Low Concentrations Of Nitric Oxide Research Articles

The association of endothelial nitric oxide synthase gene G894T polymorphism and serum nitric oxide concentration with microalbuminuria in patients with gestational diabetes

Gestational diabetes mellitus (GDM) is a glucose intolerant condition that affects 14% of all pregnancies. Diabetes mellitus (DM) occurs in 30 - 70% of patients with GDM after delivery. DM and GDM are associated with structural and functional deterioration of the renovascular system. Our aim is to investigate the association Glu- 298Asp polymorphism of the endothelial nitric oxide synthase (eNOS) gene with serum nitric oxide levels and microalbuminuria in patients with GDM and healthy pregnancies. Serum nitric oxide (NO) levels, urinary excretion of albumin and Glu298Asp polymorphism of the eNOS gene were analyzed in 68 patients with GDM and 73 healthy controls. High performance liquid chromatography (HPLC-Griess) method was used to analyze serum NO levels. Microalbuminuria was evaluated by rate nephelometry method. The Glu298Asp polymorphism of the eNOS gene was determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Nitric oxide, glucose, creatinine, and microalbuminuria were significantly different between the patients and the control subjects (p = 0.001, p = 0.001, p = 0.002, and p = 0.005, respectively). There was a significant difference between groups in terms of the ratio of GG/GT+TT of eNOS gene Glu- 298Asp (p = 0.02). The patients with GT+TT genotype had significantly higher microalbuminuria levels and lower NO concentrations (22.16 vs. 9.51, p = 0.005, and 10.56 vs. 12.73, p = 0.021, respectively). The presence of T allele of eNOS gene is an independent predictor of microalbuminuria (OR: 2.346, 95% confidence interval: 1.247 - 5.238, p = 0.02) as well as serum glucose and NO concentration. The G894T polymorphism of eNOS gene and decreased NO concentration seem to be independent predictors of increased urinary excretion of albumin in patients with GDM. Determining the frequency of eNOS gene G894T polymorphism may help to identify pregnancies at increased risk of microalbuminuria.

Study of the effects of oral zinc supplementation on peroxynitrite levels, arginase activity and NO synthase activity in seminal plasma of Iraqi asthenospermic patients

BackgroundLow concentrations of nitric oxide (NO) are necessary for the biology and physiology of spermatozoa, but high levels of NO are toxic and have negative effects on sperm functions. Although several studies have considered the relationship between infertility and semen NO concentrations, no study on the effects of asthenospermia treatments such as oral zinc supplementation on concentrations of NO, which are important in fertility, has been reported. Studies have shown that oral zinc supplementation develops sperm count, motility and the physical characteristics of sperm in animals and in some groups of infertile men. The present study was conducted to study the effect of zinc supplementation on the quantitative and qualitative characteristics of semen, along with enzymes of the NO pathway in the seminal plasma of asthenospermic patients.MethodsSemen samples were obtained from 60 fertile and 60 asthenozoospermic infertile men of matched age. The subfertile group was treated with zinc sulfate; each participant took two capsules (220 mg per capsule) per day for 3 months. Semen samples were obtained (before and after zinc sulfate supplementation). After liquefaction of the seminal fluid at room temperature, routine semen analyses were performed. The stable metabolites of NO (nitrite) in seminal plasma were measured by nitrophenol assay. Arginase activity and NO synthase activity were measured spectrophotometrically.ResultsPeroxynitrite levels, arginase activity, NO synthase activity and various sperm parameters were compared among fertile controls and infertile patients (before and after treatment with zinc sulfate). Peroxynitrite levels and NO synthase activity were significantly higher in the infertile patients compared to the fertile group. Conversely, arginase activity was significantly higher in the fertile group than the infertile patients. Peroxynitrite levels, arginase activity and NO synthase activity of the infertile patient were restored to normal values after treatment with zinc sulfate. Volume of semen, progressive sperm motility percentage and total normal sperm count were increased after zinc supplementation.ConclusionsTreatment of asthenospermic patients with zinc supplementation leads to restored peroxynitrite levels, arginase activity and NO synthase activity to normal values and gives a statistically significant improvement of semen parameters compared with controls.Trial registrationClinicalTrials.gov identifier: NCT01684059

Levels of biological markers of nitric oxide in serum of patients with squamous cell carcinoma of the oral cavity

The aim of the study was a determination of the levels of nitric oxide (NO) and its biological markers such as malonyldialdehyde (MDA) and nitrotyrosine in the serum of patients with squamous cell carcinoma (SCC) of the oral cavity and identification of the relationships between NO and those markers. These studies were performed on patients with SCC of the oral cavity before and after treatment. Griess reaction was used for the estimation of the total concentration of NO in serum. The nitrotyrosine level in serum was assessed with an enzyme-linked immunosorbent assay (ELISA) kit, and MDA level using a spectrophotometric assay. Higher concentrations of NO in blood serum were determined in patients with stage IV of the disease before treatment in comparison to the control group and patients with stages II and III of the disease. Moreover, higher concentrations of MDA and nitrotyrosine were determined in the serum of patients in all stages of the disease in comparison to healthy people. After treatment, lower concentrations of NO in the serum of patients with stage IV of the disease were observed in comparison to the amounts obtained prior to treatment. In addition, lower levels of nitrotyrosine in the serum of patients with all stages of the disease were recorded, whereas higher concentrations of MDA were determined in these patients in comparison to results obtained before treatment. The compounds formed with the contribution of NO, such as MDA and nitrotyrosine, may lead to cancer progression in patients with SCC of the oral cavity, and contribute to formation of resistance to therapy in these patients as well. Moreover, the lack of a relationship between concentrations of NO and MDA, and between NO and nitrotyrosine in serum suggests that the process of lipid peroxidation and nitration in patients with SCC does not just depend on NO.

Modulating Nitric Oxide Signaling in the Cns for Alzheimer’s Disease Therapy

Nitric oxide (NO)/solube GC (sGC)/cGMP signaling is important for modulating synaptic transmission and plasticity in the hippocampus and cerebral cortex, which are critical for learning and memory. Physiological concentrations of NO also elicit anti-apoptotic/prosurvival effects against various neurotoxic challenges and brain insults through multiple mechanisms. Depression of the NO/sGC pathway is a feature of Alzheimer's disease (AD), attributed to amyloid-β neuropathology, and altered expression and activity of NOS, sGC and PDE enzymes. Different classes of NO-releasing hybrid drugs, including nomethiazoles, NO-NSAIDs and NO-acetylcholinesterase inhibitors were designed to deliver low concentrations of exogenous NO to the CNS while targeting other underlying disease mechanisms, such as excitotoxicity, neuro-inflammation and acetylcholine deficiency, respectively. Incorporating a NO-donating moiety may also reduce gastrointestinal and liver toxicity of the parent drugs. Progress has also been made in targeting downstream sGC and PDE enzymes. The PDE9 inhibitor PF-04447943 has completed Phase II clinical trials for AD. The search for effective NO-donating hybrid drugs, CNS-targeting sGC stimulators/activators and selective PDE inhibitors is an important goal for pharmacotherapy that manipulates NO biochemical pathways involved in cognitive function and neuroprotection. Rigorous preclinical validation of target engagement, and optimization of pharmacokinetic and toxicity profiles are likely to advance more drug candidates into clinical trials for mild cognitive impairment and early stage AD.

β-galactosyl-pyrrolidinyl diazeniumdiolate: an efficient tool to investigate nitric oxide functions on promoting cell death

Nitric oxide (NO) is an active free radical gas that plays crucial roles in a broad range of biological processes. Extremely short half-life makes it difficult to use NO directly in research. It has been suggested that different concentrations of NO may lead to quite opposite results on cytotoxicity. However, the net effect of intracellular NO on tumor cell death has been controversial, partly because it is hard to precisely control the amount of NO generated exclusively within the target cells. Therefore, we have developed a cell-specific NO donor, β-galactosyl-pyrrolidinyl diazeniumdiolate (β-Gal-NONOate), in hopes of simulating the actual effects of intracellularly derived NO on the patterns of cell death as well as investigating its underlying mechanisms. In this study, by using three different tumor cell models, we showed that β-Gal-NONOate could steadily transport NO into the target cells with similar delivery efficiencies and exerted a determinative effect on cell death. In addition, β-Gal-NONOate-derived intracellular NO could provoke both apoptosis and necrosis in a concentration-dependent manner. While lower NO concentration primarily induced apoptosis, higher NO concentration mainly triggered necrosis. Moreover, the intrinsic apoptotic pathway, characterized by rapid Ca²⁺ overload and subsequent mitochondrial damage, was the collective mechanism responsible for the apoptotic death in all the three tumor cell lines. Taken together, since this cell-specifically derived NO is cheap to use and easy to quantify, β-Gal-NONOate might be used as a novel and ideal tool to standardize intracellular NO generation and evaluate its net effects in different cellular and experimental settings in the coming future.

Protective function of nitric oxide on marine phytoplankton under abiotic stresses

As an important signaling molecule, nitric oxide (NO) plays diverse physiological functions in plants, which has gained particular attention in recent years. We investigated the roles of NO in the growth of marine phytoplankton Platymonas subcordiforms and Skeletonema costatum under abiotic stresses. The growth of these two microalgae was obviously inhibited under non-metal stress (sodium selenium, Na2SeO3), heavy metal stress (lead nitrate, Pb(NO3)2), pesticide stress (methomyl) and UV radiation stress. After the addition of different low concentrations of exogenous NO (10−10–10−8molL−1) twice each day during cultivation, the growth of these two microalgae was obviously promoted. Results showed that NO could relieve the oxidative stresses to protect the growth of the two microalgae. For different environmental stress, there is a different optimum NO concentration for marine phytoplankton. It is speculated that the protective effect of NO is related to its antioxidant ability.

Synthesis of organic nitrates of luteolin as a novel class of potent aldose reductase inhibitors

Aldose reductase (AR) plays an important role in the design of drugs that prevent and treat diabetic complications. Aldose reductase inhibitors (ARIs) have received significant attentions as potent therapeutic drugs. Based on combination principles, three series of luteolin derivatives were synthesised and evaluated for their AR inhibitory activity and nitric oxide (NO)-releasing capacity in vitro. Eighteen compounds were found to be potent ARIs with IC50 values ranging from (0.099±0.008) μM to (2.833±0.102) μM. O7-Nitrooxyethyl-O3′,O4′-ethylidene luteolin (La1) showed the most potent AR inhibitory activity [IC50=(0.099±0.008) μM]. All organic nitrate derivatives released low concentrations of NO in the presence of l-cysteine. Structure–activity relationship studies suggested that introduction of an NO donor, protection of the catechol structure, and the ether chain of a 2-carbon spacer as a coupling chain on the luteolin scaffold all help increase the AR inhibitory activity of the resulting compound. This class of NO-donor luteolin derivatives as efficient ARIs offer a new concept for the development and design of new drug for preventive and therapeutic drugs for diabetic complications.

High plasma concentrations of asymmetric dimethylarginine inhibit ischemic cardioprotection in hypercholesterolemic rats

A low concentration of nitric oxide associated with a high concentration of asymmetric dimethylarginine (ADMA) can explain the lack of ischemic cardioprotection observed in the presence of hypercholesterolemia. The objective of the present study was to evaluate the effect of hypercholesterolemia on ischemic pre- and postconditioning and its correlation with plasma concentrations of ADMA. Male Wistar rats (6-8 weeks old) fed a 2% cholesterol diet (n = 21) for 8 weeks were compared to controls (n = 25) and were subjected to experimental myocardial infarction and reperfusion, with ischemic pre- and postconditioning. Total cholesterol and ADMA were measured in plasma before the experimental infarct and the infarct area was quantified. Weight, total cholesterol and plasma ADMA (means ± SE; 1.20 ± 0.06, 1.27 ± 0.08 and 1.20 ± 0.08 vs 0.97 ± 0.04, 0.93 ± 0.05 and 0.97 ± 0.04 µM) were higher in animals on the hypercholesterolemic diet than in controls, respectively. Cardioprotection did not reduce infarct size in the hypercholesterolemic animals (pre: 13.55% and post: 8% compared to 7.95% observed in the group subjected only to ischemia and reperfusion), whereas infarct size was reduced in the animals on a normocholesterolemic diet (pre: 8.25% and post: 6.10% compared to 12.31%). Hypercholesterolemia elevated ADMA and eliminated the cardioprotective effects of ischemic pre- and postconditioning in rats.

P58: Secondary amines containing one aromatic nitro group: Preparation, nitrosation, sustained nitric oxide release, and the synergistic effects of released nitric oxide and an arginase inhibitor on vascular smooth muscle cell proliferation

Background Nitric oxide (NO) is synthesized by vascular endothelial cells in vivo from l -arginine by nitric oxide synthases (NOS). Initial events in the onset of atherosclerosis lead to endothelial dysfunction and subsequent decrease of endogenous NO. Within the vasculature NO inhibits smooth muscle cell proliferation (SMC), therefore decreased concentrations lead to rapid SMC division, a key event in the formation of atherosclerotic plaques. Reestablishing physiological levels exogenously with NO-donors, aims to stop uncontrolled SMC proliferation and the progression of atherosclerosis. However current NO donors rapidly release NO in highly concentrated, single bursts, leading to cellular oxidative stress. In contrast, N-nitroso (N–NO) NO donors synthesized in our lab through the nitrosation of secondary amines, release NO in a slow, sustained manner, establishing and maintaining low NO concentrations that effectively reduce SMC proliferation, without onset of oxidative stress.Additionally, NOS competes for l -arginine with arginase, which catalyzes its conversion to l -ornithine, the precursor to various polyamines-known stimulators of SMC proliferation. Arginase inhibition would reduce SMC proliferation by decreasing l -ornithine concentrations and providing increased l -arginine for endogenous NO production. Combining arginase inhibition with NO-donor application may prevent SMC proliferation more effectively than either method individually, and may have a synergistic effect. Methods Two isomeric families of NO-donors, prepared in our laboratory, were applied to SMC cultures at various concentrations. Each secondary amine precursor was also tested for any toxic or anti-proliferative effects. Likewise the potent arginase inhibitor, ABH was tested to determine the most effective concentration for proliferation reduction. Finally, optimized concentrations of NO-donors and ABH were added in tandem. Proliferation and toxicity were monitored using a cell viability and LIVE/DEAD assay. A third assay was implemented to monitor changes in intracellular NO concentrations due to the addition of each compound. Results Cellular viability assays showed eight of ten amines exerted no toxic or anti-proliferative effects in the range tested (0.1–20 μM), nor did they increase intracellular NO concentrations. These compounds were nitrosated and tested in same concentration range. All donors exhibited proliferation inhibition without inducing cell death between 5% and 20 μM in a concentration dependent manner. At 20 μM, the decrease in viable cells ranged from 5% to 22%. Likewise, ABH decreased the number of viable cells in a concentration dependent manner from 0.001 to 5.0 mM by 9.9–30.0%, respectively. Both the NO-donors and ABH increased intracellular NO with increasing concentrations. Finally, the most effective NO-donor from each family (20 μM) in combination with ABH (5 mM) inhibited SMC proliferation in a synergistic manner, reducing the number of viable cells by an additional 2% and 16% compared to the sum of individual components alone. These results were supported by the synergistic increase of intracellular NO. Conclusion Inhibition of SMC proliferation after endothelial dysfunction could slow down the establishment of arterial plaques, reducing the occurrence of cardiovascular diseases including atherosclerosis. Reestablishing NO concentrations both exogenously with NO-donors and endogenously by arginase inhibition with ABH simultaneously has shown to more effectively reduce SMC proliferation than either method individually, while avoiding onset of oxidative stress. Disclosure NIH, Award # R15HL 106600.

Oxygen dependence of nitric oxide-mediated signaling

Nitric oxide (•NO) is a biologically important short-lived free radical signaling molecule. Both the enzymatic synthesis and the predominant forms of cellular metabolism of •NO are oxygen-dependent. For these reasons, changes in local oxygen concentrations can have a profound influence on steady-state •NO concentrations. Many proteins are regulated by •NO in a concentration-dependent manner, but their responses are elicited at different thresholds. Using soluble guanylyl cyclase (sGC) and p53 as model •NO-sensitive proteins, we demonstrate that their concentration-dependent responses to •NO are a function of the O2 concentration. p53 requires relatively high steady-state •NO concentrations (>600 nM) to induce its phosphorylation (P-ser-15), whereas sGC responds to low •NO concentrations (<100 nM). At a constant rate of •NO production (liberation from •NO-donors), decreasing the O2 concentration (1%) lowers the rate of •NO metabolism. This raises steady-state •NO concentrations and allows p53 activation at lower doses of the •NO donor. Enzymatic •NO production, however, requires O2 as a substrate such that decreasing the O2 concentration below the Km for O2 for nitric oxide synthase (NOS) will decrease the production of •NO. We demonstrate that the amount of •NO produced by RAW 264.7 macrophages is a function of the O2 concentration. Differences in rates of •NO production and •NO metabolism result in differential sGC activation that is not linear with respect to O2. There is an optimal O2 concentration (≈5–8%) where a balance between the synthesis and metabolism of •NO is established such that both the •NO concentration and sGC activation are maximal.

A Detailed Examination of Platelet Function Inhibition by Nitric Oxide in Platelet-Rich Plasma and Whole Blood

The question of whether novel instruments such as multiple electrode aggregometry (MEA) can be used for measurement of the effects of nitric oxide (NO) on platelets (PLTs) has not been examined. Therefore, we compared the effects of NO concentrations (1, 10, and 100 microM) on the PLT aggregation response to ADP, arachidonic acid (AA), collagen, ristocetin, and thrombin receptor-activating peptide 6 (TRAP6) using light transmission aggregometry (LTA) and multiple electrode aggregometry (MEA) and examined the effects of NO using the platelet function analyzer (PFA)-100. The response of PLTs to ADP and AA was strongly inhibited by all NO concentrations in LTA and MEA. The inhibition of the responses to ristocetin and collagen was detectable in MEA at lower NO concentrations than in LTA. However, the typically increasing lag phase between collagen addition and the aggregation response in the presence of NO was more obvious in LTA. TRAP caused a reproducible early response in the presence of NO in LTA which was followed by rapid PLT disaggregation, whereas even 100 microM NO did not inhibit the response to TRAP in MEA. Finally, NO prolonged the in-vitro bleeding time remarkably more in the PFA-100 collagen-epinephrin cartridge than in the collagen-ADP cartridge. Whole blood versus PLT rich plasma, citrate versus hirudin, and high versus low shear influenced the effects of NO. This shows that a careful selection of models and potentially a combination of different methods is appropriate for a differentiated evaluation of pharmacological or physiological mechanisms of NO-donors or of NO-inhibitors.

Recommendations of using at least two different methods for measuring NO

Nitric oxide (NO) is widely recognized as a signal molecule in plants. Various sources of NO were identified in plants (Moreau et al., 2010; Gupta et al., 2011; Mur et al., 2013) located in different compartments activated under various conditions. Briefly these are the mitochondrial nitrite: NO reductase reaction, the cytosolic nitrate reductase (NR), the plasma membrane nitrite: NO reductase (PM-NiNOR), xanthine oxidoreductase, NO synthase-like enzyme (putative), polyamine (PA)- and hydroxylamine (HA)-mediated pathways. NO acts as an intracellular messenger due to its diffusible capacity through various cellular compartments. After pioneering discovery of NO production in plants, scientists started digging deeply to the key function of NO and this research led to understanding of various roles of NO that include regulation of stomatal movement, root development, floral transition, response to biotic and abiotic stresses, symbiotic interactions. Despite of extensive research on NO roles in metabolism and signal transduction, its measurement remains challenging (Vandelle and Delledonne, 2008; Mur et al., 2011). We discuss below the problems associated with NO measurement and suggest some important solutions to tackle these problems. The half-life of NO depends on its concentration and usually falls is in the range of ten(s) seconds. For instance, at 10 μM concentration NO has a half-life of about 80 s whereas at 100 μM concentration NO has a half-life of about 8 s (Wink and Mitchell, 1998). This means that at low concentration NO can easily diffuse from its origin to the site of action. At higher concentrations most of the NO rapidly undergoes autoxidation. Moreover NO half-life also depends on the presence of scavengers such as non-symbiotic hemoglobin (class 1) and other major NO scavenging targets such as lipids and metal-containing proteins. Therefore, it is often very important to measure NO concentration which is crucial for understanding its function. But NO concentrations measured in same biological material by different methods often give different values (Mur et al., 2006; Planchet and Kaiser, 2006). In some cases NO production is localized to specific cells such as guard cells; therefore fluorescent probes are required to visualize NO producing sites. Here we describe why we need to use at least two different methods for measuring generation of NO in plants and suggest the best combination of methods for specific studies.

Dual Role of Nitric Oxide in Pancreatic β-Cells

An involvement of inducible nitric oxide (NO) synthase (NOS) in pancreatic β-cell degeneration during the process of type 1 diabetes has been well discussed. Recently, there is growing evidence for pivotal roles of constitutive NOS (cNOS) in β-cells; the presence of NOS1 and NOS3 in pancreatic β-cells and the effects of low-concentration NO, which is assumed to be derived from cNOS, on β-cell functions have been reported. However, the roles of cNOS-derived NO in β-cells are still under debate. One of the reasons seems to be that NO has multiple biological activities, which are dependent on its concentration. In β-cells, NO has been shown to exert positive and negative regulation of insulin secretion and anti- and pro-apoptotic activities, which is likely to be dependent on concentrations. In this review article, we will describe the current understanding of the roles of NO in pancreatic β-cells, especially focusing on cNOS-derived NO and its differential roles depending on concentrations.

Antimicrobial actions of nitric oxide

Nitric oxide (NO ) is a versatile molecular mediator that is generated enzymatically or chemically within the host and exerts broad-spectrum antimicrobial activity. Production of NO by phagocytic cells is augmented in response to inflammation. Sequential production of bactericidal reactive oxygen species and bacteriostatic NO is observed in both macrophages and mice during infection with the enteric bacterial pathogen Salmonella enterica . The reaction of NO with metal centers and thiols inhibits bacterial respiration, DNA replication and specific metabolic pathways including the tricarboxylic acid cycle. NO is predominantly detoxified by the Hmp flavohemoglobin, whose expression is exquisitely responsive to low concentrations of NO . The Gram-positive bacterium Staphylococcus aureus escapes NO inhibition by expressing Hmp in concert with inducible homolactic fermentation, which serves to maintain redox balance during nitrosative stress. This may account for the unique ability of this pathogen to withstand high NO concentrations present in the nose.

Skeletal muscle nitric oxide signaling and exercise: a focus on glucose metabolism

Nitric oxide (NO) is an important vasodilator and regulator in the cardiovascular system, and this link was the subject of a Nobel prize in 1998. However, NO also plays many other regulatory roles, including thrombosis, immune function, neural activity, and gastrointestinal function. Low concentrations of NO are thought to have important signaling effects. In contrast, high concentrations of NO can interact with reactive oxygen species, causing damage to cells and cellular components. A less-recognized site of NO production is within skeletal muscle, where small increases are thought to have beneficial effects such as regulating glucose uptake and possibly blood flow, but higher levels of production are thought to lead to deleterious effects such as an association with insulin resistance. This review will discuss the role of NO in skeletal muscle during and following exercise, including in mitochondrial biogenesis, muscle efficiency, and blood flow with a particular focus on its potential role in regulating skeletal muscle glucose uptake during exercise.

Nitric Oxide: A Double Edged Weapon for Sperm Functions

) and hydroxyl (OH) radicals. Those derived from nitrogen are called reactive nitrogen species (RNS). RNS include nitric oxide (NO ), nitrogen dioxide (NO 2 ) and peroxynitrite anion (ONOO ). RNS are often considered to be a subclass of ROS. Recent information on the NO has proved its importance as an intercellular and intracellular messenger controlling many physiological processes. It is also a mediator of cytokines and growth factors in various cell types. NO is synthesized form L-arginine by the action of nitric oxide synthase (NOS), an enzyme existing in three isoforms. Two of them, endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS), collectively called as constitutive nitric oxide synthase (cNOS), are responsible for continuous basal release of NO and both, require calcium/calmodulin for activation. The other one is called as inducible nitric oxide synthase (iNOS), is responsible for prolonged release of NO and does not require calcium/calmodulin for activation. It is expressed in response to inflammatory cytokines and lipopolysaccharides. Low concentration of NO increased the motility and viability of spermatozoa. However, high concentration of NO decreased the sperm motility and viability. The bimodal motility response to various concentrations of NO releasing compounds could be due to dual nature of NO, as both a transduction molecule at low concentration and a cytotoxic effector at high concentrations in systems. NO inhibits cellular respiration by nitrosylation of heme in mitochondrial enzymes, aconitase and glyceraldehyde-3-phosphate dehydrogenase, leading to a depletion of ATP and consequent loss of motility in the spermatozoa. The primary mechanism of NO induced sperm damage is likely to be the inhibition of mitochondrial respiration and DNA synthesis. NO induced toxicity is also mediated indirectly through its interaction with superoxide anions and formation of

Discovery of nitric oxide and cyclic GMP in cell signaling and their role in drug development

The role of nitric oxide in cellular signaling in the past three decades has become one of the most rapidly growing areas in biology. Nitric oxide is a gas and a free radical with an unshared electron that can regulate an ever-growing list of biological processes. Nitric oxide is formed from L-arginine by a family of enzymes called nitric oxide synthases. These enzymes have a complex requirement for a number of cofactors and regulators including NADPH, tetrahydrobioterin, flavins, calmodulin and heme. The enzymes are present in most cells and tissues. In many instances, nitric oxide mediates its biological effects by activating the soluble isoform of guanylyl cyclase and increasing cyclic GMP synthesis from GTP. Cyclic GMP, in turn, can activate cyclic GMP-dependent protein kinase (PKG) and can cause smooth muscles and blood vessels to relax, decrease platelet aggregation, alter neuron function, etc. These effects can decrease blood pressure, increase blood flow to tissues, alter memory and behavior, decrease blood clotting, etc. The list of effects of nitric oxide that are independent of cyclic GMP formation is also growing at a rapid rate. For example, nitric oxide can interact with transition metals such as iron, thiol groups, other free radicals, oxygen, superoxide anion, unsaturated fatty acids, and other molecules. Some of these reactions result in the oxidation of nitric oxide to nitrite and nitrate to terminate the effect, while other reactions can lead to altered protein structure function and/or catalytic capacity. These effects probably regulate bacterial infections, inflammation of tissues, tumor growth, and other disorders. These diverse effects of nitric oxide that are cyclic GMP dependent or independent can alter and regulate numerous important physiological events in cell regulation and function. Nitric oxide can function as an intracellular messenger, an antacoid, a paracrine substance, a neurotransmitter, or as a hormone that can be carried to distant sites for effects. Thus, it is a unique molecule with an array of signaling functions. However, with any messenger molecule, there can be too little or too much of the substance, resulting in pathological events. Some of the methods to regulate either nitric oxide formation metabolism, or function have been in clinical use for more than a century, as with the use of organic nitrates and nitroglycerin in angina pectoris that was initiated in the 1870s. Inhalation of low concentrations of nitric oxide can be beneficial in premature infants with pulmonary hypertension and increase survival rates. Ongoing clinical trials with nitric oxide synthase inhibitors and nitric oxide scavengers are examining the effects of these agents in septic shock, hypotension with dialysis, inflammatory disorders, cancer therapy, etc. Recognition of additional molecular targets in the areas of nitric oxide and cyclic GMP research will continue to promote drug discovery and development programs in the field. Current and future research will undoubtedly expand the clinician’s therapeutic armamentarium to manage a number of important diseases by perturbing nitric oxide formation and metabolism. Such promise and expectations have obviously fueled the interests in nitric oxide research for a growing list of potential therapeutic applications. There have been and will continue to be many opportunities from nitric oxide and cyclic GMP march to develop novel and important therapeutic agents. There are presently more than 80,000 publications in the area of nitric oxide research. The lecture will discuss our discovery of the first biological effects of nitric oxide and how the field has evolved since our original reports in 1977. The possible utility of this signaling pathway to facilitate novel drug development and the creation of numerous projects in the Pharmaceutical and biotechnology industrials will also be discussed.

The Comparative and Combined Effects of Nitric Oxide and Higher Alkanes in Sensitizing Methane Oxidation

The comparative and combined effects of nitric oxide (NO) and higher alkanes on methane oxidation were examined by experimentation and kinetic modeling. Experiments were conducted using fuel-lean, lower-alkanes mixtures with NO (0–400 ppm v/v) in an atmospheric flow reactor at residence time of 2 s over the temperature range of 820–950 K. NO was found to greatly promote methane conversion, and its sensitizing effect strengthened with the increasing concentration that was added to the system. The promoting effect of higher alkanes on methane conversion was also evident, particularly at zero or low NO concentrations. A strong dependency of the sensitizing effect on the concentration of higher alkanes present was also observed. The kinetic mechanism from Le Cong et al., performed reasonably well in reproducing the experimental trends. However, the sensitizing propensity of higher alkanes in the presence of NO could not be unambiguously ranked under all conditions. Modification to the kinetic mechanism of Le Cong et al., was attempted. Specifically, the submechanism of C3 peroxy radicals was added, but the modeling results indicated a lower impact on methane conversion than was initially expected. The most sensitive reactions were revealed, and the generalized reaction pathways for methane oxidation sensitized by higher alkanes, with or without the presence of NO, were also proposed, following detailed sensitivity analysis.

Experimental research Effect of experimentally induced metabolic acidosis on aortic endothelial permeability and serum nitric oxide concentration in normal and high-cholesterol fed rabbits

IntroductionMetabolic acidosis is present in end stage renal disease. There is a link between enhanced endothelial permeability and accelerated atherosclerosis. In this study, we investigated the effect of experimentally induced metabolic acidosis on aortic endothelial permeability and serum nitric oxide (NO) concentration in normal and high-cholesterol fed rabbits.Material and methodsTwenty-four male rabbits were divided into four groups: normal, hypercholesterolemic, acidemic, and hypercholesterolemic plus acidemic. Acidosis and hypercholesterolemia were induced by drinking water containing ammonium chloride (NH4Cl), and cholesterol-rich animal chow (1%), respectively. After 6 weeks, blood samples were taken and endothelial permeability was measured using the Evans blue dye injection method.ResultsHypercholesterolemic animals had higher aortic endothelial permeability compared with normal groups (16.18 ±0.91 µg EB/g tissue vs. 12.89 ±0.66 µg EB/g tissue, p < 0.05). Acidosis significantly increased endothelial permeability in the normal group (17.10 ±0.56 µg/g tissue vs. 12.89 ± 0.66 µg/g tissue; p < 0.05) but did not further increase endothelial permeability in hypercholesterolemic animals (16.18 ±0.91 µg EB/g tissue vs. 17.29 ±0.46 µg EB/g tissue; p > 0.05). Serum total cholesterol, low density lipoprotein (LDL) and NO concentrations in hypercholesterolemic animals were significantly higher than the normal group and acidosis could not change them either in the normal or in the high-cholesterol diet group.ConclusionsAlterations of serum lipids and NO are not the main mechanism for accelerated atherosclerosis during metabolic acidosis. Acidosis increases aortic endothelial permeability at least in a normal diet which may be a possible mechanism for progression of atherosclerosis processes in end-stage renal disease.

Gemfibrozil as a potential heme-independent sGC activator

Background Cyclic GMP producing soluble guanylyl cyclase (sGC) is highly activated by the binding of nitric oxide (NO) messenger to its heme. Besides NO, other NO-independent molecules were shown to activate sGC. These include a class of heme-replacers (e.g., protoporphyrin IX and structurally unrelated activators BAY 58-2667 and HMR1766) or sGC stimulators (e.g., BAY 41-2272 and YC-1 and aryl-acrylamide derivatives). The later class requires heme and sensitize sGC to low NO concentrations. Collectively these data suggest that various modes of allosteric sGC regulation are possible and some may have not been identified yet.