Nitric Oxide Synthase Isoforms Research Articles

Protein arginine methyltransferase 4 modulates nitric oxide synthase uncoupling and cerebral blood flow in Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of mortality, disability, and long-term care burden in the United States, with women comprising the majority of AD diagnoses. While AD-related dementia is associated with tau and amyloid beta accumulation, concurrent derangements in cerebral blood flow have been observed alongside these proteinopathies in humans and rodent models. The homeostatic production of nitric oxide synthases (NOS) becomes uncoupled in AD which leads to decreased NO-mediated vasodilation and oxidative stress via the production of peroxynitrite (ONOO-∙) superoxide species. Here, we investigate the role of the novel protein arginine methyltransferase 4 (PRMT4) enzyme function and its downstream product asymmetric dimethyl arginine (ADMA) as it relates to NOS dysregulation and cerebral blood flow in AD. ADMA (type-1 PRMT product) has been shown to bind NOS as a noncanonic ligand causing enzymatic dysfunction. Our results from RT-qPCR and protein analyses suggest that aged (9-12 months) female mice bearing tau- and amyloid beta-producing transgenic mutations (3xTg-AD) express higher levels of PRMT4 in the hippocampus when compared to age- and sex-matched C57BL6/J mice. In addition, we performed studies to quantify the expression and activity of different NOS isoforms. Furthermore, laser speckle contrast imaging analysis was indicative that 3xTg-AD mice have dysfunctional NOS activity, resulting in reduced production of NO metabolites, enhanced production of free-radical ONOO-, and decreased cerebral blood flow. Notably, the aforementioned phenomena can be reversed via pharmacologic PRMT4 inhibition. Together, these findings implicate the potential importance of PRMT4 signaling in the pathogenesis of Alzheimer's-related cerebrovascular derangement.

Sex differences in the participation of endothelial mediators and signaling pathways involved in the vasodilator effect of a selective GPER agonist in resistance arteries of gonadectomized Wistar rats

AimEndothelial mechanisms underlying the vascular effects of estrogen modulated by the G protein-coupled estrogen receptor (GPER) are not well understood, especially in gonadal sex hormone deprivation. Thus, we investigated vascular function and endothelial signaling pathways involved in the selective activation of GPER in resistance arteries of gonadectomized rats. MethodsGonadectomy was performed in Wistar rats of both sexes. After 21 days, the animals were euthanized. Concentration-response curves were obtained by cumulative additions of G-1 in third-order mesenteric arteries. The vasodilatory effects of G-1 were evaluated before and after endothelium removal or incubation with pharmacological inhibitors. Tissue protein expression was measured by western blotting. Assays with 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) and 2′,7′ dichlorodihydrofluorescein-diacetate (H2DCF-DA) were performed in the arteries investigated. Immunolocalization was assessed by immunofluorescence. ResultsG-1 induced partially endothelium-dependent relaxation in both sexes. The three isoforms of the enzyme nitric oxide synthase contributed to the production and release of nitric oxide in both gonadectomized groups, but the role of inducible nitric oxide synthase is more expressive in males. The mechanistic pathway by which endothelial nitric oxide synthase is phosphorylated appears to differ between sexes, with the rapid signaling pathway phosphatidylinositol-3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3k-Akt-eNOS) being identified for males and mitogen-activated protein kinase/extracellular signal-regulated kinase/endothelial nitric oxide synthase (MEK-ERK-eNOS) for females. The contribution of hydrogen peroxide as an endothelial relaxation mediator seems to be greater in females. ConclusionThese results provide new insights into the effects of estrogen-induced responses via GPER on vascular function in gonadal sex hormone deprivation.

Impact of nitric oxide in liver cancer microenvironment

The pro- or antitumoral properties of nitric oxide (NO) are dependent on local concentration, redox state, cellular status, duration of exposure and compartmentalization of NO generation. The intricate network of the tumor microenvironment (TME) is constituted by tumor cells, stromal and immune cells surrounded by active components of extracellular matrix that influence the biological behavior and, consequently, the treatment and prognosis of cancer. The review describes critical events in the crosstalk of cellular and stromal components in the TME, with special emphasis in the impact of NO generation in the regulation of hepatocellular carcinoma (HCC). The increased expression of nitric oxide synthase (NOS) in tumors and NO-end products in plasma have been associated with poor prognosis of cancer. We have assessed the level of the different isoforms of NOS in tumors and its relation to cell proliferation and death markers, and cell death receptor expression in tumors, and apoptotic markers and ligands of TNF-α receptor family in blood from a cohort of patients with HCC from different etiologies submitted to orthotopic liver transplantation (OLT). The high levels of NOS2 in tumors were associated with low plasma concentration of apoptotic markers (M30 and M65), FasL and TNF-α in HCV patients. By contrast, the low levels of NOS2 in tumors from alcohol-derived patients was associated with increased Trail-R1 expression in tumors, and circulating Trail levels compared to observed in plasma from HCV- and alcohol + HCV-derived patients. This study reinforces the association between increased NOS2 expression and potential risk of low patients’ survival in HCC. However, a differential functional relevance of NOS expression in HCC seems to be influenced by etiologies.

Endothelial nitric oxide synthase Glu298Asp gene polymorphism in the cases of idiopathic thrombocytopenic purpura.

BackgroundNitric oxide (NO) can induce apoptosis in megakaryocytes. Stimulatory function of NO on platelet production may be important in the pathophysiology of idiopathic thrombocytopenic purpura (ITP). NO is produced by three isoforms of NO synthase (NOS). The endothelial nitric oxide synthase (eNOS) isoform has been detected in platelets. Polymorphism of the eNOS gene, which supplies NO synthesis, changes the functions of this enzyme. In this study, the role of eNOS Glu298Asp gene polymorphism in etiopathogenesis, its course, and treatment of ITP was investigated.MethodsSixty-six patients [51 newly diagnosed ITP (ND-ITP), 15 chronic ITP (CH-ITP), and 60 healthy controls (HC)] were enrolled in this study.ResultsIn all patients, the frequency of the GT genotype was 48.5%. The frequency of the GG genotype was determined to be 40.9% and the TT genotype was 10.6%. The most common allele in all patients was the G allele. eNOS Glu298Asp gene polymorphism might be a risk factor in the etiopathogenesis of ITP. Patients with the GG genotype were thought to have a high intention for CH-ITP. Patients with the GG genotype responded effectively to medical treatment using IVIG therapy. The presence of the G allele was observed to have a positive effect on the medical treatment of patients with CH-ITP, whereas the T allele exhibited a negative effect.ConclusionIn the present study, a significant correlation was found between ITP and eNOS Glu298Asp gene polymorphism. This correlation suggested that eNOS Glu298Asp gene polymorphism might be a risk factor in the ethiopathogenesis of ITP.

Proteomic Assessment of C57BL/6 Hippocampi after Non-Selective Pharmacological Inhibition of Nitric Oxide Synthase Activity: Implications of Seizure-like Neuronal Hyperexcitability Followed by Tauopathy.

Nitric oxide (NO) is a small gaseous signaling molecule responsible for maintaining homeostasis in a myriad of tissues and molecular pathways in neurology and the cardiovasculature. In recent years, there has been increasing interest in the potential interaction between arterial stiffness (AS), an independent cardiovascular risk factor, and neurodegenerative syndromes given increasingly epidemiological study reports. For this reason, we previously investigated the mechanistic convergence between AS and neurodegeneration via the progressive non-selective inhibition of all nitric oxide synthase (NOS) isoforms with N(G)-nitro-L-arginine methyl ester (L-NAME) in C57BL/6 mice. Our previous results showed progressively increased AS in vivo and impaired visuospatial learning and memory in L-NAME-treated C57BL/6 mice. In the current study, we sought to further investigate the progressive molecular signatures in hippocampal tissue via LC–MS/MS proteomic analysis. Our data implicate mitochondrial dysfunction due to progressive L-NAME treatment. Two weeks of L-NAME treatment implicates altered G-protein-coupled-receptor signaling in the nerve synapse and associated presence of seizures and altered emotional behavior. Furthermore, molecular signatures implicate the cerebral presence of seizure-related hyperexcitability after short-term (8 weeks) treatment followed by ribosomal dysfunction and tauopathy after long-term (16 weeks) treatment.

Inducible Nitric Oxide Synthase Inhibition in the Medial Prefrontal Cortex Attenuates the Anxiogenic-Like Effect of Acute Restraint Stress via CB1 Receptors.

Stress exposure can result in several proinflammatory alterations in the brain, including overexpression of the inducible isoform of nitric oxide synthase (iNOS) in the medial prefrontal cortex (mPFC). These changes may be involved in the development of many psychiatric conditions. However, it is unknown if iNOS in mPFC plays a significant role in stress-induced behavioral changes. The endocannabinoid (ECB) system is also influenced by stress. Its activation seems to be a counter regulatory mechanism to prevent or decrease the stress-mediated neuroinflammatory consequences. However, it is unclear if the ECB system and iNOS interact to influence stress consequences. This study aimed to test the hypothesis that the anti-stress effect of iNOS inhibition in mPFC involves the local ECB system, particularly the CB1 cannabinoid receptors. Male Wistar rats with guide cannula aimed at the mPFC were submitted to acute restraint stress (RS) for 2 h. In the following morning, rats received bilateral microinjections of vehicle, AM251 (CB1 antagonist; 100 pmol), and/or 1400W (iNOS selective inhibitor; 10−4, 10−3, or 10−2 nmol) into the prelimbic area of mPFC (PL-mPFC) before being tested in the elevated plus-maze (EPM). iNOS inhibition by 1400W prevented the anxiogenic-like effect observed in animals submitted to RS. The drug did not promote behavior changes in naive animals, demonstrating a stress-dependent effect. The 1400W-anti-stress effect was prevented by local pretreatment with AM251. Our data suggest that iNOS inhibition may facilitate the local endocannabinoid signaling, attenuating stress effects.

The role of nitric oxide in sepsis-associated kidney injury.

Sepsis is one of the leading causes of acute kidney injury (AKI), and several mechanisms including microcirculatory alterations, oxidative stress, and endothelial cell dysfunction are involved. Nitric oxide (NO) is one of the common elements to all these mechanisms. Although all three nitric oxide synthase (NOS) isoforms are constitutively expressed within the kidneys, they contribute in different ways to nitrergic signaling. While the endothelial (eNOS) and neuronal (nNOS) isoforms are likely to be the main sources of NO under basal conditions and participate in the regulation of renal hemodynamics, the inducible isoform (iNOS) is dramatically increased in conditions such as sepsis. The overexpression of iNOS in the renal cortex causes a shunting of blood to this region, with consequent medullary ischemia in sepsis. Differences in the vascular reactivity among different vascular beds may also help to explain renal failure in this condition. While most of the vessels present vasoplegia and do not respond to vasoconstrictors, renal microcirculation behaves differently from nonrenal vascular beds, displaying similar constrictor responses in control and septic conditions. The selective inhibition of iNOS, without affecting other isoforms, has been described as the ideal scenario. However, iNOS is also constitutively expressed in the kidneys and the NO produced by this isoform is important for immune defense. In this sense, instead of a direct iNOS inhibition, targeting the NO effectors such as guanylate cyclase, potassium channels, peroxynitrite, and S-nitrosothiols, may be a more interesting approach in sepsis-AKI and further investigation is warranted.

P-192 ATP5G3 and VEGFC expression in cumulus cells are non-invasive markers of fertilization rate

Abstract Study question Could expression of aging- and hypoxia-related genes in cumulus cells be used as non-invasive markers of fertilization rate in ART? Summary answer The expression levels of specific aging- (ATP5G3) and hypoxia-related genes (VEGFC) in human cumulus cells correlate with oocyte ability to be fertilized. What is known already Throughout a woman reproductive life, cumulus cells (CC) protect the dormant oocyte from damage, act as sensors of the follicular microenvironment and act as gatekeeper for the oocyte developmental potential. One such mechanism relays on the hypoxia-dependent response, which controls the cell metabolic and oxidative state. With age, hypoxia tolerance decreases in the whole organism, including the ovary. Recently, alterations in CC response to the follicular microenvironment have been described in advanced maternal age (AMA) women. We investigate whether expression levels of hypoxia-responsive genes in CC could be used as non-invasive marker of fertilization rate in human oocytes. Study design, size, duration Prospective study of 94 women (41 oocyte donors and 53 patients) recruited from 2018 to 2021. The overall age was 32±8.0 (range 18-46); 25±4.3 (range 18-34) for oocyte donors and 38±4.6 (range 26-46) for patients. The mean antral follicular count (AFC) was 18±10.9 (range 1-48) and the mean MII rate at ovum pick-up (OPU) was 75±17.8% (range 20-100%). Participants/materials, setting, methods Pooled CC were collected from each woman after OPU; total RNA was extracted, reverse transcribed to cDNA using random hexamers and expression of aging- (LYZ, FGF2, ATP5G3, AMH, ANXA5) and hypoxia-related genes (HIF-1A, NOS2, NOS3, HMOX1, TXNIP, CLU, FABP3, TGFBR3, VEGFC) was detected by qPCR and normalized against TBP. Expression levels were plotted against woman age, AFC, MII rate at OPU (MR), and fertilization rates (FR) and non-parametric Spearman’s correlation was applied. Main results and the role of chance Overall, MII and FR rates did not decrease with increasing woman’s age (rs= -0.2, p = 0.06 and rs= -0.1, p = 0.39, respectively). When analyzing expression levels of aging markers (LYZ, FGF2, ATP5G3, AMH, ANXA5), none of them correlate with age, AFC, or MR (rs<│0.1│, p > 0.05) in CCs. Moreover, although abundant, the expression of HIF1A, the main hypoxia-related gene, was not correlated with age, AFC, MR or FR. However, the expression of several genes regulated by HIF1A did show correlation with age, MR or FR. CLU (a secreted chaperone involved in cell death), TXNIP (a tumor suppressor gene involved in redox regulation) and NOS2 (an inducible and calcium-independent isoform of nitric oxide synthase) correlated positively with age (rs=0.25, p = 0.014; rs=0.31, p = 0.0027; and rs=0.24, p = 0.03, respectively). We further analyzed CC gene expression against FR and we found that ATP5G3 and VEGFC levels decreased with increasing FR, independently of age (rs=-0.26, p = 0.044 and rs=-0.31, p = 0.014; respectively). Limitations, reasons for caution The analysis of pooled CC allows for cohort interpretation of results, and they cannot be extrapolated at this time to single oocyte’s reproductive outcome prediction. Wider implications of the findings We found that the fertilization potential of a cohort of oocytes is related to the ability of CC to respond to oxidative stress and hypoxia, pointing at ATP5G3 and VEGFC expression as markers for fertilization success. Further research is needed to assess their association with embryo quality and pregnancy outcomes. Trial registration number NA

Uterine Inflammation Changes the Expression of Cholinergic Neurotransmitters and Decreases the Population of AChE-Positive, Uterus-Innervating Neurons in the Paracervical Ganglion of Sexually Mature Gilts.

Simple SummaryEndometritis, both with non-infectious and infectious backgrounds, is one of the most prevalent pathological states among domestic animals. In animals, it generates severe economic problems, including lowered reproductive indices and rising medical treatment costs, and in women, it might lead to severe fertility impairment. In order to determine how the autonomic nervous system responds to such a pathological state, an experimental group of pigs were treated with Escherichia coli injection into the uterine horns, and several ganglions responsible for innervation of this organ were examined, including the paracervical ganglion located on both sides of the broad ligament of the uterus. The results clearly showed a strong impact of the inflammation on the chemical coding of neurons, some even synthesizing neurotransmitters de novo such as the GAL-expressing perikarya. Additionally, applied injections decreased the number of parasympathetic, acetylcholinesterase-expressing neurons implying the importance of the cholinergic population to keep the inflammation under control. The obtained data serve as a basis for the future implementation of modern treatment and enhancements in animal breeding.The focus of this study was based on examining the impact of endometritis on the chemical coding of the paracervical ganglion (PCG) perikaryal populations supplying pig uterus. Four weeks after the injection of Fast Blue retrograde tracer into uterine horns, either the Escherichia coli (E. coli) suspension or saline solution was applied to both horns. Laparotomy treatment was performed for the control group. Uterine cervices containing PCG were extracted on the eighth day after previous treatments. Subsequent macroscopic and histopathologic examinations acknowledged the severe form of acute endometritis in the E. coli-treated gilts, whereas double-labeling immunofluorescence procedures allowed changes to be analyzed in the PCG perikaryal populations coded with vesicular acetylcholine transporter (VAChT) and/or somatostatin (SOM), vasoactive intestinal polypeptide (VIP), a neuronal isoform of nitric oxide synthase (nNOS), galanin (GAL). The acetylcholinesterase (AChE) detection method was used to check for the presence and changes in the expression of this enzyme and further confirm the presence of cholinergic perikarya in PCG. Treatment with E. coli resulted in an increase in VAChT+/VIP+, VAChT+/VIP−, VAChT+/SOM+, VAChT+/SOM−, VAChT+/GAL− and VAChT+/nNOS− PCG uterine perikarya. An additional increase was noted in the non-cholinergic VIP-, SOM- and nNOS-immunopositive populations, as well as a decrease in the number of cholinergic nNOS-positive perikarya. Moreover, the population of cholinergic GAL-expressing perikarya that appeared in the E. coli-injected gilts and E. coli injections lowered the number of AChE-positive perikarya. The neurochemical characteristics of the cholinergic uterine perikarya of the PCG were altered and influenced by the pathological state (inflammation of the uterus). These results may indicate the additional influence of such a state on the functioning of this organ.

Measurement of Tetrahydrobiopterin in Animal Tissue Samples by HPLC with Electrochemical Detection-Protocol Optimization and Pitfalls.

Tetrahydrobiopterin (BH4) is an essential cofactor of all nitric oxide synthase isoforms, thus determination of BH4 levels can provide important mechanistic insight into diseases. We established a protocol for high-performance liquid chromatography/electrochemical detection (HPLC/ECD)-based determination of BH4 in tissue samples. We first determined the optimal storage and work-up conditions for authentic BH4 and its oxidation product dihydrobiopterin (BH2) under various conditions (pH, temperature, presence of antioxidants, metal chelators, and storage time). We then applied optimized protocols for detection of BH4 in tissues of septic (induced by lipopolysaccharide [LPS]) rats. BH4 standards in HCl are stabilized by addition of 1,4-dithioerythritol (DTE) and diethylenetriaminepentaacetic acid (DTPA), while HCl was sufficient for BH2 standard stabilization. Overnight storage of BH4 standard solutions at room temperature in HCl without antioxidants caused complete loss of BH4 and the formation of BH2. We further optimized the protocol to separate ascorbate and the BH4 tissue sample and found a significant increase in BH4 in the heart and kidney as well as higher BH4 levels by trend in the brain of septic rats compared to control rats. These findings correspond to reports on augmented nitric oxide and BH4 levels in both animals and patients with septic shock.

Вплив лапаратомії та ліпополісахаридіндукованої системної запальної відповіді на метаболічні розлади в організмі щурів

This investigation is aimed at studying the effect of abdominal surgical trauma (laparotomy) on markers of surgical stress and acute phase response as well as markers of carbohydrate and lipid metabolism under lipopolysaccharide (LPS) -induced systemic inflammatory response (SIR). Male Wistar rats were divided into 4 groups: 1st (control) group included “pseudooperated” animals (the procedure included the administration of anesthesia, epilation, fixation of animals, compression of the skin of the abdomen with Mikulicz’s clamp by one click); the 2nd group included the rats, which were injected Salmonella typhi (in a dose of 0.4 μg/kg body weight 3 times during the 1st week and once a week for the next 7 weeks) before performing the “false operation”; the 3rd group was made up of the rats after laparotomy; and the 4th group involved the rats after laparotomy performed under LPSinduced SIR. The markers were assessed in 7 days following the “pseudo-operation” or laparotomy. The results obtained have demonstrated the combined effect of laparotomy and LPS-induced SIR was accompanied by a significant increase in the marker of surgical stress, the concentration of cortisol in blood plasma, which significantly exceeded the values of the groups 2 and 3 – by 61.8 and 25.1%, respectively. However, the content of acute-phase protein ceruloplasmin, an acute phase reactant, in the serum remained at the level of the 2nd group. Under these conditions, the concentration of very low-density lipoprotein cholesterol and triglycerides significantly exceeded the relevant values in the 2nd and 3rd groups. The combined effect of surgical trauma and LPS-induced SIR considerably reduced the activity of constitutive isoforms of NO-synthase, which was significantly lower, by 41.7%, than the value in the group 2, and by 41.7% lower than in the group 3. At the same time, the total activity of this enzyme and the activity of its inducible isoform were consistent with the values of the 2nd group. This was accompanied by the development of decompensated lipid peroxidation (with a considerable decrease in the blood antioxidant potential).

2-Aminopyridines with a shortened amino sidechain as potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors

A series of potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors (hnNOS) based on the 2-aminopyridine scaffold with a shortened amino sidechain is reported. A rapid and simple protocol was developed to access these inhibitors in excellent yields. Neuronal nitric oxide synthase (nNOS) is a novel therapeutic target for the treatment of various neurological disorders. The major challenges in designing nNOS inhibitors in humans focus on potency, selectivity over other isoforms of nitric oxide synthases (NOSs), and blood–brain barrier permeability. In this context, we discovered a promising inhibitor, 6-(3-(4,4-difluoropiperidin-1-yl)propyl)-4-methylpyridin-2-amine dihydrochloride, that exhibits excellent potency for rat (Ki = 46 nM) and human nNOS (Ki = 48 nM), respectively, with 388-fold human eNOS and 135-fold human iNOS selectivity. It also displayed excellent permeability (Pe = 17.3 × 10-6 cm s-1) through a parallel artificial membrane permeability assay, a model for blood–brain permeability. We found that increasing lipophilicity by incorporation of fluorine atoms on the backbone of the inhibitors significantly increased potential blood–brain barrier permeability. In addition to measuring potency, isoform selectivity, and permeability of NOS inhibitors, we also explored structure–activity relationships via structures of key inhibitors complexed to various isoforms of nitric oxide synthases.

Reduced expression of inflammasome complex components in cluster headache.

The involvement of inflammation in the pathophysiology of cluster headache (CH) has been suggested, with a role implied for interleukin (IL)-1β. We aimed to measure peripheral blood expression levels of IL-1β-inducing systems, the inflammasome complex, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, and investigate their values as putative biomarkers in CH. In this cross-sectional study conducted in the Headache Unit of Istanbul University, Turkey, blood mononuclear cells (PBMCs) and sera were collected from 30 patients with episodic migraine, 4 with chronic CH, and 47 healthy individuals. Levels of inflammasome complex components (NLRP1, NLRP3, caspase 1, and ASC), end products of inflammasome complex activity (IL-1β, IL-18, and nitric oxide synthase isoforms), neuron-specific enolase, other inflammatory factors (NF-κB, HMGB1, and s100b), and anti-inflammatory IL-4 were measured by real-time quantitative polymerase chain reaction and/or enzyme-linked immunosorbent assay. NLRP3 expression levels were significantly reduced in PBMC samples of patients with CH, obtained during CH attacks (n=24) or headache-free (out of cycle) episodes (n=10). CH-attack patients showed greater expression levels of IL-1β (2-ΔΔCT median [25th-75th percentile], 0.96 [0.66-1.29 vs. 0.52 [0.43-0.73]) and NF-κB (1.06 [0.66-3.00] vs. 0.62 [0.43-1.19]) in PBMCs but not in sera compared with headache-free CH patients. However, these differences did not attain statistical significance (p=0.058 and p=0.072, respectively). Moreover, NLRP1 (52.52 [35.48-67.91] vs. 78.66 [54.92-213.25]; p=0.017), HMGB1 (11.51 [5.20-15.50] vs. 13.33 [8.08-18.13]; p=0.038), S100b (569.90 [524.10-783.80] vs. 763.40 [590.15-2713.00]; p=0.013), NSE (11.15 [6.26-14.91] vs. 13.93 [10.82-19.04]; p=0.021), nNOS (4.24 [3.34-12.85] vs. 12.82 [4.52-15.44]; p=0.028), and eNOS (64.83 [54.59-91.14] vs. 89.42 [61.19-228.40]; p=0.034) levels were lower in patients with three or more autonomic manifestations (n=9). No correlation was found between inflammation factors and clinical parameters of CH. Our results support the involvement of the IL-1β system in attacks of CH. However, the components of the inflammasome complex are suppressed in the peripheral blood and do not appear to play a role in the pathophysiology of CH. These findings argue against a potential biomarker value of the inflammasome complex in CH.

Characterizing CD38 Expression and Enzymatic Activity in the Brain of Spontaneously Hypertensive Stroke-Prone Rats.

Background: CD38 is a transmembrane glycoprotein that catabolizes nicotinamide adenine dinucleotide (NAD+) and is the main source for the age-dependent decrease in NAD+ levels. Increased CD38 enzymatic activity has been implicated in several neurological diseases. However, its role in the pathogenesis of cerebral small vessel disease (CSVD) remains unknown. We aimed to characterize CD38 expression and enzymatic activity in the brain of spontaneously hypertensive stroke-prone rats (SHRSP), a genetic model for hypertension and human CSVD, in comparison to age-matched normotensive Wistar Kyoto rats (WKY). Materials and Methods: Age-matched male 7- and 24-week-old WKY and SHRSP were studied. CD38 enzymatic activity was determined in the brain homogenate. Immunohistochemistry and Western Blotting (WB) were used to characterize CD38 expression and localize it in the different cell types within the brain. In addition, expression of nitric oxide synthase (NOS) isoforms and the levels of nitric oxide (NO), superoxide, nicotinamide dinucleotide (phosphate) NAD(P)H were measured the brain of in WKY and SHRSP. Results: CD38 expression and enzymatic activity were increased in SHRSP brains compared to age matched WKY starting at 7 weeks of age. CD38 expression was localized to the endothelial cells, astrocytes, and microglia. We also identified increased CD38 expression using WB with age in SHRSP and WKY. CD38 enzymatic activity was also increased in 24-week SHRSP compared to 7-week SHRSP. In association, we identified evidence of oxidative stress, reduced NO level, reduced NAD(P)H level and endothelial NOS expression in SHRSP compared to age matched WKY. NAD(P)H also decreased with age in WKY and SHRSP. Additionally, activation of astrocytes and microglia were present in SHRSP compared to WKY. Conclusions: CD38 is overexpressed, and its enzymatic activity is increased in SHRSP, a genetic model for marked hypertension and human CSVD. Our results suggest a potential role for CD38 enzymatic activation in the pathogenesis of CSVD and points to the need for future mechanistic and pharmacological studies.

Prophylactic Zinc Administration Combined with Swimming Exercise Prevents Cognitive-Emotional Disturbances and Tissue Injury following a Transient Hypoxic-Ischemic Insult in the Rat.

Exercise performance and zinc administration individually yield a protective effect on various neurodegenerative models, including ischemic brain injury. Therefore, this work was aimed at evaluating the combined effect of subacute prophylactic zinc administration and swimming exercise in a transient cerebral ischemia model. The prophylactic zinc administration (2.5 mg/kg of body weight) was provided every 24 h for four days before a 30 min common carotid artery occlusion (CCAO), and 24 h after reperfusion, the rats were subjected to swimming exercise in the Morris Water Maze (MWM). Learning was evaluated daily for five days, and memory on day 12 postreperfusion; anxiety or depression-like behavior was measured by the elevated plus maze and the motor activity by open-field test. Nitrites, lipid peroxidation, and the activity of superoxide dismutase (SOD) and catalase (CAT) were assessed in the temporoparietal cortex and hippocampus. The three nitric oxide (NO) synthase isoforms, chemokines, and their receptor levels were measured by ELISA. Nissl staining evaluated hippocampus cytoarchitecture and Iba-1 immunohistochemistry activated the microglia. Swimming exercise alone could not prevent ischemic damage but, combined with prophylactic zinc administration, reversed the cognitive deficit, decreased NOS and chemokine levels, prevented tissue damage, and increased Iba-1 (+) cell number. These results suggest that the subacute prophylactic zinc administration combined with swimming exercise, but not the individual treatment, prevents the ischemic damage on day 12 postreperfusion in the transient ischemia model.

The Endothelial Dysfunction Could Be a Cause of Heart Failure with Preserved Ejection Fraction Development in a Rat Model

50% of patients with heart failure have a preserved ejection fraction (HFpEF). Numerous studies have investigated the pathophysiological mechanisms of HFpEF and have shown that endothelial dysfunction plays an important role in HFpEF. Yet no studies answered whether endothelial dysfunction could be the cause or is the consequence of HFpEF. Recently, we have shown that the endothelial overexpression of human β3-adrenoreceptor (Tgβ3) in rats leads to the slow development of diastolic dysfunction over ageing. The aim of the study is to decipher the involvement of endothelial dysfunction in the HFpEF development. For that, we investigated endothelial and cardiac function in 15-, 30-, and 45-week-old wild-type (WT) and Tgβ3 rats. The aortic expression of •NO synthase (NOS) isoforms was evaluated by Western blot. Finally, electron paramagnetic resonance measurements were performed on aortas to evaluate •NO and O2•- production. Vascular reactivity was altered as early as 15 weeks of age in response to isoproterenol in Tgβ3 aortas and mesenteric arteries. NOS1 (neuronal NOS) expression was higher in the Tgβ3 aorta at 30 and 45 weeks of age (30 weeks: WT: 1.00 ± 0.21; Tgβ3: 6.08 ± 2.30; 45 weeks: WT: 1.00 ± 0.12; Tgβ3: 1.55 ± 0.17; p < 0.05). Interestingly, the endothelial NOS (NOS3) monomer form is increased in Tgβ3 rats at 45 weeks of age (ratio NOS3 dimer/NOS3 monomer; WT: 1.00 ± 0.37; Tgβ3: 0.13 ± 0.05; p < 0.05). Aortic •NO production was increased by NOS2 (inducible NOS) at 15 weeks of age in Tgβ3 rats (+52% vs. WT). Aortic O2•- production was increased in Tgβ3 rats at 30 and 45 weeks of age (+75% and+76%, respectively, vs. WT, p < 0.05). We have shown that endothelial dysfunction and oxidative stress are present as early as 15 weeks of age and therefore conclude that endothelial dysfunction could be a cause of HFpEF development.

Inducible Nitric Oxide Synthase/Nitric Oxide System as a Biomarker for Stress and Ease Response in Fish: Implication on Na+ Homeostasis During Hypoxia

The cellular and organismal response to stressor-driven stimuli evokes stress response in vertebrates including fishes. Fishes have evolved varied patterns of stress response, including ionosmotic stress response, due to their sensitivity to both intrinsic and extrinsic stimuli. Fishes that experience hypoxia, a detrimental stressor that imposes systemic and cellular stress response, can evoke disturbed ion homeostasis. In addition, like other vertebrates, fishes have also developed mechanisms to recover from the impact of stress by way of shifting stress response into ease response that could reduce the magnitude of stress response with the aid of certain neuroendocrine signals. Nitric oxide (NO) has been identified as a potent molecule that attenuates the impact of ionosmotic stress response in fish, particularly during hypoxia stress. Limited information is, however, available on this important aspect of ion transport physiology that contributes to the mechanistic understanding of survival during environmental challenges. The present review, thus, discusses the role of NO in Na+ homeostasis in fish particularly in stressed conditions. Isoforms of nitric oxide synthase (NOS) are essential for the synthesis and availability of NO at the cellular level. The NOS/NO system, thus, appears as a unique molecular drive that performs both regulatory and integrative mechanisms of control within and across varied fish ionocytes. The activation of the inducible NOS (iNOS)/NO system during hypoxia stress and its action on the dynamics of Na+/K+-ATPase, an active Na+ transporter in fish ionocytes, reveal that the iNOS/NO system controls cellular and systemic Na+ transport in stressed fish. In addition, the higher sensitivity of iNOS to varied physical stressors in fishes and the ability of NO to lower the magnitude of ionosmotic stress in hypoxemic fish clearly put forth NO as an ease-promoting signal molecule in fishes. This further points to the signature role of the iNOS/NO system as a biomarker for stress and ease response in the cycle of adaptive response in fish.

Endocrine Disruptor Bisphenol a Affects the Neurochemical Profile of Nerve Fibers in the Aortic Arch Wall in the Domestic Pig.

Bisphenol A (BPA) is a synthetic compound utilized in industry for the production of various plastics. BPA penetrates into the environment and adversely affects living organisms. Therefore, the influence of various BPA dosages on the neurochemical characteristics of nerve fibers located in the aortic branch wall was investigated in this study utilizing a double immunofluorescence method. It was found that BPA in concentration of 0.5 mg/kg body weight/day causes a clear increase in the density of nerves within aortic branch walls immunoreactive to cocaine- and amphetamine-regulated transcript (CART), calcitonin gene-related peptide (CGRP), neuronal isoform of nitric oxide synthase (nNOS), pituitary adenylate cyclase-activating peptide (PACAP), and vasoactive intestinal polypeptide (VIP). Nerves containing galanin (GAL) and/or somatostatin (SOM) did not change when BPA was introduced into the system. Changes noted after administration of BPA at a dose of 0.05 mg/kg body weight/day were less visible and concerned fibers immunoreactive to CART, CGRP, and/or PACAP. The obtained results show that BPA affects the neurochemical coding of nerves in the aortic branch wall. These fluctuations may be the first signs of the influence of this substance on blood vessels and may also be at the root of the disturbances in the cardiovascular system.

INDUCTION OF NRF2 TRANSCTIPTION FACTOR INHIBITS FORMATION OF RACTIVE OXYGEN AND NITROGEN SPECIES IN LIVER OF RATS UNDER MODELLING METABOLIC SYNDROME BY EXPOSURE TO ROUND-THE-CLOCK LIGHTING

Aim: To evaluate the effect of dimethyl fumarate, an Nrf2 inducer, on the production of reactive oxygen and nitrogen species in the liver of rats under the conditions of modeling the metabolic syndrome with roundthe-clock illumination. Dimethyl fumarate in 10% dimethyl sulfoxide solution in a dose of 15 mg/kg was administered intraperitoneally to white rats over the modeled metabolic syndrome (a 20% aqueous fructose solution for drinking and a diet enriched with carbohydrates and lipids). In the liver homogenate of rats, the rate of generation of the superoxide anion radical (•О 2 ), the activity of total NO synthase (NOS), its constitutive and inducible isoforms (cNOS, iNOS), and the content of peroxynitrites of alkali and alkaline earth metals were determined. The administration of dimethyl fumarate under the experimental conditions significantly restrained the •О 2 production by microsomes and NOS in the liver tissues by 48.9%, by mitochondria by 47.3%, by leukocyte NADPH oxidase by 45.6%; it also reduced NOS activity (total and iNOS) by 33.1% and 35.9%, respectively, and the concentration of peroxynitrites by 39.7% compared with the values of the control group that received only the solvent (10% dimethyl sulfoxide solution). The cNOS activity and coupling index exceeded the control result in 2.95 and 5.5 times, respectively. The introduction of the Nrf2 inductor, dimethyl fumarate, during the simulation of the metabolic syndrome by round-the-clock lighting to rats is an effective means of limiting the production of reactive oxygen and nitrogen species in the liver tissues.

THE ROLE OF AP-1 TRANSCRIPTION FACTOR ACTIVATION IN THE CHANGES OF PRODUCTION AND UTILIZATION OF NITRIC OXIDE IN THE GASTRIC MUCOSA OF RATS UNDER CONDITIONS OF CHRONIC FLUORIDE INTOXICATION

Millions of people are affected by excessive fluoride intake. The effect of fluorides on the activation or inhibition of redox-sensitive transcription factors remains poorly understood. The aim of this research is to examine the effect of activation of the transcription factor AP-1 on changes in the activity of inducible NO synthase and constitutive isoforms of NO synthase, concentrations of peroxynitrites of alkali and alkaline earth metals, concentrations of nitrites and nitrosothiols in the gastric mucosa of rats under conditions of chronic fluoride intoxication. The study was conducted on 18 adult male Wistar rats weighing 220-260 g. Experimental animals were randomly divided into 3 groups of 6 animals each: control, chronic fluoride intoxication group and AP-1 transcription factor blockade group. Chronic fluoride intoxication was simulated by the administration of sodium fluoride at a dose of 10 mg / kg for 30 days. AP-1 blockade was performed by administering SR11302 at a rate of 15 mg / kg twice a week. In the gastric mucosa, the following was studied: the activities of constitutive and inducible isoforms of NO synthase, the concentration of nitrites, peroxynitrites and nitrosothiols. Chronic fluoride intoxication reduces the activity of constitutive NO synthases by 37.73% and increases the activity of inducible NO synthase by 1.61 times. The concentration of peroxynitrites increases by 2.68 times, nitrites – by 1.74 times, and nitrosothiols – by 1.88 times. Blockade of AP-1 reduces the activity of inducible isoform by 2.11, does not affect the activity of constitutive isoforms, and reduces the concentration of peroxynitrites by 1.98 times, nitrites – by 2.10 times, and nitrosothiols – by 2.37 times. Activation of the transcription factor AP-1 under conditions of chronic excessive fluoride intake leads to increased production of nitric oxide in the gastric mucosa of rats, enhances its oxidation to nitrites, promotes the formation of nitrosyl groups in the reaction with low molecular weight donors of thiol groups and increases the peroxidation of nitric oxide with the formation of peroxynitrite.