NOS Protein Expression Research Articles

Versican accumulation drives Nos2 induction and aortic disease in Marfan syndrome via Akt activation

Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening condition associated with Marfan syndrome (MFS), a disease caused by fibrillin-1 gene mutations. While various conditions causing TAAD exhibit aortic accumulation of the proteoglycans versican (Vcan) and aggrecan (Acan), it is unclear whether these ECM proteins are involved in aortic disease. Here, we find that Vcan, but not Acan, accumulated in Fbn1C1041G/+ aortas, a mouse model of MFS. Vcan haploinsufficiency protected MFS mice against aortic dilation, and its silencing reverted aortic disease by reducing Nos2 protein expression. Our results suggest that Acan is not an essential contributor to MFS aortopathy. We further demonstrate that Vcan triggers Akt activation and that pharmacological Akt pathway inhibition rapidly regresses aortic dilation and Nos2 expression in MFS mice. Analysis of aortic tissue from MFS human patients revealed accumulation of VCAN and elevated pAKT-S473 staining. Together, these findings reveal that Vcan plays a causative role in MFS aortic disease in vivo by inducing Nos2 via Akt activation and identify Akt signaling pathway components as candidate therapeutic targets.

Silver bionanoparticles toxicity in trophoblast is mediated by nitric oxide and glutathione pathways

Silver bionanoparticles (AgNPs) biosynthesized by Pseudomonas aeruginosa culture supernatant have an important antibacterial activity mediated by ROS increase; however their toxicity in human cells is not known. Due to the high susceptibility of the developing tissues to xenobiotics, the aim of this study was to investigate the AgNPs effect on first trimester human trophoblasts. The HTR8/SVneo cell line was treated with AgNPs (0.3–1.5 pM), for 6 and 24 h. Cell viability, reactive nitrogen and oxygen species (RNS and ROS) production, nitric oxide synthase expression, antioxidant defenses and biomolecule damage were evaluated. The exposure to AgNPs produced changes in HTR8/SVneo cell morphology and decreased cell viability. Alterations in redox balance were observed, with an increase in ROS and RNS levels, and NOS2 protein expression. Superoxide dismutase and catalase augmented their activity accompanied with a decreased in glutathione content and glutathione S-transferase activity. Protein oxidation and genotoxic damage were observed at concentrations greater than 0.6 pM. The pre-incubation with l-NMMA, NAC, mannitol and peroxidase demonstrated that AgNPs-induced cytotoxicity was not mediated by HO and H2O2, but nitric oxide and glutathione pathways were implicated in cell death. Since reported AgNPs microbicidal mechanism is mediated by increasing ROS (mainly HO and H2O2) without an increase in RNS, this work indicates an interesting difference in the reactive species and oxidative pathways involved in AgNPs toxicity in eukaryotic and prokaryotic cells. Highlighting the importance of toxicity evaluation to determine the safety of AgNPs with pharmaceutical potential uses.

Phytochemicals induce apoptosis by modulation of nitric oxide signaling pathway in cervical cancer cells.

Nitric Oxide (NO) is produced by NO synthases (NOS) and is a key signaling molecule that regulates tumorigenesis, both aiding and alleviating it. Elevated NO levels are cytotoxic to cancer cells, making NOS an important target for cancer treatment. In the present study, the modulatory effects of the phytochemicals, quercetin, sulforaphane, genistein, and epigallocatechin-3-gallate on NO pathway and apoptosis were shown in HeLa cervical cancer cells. Fluorescent microscopy and flow cytometry were used to assess apoptosis. A Griess assay was used to quantitatively measure NO, quantitative PCR array was used to assess the expression levels of genes involved in the NO signaling pathway, and immunocytochemistry was used to determine NOS protein expression. The functional association among the modulated genes was evaluated using network biology analysis, gene set enrichment, and KEGG pathway analysis. Treatment with the phytochemicals elevated NO levels in HeLa cells and modulated various genes involved in nitric oxide biosynthesis, superoxide metabolism, and oxidative stress, including NOS1, NOS2, NOS3, ALOX12, and SOD2, with a concomitant increase in NOS2 and NOS3 protein expression levels; also, the phytochemicals were found to induce apoptosis. These results suggest that the phytochemical-induced cell death is partially attributed to the activation of the NO pathway and upregulation of pro-oxidant ROS generators. Further experimental studies are required to explore this mechanistic association of NO signaling pathway activation and induction of apoptosis in other types of cancer.

Effect of β3-adrenergic receptor modulation on endothelial function

Understanding endothelial dysfunction is at the heart of current concerns due to its involvement in many cardiovascular diseases such as heart failure (HF). The β3-adrenergic receptor (β3-AR) could be at the origin of this endothelial dysfunction. Indeed, it is expressed at the level of the endothelial cell and induces vasorelaxation by the release of nitric oxide (NO), a central actor in endothelial dysfunction. Our objective was therefore to study the overexpression of the receptor in endothelial cells to understand its involvement in endothelial dysfunction. We used the Tgβ3 rat at 45 weeks of age, which overexpresses the human β3-AR gene in the endothelium. Endothelium-dependent aortic relaxation versus isoproterenol was measured with or without L-VNIO an inhibitor of nNOS at 10 μM. The protein expression of NOS was evaluated by Western-Blot and the production of NO and O2- was evaluated by EPR on aorta. Vascular reactivity was significantly reduced in response to isoproterenol in Tgβ3 aorta (−10% P < 0.05 vs. WT) indicating an alteration of endothelial function without alteration in systemic arterial pressure. With L-VNIO vasorelaxation was significantly reduce in Tgβ3 rat indicating a major implication of nNOS in vascular reactivity. nNOS expression was increased in aorta (+ 56% P < 0.05 vs. WT). Vascular production of NO was increased in Tgβ3 rats (+ 53% P < 0.05 vs. WT), possibly link to nNOS increase. This production was associated with an elevation of O2- and ONOO- in Tgβ3 rats (+ 340%, respectively P < 0.05 vs. WT). The increase in β3-AR expression induces endothelial dysfunction with a key role of eNOS and nNOS. Further studies will be conducted to understand the causal links between endothelial dysfunction and the alteration of the cardiac function.

Hydrogen sulfide attenuates diabetic neuropathic pain through NO/cGMP/PKG pathway and μ-opioid receptor.

There are currently approximately 425 million diabetic patients worldwide, of which approximately 90% of patients with diabetes suffer from neuropathy. Diabetic neuropathic pain (DNP) is a common complication of diabetic neuropathy. Nearly half of the patients hospitalized with diabetes have pain symptoms or symptoms related to neurological injury, and the incidence increases with age and diabetic duration. Anti-DNP analgesics have either limited therapeutic effects or serious side effects or lack of clinical trials, which has limited their application. Physiopathological mechanisms and treatment of DNP remain a significant challenge. The present confirmed that inhalation of H2S may attenuate the diabetic neuropathic pain through NO/cGMP/PKG pathway and μ-opioid receptor. It provides us the animal study foundation for the application of H2S on the treatment of DNP and clarifies some target molecules in the pain modulation of DNP.

Downregulation of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) in a co-culture system with human stimulated X-linked CGD neutrophils.

Phagocytes in patients with chronic granulomatous disease (CGD) do not generate reactive oxidative species (ROS), whereas nitric oxide (NO) production is increased in response to the calcium ionophore A23187 in CGD phagocytes compared with healthy phagocytes. Recently, patients with X-linked CGD (X-CGD) have been reported to show higher flow-mediated dilation, suggesting that endothelial cell function is affected by NO production from phagocytes. We studied NOS3 and EDN1 mRNA and protein expression in human umbilical vein endothelial cells (HUVECs) in a co-culture system with neutrophils from X-CGD patients. HUVECs were co-cultured for 30 minutes with human neutrophils from X-CGD or healthy participants in response to A23187 without cell-to-cell contact. The expression of NOS3 and EDN1 mRNA in HUVECs was quantified by real-time polymerase chain reaction. Moreover, we demonstrated the protein expression of eNOS, ET-1, and NFκB p65, including phosphorylation at Ser1177 of eNOS and Ser536 of NFκB p65. Neutrophils from X-CGD patients showed significantly higher NO and lower H2O2 production in response to A23187 than healthy neutrophils in vitro. Compared with healthy neutrophils, X-CGD neutrophils under A23187 stimulation exhibited significantly increased NO and decreased H2O2, and promoted downregulated NOS3 and EDN1 expression in HUVECs. The total expression and phosphorylation at Ser1177 of eNOS and ET-1 expression were significantly decreased in HUVECs co-cultures with stimulated X-CGD neutrophils. Also, phosphorylation at Ser536 of NFκB p65 were significantly decreased. In conclusions, eNOS and ET-1 significantly down-regulated in co-culture with stimulated X-CGD neutrophils through their excessive NO and the lack of ROS production. These findings suggest that ROS generated from neutrophils may mediate arterial tone affecting eNOS and ET-1 expression via their NO and ROS production.

Drosophila MARF1 ensures proper oocyte maturation by regulating nanos expression.

Meiosis and oocyte maturation are tightly regulated processes. The meiosis arrest female 1 (MARF1) gene is essential for meiotic progression in animals; however, its detailed function remains unclear. In this study, we examined the molecular mechanism of dMarf1, a Drosophila homolog of MARF1 encoding an OST and RNA Recognition Motif (RRM) -containing protein for meiotic progression and oocyte maturation. Although oogenesis progressed in females carrying a dMarf1 loss-of-function allele, the dMarf1 mutant oocytes were found to contain arrested meiotic spindles or disrupted microtubule structures, indicating that the transition from meiosis I to II was compromised in these oocytes. The expression of the full-length dMarf1 transgene, but none of the variants lacking the OST and RRM motifs or the 47 conserved C-terminal residues among insect groups, rescued the meiotic defect in dMarf1 mutant oocytes. Our results indicate that these conserved residues are important for dMarf1 function. Immunoprecipitation of Myc-dMarf1 revealed that several mRNAs are bound to dMarf1. Of those, the protein expression of nanos (nos), but not its mRNA, was affected in the absence of dMarf1. In the control, the expression of Nos protein became downregulated during the late stages of oogenesis, while it remained high in dMarf1 mutant oocytes. We propose that dMarf1 translationally represses nos by binding to its mRNA. Furthermore, the downregulation of Nos induces cycB expression, which in turn activates the CycB/Cdk1 complex at the onset of oocyte maturation.

Oroxylin A Reduces Vasoconstriction in Rat Aortic Rings through Promoting NO Production and NOS Protein Expression via Estrogen Receptor Signal Pathway.

Oroxylin A, a flavonoid, is naturally produced in many medicinal plants. Our previous study identified it as a phytoestrogen. Based on this, the present study investigated its vasoconstriction reducing effects and whether the action was mediated by the estrogen receptor (ER) signal pathway. Long-term in vitro treatment with oroxylin A reduced Ach-induced vasorelaxation and NE-mediated or KCl-mediated contractile responses in rat aortic rings. These effects were interfered by an ER inhibitor ICI 182,780. Rat cardiac microvascular endothelial cells (CMECs) and aortic vascular smooth muscle cells (VSMCs) were used to study the possible underlying mechanisms. Oroxylin A activated the ER signal pathway. In CMECs, it increased NO production and eNOS protein expression. In VSMCs, it promoted NO production and iNOS protein expression. These effects were also inhibited by ICI 182,780. Besides, oroxylin A stimulated ERα and ERβ protein expression in CMECs and VSMCs. All these findings suggest that the ER signal pathway takes part in the vasoconstriction reducing effects of oroxylin A.

The glucagon-like peptide 1 receptor agonist liraglutide attenuates placental ischemia-induced hypertension.

Preeclampsia is a hypertensive disorder of pregnancy characterized by systemic perturbations of nitric oxide function, reflective of generalized endothelial dysfunction. Therapies that target the nitric oxide pathway have shown promise in both clinical and preclinical studies of preeclampsia. The glucagon-like peptide 1 agonists have been shown to increase nitric oxide and lower blood pressure in patients with diabetes, in part, through activation of nitric oxide synthase (NOS). Therefore, we hypothesized that a direct acting glucagon-like peptide 1 receptor agonist would improve stigmata of the preeclampsia syndrome. Using the reduced uterine perfusion pressure rat model, we found that treatment with liraglutide significantly lowered blood pressure, improved renal function, and upregulated NOS3 protein expression in the mesenteric arterial bed. However, there were adverse effects on pup growth that were likely related to diminished food intake in the dams. Collectively, these data support the premise that the use of drugs that improve NOS abundance, including the glucagon-like peptide 1 agonists, is a rational therapeutic approach to the treatment of preeclampsia, but suggest cautious and careful study of their safety before potential clinical use in humans.NEW & NOTEWORTHY Drugs that target the glucagon-like peptide-1 pathway such as liraglutide are already used clinically, and it has been shown to promote endothelial nitric oxide synthase (NOS3) expression. We demonstrate that liraglutide, a glucagon-like peptide 1 receptor (GLP-1R) agonist, lowers blood pressure, improves renal function, and upregulates NOS3 in a rat model of placental ischemia. These data suggest that drugs that target the nitric oxide system, including GLP-1R agonists, are a potential therapeutic option for preeclampsia.

Transcriptional Regulation of Nos2 via STAT5B Binding to Nos2 Gene Promoter Mediates Nitric Oxide Production: Relevance in β-Cell Maintenance.

Type 1 Diabetes (T1D) involves autoimmune attack due to reduced regulatory T cells as an effect of mutant Stat5b(C1462A) in non-obese diabetic (NOD) mice, a T1D model resulting in pancreatic β-cell destruction. Although reactive oxygen species are considered to orchestrate the immune attack, the role of nitric oxide (·NO) still remains debatable. Since JAK-STAT pathway is known to induce Nos2, we investigated the role of STAT5B in nitric oxide generation and oxidative stress. In this study, we have used chromatin immunoprecipitation with STAT5B antibody to explore whether STAT5B binds Nos2 promoter. Using Stat5b gene silencing and overexpression models in MIN6 mouse pancreatic β-cell line we have assayed nitric oxide and its end products, superoxide levels, H₂O₂ levels, and expression of genes related to redox pathway by immunocytochemistry, biochemical assays, quantitative real time PCR and western blotting. Our results prove that STAT5B binds to the candidate gamma-interferon-activated (GAS) element in Nos2 promoter thereby inducing Nos2 mRNA transcription resulting in NOS2 protein expression in MIN6, a mouse pancreatic β-cell line. Our findings are substantiated by reduced ·NO as well as nitric oxide end products (nitrate and nitrite), and increased superoxide production in Stat5b silenced MIN6 cells. Our results indicate that C1462A mutant STAT5B shows lack of ·NO generation ability. To detoxify excess superoxide as a consequence of lowered Nos2, an overexpressed SOD2 in Stat5b silenced cells results in increased H₂O₂ production. H₂O₂ metabolizing enzymes do not show upregulation upon Stat5b silencing, and thus oxidative stress is brought about by amassed H₂O₂. Stat5b silencing finally reduces AKT expression, a prosurvival signal. Our study enables us to conclude that β-cell stress is aggravated by the incapability of STAT5B to induce Nos2 resulting in H₂O₂ accumulation and the ensuing oxidative stress enhances β-cell damage.

Abstract P278: Flow-Induced NO Deficiency in Thick Ascending Limbs of Dahl Salt-Sensitive Rats Fed a High Salt Diet is Due to Decreased NOS3 Expression

The effects of medullary NO on Na excretion are reduced in Dahl salt-sensitive (SS) compared to salt-resistant rats (SR). We have shown that flow-induced NO production by thick ascending limbs (THALs) of SS is reduced when rats are on a normal salt diet although NO synthase 3 protein expression was the same. In Sprague-Dawley rats a high-salt diet markedly increase NOS3 expression. It is unknown whether this is also the case for both SS and SR fed a high-salt diet. We hypothesized that flow-induced NO production by SS THALs is blunted in part due to a failure to increase NOS3 expression in response to dietary salt. SS and SR were fed normal and high-salt diets for 7-9 days. We measured systolic blood pressure (SBP); flow-induced NO in isolated THALs; and NOS3 protein expression in renal medullary lysates. SBP was 164.5±3.2 mm Hg ( n = 9) in SS and 130.1±4.8 in SR when fed a high-salt diet ( n = 7 ; p &lt; 0.0001 ) . Flow-induced NO production by SS THALs from rats fed high salt was 9 ± 2 arbitrary units (AU)/min ( n = 6) while it was 44 ± 10 AU/min by SR tubules ( n = 6), significantly greater ( p &lt; 0.005). NOS3 expression was 0.74 ± 0.08 AU ( n = 5) in THALs from SS on high salt while it was 1.26 ± 0.08 AU in tubules from SR ( n = 5), significantly greater ( p &lt; 0.003). THAL NOS3 expression was unchanged by a high-salt diet in SS (1.01 ± 0.08 vs 1.11 ± 0.11; n = 5) but increased in SR. We conclude that decreased flow-induced NO production in high-salt-fed SS is at least partially due to failure to increase NOS3 expression.

Sildenafil improves vascular endothelial function in patients with cystic fibrosis.

Cystic fibrosis (CF), characterized by defective CFTR function, is associated with multiple systemic complications, including vascular dysfunction. Sildenafil, a phosphodiesterase type 5 inhibitor, not only enhances nitric oxide (NO) metabolism but has been shown to improve CFTR functionality as well. Thus, sildenafil has been proposed as a therapy to improve vascular health in CF; however, its potential therapeutic role has yet to be determined. We sought to investigate the effect of sildenafil on endothelial function in patients with CF. Patients with CF completed a randomized, double-blind, placebo-controlled, crossover study with an acute dose of sildenafil (50 mg) or placebo followed by a 4-wk open-label extension with sildenafil (20 mg/day). Flow-mediated dilation (FMD) was used to evaluate endothelial function before and after treatments. In addition, phosphorylated endothelial NO synthase (pNOS3) and total NOS3 protein expression was determined from endothelial cells that were exposed to plasma from the patients before and after 4 wk of sildenafil treatment. No changes ( P ≥ 0.110) in endothelial function were observed after the acute dose of sildenafil. However, FMD significantly ( P = 0.029) increased after 4 wk of treatment (∆FMD: 1.5 ± 2.2%). Moreover, pNOS3 protein expression significantly ( P = 0.013) increased after 4 wk of treatment (∆pNOS3: 0.31 ± 0.39 arbitrary units) and was associated ( r = 0.593, P = 0.033) with the change in FMD. These data suggest that 4 wk of sildenafil treatment can improve vascular endothelial function in patients with CF, likely through an increase in NOS3 phosphorylation. NEW & NOTEWORTHY Findings from the present study demonstrate, for the first time, significant improvement of endothelial function in patients with cystic fibrosis treated with sildenafil that is associated with greater phosphorylation of endothelial nitric oxide synthase. These results support the use of sildenafil as a potential novel therapy for this patient population.

Resveratrol treatment may preserve the erectile function after radiotherapy by restoring antioxidant defence mechanisms, SIRT1 and NOS protein expressions.

Radiotherapy (RT) for prostate cancer (PC) can cause erectile dysfunction (ED) by damaging neurovascular structures with oxidative stress. In this study, we evaluated the effects of resveratrol, an antioxidant, on post-RT ED. Fifty rats in five groups were evaluated; control (C), prostate-confined radiotherapy with short- and long-term vehicle or resveratrol treatment. Cavernosal tissues were obtained to analyze glutathione (GSH), nitric oxide (NO), cyclic guanosine monophosphate (cGMP), 8-hydroxy-2'-deoxy-guanosine (8-OHdG) levels and superoxide dismutase (SOD), caspase-3 activities, sirtuin-1, Foxo-3, nNOS, and eNOS protein expressions. Intracavernosal pressures (ICP) were measured for the long-term treatment group. In the RT + long-term vehicle treatment group, tissue GSH, NO, cGMP, and SOD activity were decreased while 8-OHdg levels and caspase-3 activities were increased. Radiotherapy caused a decrease in sirtuin-1, nNOS, and eNOS protein expressions. These parameters were reversed by resveratrol treatment. Foxo-3 protein expressions were unaltered in the RT + short-term vehicle treatment group and started to increase as a defense mechanism in the RT + long-term vehicle group; however, resveratrol treatment caused a significant increase in Foxo-3 expressions. Resveratrol preserved the metabolic pathways involved in erectile function and provided functional protection. Resveratrol can be used as a supplementary agent in patients undergoing radiotherapy to preserve erectile function.

Oleocanthal Inhibits Catabolic and Inflammatory Mediators in LPS-Activated Human Primary Osteoarthritis (OA) Chondrocytes Through MAPKs/NF-κB Pathways

Background/Aims: Oleocanthal (OC), a phenolic compound present in extra virgin olive oil (EVOO), has attracted attention since its discovery for its relevant pharmacological properties in different pathogenic processes, including inflammation. Here, we investigated the involvement of OC in LPS-activated osteoarthritis (OA) human primary chondrocytes. Methods: Human primary chondrocytes were harvested from articular cartilage samples obtained from OA patients. The effects of OC on the viability of chondrocytes were tested by MTT assay. Protein and mRNA expression of several catabolic and pro-inflammatory factors after OC treatment were measured by RT-qPCR and western blot respectively. Moreover, we analysed the NO production by Griess reaction. Finally, several pathways mediators were analysed by western blot. Results: We demonstrated that OC did not have any cytotoxic effect. Oleocanthal inhibited NO production and strongly decreased NOS2 and COX-2 protein and mRNA expression in LPS-activated human primary OA chondrocytes. Interestingly, OC also inhibits MMP-13 and ADAMTS-5. In addition, OC downregulates several pro-inflammatory factors, such as IL-6, IL-8, CCL3, LCN2 and TNF-α induced by LPS in human primary OA chondrocytes. Finally, we demonstrated that OC exerts its effects through the MAPK/P38/NF-kB pathways. Conclusion: These data show that OC is able to block LPS-mediated inflammatory response and MMP-13 and ADAMTS-5 induction in human primary OA chondrocytes via MAPKs/NF-kB pathways, suggesting that OC may be a promising agent for the treatment of inflammation in cartilage and a potential molecule to prevent disease progression by inhibiting metalloproteases and aggrecanases.

Effect of nitric oxide synthase inhibitor Nω-Nitro-L-arginine on the penile erectile function of healthy male rats

Ojective To investigate the direct effect of exogenous NOS inhibitor Nω-Nitro-L-arginine (L-NNA) on the erectile function of healthy male rats in order to clarify the mechanism of the erectile dysfunction of type 2 diabetic rats. Methods L-NNA (50 mg/kg) was administered by oral gavage to Sprague Dawley (SD) rats for 16 weeks to induce rat model of NOS inhibition. By the end of the experiment, corpora cavernosa was isolated from the rats under anesthetization and fixed in an organ chamber for the examination of relaxation function response to acetylcholine (ACh) to reflect erectile function. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the content of cyclic guanosine monophosphate (cGMP) in cavernosal tissue. NOS activity and nitric oxide (NO) content were measured by colorimetric method. Western blotting was employed to detect the protein expression of NOS and phosphodiesterase 5 (PDE5). The activity of the antioxidative enzyme superoxide dismutase (SOD) and content of the lipid peroxidative product malondialdehyde (MDA) were analyzed to reflect oxidative stress. Results In comparison with control group, the relaxation response to acetylcholine of corpus cavernosum was significan-tly impaired in L-NNA model rats, indicating penile erectile dysfunction. Either decreased contents of NO and cGMP or suppressed activity of NOS were observed in the corpus cavernosum of L-NNA model rats and in accompany with the down-regulation of endothelial NOS (eNOS) and neuronal NOS (nNOS) expression, up-regulation of inducible NOS (iNOS) and PDE5 expression as well as an increase of oxidative stress. Incubation of corpus cavernosum from control rats with L-NNA (1-10 μmol/L) ex vivo for 30 min could also inhibit the relaxation function responses to acetylcholine in a dose-dependent manner. Treatment with L-arginine ex vivo for 45 min could improve the impairments of relaxation function responses to acetylcholine of corpus cavernosum induced by L-NNA in vivo and ex vivo. Conclusions Exogenous NOS inhibitor L-NNA could induce penile erectile dysfunction in healthy male rats and the mechanism may be related to reducing NO content and increasing oxidative stress. Key words: N′-Nitro-L-arginine; Corpus cavernosum; Erectile function; Nitric oxide; Oxidative stress

Sex-specific computational models of the spontaneously hypertensive rat kidneys: factors affecting nitric oxide bioavailability

The goals of this study were to 1) develop a computational model of solute transport and oxygenation in the kidney of the female spontaneously hypertensive rat (SHR), and 2) apply that model to investigate sex differences in nitric oxide (NO) levels in SHR and their effects on medullary oxygenation and oxidative stress. To accomplish these goals, we first measured NO synthase (NOS) 1 and NOS3 protein expression levels in total renal microvessels of male and female SHR. We found that the expression of both NOS1 and NOS3 is higher in the renal vasculature of females compared with males. To predict the implications of that finding on medullary oxygenation and oxidative stress levels, we developed a detailed computational model of the female SHR kidney. The model was based on a published male kidney model and represents solute transport and the biochemical reactions among O2, NO, and superoxide ([Formula: see text]) in the renal medulla. Model simulations conducted using both male and female SHR kidney models predicted significant radial gradients in interstitial fluid oxygen tension (Po2) and NO and [Formula: see text] concentration in the outer medulla and upper inner medulla. The models also predicted that increases in endothelial NO-generating capacity, even when limited to specific vascular segments, may substantially raise medullary NO and Po2 levels. Other potential sex differences in SHR, including [Formula: see text] production rate, are predicted to significantly impact oxidative stress levels, but effects on NO concentration and Po2 are limited.

Effect of Donepezil Hydrochloride on Cognitive Function Recovery of Rats with Alzheimer’s Disease

Objective: To explore the effect of donepezil hydrochloride on cognitive function recovery of rat with Alzheimer’s disease and the positive expression of nitric oxide synthase and acetylcholinesterase (NOS and AchE) in the brain tissues of rats with Alzheimer's disease (AD).Methods: Forty SD (Sprague Dawley) rats were randomly classified into a model group, a treatment group, a control group, and a sham operation group. Each group had 10 rats. AD rat models were prepared with Aβ1-40. Donepezil hydrochloride was orally administered to rats in the treatment group two weeks after modeling. Rat recognition function was assessed. The positive expression of NOS and AchE in rat brain tissues was observed via immunohistochemistry after administration of 28 days.Results: Rat escape latency was significantly longer after modeling (p < 0.010). Compared to the model group, treatment group escape latency was significantly shortened and the difference was significant (p < 0.05). NOS and AchE positive expression mainly reflects in hippocampus region tissue. Compared with the control, and sham, operation groups, NOS positive expression decreased and AchE positive expression increased in the model group. The difference was statistically significant (p < 0.01). Compared with the model group, NOS expression in the treatment group increased and AchE expression decreased. The difference was significant (p < 0.01).Conclusion: Donepezil hydrochloride can promote AD rat cognitive function recovery. Its effects are also associated with increased NOS protein expression in hippocampus neuron cells in addition to specifically inhibiting the AchE activities.

Abstract 647: Histone Deacetylase 1 Reduces No Production In Endothelial Cells Via Lysine Deacetylation Of No Synthase 3

The lysine acetylation state of non-histone proteins may be regulated through histone deacetylases (HDACs). Evidence suggests that NO synthase 3 (NOS3; eNOS) is post-translationally lysine acetylated leading to increased NO production in the endothelium. The purpose of this study was to test the hypothesis that NOS3 is lysine-acetylated and that upregulated HDAC1-mediated deacetylation leads to reduced NO production in endothelial cells. We determined that NOS3 is basally lysine acetylated in cultured bovine aortic endothelial cells (BAECs). In BAECs, HDAC1 is expressed in the nucleus and cytosol, and forms a novel protein-protein interaction with NOS3. Overexpression of HDAC1 in BAECs resulted in a significant reduction in NOS3 lysine acetylation (control = 1.0 ± 0.1, HDAC1 = 0.59 ± 0.08 A.U., P &lt; 0.01), while HDAC1 knockdown with siRNA resulted in no significant change in NOS3 acetylation level. Moreover, overexpression or knockdown of HDAC1 resulted in no significant effect on NOS3 protein expression or on expression levels of NOS3 phosphorylation sites T497, S635, S1179. HDAC1 overexpression in BAECs significantly blunted basal nitrite production (an index of nitric oxide production; control 287.7 ± 29.1 pmol/mg p/h, HDAC1 172.4 ± 31.7 pmol/mg p/h, P &lt; 0.05) as well as attenuating endothelin-1 stimulated nitrite production (control 481.8 ± 50.3 pmol/mg/h, HDAC1 243.1 ± 48.2 pmol/mg/h, P&lt; 0.05). On the contrary, HDAC1 knockdown increased basal nitrite production (730.6 ± 99.1 pmol/mg/h), and further exaggerated increases in endothelin-1 stimulated nitrite production (1276.9 ± 288.2 pmol/mg/h). Thus, these data indicate that up-regulated HDAC1 decreases NOS3 activity, most likely, through direct lysine deacetylation of NOS3. We propose that HDAC1-mediated deacetylation of NOS3 may represent a novel target for endothelial dysfunction.

Sex-specific alterations in NOS regulation of vascular function in aorta and mesenteric arteries from spontaneously hypertensive rats compared to Wistar Kyoto rats.

The present study tested the hypothesis that spontaneously hypertensive rats (SHR) have impaired nitric oxide synthase (NOS)‐mediated regulation of vascular function versus Wistar‐Kyoto rats (WKY). Aorta and small mesenteric arteries were studied from male and female SHR (M SHR and F SHR) and WKY (M WKY and F WKY). Phenylephrine (PE)‐induced vasoconstriction was greater in aorta of M SHR versus all others (P < 0.05); there were neither sex nor strain differences in PE contraction in mesenteric arteries. The NOS inhibitor l‐Nitro‐Arginine Methyl Ester (l‐NAME) increased PE‐induced vasoconstriction in all rats, although the increase was the least in male SHR (P < 0.05), revealing a blunted vasoconstrictor buffering capacity of NOS. l‐NAME increased sensitivity to PE‐induced constriction only in mesenteric arteries of SHR, although, the maximal percent increase in contraction was comparable among groups. ACh‐induced relaxation was also less in aorta from M SHR versus all others (P < 0.05). ACh relaxation was comparable among groups in mesenteric arteries, although SHR exhibited a greater NOS component to ACh‐induced relaxation than WKY. To gain mechanistic insight into sex and strain differences in vascular function, NOS activity and NOS3 protein expression were measured. Aortic NOS activity was comparable between groups and M SHR had greater NOS3 expression than M WKY. In contrast, although vascular function was largely maintained in mesenteric arteries of SHR, NOS activity was less in SHR versus WKY. In conclusion, M SHR exhibit a decrease in NOS regulation of vascular function compared to F SHR and WKY, although this is not mediated by decreases in NOS activity and/or expression.

Female sex hormones protect against salt-sensitive hypertension but not essential hypertension.

Initial studies found that female Dahl salt-sensitive (DS) rats exhibit greater blood pressure (BP) salt sensitivity than female spontaneously hypertensive rats (SHR). On the basis of the central role played by NO in sodium excretion and BP control, we further tested the hypothesis that blunted increases in BP in female SHR will be accompanied by greater increases in renal inner medullary nitric oxide synthase (NOS) activity and expression in response to a high-salt (HS) diet compared with DS rats. Gonad-intact and ovariectomized (OVX) female SHR and DS rats were placed on normal salt (NS; 0.4% salt) or HS (4% salt) diet for 2 wk. OVX did not alter BP in SHR, and HS diet produced a modest increase in BP. OVX significantly increased BP in DS rats on NS; HS further increased BP in all DS rats, although OVX had a greater increase in BP. Renal inner medullary NOS activity, total NOS3 protein, and NOS3 phosphorylated on serine residue 1177 were not altered by salt or OVX in either strain. NOS1 protein expression, however, significantly increased with HS only in SHR, and this corresponded to an increase in urinary nitrate/nitrite excretion. SHR also exhibit greater NOS1 and NOS3 protein expression than DS rats. These data indicate that female sex hormones offer protection against HS-mediated elevations in BP in DS rats but not SHR. We propose that the relative resistance to HS-mediated increases in BP in SHR is related to greater NOS expression and the ability to increase NOS1 protein expression compared with DS rats.