Measurement of Cyclic Guanosine Monophosphate (cGMP) in Solid Tissues using Competitive Enzyme-Linked Immunosorbent Assay (ELISA).

A major pathway that produces cyclic guanosine monophosphate (cGMP) is the nitric oxide (NO)-cGMP pathway. In this pathway, NO stimulates the catalytic activity of soluble guanylyl cyclase (sGC), which converts guanosine triphosphate to cGMP. NO is of interest because, in mammals, it acts as the main vasodilator, an inhibitor of platelet aggregation, a mediator of immune function, a neurotransmitter, and to improve endurance during physical activity, among other physiological roles.

Nitrate as Warden of Nitric Oxide Homeostasis in Mammals.

Homeostasis is the self-regulating processes in cells and organisms designed to maintain stability of the internal environment while adjusting to external changes. To achieve this dynamic stability, internal conditions oscillate within tightly regulated physiological tolerance limits. In mammals, maintaining nitric oxide (NO) availability appears crucial to sustain relatively constant blood flow into all organs and tissues.

Sickle cell anaemia therapy in 2025.

New pharmacological therapies for sickle cell anaemia have not been as efficacious as hoped, while widespread application of curative stem cell and gene therapies is not likely to occur soon. This situation raises the question about whether more attention now be devoted to the use of hydroxyurea in this disease.

Hydroxyurea inhibits proliferation and stimulates apoptosis through inducible nitric oxide synthase in erythroid cells.

Hydroxyurea (hydroxycarbamide, HU) arrests cells in the S-phase by inhibiting ribonucleotide reductase and DNA synthesis, significantly contributing to the release of nitric oxide (NO). We investigated the involvement of inducible NO synthase (NOS2) in the cytostatic effect of HU using in vitro shRNA-induced knockdown of the NOS2 transcript (NOS2) or a specific NOS2 inhibitor (1400W) in human erythroleukemic HEL92.1.

Effects of low and high dietary nitrate intake on human saliva, plasma and skeletal muscle nitrate and nitrite concentrations and their functional consequences.

Dietary nitrate (NO) supplementation has been shown to reduce blood pressure (BP), improve exercise performance, and alter the oral microbiome. Following a "control" diet (CON), we manipulated dietary NO intake to examine the effect of a short-term (7-day) low NO diet (LOW) followed by a 3-day high NO diet (HIGH), compared to a 7-day standard (STD) NO diet followed by HIGH, on saliva, plasma, and muscle [NO] and nitrite ([NO]), BP, and cycling exercise performance in healthy young adults. We also examined the effect of LOW on the oral microbiome.

Skeletal Muscle, Skin, and Bone as Three Major Nitrate Reservoirs in Mammals: Chemiluminescence and N-Tracer Studies in Yorkshire Pigs.

In mammals, nitric oxide (NO) is generated either by the nitric oxide synthase (NOS) enzymes from arginine or by the reduction of nitrate to nitrite by tissue xanthine oxidoreductase (XOR) and the microbiome and further reducing nitrite to NO by XOR or several heme proteins. Previously, we reported that skeletal muscle acts as a large nitrate reservoir in mammals, and this nitrate reservoir is systemically, as well as locally, used to generate nitrite and NO. Here, we report identifying two additional nitrate storage organs-bone and skin.

High nitrate levels in skeletal muscle contribute to nitric oxide generation via a nitrate/nitrite reductive pathway in mice that lack the nNOS enzyme.

Introduction: Nitric oxide (NO) is a vasodilator gas that plays a critical role in mitochondrial respiration and skeletal muscle function. NO is endogenously generated by NO synthases: neuronal NO synthase (nNOS), endothelial NO synthase (eNOS), or inducible NO synthase (iNOS). NO in skeletal muscle is partly generated by nNOS, and nNOS deficiency can contribute to muscular dystrophic diseases.

Dietary Nitrate Metabolism in Porcine Ocular Tissues Determined Using N-Labeled Sodium Nitrate Supplementation.

Nitrate (NO) obtained from the diet is converted to nitrite (NO) and subsequently to nitric oxide (NO) within the body. Previously, we showed that porcine eye components contain substantial amounts of nitrate and nitrite that are similar to those in blood. Notably, cornea and sclera exhibited the capability to reduce nitrate to nitrite.

Nitrate and Nitrite Metabolism in Aging Rats: A Comparative Study.

Nitric oxide (NO) (co)regulates many physiological processes in the body. Its short-lived free radicals force synthesis in situ and on-demand, without storage possibility. Local oxygen availability determines the origin of NO-either by synthesis by nitric oxide synthases (NOS) or by the reduction of nitrate to nitrite to NO by nitrate/nitrite reductases.

Distribution of dietary nitrate and its metabolites in rat tissues after N-labeled nitrate administration.

The reduction pathway of nitrate (NO) and nitrite (NO) to nitric oxide (NO) contributes to regulating many physiological processes. To examine the rate and extent of dietary nitrate absorption and its reduction to nitrite, we supplemented rat diets with NaNO-containing water (1 g/L) and collected plasma, urine and several tissue samples. We found that plasma and urine showed 8.

N-labeled dietary nitrate supplementation increases human skeletal muscle nitrate concentration and improves muscle torque production.

Aim: Dietary nitrate (NO ) supplementation increases nitric oxide bioavailability and can enhance exercise performance. We investigated the distribution and metabolic fate of ingested NO at rest and during exercise with a focus on skeletal muscle.

Methods: In a randomized, crossover study, 10 healthy volunteers consumed 12.

Time course of human skeletal muscle nitrate and nitrite concentration changes following dietary nitrate ingestion.

Dietary nitrate (NO) ingestion can be beneficial for health and exercise performance. Recently, based on animal and limited human studies, a skeletal muscle NO reservoir has been suggested to be important in whole body nitric oxide (NO) homeostasis. The purpose of this study was to determine the time course of changes in human skeletal muscle NO concentration ([NO]) following the ingestion of dietary NO.

Skeletal Muscle Nitrate as a Regulator of Systemic Nitric Oxide Homeostasis.

Nonenzymatic nitric oxide (NO) generation via the reduction of nitrate and nitrite ions, along with remarkably high levels of nitrate ions in skeletal muscle, have been described recently. Skeletal muscle nitrate storage may be critical for maintenance of NO homeostasis in healthy aging, and nitrate supplementation may be useful for the treatment of specific pathophysiologies and for enhancing normal functions.

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements.

Nitrate ions (NO3) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise.

Control of rat muscle nitrate levels after perturbation of steady state dietary nitrate intake.

The roles of nitrate and nitrite ions as nitric oxide (NO) sources in mammals, complementing NOS enzymes, have recently been the focus of much research. We previously reported that rat skeletal muscle serves as a nitrate reservoir, with the amount of stored nitrate being highly dependent on dietary nitrate availability, as well as its synthesis by NOS1 enzymes and its subsequent utilization. We showed that at conditions of increased NO need, this nitrate reservoir is used in situ to generate nitrite and NO, at least in part via the nitrate reductase activity of xanthine oxidoreductase (XOR).

Potential roles of nitrate and nitrite in nitric oxide metabolism in the eye.

Nitric oxide (NO) signaling has been studied in the eye, including in the pathophysiology of some eye diseases. While NO production by nitric oxide synthase (NOS) enzymes in the eye has been characterized, the more recently described pathways of NO generation by nitrate (NO) and nitrite (NO) ions reduction has received much less attention. To elucidate the potential roles of these pathways, we analyzed nitrate and nitrite levels in components of the eye and lacrimal glands, primarily in porcine samples.

Nitrate uptake and metabolism in human skeletal muscle cell cultures.

Several studies show that dietary nitrate enhances exercise performance, presumably by increasing muscle blood flow and improving oxygen utilization. These effects are likely mediated by nitrate metabolites, including nitrite and nitric oxide (NO). However, the mechanisms of nitrate production, storage, and metabolism to nitrite and NO in skeletal muscle cells are still unclear.

Human skeletal muscle nitrate store: influence of dietary nitrate supplementation and exercise.

Key Points: Nitric oxide (NO), a potent vasodilator and a regulator of many physiological processes, is produced in mammals both enzymatically and by reduction of nitrite and nitrate ions. We have previously reported that, in rodents, skeletal muscle serves as a nitrate reservoir, with nitrate levels greatly exceeding those in blood or other internal organs, and with nitrate being reduced to NO during exercise. In the current study, we show that nitrate concentration is substantially greater in skeletal muscle than in blood and is elevated further by dietary nitrate ingestion in human volunteers.

Inhaled nebulized nitrite and nitrate therapy in a canine model of hypoxia-induced pulmonary hypertension.

Dysfunction in the nitric oxide (NO) signaling pathway can lead to the development of pulmonary hypertension (PH) in mammals. Discovery of an alternative pathway to NO generation involving reduction from nitrate to nitrite and to NO has motivated the evaluation of nitrite as an alternative to inhaled NO for PH. In contrast, inhaled nitrate has not been evaluated to date, and potential benefits include a prolonged half-life and decreased risk of methemoglobinemia.

Compensatory mechanisms in myoglobin deficient mice preserve NO homeostasis.

The mechanism for nitric oxide (NO) generation from reduction of nitrate (NO) and nitrite (NO) has gained increasing attention due to the potential beneficial effects of NO in cardiovascular diseases and exercise performance. We have previously shown in rodents that skeletal muscle is the major nitrate reservoir in the body and that exercise enhances the nitrate reduction pathway in the muscle tissue and have proposed that nitrate in muscle originates from diet, the futile cycle of nitric oxide synthase 1 (NOS1) and/or oxidation of NO by oxymyoglobin. In the present study, we tested the hypothesis that lack of myoglobin expression would decrease nitrate levels in skeletal muscle.

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation.

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation.

Decreased nitrite reductase activity of deoxyhemoglobin correlates with platelet activation in hemoglobin E/ß-thalassemia subjects.

Nitric oxide (NO) can be generated from nitrite by reductase activity of deoxygenated hemoglobin (deoxyHb) apparently to facilitate tissue perfusion under hypoxic condition. Although hemoglobin E (HbE) solutions have been shown to exhibit decreased rate of nitrite reduction to NO, this observation has never been reported in erythrocytes from subjects with hemoglobin E/ß-thalassemia (HbE/ß-thal). In this study, we investigated the nitrite reductase activity of deoxyHb dialysates from 58 non-splenectomized and 23 splenectomized HbE/ß-thal subjects compared to 47 age- and sex-matched normal subjects, and examined its correlation with platelet activity.

Phosphorylated vasodilator-stimulated phosphoprotein (P-VASPSer239) in platelets is increased by nitrite and partially deoxygenated erythrocytes.

Nitrite is recognized as a bioactive nitric oxide (NO) metabolite. We have shown that nitrite inhibits platelet activation and increases platelet cGMP levels in the presence of partially deoxygenated erythrocytes. In this study, we investigated the effect of nitrite on phosphorylation of vasodilator-stimulated phosphoprotein on residue serine 239 (P-VASPSer239), a marker of protein kinase G (PKG) activation, in human platelets.