Nitric Oxide Synthesis In Rats Research Articles

Study of the Synchronization and Transmission of Intracellular Signaling Oscillations in Cells Using Bispectral Analysis.

The oscillation synchronization analysis in biological systems will expand our knowledge about the response of living systems to changes in environmental conditions. This knowledge can be used in medicine (diagnosis, therapy, monitoring) and agriculture (increasing productivity, resistance to adverse effects). Currently, the search is underway for an informative, accurate and sensitive method for analyzing the synchronization of oscillatory processes in cell biology. It is especially pronounced in analyzing the concentration oscillations of intracellular signaling molecules in electrically nonexcitable cells. The bispectral analysis method could be applied to assess the characteristics of synchronized oscillations of intracellular mediators. We chose endothelial cells from mouse microvessels as model cells. Concentrations of well-studied calcium and nitric oxide (NO) were selected for study in control conditions and well-described stress: heating to 40 °C and hyperglycemia. The bispectral analysis allows us to accurately evaluate the proportion of synchronized cells, their synchronization degree, and the amplitude and frequency of synchronized calcium and NO oscillations. Heating to 40 °C increased cell synchronization for calcium but decreased for NO oscillations. Hyperglycemia abolished this effect. Heating to 40 °C changed the frequencies and increased the amplitudes of synchronized oscillations of calcium concentration and the NO synthesis rate. The first part of this paper describes the principles of the bispectral analysis method and equations and modifications of the method we propose. In the second part of this paper, specific examples of the application of bispectral analysis to assess the synchronization of living cells in vitro are presented. The discussion compares the capabilities of bispectral analysis with other analytical methods in this field.

In situ quantification of NO synthesis in a warm air glow discharge by WMS-based Mid-IR QCL absorption spectroscopy

Nitric oxide (NO) is one of the most crucial products in the plasma-based nitrogen fixation process. In this work, in situ measurements were performed for quantifying the NO synthesis spatially in a warm air glow discharge, through the method of Mid-infrared quantum cascade laser absorption spectroscopy (QCL-AS). Two ro-vibrational transitions at 1900.076 cm−1 and 1900.517 cm−1 of the ground-state NO(X) were probed sensitively by the help of the wavelength modulation spectroscopy (WMS) approach to increase the signal/noise (S/N) level. The results show a decline trend of NO synthesis rate along the discharge channel from the cathode to the anode. However, from the point of energy efficiency, the cathode region is of significantly low energy efficiency of NO production. Severe disproportionality was found for the high energy consumption but low NO production in the region of cathode area, compared to that in the positive column zone. Further analysis demonstrates the high energy cost of NO production in the cathode region, is ascribed to the extremely high reduced electric field therein not selectively preferable for the processes of vibrational excitation or dissociation of N2 and O2 molecules. This drags down the overall energy efficiency of NO synthesis by this typical warm air glow discharge, particularly for the ones with short electrode gaps. Limitations of further improving the energy cost of NO synthesis by variations of the discharge operation conditions, such as discharge current or airflow rate, imply other effective manners able to tune the energy delivery selectively to the NO formation process, are sorely needed.

Nitric Oxide (NO) and Arginine as Factors for Increasing Poultry Meat Productivity

Nitric oxide (NO) is intensively synthesized in the embryo of birds. There is evidence that NO mediates myogenesis at the embryonic stage. In this regard, it might be possible to control muscle development and meat productivity by modulating NO synthesis. Using high-precision and a highly sensitive enzymatic sensor, the authors found that it is not the rate of synthesis that correlates with meat productivity, but the rate of NO oxidation to nitrate, which occurs in the tissues of the embryo. In broilers, it is several orders of magnitude higher than in layers. This indicator is solely due to the characteristics of embryonic tissues and is allelically determined. In-ovo arginine supplementation, a source of NO, did not lead to a significant increase in its synthesis and oxidation, but occasionally increased live weight gain, which was likely associated with a deficiency of free arginine and was not directly related to the effect of nitric oxide. Exogenous NO donors were oxidized with the same intensity as endogenous donors. These compounds did not have a significant effect on growth rate. Also, a reduction of 50% in the rate of NO synthesis under the action of a NO synthase inhibitor did not affect this parameter. Thus, regulation of poultry meat productivity is possible by modulating gene expression related to embryonic NO oxidation and ensuring an optimal amino acid and energy equilibrium, rather than by promoting embryonic NO synthesis.
 Keywords: Nitric oxide (NO), NO donor compounds, nitrate, arginine, live weight

Irsogladine Prevents Monochloramine-Induced Gastric Mucosal Lesions by Improving the Decrease in Mucosal Blood Flow Due to the Disturbance of Nitric Oxide Synthesis in Rats

Irsogladine Prevents Monochloramine-Induced Gastric Mucosal Lesions by Improving the Decrease in Mucosal Blood Flow Due to the Disturbance of Nitric Oxide Synthesis in Rats

Mechanism and regulation of ferrous heme-nitric oxide (NO) oxidation in NO synthases

Nitric oxide (NO) synthases (NOSs) catalyze the formation of NO from l-arginine. We have shown previously that the NOS enzyme catalytic cycle involves a large number of reactions but can be characterized by a global model with three main rate-limiting steps. These are the rate of heme reduction by the flavin domain (kr ), of dissociation of NO from the ferric heme-NO complex (kd ), and of oxidation of the ferrous heme-NO complex (kox). The reaction of oxygen with the ferrous heme-NO species is part of a futile cycle that does not directly contribute to NO synthesis but allows a population of inactive enzyme molecules to return to the catalytic cycle, and thus, enables a steady-state NO synthesis rate. Previously, we have reported that this reaction does involve the reaction of oxygen with the NO-bound ferrous heme complex, but the mechanistic details of the reaction, that could proceed via either an inner-sphere or an outer-sphere mechanism, remained unclear. Here, we present additional experiments with neuronal NOS (nNOS) and inducible NOS (iNOS) variants (nNOS W409F and iNOS K82A and V346I) and computational methods to study how changes in heme access and electronics affect the reaction. Our results support an inner-sphere mechanism and indicate that the particular heme-thiolate environment of the NOS enzymes can stabilize an N-bound FeIII-N(O)OO- intermediate species and thereby catalyze this reaction, which otherwise is not observed or favorable in proteins like globins that contain a histidine-coordinated heme.

Chronic Kidney Disease Elicits an Intestinal Inflammation Resulting in Intestinal Dysmotility Associated with the Activation of Inducible Nitric Oxide Synthesis in Rat

This study was conducted to investigate whether chronic kidney disease (CKD) affects intestinal inflammation and intestinal motility and the underlying mechanisms. Rats were randomized into control group and uremic group. Uremia rats were induced by the 5/6 kidney resection, while the control went through the same procedures but without any kidney resection. Intestinal motility was assessed by charcoal transport assay; intestinal inflammation was assessed by analyses of levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 in the ileum tissue. The inducible nitric oxide synthesis (iNOS) activity was assessed in the ileum tissue. The results showed that the intestinal motility in uremic group was significantly decreased compared with that in the control group on postoperative weeks 8 and 10. Meanwhile, the uremic group presented significantly higher concentrations of TNF-α, IL-6, and IL-10 than control group on postoperative weeks 8 and/or 10, and higher gene expression on postoperative weeks 6, 8, and 10. Furthermore, the intestinal iNOS activity in the uremic group was significantly increased compared with that in control group on postoperative weeks 8 and 10. These results suggest that CKD could induce intestinal inflammation and lead to intestinal dysmotility, which may be associated with iNOS activation in the intestine.

Acute ingestion of citrulline stimulates nitric oxide synthesis but does not increase blood flow in healthy young and older adults with heart failure.

To determine if age-associated vascular dysfunction in older adults with heart failure (HF) is due to insufficient synthesis of nitric oxide (NO), we performed two separate studies: 1) a kinetic study with a stable isotope tracer method to determine in vivo kinetics of NO metabolism, and 2) a vascular function study using a plethysmography method to determine reactive hyperemic forearm blood flow (RH-FBF) in older and young adults in the fasted state and in response to citrulline ingestion. In the fasted state, NO synthesis (per kg body wt) was ∼ 50% lower in older vs. young adults and was related to a decreased rate of appearance of the NO precursor arginine. Citrulline ingestion (3 g) stimulated de novo arginine synthesis in both older [6.88 ± 0.83 to 35.40 ± 4.90 μmol · kg body wt(-1) · h(-1)] and to a greater extent in young adults (12.02 ± 1.01 to 66.26 ± 4.79 μmol · kg body wt(-1) · h(-1)). NO synthesis rate increased correspondingly in older (0.17 ± 0.01 to 2.12 ± 0.36 μmol · kg body wt(-1) · h(-1)) and to a greater extent in young adults (0.36 ± 0.04 to 3.57 ± 0.47 μmol · kg body wt(-1) · h(-1)). Consistent with the kinetic data, RH-FBF in the fasted state was ∼ 40% reduced in older vs. young adults. However, citrulline ingestion (10 g) failed to increase RH-FBF in either older or young adults. In conclusion, citrulline ingestion improved impaired NO synthesis in older HF adults but not RH-FBF, suggesting that factors other than NO synthesis play a role in the impaired RH-FBF in older HF adults, and/or it may require a longer duration of supplementation to be effective in improving RH-FBF.

Arginine metabolic endotypes in pulmonary arterial hypertension.

Decreased synthesis of nitric oxide (NO) by NO synthases (NOS) is believed to play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). Multiple factors may contribute to decreased NO bioavailability, including increased activity of arginase, the enzyme that converts arginine to ornithine and urea, which may compete with NOS for arginine; inadequate de novo arginine production from citrulline; and increased concentration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NOS. We hypothesized that PAH patients with the lowest arginine availability secondary to increased arginase activity and/or inadequate de novo arginine synthesis might have a slower rate of NO synthesis and greater pulmonary vascular resistance. Nine patients with group 1 PAH and 10 healthy controls were given primed, constant intravenous infusions of (15)N2-arginine, (13)C,(2)H4-citrulline, (15)N2-ornithine, and (13)C-urea in the postabsorptive state. The results showed that, compared with healthy controls, PAH patients had a tendency toward increased arginine clearance and ornithine flux but no difference in arginine and citrulline flux, de novo arginine synthesis, or NO synthesis. Arginine-to-ADMA ratio was increased in PAH patients. Two endotypes of patients with low and high arginase activity were identified; compared with the low-arginase group, the patients with high arginase had increased arginine flux, slower NO synthesis, and lower plasma concentrations of ADMA. These results demonstrate that increased breakdown of arginine by arginase occurs in PAH and affects NO synthesis. Furthermore, there is no compensatory increase in de novo arginine synthesis to overcome this increased utilization of arginine by arginase.

The nitric oxide pathway – evidence and mechanisms for protection against liver ischaemia reperfusion injury

Ischaemia reperfusion (IR) injury is a clinical entity with a major contribution to the morbidity and mortality of liver surgery and transplantation. A central pathway of protection against IR injury utilizes nitric oxide (NO). Nitric oxide synthase (NOS) enzymes manufacture NO from L-arginine. NO generated by the endothelial NOS (eNOS) isoform protects against liver IR injury, whereas inducible NOS (iNOS)-derived NO may have either a protective or a deleterious effect during the early phase of IR injury, depending on the length of ischaemia, length of reperfusion and experimental model. In late phase hepatic IR injury, iNOS-derived NO plays a protective role. In addition to NOS consumption of L-arginine during NO synthesis, this amino acid may also be metabolized by arginase, an enzyme whose release is increased during prolonged ischaemia, and therefore diverts L-arginine away from NOS metabolism leading to a drop in the rate of NO synthesis. NO most commonly acts through the soluble guanylyl cyclase-cyclic GMP- protein kinase G pathway to ameliorate hepatic IR injury. Both endogenously generated and exogenously administered NO donors protect against liver IR injury. The beneficial effects of NO on liver IR are not, however, universal, and certain conditions, such as steatosis, may influence the protective effects of NO. In this review, the evidence for, and mechanisms of these protective actions of NO are discussed, and areas in need of further research are highlighted.

Restoration of impaired nitric oxide production in MELAS syndrome with citrulline and arginine supplementation

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most common mitochondrial disorders. Although the pathogenesis of stroke-like episodes remains unclear, it has been suggested that mitochondrial proliferation may result in endothelial dysfunction and decreased nitric oxide (NO) availability leading to cerebral ischemic events. This study aimed to assess NO production in subjects with MELAS syndrome and the effect of the NO precursors arginine and citrulline. Using stable isotope infusion techniques, we assessed arginine, citrulline, and NO metabolism in control subjects and subjects with MELAS syndrome before and after arginine or citrulline supplementation. The results showed that subjects with MELAS had lower NO synthesis rate associated with reduced citrulline flux, de novo arginine synthesis rate, and plasma arginine and citrulline concentrations, and higher plasma asymmetric dimethylarginine (ADMA) concentration and arginine clearance. We conclude that the observed impaired NO production is due to multiple factors including elevated ADMA, higher arginine clearance, and, most importantly, decreased de novo arginine synthesis secondary to decreased citrulline availability. Arginine and, to a greater extent, citrulline supplementation increased the de novo arginine synthesis rate, the plasma concentrations and flux of arginine and citrulline, and NO production. De novo arginine synthesis increased markedly with citrulline supplementation, explaining the superior efficacy of citrulline in increasing NO production. The improvement in NO production with arginine or citrulline supplementation supports their use in MELAS and suggests that citrulline may have a better therapeutic effect than arginine. These findings can have a broader relevance for other disorders marked by perturbations in NO metabolism.

Hypoadiponectinemia is caused by chronic blockade of nitric oxide synthesis in rats, Metabolism 2005;54(4):482-487

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The editor would like to confirm the retraction of this article at the request of the corresponding author. This article contains material that has appeared in Diabetologia 2005;48:1066-1074. RETRACTED: Hypoadiponectinemia is caused by chronic blockade of nitric oxide synthesis in ratsMetabolism - Clinical and ExperimentalVol. 54Issue 4PreviewThis article has been retracted: please see Elsevier Policy on Article Withdrawal ( http://www.elsevier.com/locate/withdrawalpolicy ). The editor would like to confirm the retraction of this paper at the request of the corresponding author. This article contains material that has appeared in Diabetologia, 48 (2005) 1066–1074. Full-Text PDF

In-vivo nitric oxide synthesis is reduced in obese patients with metabolic syndrome

Nitric oxide synthesis is declined in cardiovascular and metabolic diseases associated with endothelial dysfunction such as type 2 diabetes, hypertension or congestive heart failure. The objectives were to validate a novel stable isotopic method for the determination of in-vivo nitric oxide synthesis and to evaluate differences in nitric oxide synthesis in obese patients with and without metabolic syndrome (MetSyn). The new method, called oral nitrate test (ONT), measured the decay in saliva or urine samples of an oral dose of labelled sodium nitrate. The ONT method was compared to a validated method (frequent sampling arginine test, FSAT method) in 10 healthy adult volunteers (BMI range = 20.8-27.3 kg/m). The accuracy of the saliva ONT method was then tested by measuring nitric oxide synthesis in seven healthy, normal weight individuals, seven obese patients without MetSyn and seven obese patients with MetSyn. The estimated rate of nitric oxide synthesis was 0.63 ± 0.20 μmol/h per kg from the data obtained from saliva, and 0.50 ± 0.14 μmol/h per kg from urine. The agreement of the saliva ONT method with the FSAT method (Δ = +0.02 ± 0.24; P = 0.79) was superior to the urine ONT method (Δ = -0.11 ± 0.20; P = 0.13). Obese patients with MetSyn had a significantly lower nitric oxide production rate (0.21 ± 0.13 μmol/h per kg; P = 0.009) than healthy normal weight individuals (0.63 ± 0.30 μmol/h per kg), whereas nitric oxide production rate was intermediate in obese patients without MetSyn (0.49 ± 0.22 μmol/h per kg; P = 0.33). The advantages of the new saliva ONT method are its accuracy, sensitivity and lack of invasiveness, which could make it a reference method for the assessment of in-vivo rates of whole-body nitric oxide synthesis.

P17. Oral nitrite improves myocardial fibrosis induced by chronic blockage of nitric oxide synthesis in rats

restoring nitrite and nitrate in dialysis patients should be considered. Disclosure: This study was sponsored by Neogenis Labs Inc. N.S. Bryan has a financial interest in NeoGenis Inc. His financial and research conflicts of interest are managed by UTHSCH Conflicts of Interest Management Plans, developed from and reviewed by the Research Conflicts of Interest Committee and approved by the Executive Vice President for Research at the University of Texas Health Science Center at Houston.

Low intensity light stimulates nitrite-dependent nitric oxide synthesis but not oxygen consumption by cytochrome c oxidase: Implications for phototherapy

Cytochrome c oxidase (Cco) has been reported to be a receptor for some of the beneficial effects of low intensity visible and near-infrared light on cells and tissues. Here, we have explored the role of low intensity light in affecting a newly described function of Cco, its ability to catalyze nitrite–dependent nitric oxide (NO) synthesis (Cco/NO). Using a new assay for Cco/NO we have found that both yeast and mouse brain mitochondrial Cco produce NO over a wide range of oxygen concentrations and that the rate of NO synthesis increases as the oxygen concentration decreases, becoming optimal under hypoxic conditions. Low intensity broad-spectrum light increases Cco/NO activity in an intensity-dependent fashion but has no effect on oxygen consumption by Cco. By using a series of bandpass filters and light emitting devices (LEDs) we have determined that maximal stimulation of Cco/NO activity is achieved by exposure to light whose central wavelength is 590 ± 14 nm. This wavelength of light stimulates Cco/NO synthesis at physiological nitrite concentrations. These findings raise the interesting possibility that low intensity light exerts a beneficial effect on cells and tissues by increasing NO synthesis catalyzed by Cco and offer a new explanation for the increase in NO bioavailability experienced by tissue exposed to light.

In vivo arginine production and nitric oxide synthesis in pregnant Indian women with normal and low body mass indices

Background/Objectives:Nitric oxide (NO) has been proposed as a mediator of vascular expansion during pregnancy. Inability to increase NO synthesis and/or production of its precursor, arginine, may be a contributor to pregnancy-induced hypertension or preeclampsia. Because maternal weight is associated with blood pressure and risk of preeclampsia during pregnancy, it may also influence arginine and/or NO production. The purpose of this study was to determine the in vivo arginine production and NO synthesis rate in pregnant women with normal (n=10) and low (n=10) body mass indices (BMIs).Subjects/Methods:Arginine flux and NO synthesis rate were measured in the postabsorptive state with constant infusions of 15N2-arginine and 13C,2H4-citrulline. Plasma concentrations of arginine and NO metabolites were also measured. Kinetic parameters were correlated to maternal variables, gestational age, birth weight and blood pressure.Results:Endogenous arginine flux was significantly faster in the low-BMI compared with normal-BMI women in the first trimester (63.1±3.4 vs 50.2±2.0 μmol/kg per h, P<0.01), but not in the second. Plasma NO concentration was higher (44.7±5.3 vs 30.4±1.9 μmol/l, P=0.03) and its rate of synthesis trended faster in the low-BMI compared with normal-BMI group in the second trimester. Maternal weight and BMI were negatively correlated with arginine flux in both trimesters and NO synthesis in the second trimester.Conclusions:These findings suggest, but do not prove, that maternal BMI may be a factor in the ability to produce NO during pregnancy and may be one way by which BMI influences blood pressure during pregnancy.

AtNOS/AtNOA1 Is a Functional Arabidopsis thaliana cGTPase and Not a Nitric-oxide Synthase

AtNOS1 was previously identified as a potential nitric-oxide synthase (NOS) in Arabidopsis thaliana, despite lack of sequence similarity to animal NOSs. Although the dwarf and yellowish leaf phenotype of Atnos1 knock-out mutant plants can be rescued by treatment with exogenous NO, doubts have recently been raised as to whether AtNOS1 is a true NOS. Moreover, depending on the type of physiological responses studied, Atnos1 is not always deficient in NO induction and/or detection, as previously reported. Here, we present experimental evidence showing that AtNOS1 is unable to bind and oxidize arginine to NO. These results support the argument that AtNOS1 is not a NOS. We also show that the renamed NO-associated protein 1 (AtNOA1) is a member of the circularly permuted GTPase family (cGTPase). AtNOA1 specifically binds GTP and hydrolyzes it. Complementation experiments of Atnoa1 mutant plants with different constructs of AtNOA1 show that GTP hydrolysis is necessary but not sufficient for the physiological function of AtNOA1. Mutant AtNOA1 lacking the C-terminal domain, although retaining GTPase activity, failed to complement Atnoa1, suggesting that this domain plays a crucial role in planta. cGTPases appear to be RNA-binding proteins, and the closest homolog of AtNOA1, the Bacillus subtilis YqeH, has been shown to participate in ribosome assembly and stability. We propose a similar function for AtNOA1 and discuss it in the light of its potential role in NO accumulation and plant development.

Carbon monoxide–prostaglandin E2 interaction in the hypothalamic circulation

The heme oxygenase (HO)-carbon monoxide pathway was earlier shown to increase hypothalamic blood flow after inhibition of nitric oxide synthesis in rats. We hypothesized that this effect is mediated by prostaglandin E2 (PGE2). Inhibition of constitutive HO activity decreased cerebral PGE2 production and simultaneously increased hypothalamic nitric oxide synthase (NOS) activity without changing hypothalamic blood flow. Furthermore, HO blockade induced cyclooxygenase-dependent decrease and NOS-mediated increase of the hypothalamic blood flow after inhibition of NOS and cyclooxygenase, respectively. Therefore, constitutive carbon monoxide release seems to have two indirect effects on the hypothalamic circulation: vasodilation mediated by PGE2 and vasoconstriction as a result of NOS inhibition.

10.1007/s11827-008-1002-y

Digital fluorescence imaging was used to study the effects of the amyloid-beta peptide βA (full-length peptide, βA 1–42) and its neurotoxic fragment (βA 25–35) on nitric oxide (NO) synthesis and cell viability in mixed cultures of rat hippocampal neurons and astrocytes. It was found that both βA 1–42 and βA 25–35 stimulated NO synthesis in astrocytes, but not in neurons. L-NAME, an inhibitor of the inducible NO synthase, blocked the effect of βA on NO production almost completely, reduced βA-induced mitochondrial depolarization in astrocytes, and partly prevented neuronal death. The rate of NO synthesis was decreased in Ca2+-free medium, increased in the presence of antioxidants and the NADPH oxidase inhibitor, and decreased in the presence of the SH-reagent thimerosal.

Simultaneous Assessment of Endothelial Function, Nitric Oxide Synthase Activity, Nitric Oxide–Mediated Signaling, and Oxidative Stress in Individuals with and without Hypercholesterolemia

Endothelial function is impaired in hypercholesterolemia and atherosclerosis. Based on mostly indirect evidence, this impairment is attributed to reduced synthesis or impaired biological activity of endothelium-derived nitric oxide (NO). It was the aim of this study to directly estimate and compare whole-body NO production in normo- and hypercholesterolemia by applying a nonradioactive stable isotope dilution technique in vivo. We enrolled 12 normocholesterolemic and 24 hypercholesterolemic volunteers who were all clinically healthy. To assess whole-body NO synthesis, we intravenously administered l-[guanidino-((15)N(2))]-arginine and determined the urinary excretion of (15)N-labeled nitrate, the specific end product of NO oxidation in humans, by use of gas chromatography-mass spectrometry. In addition, we measured flow-mediated vasodilation (FMD) of the brachial artery, expression of endothelial NOS (eNOS) in platelets, plasma concentration of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA), and urinary excretion of 8-isoprostaglandin F(2alpha) (8-iso-PGF(2alpha)). After infusion of l-[guanidino-((15)N(2))]-arginine, cumulative excretion of (15)N-labeled-nitrate during 48 h was 40% [95% CI 15%-66%] lower in hypercholesterolemic than normocholesterolemic volunteers [mean 9.2 (SE 0.8) micromol vs 15.4 (2.3) micromol/l, P = 0.003]. FMD was on average 36% [4%-67%] lower in hypercholesterolemic than normocholesterolemic volunteers [6.3 (4.0)% vs 9.4 (4.6)%, P = 0.027]. Normalized expression of NOS protein in platelets was also significantly lower in hypercholesterolemic volunteers, whereas there were no significant differences in plasma ADMA concentration or urinary excretion of 8-iso-PGF(2alpha) between the 2 groups. This study provides direct evidence for a decreased whole body NO synthesis rate in healthy people with hypercholesterolemia.

Electron Transfer by Neuronal Nitric-oxide Synthase Is Regulated by Concerted Interaction of Calmodulin and Two Intrinsic Regulatory Elements

The nitric-oxide synthases (NOSs) are modular, cofactor-containing enzymes, divided into a heme-containing oxygenase domain and an FMN- and FAD-containing reductase domain. The domains are connected by a calmodulin (CaM)-binding sequence, occupancy of which is required for nitric oxide (NO) production. Two additional CaM-modulated regulatory elements are present in the reductase domains of the constitutive isoforms, the autoregulatory region (AR) and the C-terminal tail region. Deletion of the AR reduces CaM stimulation of electron flow through the reductase domain from 10-fold in wild-type nNOS to 2-fold in the mutant. Deletion of the C terminus yields an enzyme with greatly enhanced reductase activity in the absence of CaM but with activity equivalent to that of wild-type enzyme in its presence. A mutant in which both the AR and C terminus were deleted completely loses CaM modulation through the reductase domain. Thus, transduction of the CaM effect through the reductase domain of nNOS is dependent on these elements. Formation of nitric oxide is, however, still stimulated by CaM in all three mutants. A CaM molecule in which the N-terminal lobe was replaced by the C-terminal lobe (CaM-CC) supported NO synthesis by the deletion mutants but not by wild-type nNOS. We propose a model in which the AR, the C-terminal tail, and CaM interact directly to regulate the conformational state of the reductase domain of nNOS.