Nitric Oxide Synthase Blocker Research Articles

Protective effect of arginine glutamate (glutargin) in chronic pancreatitis induced by nitric oxide synthase blocker

Introduction. Nitric oxide (NO) is a gaseous molecule that is a biological mediator that carries out important regulation of physiological processes necessary for the functioning of tissues. We hypothesized that nonspecific inhibition of all isoforms of NOs would cause pancreatic damage and created a model of experimental pancreatitis based on NOs inhibition. Along with the study of the mechanisms of development of chronic pancreatitis, the search for medicines to treat and inhibit the progression of this disease continues. Purpose of the research. To study the protective effect of glutargin on the pancreas of rats with chronic pancreatitis induced by a blocker of NOs. Material and methods. The work was carried out on 21 laboratory white male Wistar rats weighing 180-230 g. Chronic pancreatitis was induced by intraperitoneal administration of the NOs blocker - N-nitro-L-arginine (L-NNA) (Sigma-Aldrich, USA) at a dose of 40 mg/kg of body weight. Rats in group I (n = 7) were injected with L-NNA for 12 days, rats in group II (n = 7) were injected intraperitoneally with glutargin 20 mg/kg, after 20 minutes they were injected with L-NNA - within 12 days. Rats of the control group (n = 7) were injected intraperitoneally with 0.9% NaCl solution. The rats were euthanized on the 45th day and biochemical and morphological studies were carried out. Results. After the introduction of the NOs blocker, deterioration of the general condition of the animals was determined, a sharp increase in the level of nitrites/nitrates to 80.22±19.91 μmol/l, control 32.61±4.55 μmol/l (р < 0.05); protein-bound hydroxyproline (PBH) to 215.21±22.01μmol/l, control 178.67±26.39 μmol/l, (p < 0.05); free hydroxyproline (FH) to 14.74±1.84 μmol/l, control 9.96±0.71 μmol/l, (p < 0.05); malondialdehyde (MDA) to 5.67±0.88 nmol/ml, control 3.62±0.13, (р < 0.05). Pronounced structural changes with stasis of formed blood elements in the vessels, focal accumulation of leukocytes in the parenchyma, dystrophy of acinar cells and fibrosis in the atrophy zone were determined in the pancreas of rats. Administration of glutargin contributed to the restoration of general behavioral reactions of rats, normalization of MDA – 4.81±0.15 nmol/ml, control 4.50±0.23, (p > 0.05); ceruloplasmin (CP) – 591.71±68.07 mg/ml, control – 663.25±34.05 mg/ml, (p > 0.05); PBH 183.62±5.98 μmol/l, control – 179.28±9.19 μmol/l, (p > 0.05); FH – 9.44±1.13 μmol/l, control – 9.96±0.71 μmol/l, (p > 0.05) and prevented the development of pronounced structural changes in the pancreas. Conclusions. In chronic pancreatitis induced by a NOs blocker, glutargin can prevent chronic pancreatitis by normalizing collagen metabolism, inhibiting oxidative stress, and severity of pancreatic parenchymal damage.

Ethnomedicinal Study and Evaluation of the Anxiolytic-like and Diuretic Effects of the Orchid Stanhopea tigrina Bateman ex Lindl-(Orchidaceae).

Stanhopea tigrina Bateman ex Lindl. (Orchidaceae) is an orchid endemic to Mexico, known as "Calavera" or "calaverita", in the Huasteca Potosina (central region of Mexico). This plant species is used for the folk treatment of mental disorders and urological kidney disorders, according to the ethnomedicinal information obtained in this study. Ethanolic extracts of leaves (HE) and pseudobulb (PE) were obtained by microwave-assisted extraction (MAE). Gas Chromatography coupled with Mass Spectrometry (GC-MS) was used to carry out the chemical characterization of HE and PE. The pharmacological effects (antioxidant, diuretic, anxiolytic, locomotor, hypnotic, and sedative) of HE and PE were evaluated. The possible mechanism of action of the anxiolytic-like activity induced by HE was assessed using inhibitors of the GABAergic, adrenergic, and serotonergic systems. The possible mechanism of the diuretic action of HE was assessed using prostaglandin inhibitory antagonists and nitric oxide synthase (NOS) blockers. HE at 50 and 100 mg/kg exerted anxiolytic-like activity without inducing hypnosis or sedation. Flumazenil, prazosin, and ketanserin inhibited the anxiolytic-like activity shown by HE, which suggests the participation of GABA, α1-adrenergic receptors, and 5-HT2 receptors, respectively. The diuretic effect was reversed by the non-selective NOS inhibitor L-NAME, which caused the reduction in nitric oxide (NO). These results demonstrate that the ethanolic extract of S. tigrina leaves exhibited anxiolytic-like activity and diuretic effects without inducing hypnosis or sedation. This work validates the medicinal uses of this orchid species.

Carotid body-mediated chemoreflex drive predicts the development of HFpEF in mice

Heart failure with preserved ejection fraction (HFpEF) is a prevalent pathophysiological condition affecting millions of people worldwide. Several factors have been pointed out to play a role in the development of HFpEF; however, there are no molecular/physiological variables that can predict the incidence of HFpEF. Interestingly, carotid body (CB) chemoreceptors have been implicated in worse outcomes in reduced ejection fraction HF. Then, we hypothesized that CB chemoreflex sensitivity may offer prognostic value in HFpEF. Accordingly, we aimed to study whether high chemoreflex drive predicts the development of HFpEF in mice. A longitudinal study was performed in adult C57BLJ6 mice (P90) that underwent 16 weeks treatment with a high fat diet (60% high fat diet, HFD) and a nitric oxide synthase blocker (L-NAME 0.6 g/L) to induce HFpEF. Mice were recorded for cardiac/hemodynamic and ventilatory function before the onset of HFD and L-NAME treatment by using 2D-echocardiography, tissue doppler imaging, blood pressure and whole-body plethysmography, respectively. At 16 weeks after HFD+L-NAME all mice were recorded again. CB-chemoreflex drive was assessed by the hypoxic ventilatory response (HVR, FiO2»10%). We found that »50% of total mice (n=24) develop HFpEF after 16 weeks of HFD+L-NAME as evidenced by a significant (p<0.05) increase in the E/A ratio (1.60±0.09 vs. 1.30±0.01 mmHg, HFpEF vs. nonHFpEF), blood pressure (MAP: 98.7±1.9 vs. 88.4±1.4 mmHg, HFpEF vs. nonHFpEF) and body weight (31±1 vs. 29±0 g, HFpEF vs. nonHFpEF). Remarkably, a higher HVR before the onset of HFD+L-NAME was closely associated with the prevalence of HFpEF. Indeed, (4/5) mice that displayed a high HVR chemoreflex (³0.15 ΔVE/FiO2%) develop HFpEF after 16 weeks of HFD+L-NAME treatment. Our results showed for the first time that CB-mediated chemoreflex sensitivity predicts the development of HFpEF and support the notion that peripheral chemoreflex drive may be useful to evaluate the risk of developing HFpEF. Supported by Fondecyt 1220950. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

β3 Receptor Signaling in Pregnant Human Myometrium Suggests a Role for β3 Agonists as Tocolytics.

Preterm labor leading to preterm birth is the leading cause of infant morbidity and mortality. At the present time, nothing can reliably halt labor once it begins. The knowledge that agonists of the β2 adrenergic receptor relax airway smooth muscle and are effective in the treatment of asthma led to the notion that β2 mimetics would prevent preterm birth by relaxing uterine smooth muscle. The activation of cAMP-dependent protein kinase by β2 receptors is unable to provide meaningful tocolysis. The failure of β2 agonists such as ritodrine and terbutaline to prevent preterm birth suggests that the regulation of uterine smooth muscle is disparate from that of airway. Other smooth muscle quiescent-mediating molecules, such as nitric oxide, relax vascular smooth muscle in a cGMP-protein kinase G-dependent manner; however, nitric oxide activation of protein kinase G fails to explain the relaxation of the myometrium to nitric oxide. Moreover, nitric oxide-mediated relaxation is blunted in preterm labor, and thus, for this reason and because of the fall in maternal blood pressure, nitric oxide cannot be employed as a tocolytic. The β3 adrenergic receptor-mediated relaxation of the human myometrium is claimed to be cAMP-dependent protein kinase-dependent. This is scientifically displeasing given the failure of β2 agonists as tocolytics and suggests a non-canonical signaling role for β3AR in myometrium. The addition of the β3 agonist mirabegron to pregnant human myometrial strips in the tissue bath relaxes oxytocin-induced contractions. Mirabegron stimulates nitric oxide production in myometrial microvascular endothelial cells, and the relaxation of uterine tissue in vitro is partially blocked by the addition of the endothelial nitric oxide synthase blocker Nω-Nitro-L-arginine. Recent data suggest that both endothelial and smooth muscle cells respond to β3 stimulation and contribute to relaxation through disparate signaling pathways. The repurposing of approved medications such as mirabegron (Mybetriq™) tested in human myometrium as uterine tocolytics can advance the prevention of preterm birth.

Protective role of 3-oxypyridine derivatives in rats’ steroid-induced osteoporosis associated with reduced oxidative stress and recovery of nitric oxide formation

From the point of view of the mechanisms for the implementation of pathogenetic links in the development of steroid-induced osteoporosis considered in the paper, the increased risk of the oxidative stress in osteoblasts, as well as the development of the vessels endothelial dysfunction of the microcirculatory bloodstream in the bone tissue, are of particular interest. They lead to the impaired bone tissue trophism and progression of osteoporosis.The aim of the study was research of the osteoprotective effects of a 3-hydroxypyridine derivatives composition on the model of steroid-induced osteoporosis.Materials and methods. To model osteoporosis pathology, the animals (male Wistar rats) were injected with methylprednisolone (MP) at the dose of 5 mg/kg (intraperitoneally) every 5th day for 5 weeks. Аs a non-selective blocker of NO synthase, L-NAME was used at the dose of 25 mg/kg (intraperitoneally). Derivatives of 3-hydroxypyridine (hereinafter referred to as composition No. 1) were administrated at the dose of 50 mg/kg (per os) In all experimental groups, the level of microcirculation and the bone mineral density, as well as the analysis of histomorphological and biochemical samples, were assessed.Results. The study results showed that composition No. 1 (50 mg/kg) has an osteoprotective activity, effectively prevents a decrease in the level of the regional bone tissue microcirculation and in the development of an endothelial dysfunction. That makes it possible to increase the bone mineral density and to slow down the thinning of bone trabeculae. In addition, composition No. 1 (50 mg/kg) reduces the production of reactive oxygen species and increases the NO bioavailability.Conclusion. The data obtained indicate that the studied composition of 3-hydroxypyridine derivatives is considered a promising compound for the prevention and treatment of steroid-induced osteoporosis.

Nitric Oxide Synthase Blockade Impairs Spontaneous Calcium Activity in Mouse Primary Hippocampal Culture Cells.

Oscillation of intracellular calcium concentration is a stable phenomenon that affects cellular function throughout the lifetime of both electrically excitable and non-excitable cells. Nitric oxide, a gaseous secondary messenger and the product of nitric oxide synthase (NOS), affects intracellular calcium dynamics. Using mouse hippocampal primary cultures, we recorded the effect of NOS blockade on neuronal spontaneous calcium activity. There was a correlation between the amplitude of spontaneous calcium events and the number of action potentials (APs) (Spearman R = 0.94). There was a linear rise of DAF-FM fluorescent emission showing an increase in NO concentration with time in neurons (11.9 ± 1.0%). There is correlation between the integral of the signal from DAF-FM and the integral of the spontaneous calcium event signal from Oregon Green 488 (Spearman R = 0.58). Blockade of NOS affected the parameters of the spontaneous calcium events studied (amplitude, frequency, integral, rise slope and decay slope). NOS blockade by Nw-Nitro-L-arginine suppressed the amplitude and frequency of spontaneous calcium events. The NOS blocker 3-Bromo-7-Nitroindazole reduced the frequency but not the amplitude of spontaneous calcium activity. Blockade of the well-known regulator of NOS, calcineurin with cyclosporine A reduced the integral of calcium activity in neurons. The differences and similarities in the effects on the parameters of spontaneous calcium effects caused by different blockades of NO production help to improve understanding of how NO synthesis affects calcium dynamics in neurons.

Running exercise improves spatial learning and memory ability and enhances angiogenesis in the cerebral cortex via endogenous nitric oxide

BackgroundThe molecular mechanisms by which exercise improves brain function and capillaries in the cerebral cortex are unclear. Exercise can increase the expression of nitric oxide (NO) in the brain, and endogenous NO is thought to exert beneficial effects on proangiogenic factors, antiangiogenic factors and brain function. Therefore, we hypothesized that running exercise might improve brain function and enhance angiogenesis through endogenous NO. Methods and resultsThe following three groups of rats were administered intracerebroventricular (i.c.v.) injections before running exercise each day for 4 weeks: exercise+L-NAME group (i.c.v. L-NAME, an NO synthase blocker, dose: 1 μmol/μl and 5 μl/day; treadmill exercise, 20 min/day), exercise group (i.c.v. normal saline, 5 μl/day; treadmill exercise, 20 min/day), and sham group (i.c.v. normal saline, 5 μl/day; no treadmill exercise). Subsequently, the spatial learning and memory abilities were tested using a Morris water maze, and the nitric oxide synthase (NOS) activity in the cerebral cortex in each group of rats was measured using a method involving nitric acid reductase and metabolic chemistry. The parameters of the cortical capillaries were quantitatively investigated using an immunohistochemistry technique and stereological methods. The expression levels of proangiogenic factors (VEGF and FGF-2) and an antiangiogenic inhibitor (endostatin) in the cerebral cortex were tested using a Western blot analysis. Running exercise significantly improved the rats' spatial learning and memory abilities and increased NOS activity in the cortex. Running exercise also subsequently improved the expression of proangiogenic factors (VEGF and FGF-2) and the length, volume and surface area of capillaries and reduced the expression of antiangiogenic factors (endostatin) in the cortex. In contrast, the L-NAME treatment attenuated the effects of running exercise. ConclusionsRunning exercise regulates proangiogenic factors, antiangiogenic factors and angiogenesis in the cerebral cortex via a partially NO-dependent mechanism, and influencing endogenous NO might potentially affect the exercise-related beneficial effects on cognitive ability and cortical capillaries.

Significance of the liver arginase activity and nitrogen monoxide in the detoxification processes and development of oxidative stress in rats under alcoholic intoxication of different severity

Modern medicine faces the problem of the steady growth of alcoholic pathology. And as you know, morbidity and mortality with regular consumption of alcoholic beverages is associated with the toxic effects of ethanol on the most important human organs and, first of all, the liver. To date, a sufficient number of facts have accumulated indicating the importance of liver arginase and nitrogen monoxide (NO) in vital processes in health and disease.The aim of the study was to elucidate the significance of liver arginase activity and nitrogen monoxide in the detoxification processes and the development of oxidative stress in rats with chronic ethanol intoxication of different severity.In experiments on rats using modern physiological, biochemical research methods and a pharmacological approach, it was found that liver arginase and nitrogen monoxide participate in changes in liver detoxification function and the development of oxidative stress induced by chronic ethanol intoxication. The direction and severity of changes in arginase activity and liver detoxification function during chronic alcoholism depends on the severity of chronic alcohol intoxication. Under the influence of daily intragastric administration for 60 days, a 30 % aqueous solution of ethanol (3.5 g 92 % ethanol per kg of body weight) in animals under conditions of development of oxidative stress inhibited the activity of liver arginase and detoxification function but and the introduction of a 10 % aqueous solution of ethanol (1.0 g 92 % ethanol per kg of body weight) within 2 months leads to an increase in the activity of liver arginase and detoxification processes. The action in the body of the NO-synthase blocker methyl ester NG-nitro-L-arginine weakens, and the arginase inhibitor Nω-hydroxy-nor-L-arginine contributes to the development of characteristic changes in the processes of detoxification and lipid peroxidation in the liver during chronic alcohol intoxication caused by intragastric the introduction of ethanol at a dose of 3.5 g/kg for 60 days.

Abstract 13645: Transcutaneous Vagus Nerve Stimulation Restores the Cardiac Phenotype in Heart Failure With Preserved Ejection Fraction by Modulating the Immune Cell Profile

Introduction: A proinflammatory state plays a central role in heart failure with preserved ejection fraction (HFpEF) but the role of specific immune cells remains unclear. Transcutaneous vagus nerve stimulation (tVNS) modulates immune responses via the cholinergic anti-inflammatory pathway, which involves acetylcholine-dependent activation of α7 nicotinic acetylcholine receptors (α7nAchR) on macrophages. Hypothesis: tVNS reverses the HFpEF phenotype by reducing resident macrophage number and pro-inflammatory cytokine production in an acetylcholine-α7nAchR-dependent manner. Methods: We induced HFpEF in male C57BL/6 CCR2-RFP mice with nitric oxide synthase blocker (L-NAME) and a high-fat diet for 5 weeks. C57BL/6 CCR2-RFP mice fed with standard chow diet served as controls (n=7). We randomized the mice to sham, or tVNS with or without the α7nAchR inhibitor methyllycaconitine (MLA) (n=6 in each group) for 4 weeks, followed by euthanasia. Cardiac macrophages were analyzed using flow cytometry. Resident macrophages were further subdivided based on MHC2 and CCR2 expression. Results: HFpEF mice exposed to sham developed hypertension, diastolic dysfunction, increased heart weight normalized to tibia length, and left ventricular fibrosis compared to the control mice, while tVNS restored these parameters towards the control values. MLA partially reversed the protective effect of tVNS in these parameters. Flow cytometry data revealed a significant increase in resident macrophages in HFpEF sham compared to control hearts, mainly driven by the MHC-/CCR2- subtype (F). Notably, tVNS restored the numbers of resident macrophages to the control levels, while MLA partially attenuated this effect (Figure). Conclusions: tVNS improves the cardiac phenotype in HFpEF mice by modulating cardiac resident macrophages, in an acetylcholine-α7nAchR-dependent manner.

Participation of Nitric Oxide in the Realization of Hemostatic Effects of Glyproline Peptides.

The involvement of nitric oxide in the responses of the hemostasis system to the appearance of proline-containing peptides in the blood was studied in experiments on rats. It was shown that a single intranasal administration of peptides PGP, RPGP, and PGPL to rats led to an increase in fibrinolytic, anticoagulant, and antiplatelet potential of blood. The use of the non-selective NO-synthase blocker L-NAME almost completely inhibited the anticoagulant effects of the glyprolines. It was found that the mechanism of the anticoagulant-fibrinolytic and antiplatelet action of glyproline peptides is determined by the activation of the enzymatic pathway of nitric oxide formation. The obtained results revealed the involvement of nitric oxide in the implementation of hemostatic and vascular-endothelial functions of the organism.

Induction of Activity-Dependent Plasticity at Auditory Nerve Synapses.

Exposure to nontraumatic noise in vivo drives long-lasting changes in auditory nerve synapses, which may influence hearing, but the induction mechanisms are not known. We mimicked activity in acute slices of the cochlear nucleus from mice of both sexes by treating them with high potassium, after which voltage-clamp recordings from bushy cells indicated that auditory nerve synapses had reduced EPSC amplitude, quantal size, and vesicle release probability (P r). The effects of high potassium were prevented by blockers of nitric oxide (NO) synthase and protein kinase A. Treatment with the NO donor, PAPA-NONOate, also decreased P r, suggesting NO plays a central role in inducing synaptic changes. To identify the source of NO, we activated auditory nerve fibers specifically using optogenetics. Strobing for 2 h led to decreased EPSC amplitude and P r, which was prevented by antagonists against ionotropic glutamate receptors and NO synthase. This suggests that the activation of AMPA and NMDA receptors in postsynaptic targets of auditory nerve fibers drives release of NO, which acts retrogradely to cause long-term changes in synaptic function in auditory nerve synapses. This may provide insight into preventing or treating disorders caused by noise exposure.SIGNIFICANCE STATEMENT Auditory nerve fibers undergo long-lasting changes in synaptic properties in response to noise exposure in vivo, which may contribute to changes in hearing. Here, we investigated the cellular mechanisms underlying induction of synaptic changes using high potassium and optogenetic stimulation in vitro and identified important signaling pathways using pharmacology. Our results suggest that auditory nerve activity drives postsynaptic depolarization through AMPA and NMDA receptors, leading to the release of nitric oxide, which acts retrogradely to regulate presynaptic neurotransmitter release. These experiments revealed that auditory nerve synapses are unexpectedly sensitive to activity and can show dramatic, long-lasting changes in a few hours that could affect hearing.

Aortic Stiffness in L-NAME Treated C57Bl/6 Mice Displays a Shift From Early Endothelial Dysfunction to Late-Term Vascular Smooth Muscle Cell Dysfunction.

Introduction and Aims: Endothelial dysfunction is recognized as a cardiovascular aging hallmark. Administration of nitric oxide synthase blocker N-Ω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) constitutes a well-known small animal model of cardiovascular aging. Despite extensive phenotypic characterization, the exact aortic function changes in L-NAME treated mice are largely unknown. Therefore, this study presents a longitudinal characterization of the aortic reactivity and biomechanical alterations in L-NAME treated C57Bl/6 mice. Methods and Results: Male C57Bl/6 mice were treated with L-NAME (0.5 mg/ml drinking water) for 1, 2, 4, 8, or 16 weeks. Peripheral blood pressure measurement (tail-cuff) and transthoracic echocardiograms were recorded, showing progressive hypertension after 4 weeks of treatment and progressive cardiac hypertrophy after 8–16 weeks of treatment. Aortic stiffness was measured in vivo as aortic pulse wave velocity (aPWV, ultrasound) and ex vivo as Peterson modulus (Ep). Aortic reactivity and biomechanics were investigated ex vivo in thoracic aortic rings, mounted isometrically or dynamically-stretched in organ bath set-ups. Aortic stiffening was heightened in L-NAME treated mice after all treatment durations, thereby preceding the development of hypertension and cardiac aging. L-NAME treatment doubled the rate of arterial stiffening compared to control mice, and displayed an attenuation of the elevated aortic stiffness at high distending pressure, possibly due to late-term reduction of medial collagen types I, III, and IV content. Remarkably, endothelial dysfunction, measured by acetylcholine concentration-response stimulation in precontracted aortic rings, was only observed after short-term (1–4 weeks) treatment, followed by restoration of endothelial function which coincided with increased phosphorylation of endothelial nitric oxide synthase (S1177). In the late-disease phase (8–16 weeks), vascular smooth muscle cell (VSMC) dysfunction developed, including increased contribution of voltage-dependent calcium channels (assessed by inhibition with diltiazem), basal VSMC cytoplasmic calcium loading (assessed by removal of extracellular calcium), and heightened intracellular contractile calcium handling (assessed by measurement of sarcoplasmic reticulum-mediated transient contractions). Conclusion: Arterial stiffness precedes peripheral hypertension and cardiac hypertrophy in chronic L-NAME treated male C57Bl/6 mice. The underlying aortic disease mechanisms underwent a distinct shift from early endothelial dysfunction to late-term VSMC dysfunction, with continued disease progression.

Carbon monoxide activation of delayed rectifier potassium currents of human cardiac fibroblasts through diverse pathways.

To identify the effect and mechanism of carbon monoxide (CO) on delayed rectifier K+ currents (IK) of human cardiac fibroblasts (HCFs), we used the whole-cell mode patch-clamp technique. Application of CO delivered by carbon monoxide-releasing molecule-3 (CORM3) increased the amplitude of outward K+ currents, and diphenyl phosphine oxide-1 (a specific IK blocker) inhibited the currents. CORM3-induced augmentation was blocked by pretreatment with nitric oxide synthase blockers (L-NG-monomethyl arginine citrate and L-NG-nitro arginine methyl ester). Pretreatment with KT5823 (a protein kinas G blocker), 1H-[1,-2,-4] oxadiazolo-[4,-3-a] quinoxalin-1-on (ODQ, a soluble guanylate cyclase blocker), KT5720 (a protein kinase A blocker), and SQ22536 (an adenylate cyclase blocker) blocked the CORM3 stimulating effect on IK. In addition, pretreatment with SB239063 (a p38 mitogen-activated protein kinase [MAPK] blocker) and PD98059 (a p44/42 MAPK blocker) also blocked the CORM3’s effect on the currents. When testing the involvement of S-nitrosylation, pretreatment of N-ethylmaleimide (a thiol-alkylating reagent) blocked CO-induced IK activation and DL-dithiothreitol (a reducing agent) reversed this effect. Pretreatment with 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)-21H,23H porphyrin manganese (III) pentachloride and manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (superoxide dismutase mimetics), diphenyleneiodonium chloride (an NADPH oxidase blocker), or allopurinol (a xanthine oxidase blocker) also inhibited CO-induced IK activation. These results suggest that CO enhances IK in HCFs through the nitric oxide, phosphorylation by protein kinase G, protein kinase A, and MAPK, S-nitrosylation and reduction/oxidation (redox) signaling pathways.

The Effects of Inducible Nitric Oxide Synthase Blockers on Survival and Organ Injury in a Murine Caecal Ligation and Puncture Model of Septic Shock

The Effects of Inducible Nitric Oxide Synthase Blockers on Survival and Organ Injury in a Murine Caecal Ligation and Puncture Model of Septic Shock

Sildenafil Citrate Downregulates PDE5A mRNA Expression in Women with Recurrent Pregnancy Loss without Altering Angiogenic Factors-A Preliminary Study.

In our previous study, we showed that sildenafil citrate (SC), a selective PDE5A blocker, modulated NK cell activity in patients with recurrent pregnancy loss, which correlated with positive pregnancy outcomes. It was found that NK cells had a pivotal role in decidualization, angiogenesis, spiral artery remodeling, and the regulation of trophoblast invasion. Thus, in the current study, we determined the effects of SC on angiogenic factor expression and production, as well as idNK cell activity in the presence of nitric synthase blocker L-NMMA. Methods: NK cells (CD56+) were isolated from the peripheral blood of 15 patients and 15 fertile women on MACS columns and cultured in transformation media containing IL-15, TGF-β, and AZA—a methylation agent—for 7 days in hypoxia (94% N2, 1% O2, 5% CO2). Cultures were set up in four variants: (1) with SC, (2) without SC, (3) with NO, a synthase blocker, and (4) with SC and NO synthase blocker. NK cell activity was determined after 7 days of culturing as CD107a expression after an additional 4h of stimulation with K562 erythroleukemia cells. The expression of the PDE5A, VEGF-A, PIGF, IL-8, and RENBP genes was determined with quantitative real-time PCR (qRT-PCR) using TaqMan probes and ELISA was used to measure the concentrations of VEGF-A, PLGF, IL-8, Ang-I, Ang-II, IFN–γ proteins in culture supernatants after SC supplementation. Results: SC downregulated PDE5A expression and had no effect on other studied angiogenic factors. VEGF-A expression was increased in RPL patients compared with fertile women. Similarly, VEGF production was enhanced in RPL patients’ supernatants and SC increased the concentration of PIGF in culture supernatants. SC did not affect the expression or concentration of other studied factors, nor idNK cell activity, regardless of NO synthase blockade.

Histochemical characteristics of nitrergic neuronal system during acute alcohol intoxication and nNOS blockage in the rat lateral septum

Annotation. Alcohol is a one of the most frequently consumed substances of abuse, which can cause addiction or alcohol use disorders (AUDs). Alcohol addiction leads to decrease of the life quality of patients and considerable economic burden. Neuronal mechanisms of addiction are intensively studied. One of the most important systems involved in this process is a brain reward system that includes lateral septum (LS). Additionally alcohol consumption changes activity of the neurotransmitter systems including the nitric oxide (NO). Recent studies for blockage of nitric oxide synthase (NOS) for cocaine addiction and late stages of AUDs demonstrated that a group of the substances known as blockers of NOS can be referred to as candidates for alcohol addiction therapy. The aim of our research was to investigate histochemical characteristics of NO-system in LS, its response to acute alcohol intoxication including or excluding neuronal NOS (nNOS) blockage with selective inhibitor – 7-nitroindazole (7-NI). This study involved three experimental groups of animals (control group (n=4), group with acute alcohol intoxication (n=4), group of nNOS blockage with acute alcohol intoxication (n=4)). For statistical analysis, one-way Kruskal-Wallis test was implemented to reveal differences between groups (Matlab, Mathworks). We have identified NOS-positive structures in LS consisting of big neurons, medium/small neurons and nerve fibers. Acute alcohol intoxication activated subpopulations of NOS-positive medium/small neurons and nerve fibers. Moreover, we determined that ethanol-induced changes can be blocked with selective nNOS inhibitor 7-NI.

Investigation of the vascular effects of short chain fatty acids in rat mesenteric resistance arteries

Fiber rich diets have been shown to prevent the onset and reduce severity of hypertension1, a condition affecting 20-30% of adults in North America. High fiber diets facilitate the production of short chain fatty acids (SCFAs) by gut bacteria of which, in humans, acetate is the most abundant, followed by propionate and butyrate. It has been proposed that these three SCFAs may have direct effects on blood vessels and so may contribute to regulation of blood pressure2. Further research is needed to determine the mechanisms underlying the vascular actions of SCFAs. We tested the hypothesis that SCFAs can induce vasodilation and limit nerve-evoked vasoconstriction in rat mesenteric vasculature. Direct effects on vascular tone were assessed by constructing cumulative concentration-response curves to SCFAs in isolated third order rat mesenteric arteries mounted in a wire myograph and pre-stimulated with phenylephrine (PE) to increase tone. The effects of SCFAs on nerve-evoked vasoconstriction were assessed by constructing frequency-responses curves to perivascular nerve stimulation in a perfused rat mesenteric bed in the presence and absence of SCFAs added to the perfusate. In both experimental approaches, pharmacological tools were used to investigate the mechanism of action of SCFAs. Propionate (3 μM - 1 mM) and acetate (0.3 μM - 1 mM) caused reductions in PE-evoked tone with maximum relaxation of 32.7 ± 7% and 31.6 ± 8.2%, respectively, and attenuated nerve-evoked vasoconstriction in mesenteric arteries at 10 mM - 60 mM propionate and 30 mM - 50 mM acetate. These effects were inhibited by blockers of nitric oxide (NO) synthase (L-NG-nitroarginine methyl ester, L-NAME, 100 μM) and endothelial Ca2+-activated K+ (KCa) channels (apamin, 50 nM; TRAM-34, 1 μM). L-NAME alone significantly inhibited relaxations elicited by acetate indicating a predominant role for NO, whereas the actions of propionate were significantly inhibited by apamin and TRAM-34, indicating a predominant role for endothelial KCa channels in this response. These data indicate that SCFAs may cause vasodilation in mesenteric resistance arteries via activation of endothelial pathways and that there may be a differential contribution of NO and KCa channels to responses of individual SCFAs. 1. Appel LJ, Moore TJ, Obarzanek E, et al. A clinical trial of the effects of dietary patterns on blood pressure. DASH Collaborative Research Group. N Engl J Med. 1997;336(16):1117-1124. doi:10.1056/NEJM199704173361601 2. Pluznick J. A novel SCFA receptor, the microbiota, and blood pressure regulation. Gut Microbes. 2014;5(2):202-207. doi:10.4161/gmic.27492

Carbon monoxide activates large-conductance calcium-activated potassium channels of human cardiac fibroblasts through various mechanisms.

Carbon monoxide (CO) is a cardioprotectant and potential cardiovascular therapeutic agent. Human cardiac fibroblasts (HCFs) are important determinants of myocardial structure and function. Large-conductance Ca2+-activated K+ (BK) channel is a potential therapeutic target for cardiovascular disease. We investigated whether CO modulates BK channels and the signaling pathways in HCFs using whole-cell mode patch-clamp recordings. CO-releasing molecules (CORMs; CORM-2 and CORM-3) significantly increased the amplitudes of BK currents (IBK). The CO-induced stimulating effects on IBK were blocked by pre-treatment with specific nitric oxide synthase (NOS) blockers (L-NG-monomethyl arginine citrate and L-NG-nitroarginine methyl ester). 8-bromo-cyclic GMP increased IBK. KT5823 (inhibits PKG) or ODQ (inhibits soluble guanylate cyclase) blocked the CO-stimulating effect on IBK. Moreover, 8-bromo-cyclic AMP also increased IBK, and pre-treatment with KT5720 (inhibits PKA) or SQ22536 (inhibits adenylate cyclase) blocked the CO effect. Pre-treatment with N-ethylmaleimide (a thiol-alkylating reagent) also blocked the CO effect on IBK, and DL-dithiothreitol (a reducing agent) reversed the CO effect. These data suggest that CO activates IBK through NO via the NOS and through the PKG, PKA, and S-nitrosylation pathways.

Роль іонів калію у біосинтезі оксиду азоту в мітохондріях гладенького м’яза

The NO-synthase activity (mtNOS) in mitochondria of uterine smooth muscle was studied. The mitochondrial localization of NO synthesis in myocytes was proved using laser confocal microscopy method and specific fluorescent probes MitoTracker Orange (specific to mitochondria) and DAFFM (NO-sensitive fluorescent probe). It was demonstrated using flow cytometry that nitric oxide biosynthesis in isolated mytochondria decreased in the presence of a constitutive NOsynthase blocker 2-aminopyridine (100 μmol per l, 50% inhibition) and monoclonal antibodies (2.5 μg anti-Let m1 per 50 μg protein) against the H+-Ca2+-exchanger (Letm1 protein), but was’t sensitive to the mitochondrial permeability transition pore inhibitor cyclosporin A (5 μmol per l). A decrease of potassium ions concentration in the incubation medium and the presence of various types of potassium channel inhibitors significantly inhibited the NO-synthase reaction. We have concluded that potassium permeability of the inner mitochondrial membrane plays important role in the regulation of mtNOS activity.

Participation of NO-Dependent Mechanisms in the Effects of Increased Systemic Level of Interleukin-1β on Pial Microvessels under Conditions of Acute Hypoxia.

We studied the effect of increased systemic levels of the proinflammatory cytokine IL-1β on the vasomotor reactions of pial microvessels in anesthetized rats under conditions of experimentally simulated progressively increasing acute normobaric hypoxia. Vital microscopy showed that more pronounced dilatation of pial vessels in response to IL-1β under hypoxic conditions was almost completely prevented by pretreatment with non-specific NO synthase blocker L-NAME. These findings indicate the involvement of NO-dependent mechanisms in the vasodilator effect of proinflammatory cytokines under conditions of acute hypoxic exposure.