High Levels Of Nitric Oxide Research Articles

Synthesis and evaluation of nitric oxide-releasing derivatives of farnesylthiosalicylic acid as anti-tumor agents

Novel furoxan-based nitric oxide (NO)-releasing derivatives ( 11a– p) of farnesylthiosalicylic acid (FTA) were synthesized. Compounds 11d, 11f, 11k, and 11m– o displayed anti-tumor activities superior to FTA and sorafenib in most cancer cells tested. Analysis of six compounds revealed that 11d, 11f, 11n, 11o, and 11p, but not 11a that had low anti-tumor activity, produced high levels of NO, associated with their strong anti-tumor activity. Furthermore, the anti-tumor activity of 11f was partially mimicked by the furoxan moiety, but reduced by pre-treatment with hemoglobin. Importantly, treatment with 11f inhibited Ras-related signaling in cancer cells. Apparently, the high anti-tumor activity of 11f was attributed to the synergic effect of high levels of NO production and inhibition of Ras-related signaling in cancer cells. Our findings suggest that the furoxan/FTA hybrids may hold greater promise as therapeutic agents for the intervention of human cancers.

Leukocyte adhesion and its contribution on the local oxidative and nitrosative stresses in microcirculation

In diabetes, a number of adhesion molecules are over expressed leading to leukocyte adhesion and subsequent transmigration in the vessel wall. This is one of the early stages for vascular inflammation. These adherent or transmigrating leukocytes release high levels of nitric oxide (NO) and superoxide thereby increasing local oxidative and nitrosative stresses. In the current study, we developed a computational model to understand the effect of leukocyte adhesion and transmigration on the profiles of oxidative and nitrosative stress inducing free radicals NO, superoxide and peroxynitrite in a blood vessel. The results are presented for three cases: 1) A normal case with relatively low amount of leukocyte adhesion, 2) An oxidative stress case with leukocyte adhesion and transmigration and 3) A severe oxidative stress case with reduced SOD levels. The results indicate a significant increase in peroxynitrite concentration and decrease in NO concentration for the severe oxidative stress case with respect to the normal case. Decreasing NO formation could impair vasodilation whereas increasing peroxynitrite formation may initiate oxidation of sulfydryl groups in proteins and lipids and nitration of amino acids resulting in damage to the endothelial and smooth muscle cells of the blood vessel, which can contribute to the occurrence of cardiovascular related diseases. Supported by NIH R01 HL084337.

Human gastrointestinal stem cell (hGISC) derived non-transformed epithelial cells but not malignant colonic adenocarcinoma cell line HT29 respond to combined innate and adaptive immunostimulation by producing high levels of nitric oxide. (90.14)

Abstract Gastrointestinal (GI) inflammation is a global health burden. Yet the mechanism of GI inflammation remains undefined. Among proposed mechanisms, increased nitric oxide (NO) production by intestinal epithelial cells (EC) is thought to play a crucial role in inflammatory conditions such as IBD. However, the regulation of NO production in human intestinal epithelium are undefined, thus treatments for these diseases remain inadequate. Therefore, to gain insight about NO production regulation by intestinal EC, we studied the effects of innate (LPS) and adaptive (IFNγ) immunostimulatory agents on a novel hGISC cell system derived non-transformed primary epithelial cells (EC) and compared it to a colonic adenocarcinoma EC line HT29. A macrophage cell line MHS served as a positive control. Cells were cultured with/without LPS (1μg/ml), IFNγ (200 u/ml), or in combination for 24 hours. Culture supernatants were analyzed for NO content by Griess reaction assay. Our results show that neither LPS nor IFNγ alone affect NO production by intestinal ECs. However, NO production by hGISC derived primary ECs was dramatically increased (comparable to the NO level seen in MHS cells) when LPS and IFNγ were added in combination. In contrast, malignant HT29 cells do not show any difference in NO synthesis even when LPS (innate stimuli) and IFNγ (adaptive stimuli) were combined. These findings may help understand the intestinal inflammatory disease mechanisms and development of new treatments.

A Variant of the Endothelial Nitric Oxide Synthase Gene (NOS3) Associated with AMS Susceptibility Is Less Common in the Quechua, a High Altitude Native Population

Endothelial nitric oxide synthase (eNOS) is a vascular enzyme that produces nitric oxide, a transient signaling molecule that by vasodilatation regulates blood flow and pressure. Nitric oxide is believed to play roles in both short-term acclimatization and long-term evolutionary adaptation to environmental hypoxia. Several laboratories, including ours, have shown that variants in NOS3 (the gene encoding eNOS) are overrepresented in individuals with altitude-related illnesses such as high altitude pulmonary edema (HAPE) and acute mountain sickness (AMS), suggesting that NOS3 genotypes contribute to altitude tolerance. To further test our hypothesis that the G allele at the G894T polymorphism in NOS3 (dbSNP number: rs1799983; protein polymorphism Glu298Asp) is beneficial in hypoxic environments, we compared frequencies of this allele in an altitude-adapted Amerindian population, Quechua of the Andean altiplano, with those in a lowland Amerindian population, Maya of the Yucatan Peninsula. While common in both populations, the G allele was significantly more frequent in the highlanders. Taken together, our data suggest that this variant in NOS3, which has been previously associated with higher levels of nitric oxide, contributes to both acclimatization and adaptation to altitude.

Relationship among circulating hemoglobin, nitric oxide synthase activities and angiogenic poise in red- and white-blooded Antarctic notothenioid fishes

Nitric oxide (NO)-mediated angiogenesis may play a role in establishing dense retinal vasculatures of Antarctic hemoglobinless icefishes (suborder: Notothenioidei). We hypothesized that loss of hemoglobin (Hb) leads to elevation in [NO] due to decreased degradation of the compound when the NO-scavenger Hb is absent, thereby inducing vascular growth. We found that total mass of NO metabolites, nitrite plus nitrate (NOx), in plasma is greater in icefishes than in red-blooded notothenioids [e.g. C. aceratus (Hb−), 22.7±2.9μM; N. coriiceps (Hb+), 14.7±1.7μM], suggesting a higher NO load in hemoglobinless animals. High NO levels do not appear to be a result of greater NO synthesis; we consistently measured lower activities of the enzyme catalyzing NO production, nitric oxide synthase, in tissues of icefishes than in Hb-expressing notothenioids [e.g. 96±10 and 216±39 pmol(min g wet wt)−1 in brain tissue of C. aceratus (Hb−) and G. gibberifrons (Hb+), respectively]. Levels of mRNA for hypoxia-induced (HIF-1α and PHD2) and angiogenic genes (VEGF) were similar in red- and white-blooded species, indicating that vascular maintenance in adult animals does not require differences in angiogenic tone. This does not preclude a cause-and-effect relationship between absence of Hb and NO-mediated angiogenesis during earlier ontogenetic stages of icefishes.

Sodium nitroprusside induces autophagic cell death in glutathione‐depleted osteoblasts

Previous studies reported that high levels of nitric oxide (NO) induce apoptotic cell death in osteoblasts. We examined molecular mechanisms of cytotoxic injury induced by sodium nitroprusside (SNP), a NO donor, in both glutathione (GSH)-depleted and control U2-OS osteoblasts. Cell viability was reduced by much lower effective concentrations of SNP in GSH-depleted cells compared to normal cells. The data suggest that the level of intracellular GSH is critical in SNP-induced cell death processes of osteoblasts. The level of oxidative stress due to SNP treatments doubled in GSH-depleted cells when measured with fluorochrome H2DCFDA. Pretreatment with the NO scavenger PTIO preserved the viability of cells treated with SNP. Viability of cells treated with SNP was recovered by pretreatment with Wortmannin, an autophagy inhibitor, but not by pretreatment with zVAD-fmk, a pan-specific caspase inhibitor. Large increases of LC3-II were shown by immunoblot analysis of the SNP-treated cells, and the increase was blocked by pretreatment with PTIO or Wortmannin; this implies that under GSH-depleted conditions SNP induces different molecular signaling that lead to autophagic cell death. The ultrastructural morphology of SNP-treated cells in transmission electron microscopy showed numerous autophagic vacuoles. These data suggest NO produces oxidative stress and cellular damage that culminate in autophagic cell death of GSH-depleted osteoblasts.

Sodium nitroprusside induces autophagic cell death in glutathione-depleted osteoblasts

Previous studies reported that high levels of nitric oxide (NO) induce apoptotic cell death in osteoblasts. We examined molecular mechanisms of cytotoxic injury induced by sodium nitroprusside (SNP), a NO donor, in both glutathione (GSH)-depleted and control U2-OS osteoblasts. Cell viability was reduced by much lower effective concentrations of SNP in GSH-depleted cells compared to normal cells. The data suggest that the level of intracellular GSH is critical in SNP-induced cell death processes of osteoblasts. The level of oxidative stress due to SNP treatments doubled in GSH-depleted cells when measured with fluorochrome H2DCFDA. Pretreatment with the NO scavenger PTIO preserved the viability of cells treated with SNP. Viability of cells treated with SNP was recovered by pretreatment with Wortmannin, an autophagy inhibitor, but not by pretreatment with zVAD-fmk, a pan-specific caspase inhibitor. Large increases of LC3-II were shown by immunoblot analysis of the SNP-treated cells, and the increase was blocked by pretreatment with PTIO or Wortmannin; this implies that under GSH-depleted conditions SNP induces different molecular signalings that lead to autophagic cell death. The ultrastructural morphology of SNP-treated cells in transmission electron microscopy showed numerous autophagic vacuoles. These data suggest NO produces oxidative stress and cellular damage that culminate in autophagic cell death of GSH-depleted osteoblasts.

Neurocysticercosis, IgG immunoglobulins, and nitric oxide

This study evaluated the role of parasite load and nitric oxide on IgG levels in neurocysticercosis. Total serum IgG, IgG antibodies specific for cysticercus antigens, and nitric oxide were compared between 85 neurocysticercosis patients, 65 with solitary cysts and 20 with multiple cysts, and 13 normal healthy controls. Sixty-six percent of patients were seropositive for cysticercus IgG antibodies. Among seropositive patients, IgG levels did not differ between those infected with multiple or solitary cysts whose serum nitric oxide levels were low (<40 nmol/ml). Among seropositive solitary cyst infected patients, IgG levels were significantly higher in those whose serum nitric oxide was low compared to those with high nitric oxide levels (p < 0.001). IgG levels were significantly higher in patients with multiple compared to single cyst infections among those negative for cysticercus antibodies (p < 0.001). Parasite load and nitric oxide modulated IgG production in neurocysticercosis. IgG levels were not determined by the number of infecting cysts in seropositive patients who did not mount a nitric oxide response. IgG production correlated to parasite load in patients negative for cysticercus antibodies.

The changes in the endothelial expression of cell adhesion molecules and iNOS in the vessel wall after the short-term administration of simvastatin in rabbit model of atherosclerosis

Cell adhesion molecules P-selectin, VCAM-1 and ICAM-1 play an important role in the pathogenesis of atherosclerosis. High levels of nitric oxide (NO) produced by inducible NO synthase (iNOS) have been associated with atherosclerotic processes. Simvastatin is an HMG-CoA reductase inhibitor responsible for many clinical benefits. The aim of this study was to detect and quantify changes in endothelial expression of P-selectin, VCAM-1, ICAM-1 and iNOS in the vessel wall after the shortterm administration of simvastatin in a rabbit model of atherosclerosis. Eighteen New Zealand White rabbits were randomly divided into three groups (n=6). In the cholesterol group, rabbits consumed an atherogenic diet (0.4% cholesterol) for eight weeks. In the simvastatin group, rabbits consumed an atherogenic diet for six weeks and then consumed an atherogenic diet supplemented with simvastatin (10 mg kg(-1)) for two weeks. Biochemical analysis showed that administration of simvastatin led to an almost two-fold lowering of the total serum cholesterol, VLDL, LDL and HDL, but not triglycerides, compared with the cholesterol-fed rabbits only. Stereological analysis of the immunohistochemical staining revealed that administration of simvastatin (10 mg kg(-1) daily) in an atherogenic diet decreased the endothelial expression of P-selectin, ICAM-1 and iNOS in both aortic arch and carotid artery compared with the cholesterol fed-rabbits only. We conclude that simvastatin has beneficial effects on endothelial function by decreasing expression of P-selectin, ICAM-1 and iNOS in endothelial cells in the very early stages of atherogenesis.

Cigarette smoke enhances -defensin 2 expression in rat airways via nuclear factor- B activation

β-defensin 2 (BD-2), an antimicrobial peptide, participates in airway defence. Cigarette smoke (CS) is a major risk factor for the development of chronic obstructive pulmonary disease. This study mainly aims to investigate the effect of CS on rat BD-2 (rBD-2) expression in rat airways. Rats were exposed to CS and treated with caffeic acid phenethyl ester (CAPE), a nuclear factor (NF)-κB inhibitor, or astragaloside IV (AS-IV), an active ingredient of Astragalus mongholicus. Besides the analysis of bronchoalveolar lavage fluid (BALF) and histological changes after CS exposure, rBD-2 expression was investigated with immunohistochemistry, reverse transcription PCR and ELISA. Total glutathione and nitric oxide (NO) levels in rat lungs were also detected. CS exposure markedly increased rBD-2 immunoreactivity, as well as rBD-2 mRNA and protein levels in rat airways, which were inhibited by CAPE treatment. Moreover, associated airway inflammation induced by CS was demonstrated by histological changes, increased cell counts and pro-inflammatory cytokines in BALF, and NF-κB activation and high levels of total glutathione and NO, which were all reversed by AS-IV in a dose-dependent fashion. In conclusion, CS exposure induces rBD-2 expression in rat airways via a NF-κB-dependent pathway, and AS-IV attenuates CS-induced airway inflammation due to its anti-inflammatory and antioxidant properties, at least partly through NF-κB inactivation.

Mechanism, treatment, and evaluation of obesity-induced insulin resistance and type 2 diabetes

Obesity is a major cause of insulin resistance and type 2 diabetes. The altered glucose homeostasis is caused by faulty insulin signal transduction, which results in decreased glucose uptake by the muscle, altered lipogenesis, and increased glucose output by the liver. The etiology of this derangement in insulin signaling is related to a chronic inflammatory state, leading to the induction of inducible nitric oxide synthase and release of high levels of nitric oxide and reactive nitrogen species, which together cause posttranslational modifications in the signaling proteins. There are substantial differences in the molecular mechanisms of insulin resistance in muscle versus liver. Hormones and cytokines from adipocytes can enhance or inhibit both glycemic sensing and insulin signaling. The role of the central nervous system in glucose homeostasis also has been well established. Multi-pronged therapies aimed at rectifying obesity induced anomalies in both central nervous system and peripheral tissues may prove to be beneficial. The golden standard method to evaluate the insulin sensitivity is hyperinsulinemic euglycemic clamp.

Distinct Th1, Th2 and Treg cytokines balance in chronic periapical granulomas and radicular cysts

Periapical lesions are a host response that involves immune reaction to prevent dissemination of bacteria from an infected root canal. The purpose of this study was to evaluate the levels of nitric oxide (NO), IL-4, TGF-beta, tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in chronic periapical lesions and to determine their possible association with clinical and radiographic parameters. Seventeen human radicular cysts and 30 periapical granulomas were used in this study. Cytokines and NO were assessed by enzyme-linked immunosorbent assay and by the Griess reaction respectively confirmed by immunohistochemical. TNF-alpha and IFN-gamma were detected in 10% of granulomas and in 41.2% and 70% of radicular cysts. IL-4 was reactive in 24% of cysts, and TGF-beta was positive in all samples. Patients with tenderness showed significantly higher levels of IFN-gamma and IL-4 (P < 0.05). Swelling was associated with high levels of TNF-alpha, IFN-gamma, and IL-4 (P < 0.05). Lesions presenting bone resorption were associated with high levels of NO (P < 0.05). Periapical granulomas display a regulatory environment characterized by high TGF-beta and low inflammatory cytokine levels, while radicular cysts has mist Th1 and Th2 inflammatory reaction with the presence of IFN-gamma, TNF-alpha, and IL-4.

Nitrosative Stress as a Mediator of Apoptosis: Implications for Cancer Therapy

Nitric oxide (NO) is now recognised as one of the most important molecules influencing the development, progression and treatment of cancer. A key component of its action is as a negative and positive regulator of apoptosis. Broadly, constitutive levels of NO(nM), are capable of inhibiting numerous signalling pathways in both normal and cancer cells. These include soluble guanylate cyclase, leading to reduced Ca++ signalling, inhibition of caspases and scavenging of reactive oxygen species, all of which promote survival signalling. High concentrations (M-mM) on the other hand, generally promote apoptosis. Pathways involving cGMP, cytochrome c release, mitogen activated kinases, ceramide and poly(ADP)ribose polymerase have all been implicated. The role of p53 in NO-induced cell death has been widely studied. In many cell types p53-dependent signalling is involved, while in others, apoptosis occurs in the absence of functional p53. There is also evidence that the tumour microenvironment, where low oxygen and glucose levels prevail, enhances cell death signalling by NO and peroxynitrite, thus tumours may be more sensitive to high levels of NO than their normal tissue counterpart. The cytotoxicity of NO has been studied directly in many tumour models, both in vitro and in vivo. In all cases, high concentrations of NO, generated by donor drugs or by iNOS gene transfer caused extensive tumour cell death, which was enhanced by the ability of NO to diffuse readily from its source of generation to most cells within tumours. NO was also a very effective enhancer of conventional chemo- and radiotherapy. Thus, NO therapy has great potential to improve the treatment of cancer.

Nitric Oxide is Negatively Correlated to Pain during Acute Inflammation

BackgroundThe role that nitric oxide (NO) plays in modulating pain in the periphery is unclear. We show here, the results of two independent clinical studies (microdialysis and gene expression studies) and a pilot dose finding study (glyceryl trinitrate study), to study the role of NO in the early phase of acute inflammatory pain following oral surgery. The effect of ketorolac on NO production and nitric oxide synthase (NOS) gene expression was also studied.ResultsMicrodialysis samples showed significantly higher levels of NO at the first 100 min compared to the last 80 minutes in the placebo treated group. In the ketorolac group, on the other hand, NO levels gradually decreased over the first 60 min but were similar to placebo over the later 100-180 min, with no significant change in NO level over time. The levels of NO were negatively correlated to pain intensity scores. Local infusion of the NO donor glyceryl trinitrate at the site of surgery, showed a small analgesic effect that did not reach statistical significance in the sample size used. While the gene expression of iNOS and eNOS were not up-regulated, 3 hours after surgery, nNOS was downregulated in both treatment groups and eNOS gene expression was significantly lower in the ketorolac group compared to the placebo group. Further, there was a positive correlation between the change in gene expression of nNOS and eNOS in the placebo goup but not in the ketorolac group.ConclusionWe suggest that at this early stage of inflammatory pain in man, NO is analgesic in the periphery. Further, ketorolac down-regulates eNOS gene expression.

Comparison of hyaluronan effects among normal, osteoarthritis, and rheumatoid arthritis cartilages stimulated with fibronectin fragment

High molecular weight hyaluronan (HA) is widely used in the treatment of osteoarthritis (OA) and rheumatoid arthritis (RA) by intra-articular injection. However, comparative studies of HA actions on catalytically activated cartilages in different pathologic conditions have rarely been investigated. This study was aimed to compare the inhibitory effects of HA on nitric oxide (NO) production by COOH-terminal heparin-binding fibronectin fragment (HBFN-f) between normal and diseased cartilages. When articular cartilage explants from normal, OA, or RA joints were incubated with HBFN-f, the RA and OA cartilages produced higher levels of NO compared with normal cartilage. Pretreatment with 2700 kDa HA resulted in significant suppression of HBFN-f-stimulated NO production in OA and RA cartilages. While CD44 was up-regulated in OA and RA cartilages, anti-CD44 antibody reversed HA inhibition of HBFN-f action in those cartilages. The present results clearly demonstrated that HA blocked HBFN-f actions in OA and RA cartilages through interaction with CD44. HA, which targets CD44 highly expressed on OA and RA chondrocytes, could suppress catabolic actions by fibronectin fragments like HBFN-f in diseased cartilage.

Effect of acute exercise on the levels of salivary cortisol, tumor necrosis factor-α and nitric oxide

The aim of the present study was to assess the levels of salivary cortisol, tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) before, during and after acute exercise. Acute exercise was induced using a standard treadmill test with Bruce protocol in ten physically active male participants. Unstimulated saliva was collected before, during and after exercise. The levels of salivary cortisol and TNF-alpha were assessed by enzyme immunoassays. Salivary NO was determined by the Griess reagent. The results showed that both salivary cortisol and TNF-alpha increased and peaked at 14 min during exercise and then decreased. The levels of NO were increased up to 1 h after exercise and subsequently lowered after 24 h. The results of the present study suggest that acute exercise may induce high levels of salivary cortisol, TNF-alpha and NO.

Long-Term Adaptation of Lung Tumor Cell Lines with Increasing Concentrations of Nitric Oxide Donor

The free radical nitric oxide (NO) is known to play an important role in the biology of human cancers, including lung cancer. However, it is still not clear how elevated amounts of nitric oxide affect tumor development and propagation. Herein we develop an in vitro model system to study these effects in lung tumor cells. Two cell lines—one human lung adenocarcinoma (A549) and one mouse adenocarcinoma (LP07) cell line—were adaptively grown in increasing concentrations of the NO donor DETA-NONOate over several months. Both cell lines were successfully adapted to high levels of NO (HNO). Experiments validated the adaptation occurred as a result of the exogenous NO produced by the DETA-NONOate, and was not merely a response to the chemical composition of DETA-NONOate. No morphological differences were observed between cells that were adapted to the HNO and cells which did not undergo the adaptation process (i.e., cells). Parent cells were unable to survive when placed directly in media containing high levels of DETA-NONOate, suggesting that the adapted cells underwent a biological change enabling them to survive and grow in a HNO environment. The adapted cells were found to grow faster than the parent cells under both normal growth conditions and stressful growth conditions (serum-less media, growth on soft agar) even when the DETA-NONOate was removed from the HNO culture media. These adapted cell lines can serve as a novel tool for use in future experiments designed to better understand the role nitric oxide plays in lung cancer.

The NO donor DETA‐NONOate reversibly activates an inward current in neurones and is not mediated by the released nitric oxide

It has been previously shown that high levels of nitric oxide (NO), from NO donors, kill neurones, but the mechanisms are unclear. The effects of NO donors on the electrical properties of rat cultured cerebellar granule cells (CGC neurones) were investigated using the whole-cell patch-clamp technique. The NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate or NOC-18) caused a rapid, persistent, but fully reversible inward current that was associated with an increase in baseline noise and was concentration dependent (100 microM-10 mM). The response to 3 mM DETA-NONOate was completely inhibited by 1 mM gadolinium, but not by NO scavengers (1 mM haemoglobin or 1 mM PTIO) or glutamate receptor antagonists (10 microM MK-801 or 60 microM CNQX). Application of decomposed 3 mM DETA-NONOate or 3 mM nitrite had no effect. In contrast, the NO donor S-nitrosoglutathione (GSNO) caused a rapid, persistent, but fully reversible outward current that was also concentration dependent (1-10 mM). The 3 mM GSNO response was unaltered by NO scavengers, glutamate antagonists or gadolinium, but was mimicked by decomposed 3 mM GSNO and 3 mM oxidized glutathione. These results suggest that DETA-NONOate directly activates cation-selective channels, causing an inward current in CGCs. In contrast, GSNO causes an outward current in these cells. Some of the effects of these NO donors are independent of NO, and thus caution is required in interpreting results when using high concentrations of these compounds.

The Arabidopsis PARAQUAT RESISTANT2 gene encodes an S-nitrosoglutathione reductase that is a key regulator of cell death

Metabolism of S-nitrosoglutathione (GSNO), a major biologically active nitric oxide (NO) species, is catalyzed by the evolutionally conserved GSNO reductase (GSNOR). Previous studies showed that the Arabidopsis GSNOR1/HOT5 gene regulates salicylic acid signaling and thermotolerance by modulating the intracellular S-nitrosothiol level. Here, we report the characterization of the Arabidopsis paraquat resistant2-1 (par2-1) mutant that shows an anti-cell death phenotype. The production of superoxide in par2-1 is comparable to that of wild-type plants when treated by paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride), suggesting that PAR2 acts downstream of superoxide to regulate cell death. PAR2, identified by positional cloning, is shown to be identical to GSNOR1/HOT5. The par2-1 mutant carries a missense mutation in a highly conserved glycine, which renders the mutant protein unstable. Compared to wild type, par2-1 mutant has a higher NO level, as revealed by staining with 4,5-diaminofluorescein diacetate. Consistent with this result, wild-type plants treated with an NO donor display resistance to paraquat. Interestingly, the GSNOR1/HOT5/PAR2 protein level, other than its steady-state mRNA level, is induced by paraquat, but is reduced by NO donors. Taken together, these results suggest that GSNOR1/HOT5/PAR2 plays an important role in regulating cell death in plant cells through modulating intracellular NO level.

A Novel Pathway of Insulin Sensitivity in Chromogranin A Null Mice: A CRUCIAL ROLE FOR PANCREASTATIN IN GLUCOSE HOMEOSTASIS

Chromogranin A (CHGA/Chga), a proprotein, widely distributed in endocrine and neuroendocrine tissues (not expressed in muscle, liver, and adipose tissues), generates at least four bioactive peptides. One of those peptides, pancreastatin (PST), has been reported to interfere with insulin action. We generated a Chga knock-out (KO) mouse by the targeted deletion of the Chga gene in neuroendocrine tissues. KO mice displayed hypertension, higher plasma catecholamine, and adipokine levels and lower IL-6 and lipid levels compared with wild type mice. Liver glycogen content was elevated, but the nitric oxide (NO) level was diminished. Glucose, insulin, and pyruvate tolerance tests and hyperinsulinemic-euglycemic clamp studies established increased insulin sensitivity in liver but decreased glucose disposal in muscle. Despite higher catecholamine and ketone body levels and muscle insulin resistance, KO mice maintained euglycemia due to increased liver insulin sensitivity. Suppressed mRNA abundance of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (G6Pase) in KO mice further support this conclusion. PST administration in KO mice stimulated phosphoenolpyruvate carboxykinase and G6Pase mRNA abundance and raised the blood glucose level. In liver cells transfected with G6Pase promoter, PST caused transcriptional activation in a protein kinase C (PKC)- and NO synthase-dependent manner. Thus, PST action may be mediated by suppressing IRS1/2-phosphatidylinositol 3-kinase-Akt-FOXO-1 signaling and insulin-induced maturation of SREBP1c by PKC and a high level of NO. The combined effects of conventional PKC and endothelial NO synthase activation by PST can suppress insulin signaling. The rise in blood PST level with age and in diabetes suggests that PST is a negative regulator of insulin sensitivity and glucose homeostasis.