increase in inducible NOS mRNA and a 61% reduction in endothelial NOS mRNA were observed in the gastric corpus 4 h after endotoxin administration. 6 Evaluation of both total and Ca(2+)-dependent NOS activity by analysing the rate of conversion of L-arginine to L-citrulline in gastric corpus homogenates showed no differences between animals treated with endotoxin and those treated with saline 1 or 4 h beforehand. Ca(2+)-independent NOS activity was almost non-apparent in control as well as in endotoxin-treated rats at all the time points analysed. 7 Low endotoxemia preserves blood perfusion and protects the gastric mucosa against the deleterious effects of indomethacin through the endogenous NO release. NO synthesis in response to endotoxin does not involve the inducible NOS, but probably depends on the post-translational/biochemical regulation in vivo of a Ca(2+)-dependent NOS, most probably endothelial NOS.

Human gingival fibroblasts produce nitric oxide in response to proinflammatory cytokines.

Although nitric oxide (NO) synthesis is increased in periodontal disease (PD), little is known about the possible sources of production by gingival tissues. In fact, gingival tissues from patients with periodontitis demonstrate greater levels of inducible nitric oxide (iNOS) expression than healthy tissue. Macrophages are the source of the iNOS expression, with endothelial cells also contributing. In the present study, our hypothesis has been that human gingival fibroblasts (HGF) also have the ability to produce NO. We have established for the first time that HGF express increased levels of iNOS and modulate NO synthesis in response to proinflammatory cytokines that act synergistically. NO production under basal conditions or following incubation with tumor necrosis factor (TNF-alpha), interleukin (IL)-1beta, and interferon (IFN)-gamma was assessed by measurement of stable NO metabolites, nitrite, and nitrate, in a microplate adaptation of the Griess assay. Total RNA was isolated from HGF for determination of iNOS mRNA levels. We have shown that NO production is elevated in HGF that are stimulated simultaneously by TNF-alpha, IL-1beta, and IFN-gamma. Northern blot analysis confirmed that the production of iNOS mRNA by HGF is upregulated in the presence of these cytokines. Addition of mercaptoethyl guanidine (MEG), a specific inhibitor of iNOS, profoundly reduced the production of NO in HGF. Non specific inhibitors of iNOS, L-NG-monomethyl arginine (L-NMMA), and L-arginine-methyl ester (L-NAME) had little or no effect on NO produced in HGF. These results suggest that elevated NO production could be important in the pathogenesis of PD, and also suggest the ability of an iNOS inhibitor to modulate the disease. Treatments with drugs to block the production of nitric oxide or block its effects might be therapeutically valuable.

Necrotic tumor cells oppositely affect nitric oxide production in tumor cell lines and macrophages

Nitric oxide (NO) is a highly reactive free radical with profound tumoricidal activity, produced by both macrophages and tumor cells. While it has been postulated that necrotic tumor cells can augment macrophage anti-tumor action, we investigated the effect of tumor cell necrosis on NO synthesis and viability of L929 fibrosarcoma and C6 astrocytoma cell lines. The presence of necrotic tumor cells dose-dependently reduced NO production in IFN-γ stimulated L929 cells, and rescued them from NO-dependent autotoxicity. This effect was mediated through soluble products, since it was completely preserved after blocking the contact between the necrotic and live cells. On the other hand, apoptotic tumor cells were unable to suppress IFN-γ-triggered NO release and subsequent decrease of cell respiration in L929 cultures. Similar results were obtained with C6 astrocytoma cell line. This down-regulation of NO synthesis in response to necrotic cell products was not specific for tumor cell lines, since necrotic tumor cells markedly suppressed NO production in cytokine-stimulated primary fibroblasts and astrocytes. In contrast, both murine and rat peritoneal macrophages readily increased their basal or IFN-γ-induced NO production when incubated with necrotic tumor cells. Taken together, these results suggest that tumor cell necrosis might promote or restrict tumor growth through suppression or enhancement of NO synthesis in tumor cells and macrophages, respectively, with net effect presumably depending on the extent of macrophage infiltration.

The aqueous extract of Rhodiola sachalinensis root enhances the expression of inducible nitric oxide synthase gene in RAW264.7 macrophages

In the present study, we examined the effects of the aqueous extract of Rhodiola sachalinensis root (RSE) on the expression of inducible nitric oxide (NO) synthase (iNOS) gene in RAW264.7 macrophages. RSE synergistically increased NO synthesis in interferon-γ-primed macrophages. Reverse transcriptase polymerase chain reaction and Northern blotting analysis revealed that RSE may provide a second triggering signal for the synergistic induction of iNOS mRNA expression. Thus, iNOS-mediated NO synthesis in response to RSE may be one mechanism whereby this herbal medicine elicits its therapeutic effects.

Role of nitric oxide in the control of burn perfusion

Vascular changes following deep skin burns are characterised by vasoconstriction and progressive ischemia. Nitric oxide (NO) has been shown to be a potent regulator of vascular smooth muscle tone and tissue perfusion. We assessed the importance of NO on post-burn skin perfusion in rats using laser Doppler. The present results show that neither the NO-synthase inhibitor, NG-nitro- l-arginine ( l-NNA) ( n=6) nor the NO precursor, l-arginine, significantly influenced skin perfusion in nonburned skin compared to saline-treated animals. In the area of full-thickness skin burn, neither l-arginine ( n=6) nor l-NNA ( n=6) had significant influence on post-burn perfusion compared to saline-treated controls ( n=6). Administration of l-NNA ( n=6) significantly impaired skin perfusion in the area adjacent to the contact burn representing a partial-thickness burn, while the NO precursor, l-arginine ( n=6) had no significant effect on burn perfusion as compared to saline-treated controls ( n=6). In conclusion, impairment of perfusion in a full thickness burn following administration of NO-synthase inhibitor suggests that nitric oxide is involved in the mechanisms responsible for maintaining adequate circulation post-burn. The lack of additional improvement of perfusion in response to l-arginine may suggest that NO synthesis in response to the thermal trauma is already at a peak.

Dietary Salt Loading Increases Nitric Oxide Synthesis in Transgenic Mice Overexpressing Sodium-Proton Exchanger

We studied the role of nitric oxide (NO) synthesis in amelioration of blood pressure elevation during dietary salt loading in transgenic mice overexpressing sodium proton exchanger. Systolic blood pressure rose after starting salt loading only in the high-salt group of transgenic mice. However, this elevation of blood pressure was not continued. Urinary excretion of inorganic nitrite and nitrate in the high-salt group of transgenic mice was significantly higher than in the high-salt group of control mice. These results suggest that increased NO synthesis in response to salt loading is one of the anti-hypertensive mechanisms in transgenic mice overexpressing sodium proton exchanger.

Timing of prostaglandin exposure is critical for the inhibition of LPS- or IFN-gamma-induced macrophage NO synthesis by PGE2.

Macrophage nitric oxide (NO) synthesis is an integral component of the host defense system. We have previously found that NO and prostaglandins interact in a variety of ways. NO modulates Kupffer cell prostaglandin E2 (PGE2) production and we have recently described the inhibitory effects of PGE2 on NO synthesis in both Kupffer cells and hepatocytes. Activated macrophages produce a number of prostaglandins but studies regarding the capacity of prostaglandins to regulate macrophage NO synthesis have yielded conflicting results. We found that exogenous PGE2 decreased lipopolysaccharide (LPS)-induced NO synthesis in murine resident peritoneal macrophages and in the RAW 264.7 murine macrophage cell line. PGE2 also suppressed NO synthesis in response to interferon-gamma (IFN-gamma) alone and a combination of LPS + IFN-gamma. Inhibition of endogenous PGE2 synthesis with indomethacin or ibuprofen had no effect on NO synthesis. PGE2 added with the activating stimulus was most effective. PGE2 lost the capacity to block NO synthesis if added more than 180 min after LPS. PGE2 decreased inducible NO synthesis (iNOS) mRNA and immunoreactive iNOS protein, consistent with the hypothesis that exogenous PGE2 inhibits macrophage iNOS expression but that the inhibition depends on the time and concentration of prostaglandin exposure.

Alternative Splicing of Human Inducible Nitric-Oxide Synthase mRNA: TISSUE-SPECIFIC REGULATION AND INDUCTION BY CYTOKINES

Human inducible nitric-oxide synthase (iNOS) is responsible for nitric oxide synthesis in response to inflammatory mediators. The human iNOS gene, containing 26 exons, encodes a protein of 131 kDa. This study was aimed at investigating the presence of alternative splicing of human iNOS mRNA. Total RNA from human alveolar macrophages, nasal and bronchial epithelial cells, and several human tissues was transcribed to cDNA and analyzed using polymerase chain reaction with specific primers for segmental analysis of the iNOS gene. Four sites of alternative splicing were identified by sequence analysis; these included deletion of: (i) exon 5; (ii) exons 8 and 9; (iii) exons 9, 10, and 11; and (iv) exons 15 and 16. The deduced amino acid sequences of the novel iNOS cDNAs predict one truncated protein (resulting from exon 5 deletion) and three iNOS proteins with in-frame deletions. Southern analyses of polymerase chain reaction products were consistent with tissue-specific regulation of alternative splicing. In cultured cells, iNOS induction by cytokines and lipopolysaccharide was associated with an increase in alternatively spliced mRNA transcripts. Because iNOS is active as a dimer, the novel forms of alternatively spliced iNOS may be involved in regulation of nitric oxide synthesis.

Induction of nitric oxide synthesis in murine macrophages by Helicobacter pylori.

Intact Helicobacter pylori cells, as well as cellular components, stimulated nitric oxide (NO) synthesis in an in vitro murine macrophage system by the L-arginine-nitric oxide pathway. Macrophage-mediated NO formation was dependent on the presence of H. pylori and exhibited a dose-dependent increase at H. pylori concentrations between 10(6) and 5 and 10(7) cells/ml. H. pylori mediated NO synthesis also required L-arginine and was inhibited by NG-monomethyl-L-arginine (NMMA), a selective inhibitor of nitric oxide synthase. NO synthesis was induced by whole H. pylori cells. H. pylori media filtrate, extracted membrane proteins, and H. pylori lipopolysaccharide (LPS). Maximal NO synthesis was induced by viable H. pylori cells with media filtrate and membrane protein extracts inducing significant NO responses. NO stimulation by media filtrate and membrane protein extracts support secreted H. pylori products as potential activators of inflammatory cell NO synthesis in vivo. NO synthesis in response to H. pylori suggests that chronic H. pylori infection may increase endogenous formation of NO. Elevated NO exposure may represent an etiologic factor explaining the epidemiologic association between long-term H. pylori infection and gastric cancer.