Enzyme Nitric Oxide Synthase Research Articles

Anticonvulsant effects of pentoxifylline on seizures induced by pentylenetetrazole and maximal electroshock in male mice: The role of the nitrergic pathway

IntroductionEpilepsy remains a challenge, with one-third of patients experiencing refractory seizures despite current anti-seizure medications. The nitrergic system, which involves nitric oxide (NO) and NO synthase (NOS) enzymes, plays a complex role in seizure pathophysiology. Pentoxifylline (PTPh), an FDA-approved phosphodiesterase inhibitor, has anticonvulsant effects; however, its relationship with the pathway is unclear. This study focused at how the nitrergic system could be involved in PTPh’s anticonvulsant effects. MethodsSeizures were induced in male mice by intravenous pentylenetetrazole (PTZ) infusion (absence-like seizures), intraperitoneal PTZ injection, and maximal electroshock (generalized tonic-clonic seizures). PTPh was administered at various doses, alone or in combination with the NO precursor L-arginine, as well as non-selective (L-NAME) and selective NOS inhibitors (nNOS inhibitor 7-NI and iNOS inhibitor aminoguanidine). Seizure thresholds, latencies, incidence, and mortality were assessed. Moreover, in the next paradigm, using maximal electroshock model, we evaluate possible protective effects of PTPh against generalized tonic-clonic seizures and subsequent mortality. ResultsIn the intravenous PTZ model, PTPh (≥150 mg/kg) increased the seizure threshold, potentiated by L-arginine but reduced by L-NAME and 7-nitroindazole. In the intraperitoneal PTZ model, 150 mg/kg PTPh decreased tonic seizure frequency, which was mitigated by aminoguanidine. However, PTPh failed to prolong clonic seizure latency. In the maximal electroshock test, 100 mg/kg PTPh protected against tonic seizure incidence (reduced by aminoguanidine). Although PTPh could not reduce mortality, its combination with L-NAME or 7-nitroindazole increased mortality compared with the vehicle-treated group. ConclusionPTPh exerted anticonvulsant effects against absence-like and generalized tonic-clonic seizures, likely through modulation of the nitrergic system involving neuronal, endothelial, and inducible NOS isoform. These findings provide novel insights into the complex interplay between NO signaling and the anticonvulsant actions of PTPh, highlighting the potential therapeutic implications of targeting the NO pathway in epilepsy management.

Nitric oxide: Potential therapeutic target in Heat Stress-induced Multiple Organ Dysfunction.

As climate change intensifies, urgent action is needed to address global warming and its associated health risks, particularly in vulnerable regions. Rising global temperature and increasing frequency of heatwaves present a hidden health risk, disrupting the body's temperature regulation and leading to severe consequences such as heat stress-induced multiple organ dysfunction (HS-MOD). Multiple organ injury triggered by heat stress involves complex molecular pathways such as nitric oxide dysregulation, inflammation, oxidative stress, mitochondrial dysfunction, calcium homeostasis disruption, and autophagy impairment that contribute to cellular damage. Understanding these molecular pathways is crucial for developing targeted therapeutic interventions to alleviate the impact of heat stress (HS). As we explore numerous therapeutic strategies, a remarkable molecule captures our attention: nitric oxide (NO). This colorless gas, mainly produced by nitric oxide synthase (NOS) enzymes, plays crucial roles in various body functions. From promoting vasodilation and neurotransmission to regulating the immune response, platelet function, cell signaling, and reproductive health, NO stands out for its versatility. Exploring it as a promising treatment for heat stress-induced multiple organ injury highlights its distinctive features in the journey towards effective therapeutic interventions. This involves exploring both pharmacological avenues, considering the use of NO donors and antioxidants, and non-pharmacological strategies, such as adopting nitrate-rich diets and engaging in exercise regimens. This review highlights the concept of heat stress, the molecular framework of the disease, and treatment options based upon some new interventions.

Nitric oxide synthase system in the brain development of neonatal hypothyroid rats

Thyroid hormones play an important morphogenetic role during the fetal and neonatal periods and regulate numerous metabolic processes. In the central nervous system, they control myelination and overall brain development, regional gene expression, and regulation of oxygen consumption. Their deficiency in the fetal and neonatal periods causes severe mental retardation, due to lack of thyroid function, or to iodine deficiency. At the same time, nitric oxide is an atypical neurotransmitter that also has special relevance in neuronal development and plasticity and functions as a vasodilator, regulating cerebral blood flow. Although under physiological conditions it functions as a neuroprotector, in excess it can be neurotoxic. We have studied, by immunocytochemical and Western blot techniques, the evolution of the expression of neuronal and inducible isoforms of the enzyme nitric oxide synthase, and of nitrotyrosine as a marker of protein nitration produced by the presence of nitric oxide, during the early stages of postnatal brain development. We induced hypothyroidism by administering mercaptomethylimidazole to pregnant mothers, from the seventh day of gestation until the sacrifice of the offspring. The results show a delay in the evolution of the expression of the two isoforms of the enzyme nitric oxide synthase in hypothyroid animals, followed by an anomalous overexpression in later stages. Finally, the expression of nitrotyrosine follows an evolution that is synchronized with that shown by both isoenzymes in control and hypothyroid animals.

Skeletal Muscle, Skin, and Bone as Three Major Nitrate Reservoirs in Mammals: Chemiluminescence and 15N-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. We used bolus of ingested 15N-labeled nitrate to trace its short-term fluxes and distribution among organs. At baseline conditions, the nitrate concentration in femur bone samples was 96 ± 63 nmol/g, scalp skin 56 ± 22 nmol/g, with gluteus muscle at 57 ± 39 nmol/g. In comparison, plasma and liver contained 34 ± 19 nmol/g and 15 ± 5 nmol/g of nitrate, respectively. Three hours after 15N-nitrate ingestion, its concentration significantly increased in all organs, exceeding the baseline levels in plasma, skin, bone, skeletal muscle, and in liver 5-, 2.4-, 2.4-, 2.1-, and 2-fold, respectively. As expected, nitrate reduction into nitrite was highest in liver but also substantial in skin and skeletal muscle, followed by the distribution of 15N-labeled nitrite. We believe that these results underline the major roles played by skeletal muscle, skin, and bone, the three largest organs in mammals, in maintaining NO homeostasis, especially via the nitrate-nitrite-NO pathway.

Identification of Metabolite Compounds from A Ethanol Extract of Caulerpa racemosa using LC-MS/MS with Inhibitory Activity of Interleukin-1β and Expression Inhibitory Nitric Oxide Synthase Enzyme; In Silico Virtual Screening

Identification of Metabolite Compounds from A Ethanol Extract of Caulerpa racemosa using LC-MS/MS with Inhibitory Activity of Interleukin-1β and Expression Inhibitory Nitric Oxide Synthase Enzyme; In Silico Virtual Screening

Erectile dysfunction in cardiovascular patients: A prospective study of the eNOS gene T‐786C, G894T, and INTRON variable number of the tandem repeat functional interaction

AbstractBackgroundCardiovascular disease induces erectile dysfunction modulated by endothelial nitric oxide synthase enzyme and an impaired ejection fraction that restricts penis vascular congestion. However, the mechanisms regulating endothelial dysfunction are not understood.ObjectivesExploring the functional impact of endothelial nitric oxide synthase genetic polymorphisms on erectile dysfunction and drug therapy optimization in high‐risk cardiovascular disease patients.Materials and methodsPatients with erectile dysfunction symptoms and candidates for andrology therapy were included (n = 112). Clinical data and endothelial nitric oxide synthase rs1799983 (G894T) and rs2070744 (T‐786C), genotyped by fluorescence polarization assays, were registered. The 27‐bp variable number of the tandem repeat polymorphism in intron 4 (intron4b/a) was analyzed by polymerase chain reaction‐restriction fragment length polymorphism. Association analyses were run with the R‐3.2.0 software.ResultsA significant association between endothelial nitric oxide synthase 786‐TT (p = 0.005) and the aa/ac of intron 4 variable number of the tandem repeat (p = 0.02) with higher erectile dysfunction susceptibility was observed in cardiovascular disease patients (60 ± 9 years, 66% severe erectile dysfunction, 56% ejection fraction). After 3‐months of phosphodiesterase type 5 inhibitors, erectile dysfunction (International Index of Erectile Function, 50 ± 16 scores, the International Index of Erectile Function‐Erectile Function 21 ± 10 scores, p < 0.001) and sexual quality of life (modified Sexual Life Quality Questionnaire 55 ± 23 scores, p < 0.001) had significantly improved. The cardiovascular ejection fraction was influenced positively with better sexual quality of life (0.1941), and also in the endothelial nitric oxide synthase G894‐T allele (p = 0.076) carriers, which could merit future analyses. Erectile dysfunction was present as the primary clinical manifestation in 62% of cases, with cardiovascular disease occurring concurrently. Only former smokers and obese subjects debuted prior to cardiovascular disease than to erectile dysfunction.ConclusionsOur study provides comprehensive insights into the functional interaction linking endothelial nitric oxide synthase gene polymorphisms, erectile function, and ejection fraction in high‐risk cardiovascular disease patients. Future therapeutic strategies could target endothelial nitric oxide synthase activity by including lifestyle changes and epigenetic modulations.

Anti-inflammatory activity of phenolics from Dianella ensifolia

A new diarylpropane (1) along with seventeen known compounds (2–18), were extracted from the entire plant of Dianella ensifolia. The compounds 1–3 were identified as a distinctive group of diarylpropanes. Their structures were determined through comprehensive spectral analysis. An anti-inflammatory activity assay revealed that compound 7 demonstrated significant inhibitory effects on NO generation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, with an IC50 value of 9.93 μg/mL. Further in-depth mechanistic studies indicated that compound 7 suppresses LPS-induced NO production by inhibiting the activation of NF-κB, which subsequently led to a decrease in the expression of nitric oxide synthase enzymes (iNOS).

Comparing Nitric Oxide Generation Pathways using Dietary L-Arginine and Dietary Nitrate: A 15N tracer study in Wistar Rats

Mammals generate nitric oxide (NO) through two main pathways. In the direct production pathway, endogenous nitric oxide synthase (NOS) enzymes catalyze the reaction of L-arginine with oxygen, producing nitric oxide and citrulline. When oxygen accessibility is low, the reductive pathway supplies nitric oxide through the nitrate-nitrite-NO reduction cascade. Additional research is needed to understand whether the two NO generation pathways interact and what effects the two pathways have on the body and each other. We investigated how dietary supplementation of 15N-L-arginine (0.5 g/kg), 15N-nitrate (0.013 g/kg), or a combination of L-arginine and nitrate influences nitrate concentrations in plasma, skeletal muscle, and liver tissue from Wistar rats. We administered treatments via oral gavage. Two hours after treatment administration, we euthanized the rats and collected tissue samples. The nitrate concentrations were measured using chemiluminescence, and future mass spectrometry analysis for 15N/14N ratios will be performed. The calculated concentrations were compared between treatment groups using single factor analysis of variance. Firstly, we observed that nitrate administration significantly increased total nitrate levels in plasma, liver, and muscle tissues compared to the control group. Interestingly, L-arginine alone did not result in a significant rise in total nitrate concentrations. Notably, liver tissue from the L-arginine-treated group had slightly lower concentrations of nitrate than the control group. The combination of L-arginine and nitrate showed no statistically significant difference when compared with the nitrate-treated group in all three tissues. These results suggest that a bolus of L-arginine does not have a substantial short-term effect (within 2 hours after ingestion) on total nitrate concentrations in rat tissues. Future mass spectrometry analysis will indicate how the tissues handle acute ingestion of two different NO sources: L-arginine and nitrate. Further research must be performed for a more detailed understanding of the kinetics of arginine and nitrate, as well as for the effects of these dietary supplements on improving NO homeostasis. This work was supported by intramural NIDDK/NIH grant ZIA DK 025104-15 to Alan N. Schechter. 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.

A Role for iNOS in Erastin Mediated Reduction of P-Glycoprotein Transport Activity.

The blood-brain barrier is composed of both a physical barrier and an enzymatic barrier. Tight junction (TJ) proteins expressed between endothelial cells of brain capillaries provide the physical barrier to paracellular movement of ions and molecules to the brain, while luminal-facing efflux transporters enzymatically restrict the entry of blood-borne molecules from entering the brain. The expression and activity of ATP Binding Cassette transporters or "ABC" transporters in endothelial cells of the BBB and in human tumor cells are dynamically regulated by numerous signaling pathways. P-glycoprotein (P-gp), (ABCB1), is arguably the most studied transporter of the BBB, and in human cell lines. P-glycoprotein transport activity is rapidly inhibited by signaling pathways that call for the rapid production of nitric oxide (NO) from the inducible nitric oxide synthase enzyme, iNOS. This study investigated how nano-molar levels of the selective chemotherapeutic erastin affect the activity or expression of P-glycoprotein transporter in brain capillaries and in human tumor cell lines. We chose erastin because it signals to iNOS for NO production at low concentrations. Furthermore, erastin inhibits the cellular uptake of cystine through the XC- cystine/glutamate antiporter. Since previous reports indicate that NO production from iNOS can rapidly inhibit P-gp activity in tumor cells, we wondered if induction of iNOS by erastin could also rapidly reduce P-glycoprotein transport activity in brain endothelial cells and in human tumor cell lines. We show here that low concentrations of erastin (1 nM) can induce iNOS, inhibit the activity of P-glycoprotein, and reduce the intracellular uptake of cystine via the Xc- cystine/glutamate antiporter. Consistent with reduced P-glycoprotein activity in rat brain capillary endothelial cells, we show that human tumor cell lines exposed to erastin become more sensitive to cytotoxic substrates of P-glycoprotein.

Activation of hepatic acetyl-CoA carboxylase by S-nitrosylation in response to diet

Nitric oxide (NO), produced primarily by nitric oxide synthase enzymes, is known to influence energy metabolism by stimulating fat uptake and oxidation. The effects of NO on de novo lipogenesis (DNL), however, are less clear. Here we demonstrate that hepatic expression of endothelial nitric oxide synthase is reduced following prolonged administration of a hypercaloric high-fat diet. This results in marked reduction in the amount of S-nitrosylation of liver proteins including notably acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in DNL. We further show that ACC S-nitrosylation markedly increases enzymatic activity. Diminished endothelial nitric oxide synthase expression and ACC S-nitrosylation may thus represent a physiological adaptation to caloric excess by constraining lipogenesis. Our findings demonstrate that S-nitrosylation of liver proteins is subject to dietary control and suggest that DNL is coupled to dietary and metabolic conditions through ACC S-nitrosylation.

Current insights and future perspectives of flavonoids: A promising antihypertensive approach.

Hypertension, or high blood pressure (BP), is a complex disease influenced by various risk factors. It is characterized by persistent elevation of BP levels, typically exceeding 140/90 mmHg. Endothelial dysfunction and reduced nitric oxide (NO) bioavailability play crucial roles in hypertension development. L-NG-nitro arginine methyl ester (L-NAME), an analog of L-arginine, inhibits endothelial NO synthase (eNOS) enzymes, leading to decreased NO production and increased BP. Animal models exposed to L-NAME manifest hypertension, making it a useful design for studying the hypertension condition. Natural products have gained interest as alternative approaches for managing hypertension. Flavonoids, abundant in fruits, vegetables, and other plant sources, have potential cardiovascular benefits, including antihypertensive effects. Flavonoids have been extensively studied in cell cultures, animal models, and, to lesser extent, in human trials to evaluate their effectiveness against L-NAME-induced hypertension. This comprehensive review summarizes the antihypertensive activity of specific flavonoids, including quercetin, luteolin, rutin, troxerutin, apigenin, and chrysin, in L-NAME-induced hypertension models. Flavonoids possess antioxidant properties that mitigate oxidative stress, a major contributor to endothelial dysfunction and hypertension. They enhance endothelial function by promoting NO bioavailability, vasodilation, and the preservation of vascular homeostasis. Flavonoids also modulate vasoactive factors involved in BP regulation, such as angiotensin-converting enzyme (ACE) and endothelin-1. Moreover, they exhibit anti-inflammatory effects, attenuating inflammation-mediated hypertension. This review provides compelling evidence for the antihypertensive potential of flavonoids against L-NAME-induced hypertension. Their multifaceted mechanisms of action suggest their ability to target multiple pathways involved in hypertension development. Nonetheless, the reviewed studies contribute to the evidence supporting the useful of flavonoids for hypertension prevention and treatment. In conclusion, flavonoids represent a promising class of natural compounds for combating hypertension. This comprehensive review serves as a valuable resource summarizing the current knowledge on the antihypertensive effects of specific flavonoids, facilitating further investigation and guiding the development of novel therapeutic strategies for hypertension management.

Synthesis of 2-amino-5-methylpyridinium tetrachloridocadmate(II) (C6H9N2)2[CdCl4]: Structure, DFT-calculated descriptors and molecular docking study

In this research paper, (C6H9N2)2[CdCl4], was effectively synthesized using the slow solvent evaporation procedure. Single crystal X-ray diffraction (scXRD) analysis revealed that the compound crystallizes in the triclinic system, specifically in the space group P1¯. Powder XRD (PXRD) of the bulk material showed some minor impurities. The atomic arrangement of the title structure comprises discrete tetrahedral groups [CdCl4]2− linked to the organic entities through weak N(C)H…Cl hydrogen bonds. Solid-state contacts were further studied through Hirshfeld surface analyses, complemented by 2D fingerprint plots. Computational results, obtained using the B3LYP tool with 6-311++G(d,p) + LANL2DZ mixed basis set, demonstrated consistent geometrical, vibrational, and electronic features to the experimental data. Non-covalent interactions were explored in depth using Atoms-In-Molecule (AIM) and Reduced Density Gradient (RDG) analyses. Thermogravimetry (TG) and Differential Scanning Calorimetry (DSC) analyses showed melting at 378 K and decomposition at around 540 K. Furthermore, the inhibition activity of the examined compound was explored in-silico through molecular docking studies targeting the inducible Nitric Oxide Synthase (iNOS) enzymes.

Activation of mitoKATP channels induces penile vasodilation and inhibits mitochondrial respiration and ROS production: Role of NO

ObjectiveMitochondrial ATP-sensitive K+ (mitoKATP) channels are involved in neuronal and cardiac protection from ischemia and oxidative stress. Penile erection is a neurovascular event mediated by relaxation of the erectile tissue via nitric oxide (NO) released from nerves and endothelium. In the present study, we investigated whether mitoKATP channels play a role in the control of penile vascular tone and mitochondrial dynamics, and the involvement of NO. MethodsThe effect of the selective mitoKATP activator BMS191095 was examined on vascular tone, on mitochondrial bioenergetics by real-time measurements with Agilent Seahorse and on ROS production by MitoSOX fluorescence in freshly isolated microarteries. ResultsBMS191095 and diazoxide relaxed penile arteries, BMS191095 being one order of magnitude more potent. BMS191095-induced relaxations were reduced by mechanical endothelium removal and by inhibitors of the nitric oxide synthase (NOS) and PI3K enzymes. The NO-dependent component of the relaxation to BMS191095 was impaired in penile arteries from insulin resistant obese rats. The blockers of mitoKATP channel 5-HD, sarcolemma KATP (sarcKATP) channel glibenclamide, and large conductance Ca2+-activated K+ (BKCa) channel iberiotoxin, inhibited relaxations to BMS191095 and to the NO donor SNAP. BMS191095 reduced the mitochondrial bioenergetic profile of penile arteries and attenuated mitochondrial ROS production. Blockade of endogenous NO impaired and exogenous NO mimicked, respectively, the inhibitory effects of BMS191095 on basal respiration and oxygen consumed for ATP synthesis. Exogenous NO exhibited dual inhibitory/stimulatory effects on mitochondrial respiration. ConclusionsThese results demonstrate that selective activation of mitoKATP channels causes penile vasodilation, attenuates ROS production and inhibits mitochondrial respiration in part by releasing endothelial NO. These mechanisms couple blood flow and metabolism in penile arterial wall and suggest that activation of vascular mitoKATP channels may protect erectile tissue against ischemic injury.

Down-regulation of neuronal form of Nitric oxide synthase in the Nurse cell of Trichinella spiralis.

The free radical nitric oxide (NO) and Ca2+ are critical regulators of skeletal muscle exercise performance and fatigue. The major source of NO in skeletal muscle cells is the neuronal form of the enzyme Nitric oxide synthase (nNOS). One of the most peculiar characteristics of the Nurse cell of Trichinella spiralis (T. spiralis) is the complete loss of the contractile capabilities of its derivative striated muscle fiber. The aim of the present study was to clarify the expression of nNOS protein and mRNA in striated muscles during the muscle phase of T. spiralis infection in mice. Muscle tissue samples were collected from mice at days 0, 14, 24, and 35 post infection (d.p.i.). The expression of nNOS was investigated by immunohistochemistry, and the expression levels of mRNA of mouse Nitric oxide synthase 1 (Nos1) by real-time PCR. The presence of nNOS protein was still well observable in the disintegrated sarcoplasm at the early stage of infection. The cytoplasm of the developing and mature Nurse cell showed the absence of this protein. At least at the beginning of the Nurse cell development, Trichinella uses the same repairing process of skeletal muscle cell, induced after any trauma and this corroborates very well our results concerning the nNOS expression on day 14 p.i. At a later stage, however, we could suggest that the down-regulation of nNOS in the Nurse cell of T. spiralis either serves a protective function or is an outcome of the genetic identity of the Nurse cell.

Extracellular ATP- and adenosine-mediated purinergic signaling modulates inducible nitric oxide synthase (iNOS) gene expression, enzyme activity and nitric oxide production in common carp (Cyprinus carpio) head kidney macrophages

Inducible nitric oxide (NO) synthase (iNOS) is a key immune mediator for production of inflammatory mediator NO from l-arginine. Tight regulation of iNOS expression and enzyme activity is critical for proper NO productions under inflammation and infection conditions. However, the regulatory mechanism for iNOS expression and enzyme activity in fish remains largely unknown. Here, we show that extracellular ATP treatment significantly up-regulates iNOS gene expression and enzyme activity, and consequently leads to enhanced NO production in Cyprinus carpio head kidney macrophages (HKMs). We further show that the extracellular ATP-induced iNOS enzyme activity and NO production can be attenuated by pharmacological inhibition of the ATP-gated P2X4 and P2X7 receptors with their respective specific antagonists, but enhanced by overexpression of P2X4 and P2X7 receptors in grass carp ovary cells. In contrast, adenosine administration significantly reduces iNOS gene expression, enzyme activity and NO production in carp HKMs, and these inhibitory effects can be reversed by pharmacological inhibition of adenosine receptors with the antagonist XAC. Furthermore, LPS- and poly(I:C)-induced iNOS gene expression, enzyme activity, and NO production are significantly attenuated by blockade of P2X4 and P2X7 receptors with their respective specific antagonists in carp HKMs, while overexpression of P2X and P2X7 receptors results in enhanced iNOS gene expression, enzyme activity and NO production in LPS- and poly(I:C)-treated grass carp ovary cells. Taken together, we firstly report an opposite role of extracellular ATP/adenosine-mediated purinergic signaling in modulating iNOS-NO system activity in fish.

Alterations in glutamate and nitric oxide metabolism in the RdS mice

Aims/Purpose: Nitric oxide (NO) is an essential signalling molecule, with gaseous nature. NO is synthesized by the conversion of L‐arginine to L‐citrulline. This reaction is catalysed by the enzyme nitric oxide synthase (NOS). There are three NOS isoforms, but nNOS reflects most of the production of NO in the retina. Although NO is employed in the retina to maintain ocular function, it has also been related to different visual diseases. Increased levels of NO have been associated with ocular diseases involving increased glutamate concentrations, ischaemia, oxidative stress and inflammation (such as retinitis pigmentosa (RP)). The main purpose of this work was to determine glutamate retinal concentrations at different stages of the retinal degeneration that occurs in the rds mice and to study possible NO metabolism alterations.Methods: Rds mice were treated in accordance with the ARVO statement for the use of animals in ophthalmic and vision research. Mice were sacrificed at different postnatal days (11, 17, 21 and 28). Retinal glutamate concentrations were determined by high pressure liquid chromatography (HPLC). The expression of different enzymes related with NO metabolism (nNOS, guanylate cyclase (GC) and arginase) was determined by histochemistry.Results: Glutamate as well as nNOS was observed to be increased in the retina of rds mice mainly between the peak of rods and cones death. GC was observed to be decreased in the rds mice compared to control ones and no significant changes were observed in arginase expression when we compared rds and control mice.Conclusions: There are alterations in the expression of glutamate, nNOS and GC in rds mice retina compared to control mice. RP is a disease with no effective treatment to date and NO metabolism may be a new target in RP treatment.

Potentiation of anti-angiogenic eNOS-siRNA transfection by ultrasound-mediated microbubble destruction in ex vivo rat aortic rings.

Nitric oxide (NO) regulates vascular homeostasis and plays a key role in revascularization and angiogenesis. The endothelial nitric oxide synthase (eNOS) enzyme catalyzes NO production in endothelial cells. Overexpression of the eNOS gene has been implicated in pathologies with dysfunctional angiogenic processes, such as cancer. Therefore, modulating eNOS gene expression using small interfering RNAs (siRNAs) represents a viable strategy for antitumor therapy. siRNAs are highly specific to the target gene, thus reducing off-target effects. Given the widespread distribution of endothelium and the crucial physiological role of eNOS, localized delivery of nucleic acid to the affected area is essential. Therefore, the development of an efficient eNOS-siRNA delivery carrier capable of controlled release is imperative for targeting specific vascular regions, particularly those associated with tumor vascular growth. Thus, this study aims to utilize ultrasound-mediated microbubble destruction (UMMD) technology with cationic microbubbles loaded with eNOS-siRNA to enhance transfection efficiency and improve siRNA delivery, thereby preventing sprouting angiogenesis. The efficiency of eNOS-siRNA transfection facilitated by UMMD was assessed using bEnd.3 cells. Synthesis of nitric oxide and eNOS protein expression were also evaluated. The silencing of eNOS gene in a model of angiogenesis was assayed using the rat aortic ring assay. The results showed that from 6 to 24 h, the transfection of fluorescent siRNA with UMMD was twice as high as that of lipofection. Moreover, transfection of eNOS-siRNA with UMMD enhanced the knockdown level (65.40 ± 4.50%) compared to lipofectamine (40 ± 1.70%). Silencing of eNOS gene with UMMD required less amount of eNOS-siRNA (42 ng) to decrease the level of eNOS protein expression (52.30 ± 0.08%) to the same extent as 79 ng of eNOS-siRNA using lipofectamine (56.30 ± 0.10%). NO production assisted by UMMD was reduced by 81% compared to 67% reduction transfecting with lipofectamine. This diminished NO production led to higher attenuation of aortic ring outgrowth. Three-fold reduction compared to lipofectamine transfection. In conclusion, we propose the combination of eNOS-siRNA and UMMD as an efficient, safe, non-viral nucleic acid transfection strategy for inhibition of tumor progression.

Synergistic gastroprotective and antioxidative effects of natural olive oil and usnic acid isolated from Usnea longissima, a lichen species in Anatolia (Türkiye), in the indomethacin ulcer model created in rats

Usnea longissima, a medically important lichen growing up in forests in Anatolia (Turkey). In this study, the gastroprotective effect of usnic acid (UA) was investigated using an indomethacin (IND)-induced gastric ulcer model in rats. While 25, 50, 100 and 200 mg/kg UA doses were dissolved in 2 ml of olive oil (OO) and administered to rats, only OO was given to one group. In addition, lansoprazole (LAN) and ranitidine (RAN) and IND were dissolved in water and administered to rat groups. IND administration caused very high levels of damage to rat stomachs. On the other hand, when four doses of UA, OO, RAN and LAN were administered, it was determined that hyperemias in the stomach of rats was significantly reduced. After macroscopic analysis of gastric tissues, the activities of superoxide dismutase (SOD), catalase (CAT), myeloperoxidase (MPO) and nitric oxide synthase (iNOS and cNOS) enzymes as well as glutathione (GSH) and lipid peroxidation (LPO) levels were determined in these tissues. After IND application, it was detected increases in MPO, CAT and iNOS activities in gastric tissues and decreases in SOD, cNOS and GSH amounts. Four doses of UA, OO, RAN and LAN applications reversed the trend, bringing them closer to healthy levels.

Association of Endothelial Nitric Oxide Synthase and Angiotensin-Converting Enzyme Genes Polymorphism With In-Sent Restenosis of Bare Metal Stents vs Drug-Eluting Stents in Egyptians.

Despite its unequivocal superiority compared with balloon angioplasty, coronary stenting did not abolish restenosis. We aimed to evaluate the associations between a common single nucleotide polymorphism occurring in endothelial nitric oxide synthase (eNOS) and angiotensin-converting enzyme (ACE) genes and the risk of in-stent restenosis (ISR) of bare metal stents vs drug-eluting stents (BMS vs DES) implanted in Egyptian patients. Two hundred patients who had coronary stenting were divided into group I (n = 98) who received a BMS and group II (n = 102) who received a DES. eNOS and ACE genes polymorphism were analyzed by polymerase chain reaction (PCR). We found that the GA and AA genotypes of the eNOS gene were associated with the ISR with both BMS and DES. However, the ACE gene was not associated with ISR. We concluded that eNOS gene polymorphism is associated with ISR. Hypertension, stent length, and AA genotype of the eNOS gene were found to be independent predictors of the occurrence of ISR after both BMS and DES use.

Zebrafish nos2a benefits bacterial proliferation via suppressing ROS and inducing NO production to impair the expressions of inflammatory cytokines and antibacterial genes

The enzyme nitric oxide synthase 2 or inducible NOS (NOS2), reactive oxygen species (ROS) and nitric oxide (NO) are important participants in various inflammatory and immune responses. However, the functional significances of the correlations among piscine NOS2, ROS and NO during pathogen infection remain unclear. In teleost, there are two nos2 genes (nos2a and nos2b). It has been previously reported that zebrafish nos2a behaves as a classical inducible NOS, and nos2b exerts some functions similar to mammalian NOS3. In the present study, we reported the functional characterization of zebrafish nos2a during bacterial infection. We found that zebrafish nos2a promoted bacterial proliferation, accompanied by an increased susceptibility to Edwardsiella piscicida infection. The nagative regulation of zebrafish nos2a during E. piscicida infection was characterized by the impaired ROS levels, the induced NO production and the decreased expressions of proinflammatory cytokines, antibacterial genes and oxidant factors. Furthermore, although both inducing ROS and inhibiting NO production significantly inhibited bacterial proliferation, only inhibiting NO production but not inducing ROS significantly increased resistance to E. piscicida infection. More importantly, ROS supplementation and inhibition of NO completely abolished this detrimental consequence mediated by zebrafish nos2a during E. piscicida infection. All together, these results firstly demonstrate that the innate response mediated by zebrafish nos2a in promoting bacterial proliferation is dependent on the lower ROS level and higher NO production. The present study also reveals that inhibition of NO can be effective in the protection against E. piscicida infection.