Excessive Nitric Oxide Research Articles

Comparative proteomic analysis on wild type and nitric oxide-overproducing mutant (nox1) of Arabidopsis thaliana

Nitric oxide (NO) as a ubiquitous signal molecule plays an important role in plant development and growth. Here, we compared the proteomic changes between NO-overproducing mutant (nox1) and wild-type (WT) of Arabidopsis thaliana using two-dimensional electrophoresis coupled with MALDI-TOF MS. We successfully identified 59 differentially expressed proteins in nox1 mutant, which are predicted to play potential roles in specific cellular processes, such as post-translational modification, energy production and conversion, metabolism, transcription and signal transduction, cell rescue and defense, development and differentiation. Particularly, expression levels of five anti-oxidative enzymes were altered by the mutation; and assays of their respective enzymatic activities indicated an enhanced level of oxidative stress in nox1 mutant. Finally, some important proteins were further confirmed at transcriptional level using quantitative real-time PCR revealing the systemic changes between WT and nox1. The result suggests that obvious morphological changes in the nox1 mutant may be regulated by different mechanisms and factors, while excess endogenous NO maybe one of the possible reasons.

Hyperuricemia Causes Pancreatic β-Cell Death and Dysfunction through NF-κB Signaling Pathway

Accumulating clinical evidence suggests that hyperuricemia is associated with an increased risk of type 2 diabetes. However, it is still unclear whether elevated levels of uric acid can cause direct injury of pancreatic β-cells. In this study, we examined the effects of uric acid on β-cell viability and function. Uric acid solution or normal saline was administered intraperitoneally to mice daily for 4 weeks. Uric acid-treated mice exhibited significantly impaired glucose tolerance and lower insulin levels in response to glucose challenge than did control mice. However, there were no significant differences in insulin sensitivity between the two groups. In comparison to the islets in control mice, the islets in the uric acid–treated mice were markedly smaller in size and contained less insulin. Treatment of β-cells in vitro with uric acid activated the NF-κB signaling pathway through IκBα phosphorylation, resulting in upregulated inducible nitric oxide synthase (iNOS) expression and excessive nitric oxide (NO) production. Uric acid treatment also increased apoptosis and downregulated Bcl-2 expression in Min6 cells. In addition, a reduction in insulin secretion under glucose challenge was observed in the uric acid–treated mouse islets. These deleterious effects of uric acid on pancreatic β-cells were attenuated by benzbromarone, an inhibitor of uric acid transporters, NOS inhibitor L-NMMA, and Bay 11–7082, an NF-κB inhibitor. Further investigation indicated that uric acid suppressed levels of MafA protein through enhancing its degradation. Collectively, our data suggested that an elevated level of uric acid causes β-cell injury via the NF-κB-iNOS-NO signaling axis.

Partial neuroprotection by nNOS inhibition during profound asphyxia in preterm fetal sheep

Preterm brain injury is partly associated with hypoxia–ischemia starting before birth. Excessive nitric oxide production during HI may cause nitrosative stress, leading to cell membrane and mitochondrial damage. We therefore tested the hypothesis that therapy with a new, selective neuronal nitric oxide synthase (nNOS) inhibitor, JI-10 (0.022mg/kg bolus, n=8), given 30min before 25min of complete umbilical cord occlusion was protective in preterm fetal sheep at 101–104day gestation (term is 147days), compared to saline (n=8). JI-10 had no effect on fetal blood pressure, heart rate, carotid and femoral blood flow, total EEG power, nuchal activity, temperature or intracerebral oxygenation on near-infrared spectroscopy during or after occlusion. JI-10 was associated with later onset of post-asphyxial seizures compared with saline (p<0.05), and attenuation of the subsequent progressive loss of cytochrome oxidase (p<0.05). After 7days recovery, JI-10 was associated with improved neuronal survival in the caudate nucleus (p<0.05), but not the putamen or hippocampus, and more CNPase positive oligodendrocytes in the periventricular white matter (p<0.05). In conclusion, prophylactic nNOS inhibition before profound asphyxia was associated with delayed onset of seizures, slower decline of cytochrome oxidase and partial white and gray matter protection, consistent with protection of mitochondrial function.

Critical role of nitric oxide in the modulation of prepulse inhibition in Swiss mice

Nitric oxide (NO) modulates the dopamine uptake and release processes and appears to be implicated in dopamine-related pathologies, such as schizophrenia. However, it is unclear whether there is excess or deficient NO synthesis in schizophrenia pathophysiology. Analyses of the intracellular pathways downstream of NO system activation have identified the cyclic nucleotide cyclic guanosine monophosphate (cGMP) as a possible target for drug development. Defects in the sensorimotor gating of the neural mechanism underlying the integration and processing of sensory information have been detected across species through prepulse inhibition (PPI). The aim of this study was to investigate the effects of NO/cGMP increase on sensorimotor gating modulation during dopamine hyperfunction. Mice were treated with NO donors and subjected to the PPI test. Treatment with the NO donor sodium nitroprusside was preceded by pretreatment with a soluble guanylate cyclase (sGC) inhibitor. Additionally, the mice were treated with NO donors and phosphodiesterases inhibitors prior to amphetamine treatment. Pretreatment with the NO donors enhanced the PPI response and attenuated the amphetamine-disruptive effects on the PPI. The sGC inhibitor did not modify the sodium nitroprusside effects. Additionally, the cGMP increase induced by a specific phosphodiesterase inhibitor did not modify the amphetamine-disruptive effect. This study provides the first demonstration that an increase in NO can improve the PPI response and block the amphetamine-disruptive effects on the PPI response. Our data are consistent with recent clinical results. However, these effects do not appear to be related to an increase in cGMP levels, and further investigation is thus required.

Relationship between alveolar nitric oxide concentration in exhaled air and small airway function in COPD

Nitrative stress is thought to be involved in the inflammatory process in COPD airways, and the alveolar nitric oxide concentration (CAlv) has been reported to be increased. However, the CAlv levels are also regulated by gas diffusion at alveolar sites. The aim of the study was to assess the relationship between the CAlv and pulmonary function in COPD patients, while taking into account the lung diffusion capacity. Twenty stable COPD patients (GOLD stage1/2/3/4 = 6/8/6/0) and 16 healthy subjects took part in this cross-sectional study. Fractional exhaled nitric oxide (FENO), CAlv, and pulmonary functions were measured. Pulmonary function, including single nitrogen washout curve (dN2) and diffusion capacity for carbon monoxide (DLCO), was also evaluated in patients with COPD. The mean FENO levels (20.7 ppb versus 16.3 ppb, p < 0.05) and the mean CAlv levels (6.4 ppb versus 4.2 ppb, p < 0.01) in COPD patients were significantly increased compared to those in HS. The CAlv level in COPD was significantly correlated with dN2, %DLCO/alveolar volume (VA). Using the standard entry method of multivariate analysis to adjust for dN2 and %DLCO/VA, dN2 (β = 0.54, p = 0.005) and %DLCO/VA (β = −0.44, p = 0.018) still showed significant correlations with the CAlv levels. These results suggest that the CAlv could be a useful marker for the small airway dysfunction in COPD. Airway inflammation, including excess nitric oxide generation in the peripheral airways, might be related to the pathophysiology of COPD.

TXNIP Deficiency Exacerbates Endotoxic Shock via the Induction of Excessive Nitric Oxide Synthesis

Thioredoxin-interacting protein (TXNIP) has multiple functions, including tumor suppression and involvement in cell proliferation and apoptosis. However, its role in the inflammatory process remains unclear. In this report, we demonstrate that Txnip−/− mice are significantly more susceptible to lipopolysaccharide (LPS)-induced endotoxic shock. In response to LPS, Txnip−/− macrophages produced significantly higher levels of nitric oxide (NO) and inducible nitric oxide synthase (iNOS), and an iNOS inhibitor rescued Txnip−/− mice from endotoxic shock-induced death, demonstrating that NO is a major factor in TXNIP-mediated endotoxic shock. This susceptibility phenotype of Txnip−/− mice occurred despite reduced IL-1β secretion due to increased S-nitrosylation of NLRP3 compared to wild-type controls. Taken together, these data demonstrate that TXNIP is a novel molecule that links NO synthesis and NLRP3 inflammasome activation during endotoxic shock.

Inhibition of autophagy and glycolysis by nitric oxide during hypoxia–reoxygenation impairs cellular bioenergetics and promotes cell death in primary neurons

Excessive nitric oxide (NO) production is known to damage mitochondrial proteins and the autophagy repair pathway and so can potentially contribute to neurotoxicity. Accordingly, we hypothesized that protection against protein damage from reactive oxygen and nitrogen species under conditions of low oxygen by the autophagy pathway in neurons would be impaired by NO and enhance bioenergetic dysfunction. Rat primary cortical neurons had the same basal cellular respiration in hypoxia as in normoxia, whereas NO-exposed cells exhibited a gradual decrease in mitochondrial respiration in hypoxia. Upon reoxygenation, the respiration in NO-treated cells did not recover to prehypoxic levels. Hypoxia–reoxygenation in the presence of NO was associated with inhibition of autophagy, and the inability to recover during reoxygenation was exacerbated by an inhibitor of autophagy, 3-methyladenine. The effects of hypoxia could be recapitulated by inhibiting glycolytic flux under normoxic conditions. Under both normoxic and hypoxic conditions NO exposure induced immediate stimulation of glycolysis, but prolonged NO exposure, associated with irreversible inhibition of mitochondrial respiration in hypoxia, inhibited glycolysis. Importantly, we found that NO inhibited basal respiration under normoxic conditions only when glucose was absent from the medium or glycolysis was inhibited by 2-deoxy-d-glucose, revealing a novel NO-dependent mechanism for the inhibition of mitochondrial respiration that is modulated by glycolysis. Taken together these data suggest an oxygen-dependent interaction between mitochondrial respiration, glycolysis, and autophagy in protecting neuronal cells exposed to NO. Importantly, they indicate that mitochondrial dysfunction is intimately linked to a failure of glycolytic flux induced by exposure to NO. In addition, these studies provide new insights into the understanding of how autophagy and NO may play interactive roles in neuroinflammation-induced cellular damage, which is pertinent to our understanding of the pathology of neurodegenerative diseases in which excessive NO is generated.

Clematichinenoside Attenuates Myocardial Infarction in Ischemia/Reperfusion Injury both In Vivo and In Vitro

Clematichinenoside is a triterpenoid saponin isolated from the roots of Clematis chinensis. Oxidative stress and excessive nitric oxide production are thought to play considerable roles in ischemia/reperfusion injury that impairs cardiac function. The present study investigated the protective effect of clematichinenoside on regional and global ischemia/reperfusion injury and ventricular myocytes. In vivo, regional myocardial ischemia/reperfusion injury of rats was induced by the occlusion of the left anterior descending coronary artery, and isolated guinea pigs heart using Langendorff apparatus served as a global ischemia/reperfusion injury model ex vivo. Primary cultured neonatal ventricular myocytes were further applied to explore the anti-ischemia/reperfusion injury property in vitro. Infarct size was measured with TTC stain; enzyme activities such as lactate dehydrogenase, creatine kinase, superoxide dismutase, malondialdehyde, and nitric oxide were analyzed with assay kits; inducible nitric oxide synthase and endothelial nitric oxide synthase expressions were determined by Western blot. Clematichinenoside attenuated infarct size, decreased lactate dehydrogenase, creatine kinase, and malondialdehyde levels and enhanced superoxide dismutase activity. Clematichinenoside improved hemodynamics indexes, such as left ventricular developed pressure, maximum left ventricular developed pressure, and increase/decrease rate (± dp/dtmax) in the isolated guinea pig heart after reperfusion. Clematichinenoside also inhibited excessive production of nitric oxide through downregulating inducible nitric oxide synthase as well as upregulating endothelial nitric oxide synthase during ischemia/reperfusion injury. Clematichinenoside attenuates ischemia/reperfusion injury in vivo, ex vivo, and in vitro via an antioxidant effect and by restoring the balance between inducible nitric oxide synthase and endothelial nitric oxide synthase.

Morphofunctional Changes of Rat Pancreas Under Conditions оf Nitric Oxyde Excess

The study was carried out on 40 male Wistar rats after daily intraperitoneal injection of donator of nitric oxide (NO) molecules sodium nitroprusside in a dose of 1.5 mg/kg in 1 day 1 (n = 6); 2 days (n = 6); 6 days (n = 6); 12 days (n = 6); 30 days (n = 6). It is established that an excessive NO intake leads to morphological changes in the pancreas: vasodilation with stasis of blood cells after 1 and 2 days, focal necrosis, destruction of acinar tissue, ductal dilatation with worsening outflow of pancreatic secretion after 6 days; fatty degeneration, segmental apoptosis in 12 days; compensated microcirculatory changes in the body, the formation of fibrous tissue in periductal and perivasal areas with penetration in the interlobular space in 30 days. In the blood serum we detected impaired exocrine pancreatic function: increased activity of pancreatic enzymes — α-amylase and trypsin up to 6 days, and in 30 days — they significantly reduced; increasing the levels of biochemical markers of fibrosis (protein-bound hydroxyproline and hexosamine); failure of endocrine function — increase of glucose level, that is, changes typical for chronic experimental pancreatitis.

GW24-e1289 Atorvastatin decreases nitric oxide production and restore vascular responsiveness impaired by LPS in rats

ObjectivesLipopolysaccharides (LPS) were found in the outer membrane of Gram-negative bacteria and be used to induce experimental endotoxic shock, which could decrease vasoconstrictor response, produce excessive nitric oxide and decrease...

Nitric oxide modulates mitochondrial activity and apoptosis through protein S-nitrosylation for preimplantation embryo development

Previous studies reported that patients with endometriosis had excess nitric oxide (NO) in the reproductive tract and poor embryo development in IVF cycles. This study aims to elucidate the effects of NO on early embryo development. Zygotes from superovulated B6CBF1 mice were cultured to blastocysts in a variety of media. Sodium nitroprusside (SNP) and N(G)-nitro-L-arginine (LNA) were added to the culture medium as a NO donor and a NO synthase inhibitor, respectively. The localization and fluorescence intensity of S-nitrosylated (SNO) proteins within 2-cell stage embryos were analyzed with confocal microscopy. Apoptosis and ATP production in the blastocysts were measured. Subsequent to NO exposure, the SNO proteins mainly colocalized with the mitochondria and endoplasmic reticulum and the intensity of SNO proteins increased. The addition of a quanylate cyclase inhibitor and a cyclic GMP mimic agent induced nonsignificant changes in SNO proteins, whereas addition of a superoxide scavenger or a reduced form of glutathione rescued the embryos from the effects of NO. However, superoxide scavenger supplementation resulted in decreased blastocyst ATP production. Elevated NO exerts deleterious effects on embryo development, possibly through protein S-nitrosylation in the mitochondria and endoplasmic reticulum. Including glutathione as a component in the culture medium might counteract this effect.

Pentylentetrazole-induced loss of blood–brain barrier integrity involves excess nitric oxide generation by neuronal nitric oxide synthase

Dysfunction of the blood–brain barrier (BBB) is one of the major pathophysiological consequences of epilepsy. The increase in the permeability caused by BBB failure is thought to contribute to the development of epileptic outcomes. We developed a method by which the BBB permeability can be demonstrated by gadolinium-enhanced T1 weighted imaging (GdET1WI). The present study examined the changes in the BBB permeability in mice with generalized convulsive seizures (GCS) induced by acute pentylentetrazole (PTZ) injection. At 15min after PTZ-induced GCS, the BBB temporarily leaks BBB-impermeable contrast agent into the parenchyma of the diencephalon, hippocampus and cerebral cortex in mice, and the loss of BBB integrity was gradually recovered by 24h. The temporary BBB failure is a critical link to the glutamatergic activities that occur following the injection of PTZ. PTZ activates the glutamatergic pathway via the NMDA receptor, then nitric oxide (NO) is generated by NMDA receptor-coupled neuronal NO synthase (nNOS). To examine the influence of nNOS-derived NO induced by PTZ on the increases of the BBB permeability, GdET1WI was performed using conventional nNOS gene-deficient mice with or without PTZ injection. The failure of the BBB induced by PTZ was completely protected by nNOS deficiency in the brain. These results suggest that nNOS-derived excess NO in the glutamatergic pathway plays a key role in the failure of the BBB during PTZ-induced GCS. The levels of NO synthetized by nNOS in the brain may represent an important target for the future development of drugs to protect the BBB.

Excess nitric oxide impairs liver X receptor α-ATP-binding cassette transporter A1-dependent cholesterol efflux in macrophage foam cells

Excess nitric oxide (NO) promotes the progression of atherosclerosis by increasing the oxidation of low-density lipoprotein (LDL) and inflammatory responses. However, little is known about the impact of NO and its underlying molecular mechanism on lipid metabolism of macrophage foam cells. In this study, Oil-red O staining, cholesterol and triglyceride assay, Dil-oxidized LDL (oxLDL) binding assay, cholesterol efflux assay, real-time RT-PCR and Western blot analysis were used for in vitro experiments. Apolipoprotein E-deficient (apoE(-/-) ) and apoE and inducible nitric oxide synthase-deficient (apoE(-/-) iNOS(-/-) ) mice were as our in vivo models. Treatment with S-nitroso-N-acetyl-D,L-penicillamine (SNAP), an NO donor, exacerbated oxLDL-induced cholesterol accumulation in macrophages, because of reduced efficacy of cholesterol efflux. In addition, SNAP decreased the protein level of ATP-binding cassette transporter A1 (ABCA1) without affecting scavenger receptor type A (SR-A), CD36, ABCG1, or SR-B1 levels. This SNAP-mediated downregulation of ABCA1 was mainly through the effect of NO but not peroxynitrite. Furthermore, the SNAP-downregulated ABCA1 was due to the decrease in the liver X receptor α (LXRα)-dependent transcriptional regulation. Moreover, genetic deletion of iNOS increased the serum capacity of reverse cholesterol efflux and protein expression of LXRα, ABCA1, and SR-BI in aortas and retarded atherosclerosis in apoE(-/-) mice. Our findings provide new insights in the pro-atherogenic effect of excess NO on cholesterol metabolism in macrophages.

TLR2 activation causes no morbidity or cardiovascular failure, despite excessive systemic nitric oxide production

Septic shock is the leading cause of death in intensive care units worldwide, resulting from a progressive systemic inflammatory reaction causing cardiovascular and organ failure. Nitric oxide (NO) is a potent vasodilator and inhibition of NO synthases (NOS) can increase blood pressure in septic shock. However, NOS inhibition does not improve outcome, on the contrary, and certain NO donors may even provide protection. In addition, NOS produce superoxide in case of substrate or cofactor deficiency or oxidation. We hypothesized that excessive systemic iNOS-derived NO production is insufficient to trigger cardiovascular failure and shock. We found that the systemic injection with various synthetic Toll-like receptor-2 (TLR2), TLR3, or TLR9 agonists triggered systemic NO production identical to that of lipopolysaccharide (LPS) or tumour necrosis factor. In contrast to the latter, however, these agonists did not cause hypothermia or any other signs of discomfort or morbidity, and inflammatory cytokine production was low. TLR2 stimulation with the triacylated lipopeptide Pam3CSK4 not only caused identical NO levels in circulation, but also identical iNOS expression patterns as LPS. Nevertheless, Pam3CSK4 did not cause hypotension, bradycardia, reduced blood flow, or inadequate tissue perfusion in the kidney or the liver. We demonstrate that excessive iNOS-derived NO in circulation is not necessarily linked to concomitant cardiovascular collapse, morbidity, or mortality. As such, our data indicate that the central role of iNOS-derived NO in inflammation-associated cardiovascular failure may be overestimated.

Field testing of new-technology ambient air ozone monitors

Multibillion-dollar strategies control ambient air ozone (O3) levels in the United States, so it is essential that the measurements made to assess compliance with regulations be accurate. The predominant method employed to monitor O3 is ultraviolet (UV) photometry. Instruments employ a selective manganese dioxide or heated silver wool “scrubber” to remove O3 to provide a zero reference signal. Unfortunately, such scrubbers remove atmospheric constituents that absorb 254-nm light, causing measurement interference. Water vapor also interferes with the measurement under some circumstances. We report results of a 3-month field test of two new instruments designed to minimize interferences (2B Technologies model 211; Teledyne-API model 265E) that were operated in parallel with a conventional Thermo Scientific model 49C O3 monitor. The field test was hosted by the Houston Regional Monitoring Corporation (HRM). The model 211 photometer scrubs O3 with excess nitric oxide (NO) generated in situ by photolysis of added nitrous oxide (N2O) to provide a reference signal, eliminating the need for a conventional O3 scrubber. The model 265E analyzer directly measures O3–NO chemiluminescence from added excess NO to quantify O3 in the sample stream. Extensive quality control (QC) and collocated monitoring data are assessed to evaluate potential improvements to the accuracy of O3 compliance monitoring. Implications: Two new-technology ozone monitors were compared with a conventional monitor under field conditions. Over 3 months the conventional monitor reported more exceedances of the current standard than the new instruments, which could potentially result in an area being misjudged as “nonattainment.” Instrument drift can affect O3 data accuracy, and the same degree of drift has a proportionally greater compliance effect as standard stringency is increased. Enhanced data quality assurance and data adjustment may be necessary to achieve the improved accuracy required to judge compliance with tighter standards.

The suppressor of cytokine signaling 2 (SOCS2) modulating the neurotransmitters release in Eriocheir sinensis

The SOCS proteins appear to define an important mechanism for the negative regulation of the cytokine–JAK–STAT pathway. In the present study, the mRNA expression profiles of a SOCS2 from Chinese mitten crab Eriocheir sinensis (EsSOCS2) after pentachlorophenol (PCP) treatment or RNA interference (RNAi) were analyzed to understand its possible regulatory roles in modulating the neurotransmitter release. The EsSOCS2 expression level in the PCP treated group was significantly higher than that of blank at 1.5, 3, 12 and 24 h after exposure, suggesting that EsSOCS2 might be involved in controlling and reducing neuronal cell damage resulted from PCP treatment. After the expression of EsSOCS2 gene was silenced by RNAi, the concentrations of catecholamines and nitric oxide (NO) were examined to evaluate the modulation of EsSOCS2 on the release of neurotransmitters. At 48 h after the treatment with sequence-specific dsRNA targeting EsSOCS2, the expression of EsSOCS2 was reduced to half compared to the original level, and the concentrations of norepinephrine and NO increased, while dopamine decreased significantly in haemolymph. The preliminary results indicated that EsSOCS2 regulated catecholaminergic neuroendocrine system to release catecholamines into haemolymph and might be an important feedback inhibitor of tyrosine kinase signaling pathways in crab, which subsequently regulated NO synthesis and prevented excessive NO release. This information is helpful to further understand the modulation of EsSOCS2 on neurotransmitter release in crab.

Overexpressed neuroglobin raises threshold for nitric oxide-induced impairment of mitochondrial respiratory activities and stress signaling in primary cortical neurons

Surges of nitric oxide compromise mitochondrial respiration primarily by competitive inhibition of oxygen binding to cytochrome c oxidase (complex IV) and are particularly injurious in neurons, which rely on oxidative phosphorylation for all their energy needs. Here, we show that transgenic overexpression of the neuronal globin protein, neuroglobin, helps diminish protein nitration, preserve mitochondrial function and sustain ATP content of primary cortical neurons challenged by extended nitric oxide exposure. Specifically, in transgenic neurons, elevated neuroglobin curtailed nitric oxide-induced alterations in mitochondrial oxygen consumption rates, including baseline oxygen consumption, consumption coupled with ATP synthesis, proton leak and spare respiratory capacity. Concomitantly, activation of genes involved in sensing and responding to oxidative/nitrosative stress, including the early-immediate c-Fos gene and the phase II antioxidant enzyme, heme oxygenase-1, was diminished in neuroglobin-overexpressing compared to wild-type neurons. Taken together, these differences reflect a lesser insult produced by similar concentrations of nitric oxide in neuroglobin-overexpressing compared to wild-type neurons, suggesting that abundant neuroglobin buffers nitric oxide and raises the threshold of nitric oxide-mediated injury in neurons.

Up-regulation of protein tyrosine nitration in methamphetamine-induced neurotoxicity through DDAH/ADMA/NOS pathway

Protein tyrosine nitration is an important post-translational modification mediated by nitric oxide (NO) associated oxidative stress, occurring in a variety of neurodegenerative diseases. In our previous study, an elevated level of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protein was observed in different brain regions of acute methamphetamine (METH) treated rats, indicating the possibility of an enhanced expression of protein nitration that is mediated by excess NO through the DDAH1/ADMA (Asymmetric Dimethylated l-arginine)/NOS (Nitric Oxide Synthase) pathway. In the present study, proteomic methods, including stable isotope labeling with amino acids in cell culture (SILAC) and two dimensional electrophoresis, were used to determine the relationship between protein nitration and METH induced neurotoxicity in acute METH treated rats and PC12 cells. We found that acute METH administration evokes a positive activation of DDAH1/ADMA/NOS pathway and results in an over-production of NO in different brain regions of rat and PC12 cells, whereas the whole signaling could be repressed by DDAH1 inhibitor Nω-(2-methoxyethyl)-arginine (l-257). In addition, enhanced expressions of 3 nitroproteins were identified in rat striatum and increased levels of 27 nitroproteins were observed in PC12 cells. These nitrated proteins are key factors for Cdk5 activation, cytoskeletal structure, ribosomes function, etc. l-257 also displayed significant protective effects against METH-induced protein nitration, apoptosis and cell death. The overall results illustrate that protein nitration plays a significant role in the acute METH induced neurotoxicity via the activation of DDAH1/ADMA/NOS pathway.

Plasma membrane calcium ATPase 4b inhibits nitric oxide generation through calcium-induced dynamic interaction with neuronal nitric oxide synthase

The activation and deactivation of Ca(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca(2+) concentrations ([Ca(2+)]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca(2+)]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.

Autophagy in neuronal bioenergetics and survival

Neurodegeneration are associated with multiple hits at metabolic pathways including those involved in metabolism (mitochondria and glycolysis) cell signaling (Keap1‐Nrf2 or apoptosis) or cell repair pathways (fission/fusion and autophagy). We investigated the regulation of autophagy in neuronal response to oxidative and nitrative stress. 4‐hydroxynonenal (HNE) is a lipid peroxidation product formed during oxidative stress, accumulated in neurodegenerative diseases, and can further induce formation of other reactive species. DETA‐NONOate is a nitric oxide donor, used to mimick excessive nitric oxide produced by both neurons and glia in neurodegeneration. We found that HNE and DETA‐NONOate decrease mitochondrial oxygen consumption. Interestingly, HNE inhibited, and DETA‐NONOate stimulated glycolysis. The glycolysis response was inversely correlated with autophagy, as HNE stimulated and DETA‐NONOate inhibited autophagy. To investigate how glucose metabolism and autophagy play a role in mitochondrial quality control and cell death, we blocked glycolysis using 2‐DG, and found that 2‐DG abolished glycolysis, decreased mitochondrial reserve capacity, inhibited autophagy and exacerbated HNE‐ and DETA‐NONOate‐induced cell death. Inhibition of autophagy by 3‐MA, while increased glycolysis which is in contrary to 2‐DG, decreased mitochondrial reserve capacity and exacerbated HNE‐ and DETA‐NONOate‐induced cell death, effects similar to 2‐DG. Moreover, we found that 2‐DG played an important role in regulation of Bcl‐2 expression and 3‐MA played an important role in regulation of Drp1 and JNK activity. Our studies provide novel insights into how regulation of autophagy and glucose metabolism plays an important role in neuronal bioenergetics and survival in response to oxidative and nitrative stress. Funded by NIH and VA.