Activation Of Nitric Oxide Synthesis Research Articles

Endothelial cell phenotype is linked to endothelial dysfunction in individuals with a family history of type 2 diabetes.

The patient's family history of type 2 diabetes (FH-DM2) has been negatively associated with the functionality of endothelial cells (ECs). Our objectives in this work were to use human umbilical vein endothelial cells (HUVECs) as a model, to substantiate whether FH-DM2 influences endothelial phenotype and impairs NO and ROS synthesis, cell metabolism, and mitochondrial activity of ECs from individuals with FH-DM2. In this study were evaluated the synthesis of reactive oxygen species (ROS) and nitric oxide (NO), mitochondrial membrane potential (MMP), mRNA of eNOS, glucose consumption, and lactate synthesis in HUVECs from newborns with FH-DM2. Furthermore, we also evaluated EC complexity and cell size through flow cytometry. Our results showed significant differences in HUVECs with FH-DM2, regarding their complexity and cell size, in the synthesis of ROS (p<0.01), and NO (p<0.05); they also reflected diminished glucose consumption and slight changes in the lactate levels. In conclusion, our results showed that HUVECs from children with FH-DM2 have a reduced capability of synthesizing ROS and NO, which might be linked to the metabolism of endothelial cells. These results are relevant since early endothelial dysfunction has been reported in individuals with FH-DM2, and could be used to establish preventive measures to reduce the risk of developing atherosclerosis or cardiovascular diseases in healthy individuals, but with this family background.

Nitric oxide in the mechanisms of inhibitory effects of sodium butyrate on colon contractions in a mouse model of irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a multifactorial disorder, with altered intestinal motility, visceral hypersensitivity, and dysfunction of the gut-brain axis. The aim of our study was to analyze the role of nitric oxide (NO) in the inhibitory effects of sodium butyrate on spontaneous contractility of proximal colon in a mouse model of IBS. IBS was induced by intracolonic infusion of acetic acid in the early postnatal period. Spontaneous contractions of proximal colon segments were studied in isometric conditions. The amplitude and frequency of colon contractions were higher in the IBS group. Sodium butyrate exerted inhibitory effects on colon contractions, which were less pronounced in IBS group. NO donors decreased spontaneous colon contractility and prevented the inhibitory effects of sodium butyrate in control and IBS groups. Nitric oxide synthase (NOS) inhibition by L-NAME increased contractile activity more effective in the control group and decreased the inhibitory action of sodium butyrate. In IBS group, preliminary application of L-NAME did not prevent sodium butyrate action. Our data indicate that butyrate exerts its inhibitory effects on colon motility at least partially through activation of NO synthesis. In the IBS model group, the NO-dependent mechanisms were less effective probably due to downregulation of NOS.

Methabolic reaction on the cerebral ischemia during carotid artery surgery

One of the ischemic stroke prevention methods is reconstructive surgery on the carotid arteries, during which there is a risk of ischemia-reperfusion phenomenon. Nevertheless, there is still a high risk of stroke at all stages of the surgery, which equally depends on the atherosclerotic plaque structure and on the technique and tactics of surgery. The aim of this study was to investigate the dynamics of redox homeostasis and nitric oxide production in the blood taken from the internal jugular vein in patients with carotid arteries pathology against the episode of cerebral blood flow restriction during carotid surgery. This prospective cohort study involved 56 patients which required carotid reconstructive surgery. The markers dynamics of oxygen metabolism, redox homeostasis and nitric oxide production in blood from the internal jugular vein on the side of surgery were studied. It was found that the activity of free radical processes is proportional to the level of jugular hypoxemia. Bidirectional dynamics of lactate concentration during intraoperative transient cerebral ischemia was revealed. In some patients, lactate production activates with an increase in free radical processes activity in proportion to the intensification of nitric oxide synthesis against a decrease in peroxynitrite production and activation of molecular compensatory mechanisms increasing concentrations of L-arginine and reduced glutathione. In others, the extraction of oxygen from the blood increases without lactate production, activation of nitric oxide synthesis and development of oxidative stress. This indicates good adaptive reserves against acute restriction of cerebral blood flow.

Hypochlorous acid facilitates inducible nitric oxide synthase subunit dissociation: The link between heme destruction, disturbance of the zinc-tetrathiolate center, and the prevention by melatonin

Inducible nitric oxide synthase (iNOS) is a zinc-containing hemoprotein composed of two identical subunits, each containing a reductase and an oxygenase domain. The reductase domain contains binding sites for NADPH, FAD, FMN, and tightly bound calmodulin and the oxygenase domain contains binding sites for heme, tetrahydrobiopterin (H4B), and l-arginine. The enzyme converts l-arginine into nitric oxide (NO) and citrulline in the presence of O2. It has previously been demonstrated that myeloperoxidase (MPO), which catalyzes formation of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride (Cl−), is enhanced in inflammatory diseases and could be a potent scavenger of NO. Using absorbance spectroscopy and gel filtration chromatography, we investigated the role of increasing concentrations of HOCl in mediating iNOS heme destruction and subsequent subunit dissociation and unfolding. The results showed that dimer iNOS dissociation between 15 and 100 μM HOCl was accompanied by loss of heme content and NO synthesis activity. The dissociated subunits-maintained cytochrome c and ferricyanide reductase activities. There was partial unfolding of the subunits at 300 μM HOCl and above, and the subunit unfolding transition was accompanied by loss of reductase activities. These events can be prevented when the enzyme is preincubated with melatonin prior to HOCl addition. Melatonin supplementation to patients experiencing low NO levels due to inflammatory diseases may be helpful to restore physiological NO functions.

Facial Stimulation Induces Long-Term Potentiation of Mossy Fiber-Granule Cell Synaptic Transmission via GluN2A-Containing N-Methyl-D-Aspartate Receptor/Nitric Oxide Cascade in the Mouse Cerebellum.

Long-term synaptic plasticity in the cerebellar cortex is a possible mechanism for motor learning. Previous studies have demonstrated the induction of mossy fiber-granule cell (MF-GrC) synaptic plasticity under in vitro and in vivo conditions, but the mechanisms underlying sensory stimulation-evoked long-term synaptic plasticity of MF-GrC in living animals are unclear. In this study, we investigated the mechanism of long-term potentiation (LTP) of MF-GrC synaptic transmission in the cerebellum induced by train of facial stimulation at 20 Hz in urethane-anesthetized mice using electrophysiological recording, immunohistochemistry techniques, and pharmacological methods. Blockade of GABAA receptor activity and repetitive facial stimulation at 20 Hz (240 pulses) induced an LTP of MF-GrC synapses in the mouse cerebellar cortical folium Crus II, accompanied with a decrease in paired-pulse ratio (N2/N1). The facial stimulation-induced MF-GrC LTP was abolished by either an N-methyl-D-aspartate (NMDA) receptor blocker, i.e., D-APV, or a specific GluNR2A subunit-containing NMDA receptor antagonist, PEAQX, but was not prevented by selective GluNR2B or GluNR2C/D subunit-containing NMDA receptor blockers. Application of GNE-0723, a selective and brain-penetrant-positive allosteric modulator of GluN2A subunit-containing NMDA receptors, produced an LTP of N1, accompanied with a decrease in N2/N1 ratio, and occluded the 20-Hz facial stimulation-induced MF-GrC LTP. Inhibition of nitric oxide synthesis (NOS) prevented the facial stimulation-induced MF-GrC LTP, while activation of NOS produced an LTP of N1, with a decrease in N2/N1 ratio, and occluded the 20-Hz facial stimulation-induced MF-GrC LTP. In addition, GluN2A-containing NMDA receptor immunoreactivity was observed in the mouse cerebellar granular layer. These results indicate that facial stimulation at 20 Hz induced LTP of MF-GrC synaptic transmission via the GluN2A-containing NMDA receptor/nitric oxide cascade in mice. The results suggest that the sensory stimulation-evoked LTP of MF-GrC synaptic transmission in the granular layer may play a critical role in cerebellar adaptation to native mossy fiber excitatory inputs and motor learning behavior in living animals.

Assessment of leukocyte activity in mice devoid of the glucocorticoid receptor in the noradrenergic system (GRDBHCre)

Disturbances in brain monoamines, overactivity of the hypothalamo-pituitary adrenal (HPA) axis and pro-inflammatory tendency in the immune system are the key features of depressive disorders. Recently, several murine lines with mutations in glucocorticoid receptors (GRs) have been generated and these animals may be utilized for study depressive-like disorders. In the present study, we have investigated whether selective ablation of GRs in noradrenergic neurons affects functional properties of leukocytes and redirects them towards pro-inflammatory activity.Transgenic mice selectively devoid of GRs on noradrenergic cells were constructed using the Cre/loxP approach. Peritoneal leukocytes were collected from mutant and wild type (WT) animals of both sexes and were cultured in vitro for 24h both in basal conditions and after application of selected pro- or anti-inflammatory stimuli. Metabolic activity and adherence were measured in basal conditions. Nitric oxide (NO) synthesis and arginase (ARG) activity were assessed as the markers of functional status of the cells. Because adult mutant mice lack adrenal medulla and thereby peripheral adrenaline, we modulated pro- and anti-inflammatory culture conditions by addition of noradrenaline (10–6M). Finally, effects of in vivo pro-inflammatory challenge (with intraperitoneal administration of lipopolysaccharide) on properties of leukocytes were assessed 24h (in both sexes) and 48h later (in males only).The experiments indicated that selective ablation of GR in noradrenergic neurons did not affect fundamental properties of peritoneal leukocytes and exerted effects only under conditions of selected pro- or anti-inflammatory stimuli in vitro. Stronger response to pro-inflammatory stimulation in terms of NO synthesis and ARG activity may suggest pro-inflammatory tendency in mutant mice. In vivo inflammatory challenge failed to show any effect of GR ablation on selected parameters of leukocyte activity. Both in vitro studies and in vivo challenge revealed mainly sex-related differences in leukocyte activity.

Dystrophin–glycoprotein complex regulates muscle nitric oxide production through mechanoregulation of AMPK signaling

Patients deficient in dystrophin, a protein that links the cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex (DGC), exhibit muscular dystrophy, cardiomyopathy, and impaired muscle nitric oxide (NO) production. We used live-cell NO imaging and in vitro cyclic stretch of isolated adult mouse cardiomyocytes as a model system to investigate if and how the DGC directly regulates the mechanical activation of muscle NO signaling. Acute activation of NO synthesis by mechanical stretch was impaired in dystrophin-deficient mdx cardiomyocytes, accompanied by loss of stretch-induced neuronal NO synthase (nNOS) S1412 phosphorylation. Intriguingly, stretch induced the acute activation of AMP-activated protein kinase (AMPK) in normal cardiomyocytes but not in mdx cardiomyocytes, and specific inhibition of AMPK was sufficient to attenuate mechanoactivation of NO production. Therefore, we tested whether direct pharmacologic activation of AMPK could bypass defective mechanical signaling to restore nNOS activity in dystrophin-deficient cardiomyocytes. Indeed, activation of AMPK with 5-aminoimidazole-4-carboxamide riboside or salicylate increased nNOS S1412 phosphorylation and was sufficient to enhance NO production in mdx cardiomyocytes. We conclude that the DGC promotes the mechanical activation of cardiac nNOS by acting as a mechanosensor to regulate AMPK activity, and that pharmacologic AMPK activation may be a suitable therapeutic strategy for restoring nNOS activity in dystrophin-deficient hearts and muscle.

Traditional Chinese medicine's intervention in endothelial nitric oxide synthase activation and nitric oxide synthesis in cardiovascular system.

Cardiovascular disease (CVD) is one of the most dangerous diseases which has become a major cause of human death. Many researches evidenced that nitric oxide (NO)/endothelial nitric oxide synthase (eNOS) system plays a significant role in the occurrence and development of CVD. NO, an important signaling molecule, closely associated with the regulation of vasodilatation, blood rheology, blood clotting and other physiological and pathological processes. The synthesis of NO in the endothelial cells primarily depends on the eNOS activity, thus the exploration of the mechanisms and effects of the eNOS activation on NO production is of great significance. Recently, studies on the effects of traditional Chinese medicine (TCM) and its extracts on eNOS activation and NO synthesis have gradually attracted more and more attentions. In this paper, we reviewed the mechanisms of NO synthesis and eNOS activation in the vascular endothelial cells (VECs) and intervention of TCM, so as to provide reference and train of thought to the intensive study of NO/eNOS system and the research and development of new drug for the treatment of CVD.

Citrulline uptake in rat cerebral cortex slices: Modulation by Thioacetamide -Induced hepatic failure

L-citrulline (Cit) is a co-product of NO synthesis and a direct L-arginine (Arg) precursor for de novo NO synthesis. Acute liver failure (ALF) is associated with increased nitric oxide (NO) and cyclic GMP (cGMP) synthesis in the brain, indirectly implicating a role for active transport of Cit. In the present study we characterized [3H]Cit uptake to the cortical brain slices obtained from control rats and rats with thioacetamide (TAA)-induced ALF (“TAA slices”). In both control and TAA slices the uptake was partially Na+-dependent and markedly inhibited by substrates of systems L and N, including L-glutamine (Gln), which accumulates in excess in brain during ALF. Cit uptake was not affected by Arg, the y+/y+L transport system substrate, nor by amino acids taken up by systems A, xc −or XAG. The Vmax of the uptake in TAA slices was ~60 % higher than in control slices. Chromatographic (HPLC) analysis revealed a ~30 % increase of Cit concentration in the cerebral cortical homogenates of TAA rats. The activity of argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL), the two enzymes of Cit-NO cycle catalyzing synthesis of Arg, showed an increase in TAA rats, consistent with increased ASS and ASL protein expression, by ~30 and ~20 %, respectively. The increased Cit-NO cycle activity was paralleled by increased expression of mRNA coding for inducible nitric oxide synthase (iNOS). Taken together, the results suggest a role for Cit in the activation of cerebral NO synthesis during ALF.

Tumor Cells Upregulate Normoxic HIF-1α in Response to Doxorubicin

Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor that controls cellular homeostasis. Although its activation benefits normal tissue, HIF-1 activation in tumors is a major risk factor for angiogenesis, therapeutic resistance, and poor prognosis. HIF-1 activity is usually suppressed under normoxic conditions because of rapid oxygen-dependent degradation of HIF-1α. Here, we show that, under normoxic conditions, HIF-1α is upregulated in tumor cells in response to doxorubicin, a chemotherapeutic agent used to treat many cancers. In addition, doxorubicin enhanced VEGF secretion by normoxic tumor cells and stimulated tumor angiogenesis. Doxorubicin-induced accumulation of HIF-1α in normoxic cells was caused by increased expression and activation of STAT1, the activation of which stimulated expression of iNOS and its synthesis of nitric oxide (NO) in tumor cells. Mechanistic investigations established that blocking NO synthesis or STAT1 activation was sufficient to attenuate the HIF-1α accumulation induced by doxorubicin in normoxic cancer cells. To our knowledge, this is the first report that a chemotherapeutic drug can induce HIF-1α accumulation in normoxic cells, an efficacy-limiting activity. Our results argue that HIF-1α-targeting strategies may enhance doxorubicin efficacy. More generally, they suggest a broader perspective on the design of combination chemotherapy approaches with immediate clinical impact.

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.

Role of equilibrative adenosine transporters and adenosine receptors as modulators of the human placental endothelium in gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is a diseases that alters human placenta macro and microvascular reactivity as a result of endothelial dysfunction. The human placenta is a highly vascularized organ which lacks innervation, so blood flux is governed by locally released vasoactive molecules, including the endogenous nucleoside adenosine and the free radical nitric oxide (NO). Altered adenosine metabolism and uptake by the endothelium leads to increased NO synthesis which then turns-off the expression of genes coding for a family of nucleoside membrane transporters belonging to equilibrative nucleoside transporters, particularly isoforms 1 (hENT1) and 2 (hENT2). This mechanism leads to increased extracellular adenosine and, as a consequence, activation of adenosine receptors to further sustain a tonic activation of NO synthesis. This is a phenomenon that seems operative in the placental macro and microvascular endothelium in GDM. We here summarize the findings available in the literature regarding these mechanisms in the human feto-placental circulation. This phenomenon is altered in the feto-placental vasculature, which could be crucial for understanding GDM deleterious effects in fetal growth and development.

Green Tea Polyphenols Decrease Enzyme Activity of Nitric Oxide Synthase

Oxidative stress is found to be involved in a number of diseases including cancer, Parkinson and Alzheimer disease. It is due to the unbalanced higher activities of endogenous pro-oxidative enzymes over anti-oxidative enzymes which increase the production of reactive oxygen species (ROS). The accumulation of highly reactive ROS can damage nucleic acids, proteins, lipids and impair mitochondrial function, ultimately leading to cell death. Recent studies have shown that dietary polyphenolic antioxidants can reduce oxidative stress by directly scavenging ROS, inhibiting enzymatic sources of oxidative stress and stimulating endogenous antioxidant enzymes. We investigated how green tea catechins (small molecule polyphenolic antioxidants) affect the free radical nitric oxide (NO) production from Nitric Oxide Synthase (NOS). NO production was determined by oxy-hemoglobin assay and Griess reagent assay. We found that both (−)-epigallocatechin (EC) and (−)-epigallocatechin-3-gallate (EGCG) greatly reduced the NO production from three different NOS isoforms (neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS)), although their abilities of inhibition were slightly different among three NOS isoforms. We also observed that EC decreased the NO synthesis activities more than EGCG in nNOS, suggesting the gallate structure plays an important role in the inhibition. When nNOS enzyme was incubated with increasing concentration of EGCG from 0 μM to 100 μM, its activities gradually decreased, with only 50% activity remaining when 100 μM EGCG in presence. We propose that the two catechins may inhibit the enzyme activities by binding to the enzyme's active site, decreasing the percentage of its active dimeric form, reducing the enzymatic coupling and affecting the ROS generation during the reaction. Chemical Structure of EGCG

Gestational Diabetes Mellitus and the Role of Adenosine in the Human Placental Endothelium and Central Nervous System

Diabetes mellitus is a syndrome that alters macro and microvascular reactivity as a result of endothelial dysfunction. This phenomenon occurs in vascular beds from different regions in the adult human body, as well as the developing fetus including the placenta and the brain vasculature. The human placenta is highly vascularized lacking of innervation, so that blood flux is governed by locally released vasoactive moleculles, including the endogenous nucleoside adenosine and the gas nitric oxide (NO). Altered adenosine uptake by the endothelium leads to increased synthesis of NO resulting in repression of equilibrative nucleoside membrane transporters isoforms 1 (hENT1) and 2 (hENT2). The latter result in higher extracellular adenosine and activation of adenosine receptors (ARs) sustaining tonic activation of NO synthesis in gestational diabetes mellitus (GDM). Similar alterations are apparent in the fetus brain. Because GDM associates with serious alterations in the CNS functions leading to abnormal higher cognitive functions, among others complications, it is proposed that altered adenosine metabolism, uptake and dynamics of ARs activation in the brain is a factor leading to these pathologies. We here summarize the findings available in the literature regarding these mechanisms in the human fetus brain with reference to that in the feto-placental circulation. We emphasize the possibility that adenosine plays a key role in the functional link established by astrocytes connecting neurons and the brain vasculature. This phenomenon is altered in the brain and the feto-placental vasculature, which could be crucial for understanding GDM deleterous effects in fetal growth and development, as well as CNS abnormal function in adulthood.

L-Arginine infusion during resuscitation for hemorrhagic shock

Our previous work showed a survival advantage with L-arginine (L-Arg) pretreatment in a swine model of severe hemorrhagic shock. This study was designed to evaluating whether the benefit is sustained when L-Arg is given during resuscitation and whether the mechanism is mediated by enzymatic activation of nitric oxide (NO) synthesis. Adult rats (n = 30) underwent 40% blood volume loss and were resuscitated with saline (3 shed blood volume). Animals were divided into five treatment groups of six animals each: (1) Sham, (2) Control (resuscitation alone), (3) L-Arg (300 mg/kg)with resuscitation, (4) L-Arg + L-nitroarginine methyl ester pretreatment, and (5) D-arginine (300 mg/kg) with resuscitation.Animals were observed for 240 minutes postresuscitation or until death. Hemodynamic, metabolic, histologic, and survival outcomes were measured. Administration of L-Arg after hemorrhage and before resuscitation significantly improved outcomes, relative to the control group.The L-Arg infusion improved terminal arterial pressures, lowered lactate, improved small bowel histologic signs of reperfusion injury, and increased survival (p < 0.05). Endpoints of the L-Arg group were similar to the Sham group. The benefits of L-Arg infusion were abolished or attenuated when animals were pretreated with L-nitro arginine methyl ester and potentiated with D-arginine, suggesting a NO-specific mechanism of L-Arg. Finally, severe shock and resuscitation injury significantly elevated circulating asymmetric dimethylarginine levels, which are potent competitive inhibitors of NO synthetase. L-Arg infusion during resuscitation offers a significant functional, metabolic, and survival benefit after severe hemorrhagic shock.The mechanism seems to be by activation of NO synthesis with its attendant benefits to local perfusion and inflammation after global reperfusion.

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.

Surface Charges and Regulation of FMN to Heme Electron Transfer in Nitric-oxide Synthase

The nitric-oxide synthases (NOS, EC 1.14.13.39) are modular enzymes containing attached flavoprotein and heme (NOSoxy) domains. To generate nitric oxide (NO), the NOS FMN subdomain must interact with the NOSoxy domain to deliver electrons to the heme for O(2) activation during catalysis. The molecular basis and how the interaction is regulated is unclear. We explored the role of eight positively charged residues that create an electropositive patch on NOSoxy in enabling the electron transfer by incorporating mutations that neutralized or reversed their individual charges. Stopped-flow and steady-state experiments revealed that individual charges at Lys(423), Lys(620), and Lys(660) were the most important in enabling heme reduction in nNOS. Charge reversal was more disruptive than neutralization in all cases, and the effects on heme reduction were not due to a weakening in the thermodynamic driving force for heme reduction. Mutant NO synthesis activities displayed a complex pattern that could be simulated by a global model for NOS catalysis. This analysis revealed that the mutations impact the NO synthesis activity only through their effects on heme reduction rates. We conclude that heme reduction and NO synthesis in nNOS is enabled by electrostatic interactions involving Lys(423), Lys(620), and Lys(660), which form a triad of positive charges on the NOSoxy surface. A simulated docking study reveals how electrostatic interactions of this triad can enable an FMN-NOSoxy interaction that is productive for electron transfer.

Lys842 in Neuronal Nitric-oxide Synthase Enables the Autoinhibitory Insert to Antagonize Calmodulin Binding, Increase FMN Shielding, and Suppress Interflavin Electron Transfer

Neuronal nitric-oxide synthase (nNOS) contains a unique autoinhibitory insert (AI) in its FMN subdomain that represses nNOS reductase activities and controls the calcium sensitivity of calmodulin (CaM) binding to nNOS. How the AI does this is unclear. A conserved charged residue (Lys(842)) lies within a putative CaM binding helix in the middle of the AI. We investigated its role by substituting residues that neutralize (Ala) or reverse (Glu) the charge at Lys(842). Compared with wild type nNOS, the mutant enzymes had greater cytochrome c reductase and NADPH oxidase activities in the CaM-free state, were able to bind CaM at lower calcium concentration, and had lower rates of heme reduction and NO synthesis in one case (K842A). Moreover, stopped-flow spectrophotometric experiments with the nNOS reductase domain indicate that the CaM-free mutants had faster flavin reduction kinetics and had less shielding of their FMN subdomains compared with wild type and no longer increased their level of FMN shielding in response to NADPH binding. Thus, Lys(842) is critical for the known functions of the AI and also enables two additional functions of the AI as newly identified here: suppression of electron transfer to FMN and control of the conformational equilibrium of the nNOS reductase domain. Its effect on the conformational equilibrium probably explains suppression of catalysis by the AI.

Systemic Induction and Role of Mitochondrial Alternative Oxidase and Nitric Oxide in a Compatible Tomato–Tobacco mosaic virus Interaction

The role of mitochondrial alternative oxidase (AOX) and the relationship between AOX and nitric oxide (NO) in virus-induced systemic defense to Tobacco mosaic virus (TMV) were investigated in susceptible tomato (Solanum lycopersicum) plants. TMV inoculation to the lower leaves induced a rapid NO synthesis and AOX activation in upper uninoculated leaves as early as 0.5 day postinoculation. Application of exogenous potassium cyanide (KCN, a cytochrome pathway inhibitor) at nonlethal concentrations and NO donor diethylamine NONOate (DEA/NO) to the upper uninoculated leaves greatly induced accumulation of AOX transcript, reduced TMV viral RNA accumulation, and increased the leaf photochemical quantum yield at photosystem II. Pretreatment with NO scavenger almost completely blocked TMV-induced AOX induction and substantially increased TMV susceptibility. Salicylhydroxamic acid (SHAM, an AOX inhibitor) pretreatment reduced the DEA/NO-induced cyanide-resistant respiration and partially compromised induced resistance to TMV. Conversely, KCN and SHAM pretreatment had very little effect on generation of NO, and pretreatment with NO scavenger did not affect KCN-induced AOX induction and TMV resistance. These results suggest that TMV-induced NO generation acts upstream and mediates AOX induction which, in turn, induces mitochondrial alternative electron transport and triggers systemic basal defense against the viral pathogen.

How does a valine residue that modulates heme-NO binding kinetics in inducible NO synthase regulate enzyme catalysis?

Nitric oxide (NO) release from nitric oxide synthases (NOSs) depends on the dissociation of a ferric heme-NO product complex (Fe(III)NO) that forms immediately after NO is made in the heme pocket. The NOS-like enzyme of Bacillus subtilis (bsNOS) has 10-20 fold slower Fe(III)NO dissociation rate (k(d)) and NO association rate (k(on)) compared to mammalian NOS counterparts. We previously showed that an Ile for Val substitution at the opening of the heme pocket in bsNOS contributes to these differences. The complementary mutation in mouse inducible NOS oxygenase domain (Val346Ile) decreased the NO k(on) and k(d) by 8 and 3-fold, respectively, compared to wild-type iNOSoxy, and also slowed the reductive processing of the heme-O(2) catalytic intermediate. To investigate how these changes affect steady-state catalytic behaviors, we generated and characterized the V346I mutant of full-length inducible NOS (iNOS). The mutant exhibited a 4-5 fold lower NO synthesis activity, an apparent uncoupled NADPH consumption, and formation of a heme-NO complex during catalysis that was no longer sensitive to solution NO scavenging. We found that these altered catalytic behaviors were not due to changes in the heme reduction rate or in the stability of the enzyme heme-O(2) intermediate, but instead were due to the slower NO k(on) and k(d) and a slower oxidation rate of the enzyme ferrous heme-NO complex. Computer simulations that utilized the measured kinetic values confirmed this interpretation, and revealed that the V346I iNOS has an enhanced NADPH-dependent NO dioxygenase activity that converts almost 1 NO to nitrate for every NO that the enzyme releases into solution. Together, our results highlight the importance of heme pocket geometry in tuning the NO release versus NO dioxygenase activities of iNOS.