Nitric Oxide Synthase Isoforms Research Articles

Comparison of the Influence of Bisphenol A and Bisphenol S on the Enteric Nervous System of the Mouse Jejunum.

Bisphenols are dangerous endocrine disruptors that pollute the environment. Due to their chemical properties, they are globally used to produce plastics. Structural similarities to oestrogen allow bisphenols to bind to oestrogen receptors and affect internal body systems. Most commonly used in the plastic industry is bisphenol A (BPA), which also has negative effects on the nervous, immune, endocrine, and cardiovascular systems. A popular analogue of BPA-bisphenol S (BPS) also seems to have harmful effects similar to BPA on living organisms. Therefore, with the use of double immunofluorescence labelling, this study aimed to compare the effect of BPA and BPS on the enteric nervous system (ENS) in mouse jejunum. The study showed that both studied toxins impact the number of nerve cells immunoreactive to substance P (SP), galanin (GAL), vasoactive intestinal polypeptide (VIP), the neuronal isoform of nitric oxide synthase (nNOS), and vesicular acetylcholine transporter (VAChT). The observed changes were similar in the case of both tested bisphenols. However, the influence of BPA showed stronger changes in neurochemical coding. The results also showed that long-term exposure to BPS significantly affects the ENS.

Nitric oxide in oncology: a two-faced Janus

Nitric oxide (NO) plays a significant role in the development of tumor processes, offering potential therapeutic options and serving as a target for influencing tumor growth.The aim of this review was to study the role of nitric oxide in the development of cancer, identify key signaling pathways in which NO is involved, mechanisms and methods of nitric oxide delivery in tumor therapy, as well as its impact as a therapeutic target.Results. Different isoforms of nitric oxide synthase (NOS) regulate nervous functions, vascular functions, and inflammatory processes. Elevated levels of induced nitric oxide synthase (iNOS) are associated with tumor development, and its inhibitors can suppress tumor growth. Research indicates that the effect of NO on tumors depends on the concentration and duration of exposure. Low concentrations stimulate the growth and metastases of tumor cells, whereas high concentrations exert an anti-tumor effect, enhancing therapy sensitivity. NO also impacts angiogenesis, metastases, and immune response. The application of NO in tumor treatment is challenging due to its short half-life and rapid diffusion. Developing various NO delivery methods, like gaseous NO and nanoparticles, holds promise for improving the effectiveness and control of distribution. Silicon and goldbased nanoparticles demonstrate potential for NO delivery, enhancing immune response and showing synergism with chemotherapy. Inhibitors of iNOS suppress tumor growth. Their combined usage with other agents, such as chemotherapy, displays promising results in tumor growth control. Further research and clinical trials are essential to determine optimal conditions for employing NO and iNOS inhibitors in cancer treatment. Overall, studying the influence of NO and iNOS on tumor processes represents a crucial area for developing novel treatment methods, underscoring the therapeutic potential of these molecules as agents and targets to enhance oncological outcomes.Conclusion. Nitric oxide and various isoforms of NOS, in particular induced NOS, play a vital role in the regulation of oncological processes. Research confirms the promise of nitric oxide in oncology as a potential antitumor agent. The use of iNOS inhibitors shows potential in controlling tumor growth, especially when combined with other drugs such as chemotherapy. In addition, the development of NO delivery methods is an area of active research that may improve the efficiency of NO distribution in the body and tumor.

Effects of different nitric oxide synthases on pulmonary and systemic hemodynamics in hypoxic stress rat model.

Under hypoxia, exaggerated compensatory responses may lead to acute mountain sickness. The excessive vasodilatory effect of nitric oxide (NO) can lower the hypoxic pulmonary vasoconstriction (HPV) and peripheral blood pressure. While NO is catalyzed by various nitric oxide synthase (NOS) isoforms, the regulatory roles of these types in the hemodynamics of pulmonary and systemic circulation in living hypoxic animals remain unclear. Therefore, this study aims to investigate the regulatory effects of different NOS isoforms on pulmonary and systemic circulation in hypoxic rats by employing selective NOS inhibitors and continuously monitoring hemodynamic parameters of both pulmonary and systemic circulation. Forty healthy male Sprague-Dawley (SD) rats were randomly divided into four groups: Control group (NG-nitro-D-arginine methyl ester, D-NAME), L-NAME group (non-selective NOS inhibitor, NG-nitro-L-arginine methyl ester), AG group (inducible NOS inhibitor group, aminoguanidine), and 7-NI group (neurological NOS inhibitor, 7-nitroindazole). Hemodynamic parameters of rats were monitored for 10 min after inhibitor administration and 5 min after induction of hypoxia [15% O2, 2200 m a. sl., 582 mmHg (76.5 kPa), Xining, China] using the real-time dynamic monitoring model for pulmonary and systemic circulation hemodynamics invivo. Serum NO concentrations and blood gas analysis were measured. Under normoxia, mean arterial pressure and total peripheral vascular resistance were increased, and ascending aortic blood flow and serum NO concentration were decreased in the L-NAME and AG groups. During hypoxia, pulmonary arterial pressure and pulmonary vascular resistance were significantly increased in the L-NAME and AG groups. This compensatory mechanism activated by inducible NOS and endothelial NOS effectively counteracts the pulmonary hemodynamic changes induced by hypoxic stress. It plays a crucial role in alleviating hypoxia-induced pulmonary arterial hypertension.

RETRACTION: Melatonin Inhibits the Expression of the Inducible Isoform of Nitric Oxide Synthase and Nuclear Factor Kappa B Activation in Rat Skeletal Muscle.

Alonso M, Collado PS, González-Gallego J. Melatonin inhibits the expression of the inducible isoform of nitric oxide synthase and nuclear factor kappa B activation in rat skeletal muscle. J Pineal Res 2006;41:8-14. https://doi.org/10.1111/j.1600-079X.2006.00323.x The above article, published online on 28 June 2006 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief, Gianluca Tosini, and John Wiley and Sons Ltd. Following publication, concerns were raised by third parties regarding Figure 1. The authors could not provide the original data for this figure, and were unable to provide a satisfactory explanation to resolve the concerns. The retraction has been agreed because of concerns that portions of the figure were duplicated, affecting the interpretation of the data and results presented. The authors disagree with this decision.

Phenformin attenuates the oxidative-nitrosative stress in the liver of rats under long-term ethanol administration

Modulation of the AMP-activated protein kinase (AMPK) pathway activity is considered to be a promi­sing option in the development of approaches to chronic alcoholic hepatitis treatment. Phenformin, which is a biguanide, has been reported to increase AMPK activity. The aim of this work was to estimate the effect of phenformin as AMPK activator on the development of oxidative-nitrosative stress in the liver of rats under conditions of long-term ethanol administration. The experiments were performed on 24 male Wistar rats, divided into 4 groups: control; animals, which received phenformin hydrochloride orally at a dose of 10 mg/kg daily for 63 days; animals with a forced intermittent alcoholization for 5 days by intraperitoneal administration of 16.5% ethanol solution in 5% glucose at the rate of 4 ml/kg b.w. and subsequent transfer to 10% ethanol as the only source of drinking; animals with chronic alcohol hepatitis simulation and phenformin administration. Superoxide dismutase, catalase, NO synthase isoforms activity, superoxide anion radical production, concentration of malonic dialdehyde, peroxynitrite, nitrites, nitrosothiols concentration and oxidative modification of proteins (OMP) were estimated in liver homogenates. The increased production of oxygen and nitrogen active forms and OMP intensification in the liver of rats under long-term administration of ethanol was detected. Phenformin introduction under long-term ethanol administration was shown to limit the excess peroxynitrite formation and to prevent oxidative damage to rat liver proteins. Keywords: AMP-activated protein kinase., chronic alcoholic hepatitis, liver, oxidative and nitrosative stress, phenformin

Nitric oxide mediated kisspeptin regulation of steroidogenesis and gametogenesis in the catfish, Clarias batrachus.

Nitric oxide (NO) is a gaseous molecule that regulates various reproductive functions. It is a well-recognized regulator of GnRH-FSH/LH-sex steroid secretion in vertebrates including fish. Kisspeptin is a recently discovered neuropeptide which also regulates GnRH secretion. Nitrergic and kisspeptin neurons are reported in close physical contact in the mammalian brain suggesting their interactive role in the release of GnRH. The existence of kisspeptin and NOS is also demonstrated in vertebrate gonads, but information on their reciprocal relation in gonads, if any, is obscure. Therefore, attempts were made to evaluate the functional reciprocal relation between nitric oxide and kisspeptin in the catfish gonads, if any, by administering the nitric oxide synthase (NOS) inhibitor, L-NAME {N(G)-nitro-L-arginine methyl ester}, which reduces NO production, and kisspeptin agonist (KP-10) and assessing their impacts on the expressions of kisspeptin1, different NOS isoforms, NO and steroid production in the gonadal tissue. The results revealed that L-NAME suppressed the expression of kiss1 in gonads of the catfish establishing the role of NO in kisspeptin expression. However, KP-10 increased the expression of all the isoforms of NOSs (iNOS, eNOS,nNOS) and concurrently NO and steroids in the ovary and testis. In vitro studies also indicate that kisspeptin stimulates the production of NO and estradiol and testosterone levels in the gonadal explants and medium. Thus, in vivo results clearly suggest a reciprocal interaction between kisspeptin and NO to regulate the gonadal activity of the catfish. The in vitro findings further substantiate our contention regarding the interactive role of kisspeptin and NO in gonadal steroidogenesis.

Available Therapeutic Options for Corneal Neovascularization: A Review

Corneal neovascularization can impair vision and result in a poor quality of life. The pathogenesis involves a complex interplay of angiogenic factors, notably vascular endothelial growth factor (VEGF). This review provides a comprehensive overview of potential therapies for corneal neovascularization, covering tissue inhibitors of metalloproteinases (TIMPs), transforming growth factor beta (TGF-β) inhibitors, interleukin-1L receptor antagonist (IL-1 Ra), nitric oxide synthase (NOS) isoforms, galectin-3 inhibitors, retinal pigment epithelium-derived factor (PEDF), platelet-derived growth factor (PDGF) receptor inhibitors, and surgical treatments. Conventional treatments include anti-VEGF therapy and laser interventions, while emerging therapies such as immunosuppressive drugs (cyclosporine and rapamycin) have been explored. Losartan and decorin are potential antifibrotic agents that mitigate TGF-β-induced fibrosis. Ocular nanosystems are innovative drug-delivery platforms that facilitate the targeted release of therapeutic agents. Gene therapies, such as small interfering RNA and antisense oligonucleotides, are promising approaches for selectively inhibiting angiogenesis-related gene expression. Aganirsen is efficacious in reducing the corneal neovascularization area without significant adverse effects. These multifaceted approaches underscore the corneal neovascularization management complexity and highlight ideas for enhancing therapeutic outcomes. Furthermore, the importance of combination therapies and the need for further research to develop specific inhibitors while considering their therapeutic efficacy and potential adverse effects are discussed.

Constitutive NOS Production Is Modulated by Alzheimer's Disease Pathology Depending on APOE Genotype.

Both the endothelial (eNOS) and the neuronal (nNOS) isoforms of constitutive Nitric Oxide Synthase have been implicated in vascular dysfunctions in Alzheimer's disease (AD). We aimed to explore the relationship between amyloid pathology and NO dynamics by comparing the cerebrospinal fluid (CSF) levels of nNOS and eNOS of 8 healthy controls (HC) and 27 patients with a clinical diagnosis of Alzheimer's disease and isolated CSF amyloid changes, stratified according to APOE ε genotype (APOE ε3 = 13, APOE ε4 = 14). Moreover, we explored the associations between NOS isoforms, CSF AD biomarkers, age, sex, cognitive decline, and blood-brain barrier permeability. In our cohort, both eNOS and nNOS levels were increased in APOE ε3 with respect to HC and APOE ε4. CSF eNOS inversely correlated with CSF Amyloid-β42 selectively in carriers of APOE ε3; CSF nNOS was negatively associated with age and CSF p-tau only in the APOE ε4 subgroup. Increased eNOS could represent compensative vasodilation to face progressive Aβ-induced vasoconstriction in APOE ε3, while nNOS could represent the activation of NO-mediated plasticity strategies in the same group. Our results confirm previous findings that the APOE genotype is linked with different vascular responses to AD pathology.

THE INFLUENCE OF QUERCETIN ON THE PRODUCTION OF REACTIVE OXYGEN-NITROGEN FORMS IN THE STRUCTURE OF THE INTERSTITIAL SPACE OF RAT TESTICLES WITH LONG-TERM EFFECT OF TRIPTORELIN

Quercetin is a flavonoid commonly found in fruits, vegetables, and seeds. Flavonoids can help prevent cardiovascular diseases, reduce the risk of degenerative brain processes and cancer. Studies have shown that blocking the synthesis of luteinising hormone can lead to oxidative stress in the liver, heart, and salivary glands of rats. The purpose of this study was to assess alterations in the interstitial space of rat testes, identify sources of nitric oxide production, and measure the intensity of oxidative stress in the testes during long-term experimental central blocking of luteinising hormone synthesis by tryptorelin. The study was conducted on 15 sexually mature male white rats. The animals were divided into two groups. The first group (control) received a subcutaneous injection of 0.9% sodium chloride solution. The second group (experimental) received a subcutaneous injection of tryptorelin at a dose of 0.3 mg/kg of active ingredient for 365 days, with simultaneous addition of quercetin to the diet, which was administered orally through a gastric tube three times a day. Total nitric oxide production was assessed by measuring the activity of total NO synthase (gNOS). The activity of gNOS was determined by the increase in nitrite (NO2-) after incubation in Tris-buffered saline. The nitrite concentration was determined using the Gris-Ilosvay reagent at a wavelength of 540 nm. The activity of the inducible (iNOS) and constitutive (cNOS) isoforms was also determined using a selective iNOS inhibitor, aminoguanidine hydrochloride. The production of superoxide anion radical (O2--) was measured by the increase in the formation of diformazan resulting from the reaction of O2-- with nitroblue tetrazolium after incubation in a buffered solution containing sodium hydroxide. The administration of quercetin mitigates the adverse effects of tryptorelin on the structural and functional components of the connective tissue in the rat testes. This effect is observed on day 365 and is characterised by fibrosis and impaired nitric oxide production by constitutive isoforms of NO synthase.

NO Deficiency Compromises Inter- and Intrahemispheric Blood Flow Adaptation to Unilateral Carotid Artery Occlusion.

Carotid artery stenosis (CAS) affects approximately 5-7.5% of older adults and is recognized as a significant risk factor for vascular cognitive impairment (VCI). The impact of CAS on cerebral blood flow (CBF) within the ipsilateral hemisphere relies on the adaptive capabilities of the cerebral microcirculation. In this study, we aimed to test the hypothesis that the impaired availability of nitric oxide (NO) compromises CBF homeostasis after unilateral carotid artery occlusion (CAO). To investigate this, three mouse models exhibiting compromised production of NO were tested: NOS1 knockout, NOS1/3 double knockout, and mice treated with the NO synthesis inhibitor L-NAME. Regional CBF changes following CAO were evaluated using laser-speckle contrast imaging (LSCI). Our findings demonstrated that NOS1 knockout, NOS1/3 double knockout, and L-NAME-treated mice exhibited impaired CBF adaptation to CAO. Furthermore, genetic deficiency of one or two NO synthase isoforms increased the tortuosity of pial collaterals connecting the frontoparietal and temporal regions. In conclusion, our study highlights the significant contribution of NO production to the functional adaptation of cerebrocortical microcirculation to unilateral CAO. We propose that impaired bioavailability of NO contributes to the impaired CBF homeostasis by altering inter- and intrahemispheric blood flow redistribution after unilateral disruption of carotid artery flow.

Evaluation of serum nitric oxide synthase levels in patients with coronary slow flow based on corrected TIMI frame count.

The coronary slow flow phenomenon (CSFP) finding in angiography is characterized by the delayed filling of the terminal vessels without significant epicardial coronary disease. The endothelium performs a vital role in cardiovascular homeostasis by releasing vasoactive substances. Endothelial cells produce nitric oxide (NO) as one of these essential compounds. Three isoforms of nitric oxide synthase (NOS) are endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and induced nitric oxide synthase (iNOS). We aimed to determine the role of NOS in the development of CSFP as the first human study. A total of 129 patients who met the inclusion criteria were enrolled in the study. The patients were classified into five groups based on the results of coronary angiography: Group 1 without coronary artery disease (CAD) and without CSF, group 2 without CAD and with CSF, group 3 with CAD (< 50%) and without CSF, group 4 with CAD (50-90%) and without CSF, and group 5 with CAD and CSF. The serum level of NOS was determined in the participants. Coronary flow was quantified in patients with CSFP using the corrected TIMI frame count (CTFC) method, and the correlation between the levels of this biomarker and CTFC was investigated. In this study, the NOS serum levels were not significantly correlated with the mean CTFC. Since the total amount of NOS was measured as a result of 3 isoforms of this enzyme, the lack of correlation could be related to increased iNOS level and decreased eNOS concentration. These results should be confirmed by more human studies.

Influence of pallet rich plasma, quercetin and their combination on activity of nitric oxide cycle enzymes in nasal mucosa of patients with atrophic rhinitis.

Aim: To study the general activity of NO synthases (gNOS), the activity of inducible and constitutive isoforms of NO synthase, the activity of arginases, and the concentration of nitrites in the nasal mucosa under the conditions of local treatment of chronic atrophic rhinitis (AR) with quercetin and platelet-rich plasma (PRP therapy).. Materials and Methods: The study was conducted on 118 patients divided into two groups: control (n=20) and experimental (patients with AR, n=98). Experimental group was divided into 4 subgroups: standard treatment (n=29), PRP therapy (6 injections for 28 day course, n=19), Quercetin (40 mg 3 times a day for 28 days, n=26) and PRP+Quercetin (n=24) groups. Results: Standard therapy of SaR increases gNOS by 278.38% and arginase activity increases by 222.73%. PRP therapy increases gNOS by 211.43% and arginase by 540.91%. Quercetin elevates gNOS by 108.33% and arginase by 250%. PRP therapy and quercetin increases gNOS by 146.15% and arginase by 536.36%. Conclusions: The use of standard therapy of SaR and addition of PRP therapy, quercetin and their combination effectively restores the production of nitric oxide and the arginase activity in the nasal mucosa.

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.

Indicators of oxidative-nitrosative stress of men injured as a result of combat actions

The article presents data on the state of lipid peroxidation and the activity of NO-synthase isoforms and arginase in lymphocytes and peripheral blood serum in men injured in combat (bullet and shrapnel wounds). 68 men injured as a result of hostilities (shrapnel and bullet wounds), who made up the main group, were examined. 48 practically healthy men were included in the control group. Patients of the main and control groups were divided into two age subgroups each: 20–39 years and 40–60 years. The MDA content in the blood serum of patients of both subgroups of the main group was 1.4 times higher than in the individuals of the corresponding subgroups of the control group (p&lt;0.05). In peripheral blood lymphocytes, the MDA content in patients of both age subgroups of the main group was 1.3–1.4 times higher than in individuals of the corresponding subgroups of the control group (p&lt;0.05; p&lt;0.01). The activity of inducible (Ca2+-independent) de novo synthesis of NO significantly increases in blood serum and lymphocytes of men injured as a result of hostilities. At the same time, the activity of Ca2+-dependent NO-synthase in blood lymphocytes of men injured as a result of hostilities was 2.4 times lower compared to the values in practically healthy men (p&lt;0.001), while it almost did not change in blood serum. It was confirmed that the inducible NOS can activate the processes of lipid peroxidation in men injured as a result of hostilities, as a direct significant correlation of high strength was recorded between the activity of Ca2+-independent iNOS and the MDA content (r=0.74; p&lt;0.05). A significant correlation of medium strength was also established between arginase activity and MDA content (r=0.52; p&lt;0.05). Keywords: malondialdehyde, NO-synthase, arginase.

Chronic Inhibition of Nitric Oxide Synthases Impairs Spatiotemporal Learning and Memory to a Similar Extent in C57BL/6 and hAPP23+/− Mice

Due to global population growth, age-related disorders like cardiovascular disease and dementia are anticipated to increase. Recent data suggests a connection between cardiovascular disease and neurodegeneration, especially focusing on arterial stiffness (AS) and Alzheimer’s disease (AD). In light of this, we conducted a study to explore the impact of long-term nitric oxide synthase (NOS) isoform inhibition, which leads to AS, on neurobehavioral performance. We also compared these effects in an AD model and control mice. C57BL/6 and hAPP23+/− mice (an established AD model) were given 0.5 mg/mL N(G)-Nitro-L-Arginine Methyl Ester (L-NAME) in their drinking water for 16 weeks. Our findings indicate that chronic non-selective NOS inhibition increased AS and reduced spatiotemporal learning and memory in both C57BL/6 and hAPP23+/− mice. These effects were consistent across both groups, emphasizing the role of neuronal NOS (nNOS) in cognitive aging, regardless of genetic predisposition to AD.

Folic acid antioxidant supplementation to binge drinking adolescent rats improves hydric-saline balance and blood pressure, but fails to increase renal NO availability and glomerular filtration rate.

Binge drinking (BD) is an especially pro-oxidant pattern of alcohol consumption, particularly widespread in the adolescent population. In the kidneys, it affects the glomerular filtration rate (GFR), leading to high blood pressure. BD exposure also disrupts folic acid (FA) homeostasis and its antioxidant properties. The aim of this study is to test a FA supplementation as an effective therapy against the oxidative, nitrosative, and apoptotic damage as well as the renal function alteration occurred after BD in adolescence. Four groups of adolescent rats were used: control, BD (exposed to intraperitoneal alcohol), control FA-supplemented group and BD FA-supplemented group. Dietary FA content in control groups was 2 ppm, and 8 ppm in supplemented groups. BD provoked an oxidative imbalance in the kidneys by dysregulating antioxidant enzymes and increasing the enzyme NADPH oxidase 4 (NOX4), which led to an increase in caspase-9. BD also altered the renal nitrosative status affecting the expression of the three nitric oxide (NO) synthase (NOS) isoforms, leading to a decrease in NO levels. Functionally, BD produced a hydric-electrolytic imbalance, a low GFR and an increase in blood pressure. FA supplementation to BD adolescent rats improved the oxidative, nitrosative, and apoptotic balance, recovering the hydric-electrolytic equilibrium and blood pressure. However, neither NO levels nor GFR were recovered, showing in this study for the first time that NO availability in the kidneys plays a crucial role in GFR regulation that the antioxidant effects of FA cannot repair.

Distinctive Roles of Nitric Oxide Synthase Isoforms in Hydroxyurea-Mediated Cytostasis of Erythroid Progenitors

Chemotherapeutic hydroxyurea (HU) is used in the therapy of sickle cell anemia, thalassemia and myeloproliferative neoplasms according to its stimulation of fetal hemoglobin and myelosuppression. We reported that most nitric oxide (NO) in mammals is produced by the reduction of nitrate and nitrite. The NO properties of HU have been widely explored, while its enzymatic NO synthase (NOS) dependence is not completely defined. We have already demonstrated concomitant inhibition of erythroid cell proliferation by HU and NO donors. In this study, we investigated the NOS-mediation in HU regulation of proliferation, differentiation and cell cycle in HEL92.1.7 human erythroleukemic cell line and / or erythroid progenitors of Nos2- or Nos3-null mice. HU induced a dose-dependent increase in NOS2 and NOS3 protein levels and their enzymatic activity by increasing production of NO and citrulline in erythroleukemic cells. Inhibition of the NOS enzymes by pan-specific L-NAME, NOS2 inhibitor 1400W, or NOS2/NOS3 inhibitor DPI was sufficient to prevent the HU-mediated decrease in HEL92.1.7 proliferation assessed by Ki67 immunofluorescence. HU increased the frequency of cells in S-phase (66.58±2.44 vs. 55.18±1,06%) of the cycle at the expense of G0/G1 (17.07±1.67 vs. 28.55±1.01%) due to blocked DNA synthesis. The combined treatment of HU with 10μM DPI decreased the number of cells in G0/G1 (3.34±0.98 vs. 17.07±1.67%) and increased in S (72.3±0.96 vs. 66.58±%) and G2/M (24.36±0.19 vs. 16.35±2.28%) compared with HU alone. Moreover, NOS3 knock-down by shRNA decreased the percentage of cells in S phase from 69,19±2.68 to 34.23±1.07% (p&amp;lt;0.001) and increased in G2/M from 15.62±2.17 to 34.71±3.76 (p&amp;lt;0.001). HU-induced apoptosis was prevented by NOS2 (1400W) inhibition, determined by annexin V, decreasing the number of cells in early and late apoptosis in erythroleukemic cells. In accordance, NOS3 knock-down was not able to rescue HU-induced apoptosis. Furthermore, by performing in vitro enzymatic assay we showed that regulation of NOS2 activity seems to be achieved by direct interaction with HU leading to a dose- and time-dependent increase in NO and citrulline products. HU increased the expression of Nos1/2 isoforms by 20%, and Nos3 by 10% in mouse erythroid progenitors (mERP) of wild-type mice. Bone marrow cells isolated from Nos2- or Nos3-null mice treated orally with HU showed decreased protein nitrosylation in bone marrow compared to HU-treated wild-type mice. In ex vivo colony formation assay, both Nos2-/- and Nos3-/- bone marrow cells formed significantly more late erythroid colonies (CFU-E, 34- and 24-fold, respectively, p&amp;lt;0.001), and Nos2-/- also yielded more of the early erythroid (BFU-E, 9- and 7-fold, p&amp;lt;0.01) and granulocyte/macrophage (CFU-GM, 14- and 15-fold, p&amp;lt;0.01) colonies compared to wild-type mice untreated or HU-treated. HU treatment of mERP, of Nos2-/- and Nos3-/- origin, demonstrated decreased frequency of S-phase cells to levels comparable to untreated wild-type mice. Nos2 and Nos3 deficiency was able to rescue Caspase-3 expression in the presence of HU in mERP. Nevertheless, HU increased the quantity of late apoptotic cells in both Nos2 and Nos3 deficient mice. Our results demonstrated that HU induces the enzymatic activity of NOS proteins that mediate the cytostatic effect. NOS2/3 enzymes are involved in HU-inhibition of proliferation of human erythroleukemic cells and ex vivo differentiation of mERP. The observed NOS enzymes were fully and partially involved in HU-induced apoptosis of HEL92.1.7 cells and mERP, respectively. Uncovering all aspects of HU mechanism of action may help to increase the responsiveness or reduce the negative effects of cytotoxic chemotherapy.

Bisphenol A Effects on Neurons’ Neurochemical Character in the Urinary Bladder Intramural Ganglia of Domestic Pigs

Bisphenol A (BPA), a substance globally used to produce plastics, is part of many everyday items, including bottles, food containers, electronic elements, and others. It may penetrate the environment and living organisms, negatively affecting, among others, the nervous, immune, endocrine, and cardiovascular systems. Knowledge of the impact of BPA on the urinary bladder is extremely scarce. This study investigated the influence of two doses of BPA (0.05 mg/kg body weight (b.w.)/day and 0.5 mg/kg b.w./day) given orally for 28 days on the neurons situated in the ganglia located in the urinary bladder trigone using the typical double immunofluorescence method. In the study, an increase in the percentage of neurons containing substance P (SP), galanin (GAL), a neuronal isoform of nitric oxide synthase (nNOS—used as the marker of nitrergic neurons), and/or cocaine- and amphetamine-regulated transcript (CART) peptide was noted after BPA administration. The severity of these changes depended on the dose of BPA and the type of neuronal factors studied. The most visible changes were noted in the cases of SP- and/or GAL-positive neurons after administering a higher dose of BPA. The results have shown that oral exposure to BPA, lasting even for a short time, affects the intramural neurons in the urinary bladder wall, and changes in the neurochemical characterisation of these neurons may be the first signs of BPA-induced pathological processes in this organ.

Patients with Chronic Coronary Syndrome Can Benefit from Consumption of Enriched Chicken Eggs: The Effects on Microvascular Function, Inflammatory Biomarkers, and Oxidative Status—Randomized Clinical Study

The aim of this clinical study was to determine the impact of the consumption of chicken eggs enriched with n-3 polyunsaturated fatty acids, selenium, vitamin E, and lutein on micro- and macrovascular endothelium-dependent dilation, inflammation biomarkers, and oxidative stress levels in participants with chronic coronary syndrome (CCS). This was a double-blind, placebo-controlled clinical study that included 30 CCS participants (9 women, 21 men) randomized into the control group (N = 15), who ate ordinary chicken eggs (three per day), and the Nutri4 group (N = 15), who ate enriched eggs (three per day) for 21 days. Microvascular and macrovascular endothelium-dependent vasodilation was evaluated by measuring forearm skin post-occlusive reactive hyperemia (PORH) and acetylcholine-induced dilation (AChID) and the flow-mediated dilation (FMD) of the brachial artery, respectively. The serum lipid profile, anti- and proinflammatory cytokine levels, serum concentration of nitric oxide synthase (NOS) isoforms, and oxidative stress biomarkers were measured before and after the diet protocols. Enriched, but not regular, chicken eggs significantly improved microvascular PORH and AChID and macrovascular FMD, increased the serum concentration of inducible NOS, decreased serum triglyceride levels, and decreased proinflammatory cytokine IL-17A and TGF-1β levels compared to initial measurements. Patients with CCS can benefit from the consumption of enriched chicken eggs due to improved lipid biomarkers, a more favorable anti-inflammatory milieu, and improved vascular relaxation at micro- and macrovascular levels.

MECHANISMS OF NITROSATIVE STRESS AND DESTRUCTION OF THE ORGANIC MATRIX OF THE RAT MANDIBLE IN THE RECOVERY PERIOD AFTER ITS INCOMPLETE FRACTURE UNDER CONDITIONS OF CHRONIC ALCOHOL INTOXICATION

The mechanisms of nitrosative stress and depolymerization of biopolymers of the mandibular bone tissue after its incomplete fracture under conditions of chronic alcohol intoxication were investigated. The experiments were conducted on 20 white Wistar rats weighing 190-240 g, divided into 4 groups: Group 1 - intact animals, Group 2 - after reproduction of chronic alcohol intoxication, Group 3 - after modeling of mandibular fracture, Group 4 - after reproduction of mandibular fracture in the setting of chronic alcohol intoxication. The activity of total NO synthase, its constitutive and inducible isoforms, ornithine decarboxylase, concentrations of peroxynitrites of alkaline and alkaline-earth metals, free hydroxyproline, N-acetylneuraminic and hexuronic acids were determined in the homogenate of the standard mandible area by spectrophotometric method. It has been shown that the reproduction of chronic alcohol intoxication significantly increases the total and inducible NO synthase activity in the mandibular homogenate, accompanied by an increase in the concentration of the key marker of nitrosative stress – peroxynitrite. On the 14th day after the reconstruction of a mandibular fracture on the background of chronic alcohol intoxication, NO synthase activity and the content of peroxynitrites of alkaline and alkaline-earth metals in the bone homogenate exceed the results of groups with separate action of pathogenic factors. Under these conditions, the activity of the key enzyme of polyamine biosynthesis, ornithine decarboxylase, significantly decreases, and the depolymerization of bone biopolymers (collagen, glycoproteins, and proteoglycans) increases, which creates prerequisites for disruption of reparative processes in the fracture site.