Alterations In Nitric Oxide Synthase Research Articles

Nitric oxide modulates tapentadol antinociceptive tolerance and physical dependence

Tapentadol, an analgesic with a dual mechanism of action, involving both μ-opioid receptor agonism and noradrenaline reuptake inhibition (MOP-NRI), was designed for the treatment of moderate to severe pain. However, the widely acknowledged risk of analgesic tolerance and development of physical dependence following sustained opioid use may hinder their effectiveness. One of the possible mechanisms behind these phenomena are alterations in nitric oxide synthase (NOS) system activity. The aim of the study was to investigate the tolerance and dependence potential of tapentadol in rodent models and to evaluate the possible role of nitric oxide (NO) in these processes. Our study showed that chronic tapentadol treatment resulted in tolerance to its antinociceptive effects to an extent similar to tramadol, but much less than morphine. A single injection of a non-selective NOS inhibitor, NG-nitro-L-arginine (L-NOArg), reversed the tapentadol tolerance. In dependence studies, repeated administration of L-NOArg attenuated naloxone-precipitated withdrawal in tapentadol-treated mice, whereas a single injection of L-NOArg was ineffective. Biochemical analysis revealed that tapentadol decreased nNOS protein levels in the dorsal root ganglia of rats following 31 days of treatment, while no significant changes were found in iNOS and eNOS protein expression. Moreover, pre-treatment with L-NOArg augmented tapentadol antinociception in an opioid- and α2-adrenoceptor-dependent manner. In conclusion, our data suggest that the NOS system plays an important role in the attenuation of tapentadol-induced tolerance and withdrawal. Thus, inhibition of NOS activity can serve as a promising treatment option for long-term tapentadol use by extending its effectiveness and improving the side-effects profile.

Effects of short-term heat acclimation on whole-body heat exchange and local nitric oxide synthase- and cyclooxygenase-dependent heat loss responses in exercising older men.

What is the central question of this study? Does short-term heat acclimation enhance whole-body evaporative heat loss and augment nitric oxide synthase (NOS)-dependent cutaneous vasodilatation and NOS- and cyclooxygenase (COX)-dependent sweating, in exercising older men? What is the main finding and its importance? Our preliminary data (n=8) demonstrated that short-term heat acclimation improved whole-body evaporative heat loss, but it did not influence the effects of NOS and/or COX inhibition on cutaneous vasodilatation or sweating in older men during an exercise-heat stress. These outcomes might imply that although short-term heat acclimation enhances heat dissipation in older men, it does not modulate NOS- and COX-dependent control of cutaneous vasodilatation or sweating on the forearm. Ageing is associated with decrements in whole-body heat loss (evaporative+dry heat exchange), which might stem from alterations in nitric oxide synthase (NOS)- and cyclooxygenase (COX)-dependent cutaneous vasodilatation and sweating. We evaluated whether short-term heat acclimation would (i) enhance whole-body heat loss primarily by increasing evaporative heat loss, and (ii) augment NOS-dependent cutaneous vasodilatation and NOS- and COX-dependent sweating, in exercising older men. Eight older men [mean (SD) age, 59(8)years] completed a calorimetry and microdialysis trial before and after 7days of exercise-heat acclimation. For the calorimetry trials, whole-body evaporative and dry heat exchange were assessed using direct calorimetry during 30min bouts of cycling at light, moderate and vigorous metabolic heat productions (150, 200 and 250W/m2 , respectively) in dry heat (40°C, 20% relative humidity). For the microdialysis trials, local cutaneous vascular conductance and sweat rate were assessed during 60min exercise in the heat (35°C, 20% relative humidity) at four dorsal forearm skin sites treated with lactated Ringer solution (control), NOS inhibitor, COX inhibitor or combined NOS and COX inhibitors, via microdialysis. Evaporative heat loss during moderate (P=0.036) and vigorous (P=0.021) exercise increased after acclimation. Inhibition of NOS alone reduced cutaneous vascular conductance to a similar extent before and after acclimation (P<0.040), whereas separate and combined NOS and COX inhibition had no significant effects on sweating relative to the control site (P=0.745). Our preliminary results might suggest that short-term heat acclimation improves evaporative heat loss, but does not significantly modulate the contributions of NOS or COX to cutaneous vasodilatation or sweating on the forearm in older men during an exercise-heat stress.

Structural and functional alterations of nitric oxide synthase 3 due to missense variants associate with high-altitude pulmonary edema through dynamic study

The human endothelial nitric oxide synthase (NOS3) is 28 Kbp at 7q36.1 and encodes protein comprising of 1280 amino acids. Being a major source of nitric oxide, the enzyme is crucial to the vascular homeostasis and thereby to be an important pharmaceutical target. We hence have been investigating this molecule in a high-altitude disorder namely, high-altitude pulmonary edema (HAPE). We performed a genome-wide association study (GWAS) in a case-control design of sojourners that included healthy controls and HAPE patients (n = 200) each. Four NOS3 missense SNPs i.e. rs1799983 (E298D), rs3918232 (V827M), rs3918201 (R885M) and rs3918234 (Q982L), were associated significantly with HAPE (P-value < 0.05). Furthermore, extensive in silico analyses were performed to predict the detrimental effect of the four variant types and their three most relevant co-factors namely, heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) that are accountable for amendment of protein stability leading to structural de-construction. Subsequently, we validated the findings in a larger sample size of the two study groups. HAPE patients had a higher frequency of the four variants and significantly decreased levels of circulating nitric oxide (NO) (P-value < 0.001). The in silico and human subjects findings complement each other. This study explored the impact of HAPE-associated NOS3 variants with its protein structure stability and holds promise to be current and future drug targets.Communicated by Ramaswamy H. Sarma

Prevention of Diabetic Nephropathy by Modified Acidic Fibroblast Growth Factor

Background/Aims: Oxidative stress (OS) contributes to all chronic diabetic complications, including diabetic nephropathy (DN). Acidic fibroblast growth factor (aFGF) has shown to confer protection from OS. However, it also has potent angiogenic activity. We hypothesized that a modified human aFGF (maFGF), with antioxidant properties but devoid of angiogenic activity, has preventative action in DN. Methods: Streptozotocin-induced diabetic mice were treated with maFGF (intraperitoneally) daily for 1 or 6 months and were compared with untreated diabetic and non-diabetic controls. Microalbuminuria was assessed to determine functional damage. Renal cortical tissues were examined for multiple extracellular matrix proteins, vasoactive factors and OS markers. For mechanistic studies, immortalized mouse podocytes and human microvascular endothelial cells were exposed to high (25 m<smlcap>M</smlcap>) or low glucose (5 m<smlcap>M</smlcap>). OS, vasoactive factors, fibrosis and apoptosis-related gene expression were tested by real-time qPCR and Enzyme-Linked Immunosorbent Assay. Nitric oxide (NO) analyses were also performed. Results: maFGF did not affect body weight and glycemia but prevented renal hypertrophy and functional changes in DN. It also prevented diabetes-induced DNA damage, nitrosative stress, vasoactive factors, angiotensinogen and endothelial NO synthase alterations. Although it failed to prevent transforming growth factor (TGF)-β1 mRNA upregulation, it prevented fibronectin production. Similar results were obtained in vitro. Decreased NO production in vivo and in vitro was also prevented by maFGF. Conclusions: maFGF treatment prevents DN. This prevention probably involves NO production.

Subacute Hemolysis in Sickle Cell Mice Causes Priapism Secondary to NO Imbalance and PDE5 Dysregulation.

Recent research suggests that priapism in sickle cell disease (SCD) is due to dysregulation of penile erection homeostasis including alteration of nitric oxide synthase (NOS) and phosphodiesterase type 5 (PDE5) activities by excessive levels of reactive oxygen species (ROS) released during hemolysis. It is unknown if subacute exposure to hemolysis is sufficient or if chronic reconditioning of erectile tissues is required for perturbation of homeostatic pathways and whether PDE5 inhibitor (PDE5I) treatment can restore erectile homeostasis in the subacute setting. The aim of this study was to investigate the effects of subacute hemolysis (3-month exposure) on priapism and NO pathway regulation. Mice underwent bone marrow transplantation with either SCD (BM-SS) or wild-type (WT) bone marrow. BM-SS mice were treated with sildenafil 100 mg/kg/day. We measured intracavernous pressure (ICP) measurements with or without cavernous nerve stimulation following bone marrow transplantation to assess for priapism. ICP and frequency of erections were assessed. Penile tissues were analyzed for NOS, protein kinase G (PKG), PDE5, and ROS activities. BM-SS mice demonstrated a priapism phenotype. PDE5I treatment reduced the frequency of erections in BM-SS mice (1.7 ± 1.1 vs. 5.5 ± 2.8 erections per hour, P < 0.05). Penile tissues from BM-SS mice demonstrated decreased NOS, PKG, PDE5 and elevated ROS activities compared with that of control mice. PDE5I treatment increased NOS (11.6 ± 1.3% vs. 7.8 ± 2.3%, P < 0.05) and PDE5 (76.3 ± 9.8% vs. 52.3 ± 11.1%, P < 0.05) activities and decreased ROS activity (137.8 ± 12.1% vs. 199.1 ± 11.3%, P < 0.05) compared with non-PDE5I treated BM-SS mice. PKG activity was increased beyond control levels with PDE5I treatment (158.4 ± 10.3%, P < 0.05). Short-term hemolysis is sufficient to establish a priapism phenotype and results in loss of erectile function. PDE5I treatment ameliorates priapism, in part, because of restored NO balance with decreased ROS generation and increased PDE5 activity.

Expression of Nitric Oxide Synthase Isoenzyme in Lung Tissue of Smokers with and without Chronic Obstructive Pulmonary Disease.

Background:It has been demonstrated that only 10%–20% cigarette smokers finally suffer chronic obstructive pulmonary disease (COPD). The underlying mechanism of development remains uncertain so far. Nitric oxide (NO) has been found to be closely associated with the pathogenesis of COPD, the alteration of NO synthase (NOS) expression need to be revealed. The study aimed to investigate the alterations of NOS isoforms expressions between smokers with and without COPD, which might be helpful for identifying the susceptibility of smokers developing into COPD.Methods:Peripheral lung tissues were obtained from 10 nonsmoker control subjects, 15 non-COPD smokers, and 15 smokers with COPD. Neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) mRNA and protein levels were measured in each sample by using real-time polymerase chain reaction and Western blotting.Results:INOS mRNA was significantly increased in patients with COPD compared with nonsmokers and smokers with normal lung function (P < 0.001, P = 0.001, respectively). iNOS protein was also higher in COPD patients than nonsmokers and smokers with normal lung function (P < 0.01 and P = 0.01, respectively). However, expressions of nNOS and eNOS did not differ among nonsmokers, smokers with and without COPD. Furthermore, there was a negative correlation between iNOS protein level and lung function parameters forced expiratory volume in 1 s (FEV1) (% predicted) (r = −0.549, P = 0.001) and FEV1/forced vital capacity (%, r = −0.535, P = 0.001).Conclusions:The expression of iNOS significantly increased in smokers with COPD compared with that in nonsmokers or smokers without COPD. The results suggest that iNOS might be involved in the pathogenesis of COPD, and may be a potential marker to identify the smokers who have more liability to suffer COPD.

Endothelin-1 Activation of the Endothelin B Receptor Modulates Pulmonary Endothelial CX3CL1 and Contributes to Pulmonary Angiogenesis in Experimental Hepatopulmonary Syndrome

Hepatic production and release of endothelin-1 (ET-1) binding to endothelin B (ETB) receptors, overexpressed in the lung microvasculature, is associated with accumulation of pro-angiogenic monocytes and vascular remodeling in experimental hepatopulmonary syndrome (HPS) after common bile duct ligation (CBDL). We have recently found that lung vascular monocyte adhesion and angiogenesis in HPS involve interaction of endothelial C-X3-C motif ligand 1 (CX3CL1) with monocyte CX3C chemokine receptor 1 (CX3CR1), although whether ET-1/ETB receptor activation influences these events is unknown. Our aim was to define if ET-1/ETB receptor activation modulates CX3CL1/CX3CR1 signaling and lung angiogenesis in experimental HPS. A selective ETB receptor antagonist, BQ788, was given for 2 weeks to 1-week CBDL rats. ET-1 (±BQ788) was given to cultured rat pulmonary microvascular endothelial cells overexpressing ETB receptors. BQ788 treatment significantly decreased lung angiogenesis, monocyte accumulation, and CX3CL1 levels after CBDL. ET-1 treatment significantly induced CX3CL1 production in lung microvascular endothelial cells, which was blocked by inhibitors of Ca(2+) and mitogen-activated protein kinase (MEK)/ERK pathways. ET-1-induced ERK activation was Ca(2+) independent. ET-1 administration also increased endothelial tube formation in vitro, which was inhibited by BQ788 or by blocking Ca(2+) and MEK/ERK activation. CX3CR1 neutralizing antibody partially inhibited ET-1 effects on tube formation. These findings identify a novel mechanistic interaction between the ET-1/ETB receptor axis and CX3CL1/CX3CR1 in mediating pulmonary angiogenesis and vascular monocyte accumulation in experimental HPS.

Pregnancy increases myometrial artery myogenic tone via NOS- or COX-independent mechanisms

Myogenic tone (MT) is a primary modulator of blood flow in the resistance vasculature of the brain, kidney, skeletal muscle, and perhaps in other high-flow organs such as the pregnant uterus. MT is known to be regulated by endothelium-derived factors, including products of the nitric oxide synthase (NOS) and/or the cyclooxygenase (COX) pathways. We asked whether pregnancy influenced MT in myometrial arteries (MA), and if so, whether such an effect could be attributed to alterations in NOS and/or COX. MA (200-300 μm internal diameter, 2-3 mm length) were isolated from 10 nonpregnant and 12 pregnant women undergoing elective hysterectomy or cesarean section, respectively. In the absence of NOS and/or COX inhibition, pregnancy was associated with increased MT in endothelium-intact MA compared with MA from nonpregnant women (P < 0.01). The increase in MT was not due to increased Ca(2+) entry via voltage-dependent channels since both groups of MA exhibited similar levels of constriction when exposed to 50 mM KCl. NOS inhibition (N(ω)-nitro-L-arginine methyl ester, L-NAME) or combined NOS/COX inhibition (L-NAME/indomethacin) increased MT in MA from pregnant women (P = 0.001 and P = 0.042, respectively) but was without effect in arteries from nonpregnant women. Indomethacin alone was without effect on MT in MA from either nonpregnant or pregnant women. We concluded that MT increases in MA during human pregnancy and that this effect was partially opposed by enhanced NOS activity.

Detrusor Overactivity in Hyperchoelsterolemia Rats.

Many theories attempt to explain the complex etiology of overactive bladder syndrome (OAB), but the exact mechanisms of the pathophysiology have yet to be fully understood. Recent findings have suggested that hypercholesterolemia is related with detrusor overactivity (DO), which, in turn, is usually associated with OAB. The present report examines published studies that have associated hypercholesterolemia with DO to determine the grounds on which such studies were based. According to our analysis, OAB and DO are closely related with hypercholesterolemia. Furthermore, DO and OAB may be affected not just by a single factor like hypercholesterolemia, but rather by all components of metabolic syndrome. Several mechanisms, including autonomic overactivity, artherosclerosis, ischemic change, alteration of nitric oxide synthase (NOS)/NO system and increased Rho-kinase activity may have a role in the relationship between OAB and hypercholesterolemia. Further studies are warranted, however, to evaluate more about the pathophysiology of OAB.

Nitric oxide synthase derangements and hypertension in kidney disease

Nitric oxide deficiency occurs by multiple mechanisms and contributes to the pathogenesis of progression of chronic kidney disease (CKD) and its cardiovascular complications. This article concentrates on recent developments on the regulation of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) in CKD and on the importance of the nitric oxide synthases in kidney disease progression, particularly in diabetic nephropathy. The increased plasma ADMA seen in renal disease is generally predictive of severity of CKD progression and cardiovascular risk. However, some assumptions about the control of ADMA have been challenged: the primacy of the kidney as a metabolic organ for plasma ADMA regulation has come under scrutiny and the relative importance of the two isoforms of the ADMA-metabolizing enzymes dimethylarginine dimethylaminohydrolases (DDAHs) is being re-evaluated. Alterations in NOS also contribute to CKD progression with the endothelial isoform playing a major role in diabetic nephropathy. Improving our understanding of ADMA regulation is important since pharmacologic targeting of DDAH is underway. The major role of endothelial NOS-derived nitric oxide in diabetic nephropathy should lead to novel therapies. The beneficial actions of dietary nitrate supplementation on blood pressure and kidney disease are of considerable clinical relevance.

Alterations in nitric oxide synthase in the aged CNS.

Aging is associated with neuronal loss, gross weight reduction of the brain, and glial proliferation in the cortex, all of which lead to functional changes in the brain. It is known that oxidative stress is a critical factor in the pathogenesis of aging; additionally, growing evidence suggests that excessive nitric oxide (NO) production contributes to the aging process. However, it is still unclear how NO plays a role in the aging process. This paper describes age-related changes in the activity of NADPH-diaphorase (NADPH-d), a marker for neurons containing nitric oxide synthase (NOS), in many CNS regions. Understanding these changes may provide a novel perspective in identifying the aging mechanism.

Impact of Nitric Oxide Synthase Glu298Asp Polymorphism on the Development of End-Stage Renal Disease in Type 2 Diabetic Egyptian Patients

Background: Nitric oxide is an important regulator of renal hemodynamics. This study aimed to investigate the role of endothelial nitric oxide synthase (eNOS) gene polymorphism in type 2 diabetic patients with end-stage renal disease (ESRD) and to elucidate any alteration of nitric oxide synthase (NOS) activity caused by this polymorphism. Methods: The study included 80 patients with type 2 diabetes of >10 years duration (40 with diabetes-derived ESRD, 40 without nephropathy) and 20 healthy controls. Plasma nitrate/nitrite level, and serum NOS activity were measured and eNOS Glu298Asp genotypes were determined. Results: The frequency of Glu/Glu (GG) genotype in diabetics with ESRD was lower than controls. However, the frequency of Asp/Asp (TT) genotype was increased in diabetics with ESRD as compared to those without nephropathy and controls. Diabetics with ESRD had significantly lower nitrate/nitrite level and NOS activity than those without nephropathy. Diabetic patients with TT genotype are at a significant risk for ESRD. Moreover, subjects carrying TT genotype had lower nitrate/nitrite level and NOS activity than those carrying GG genotype. In diabetics with ESRD, creatinine clearance was positively correlated with both nitrate/nitrite level and NOS activity. Conclusions: These results imply that TT genotype of eNOS may be associated with an increased risk of ESRD in Egyptian type 2 diabetics. It could represent a useful genetic marker to identify diabetics at high risk for the development of ESRD. However, larger future prospective studies are required to confirm the role of eNOS gene polymorphism in the progression of diabetic nephropathy to ESRD.

Effects of N-hexacosanol on nitric oxide synthase system in diabetic rat nephropathy

We attempted to clarify the effects of cyclohexenonic long-chain fatty alcohol (N-hexacosanol) on nitric oxide synthase (NOS) in streptozotocin-induced diabetic nephropathy. After induction of experimental diabetes with streptozotocin, rats were maintained for 8 weeks with or without treatment by N-hexacosanol (8 mg/kg i.p. every day). Urinary albumin excretion, blood chemistry, immunoblot analysis, and real-time polymerase chain reactions (real-time PCR) of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were investigated. Although N-hexacosanol had no effects on serum glucose or insulin level, it normalized serum creatinine and urinary albumin excretion. N-hexacosanol was found to improve the diabetes-induced alterations in the eNOS, iNOS, and nNOS protein and their mRNA levels. Histologically, N-hexacosanol inhibited the progression to glomerular sclerosis. Our data suggest that N-hexacosanol improves diabetes-induced NOS alterations in the kidney, resulting in the amelioration of diabetic nephropathy.

Nitric oxide synthase in critically ischaemic muscle and alterations in isoform expression during revascularization surgery

Dysfunction of the nitric oxide pathway is implicated in peripheral arterial disease. Nitric oxide synthase (NOS) isoforms and NOS activity were studied in muscle from patients with critical leg ischaemia (CLI). Alterations in NOS during revascularization surgery were also assessed. Muscle biopsies were taken from patients with CLI undergoing amputation and also from patients undergoing femorodistal bypass at the start of surgery, after arterial clamping and following reperfusion. The presence of NOS within muscle sections was confirmed using reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. NOS isoform distribution was studied by immunohistochemistry. NOS mRNA and protein levels were measured using real-time reverse transcriptase-polymerase chain reaction and western blotting. NOS activity was assessed with the citrulline assay. All three NOS isoforms were found in muscle, associated with muscle fibres and microvessels. NOS I and III protein expression was increased in CLI (P = 0.041). During revascularization, further ischaemia and reperfusion led to a rise in NOS III protein levels (P = 0.008). NOS activity was unchanged. Alterations in NOS I and III occurred in muscle from patients with CLI and further changes occurred during bypass surgery.

Alterations in Nitric Oxide Synthase Isoforms in Acute Lower Limb Ischemia and Reperfusion

Alterations in nitric oxide synthase (NOS) are implicated in ischemia and ischemia-reperfusion injury. Changes in the 3 NOS isoforms in human skeletal muscle subjected to acute ischemia and reperfusion were studied. Muscle biopsies were taken from patients undergoing total knee replacement. Distribution of the specific NOS isoforms within muscle sections was studied using immunohistochemistry. NOS mRNA levels were measured using real-time reverse transcription-polymerase chain reaction and protein levels studied using Western blotting. NOS activity was also assessed using the citrulline assay. All 3 NOS isoforms were found in muscle sections associated with muscle fibers and microvessels. In muscle subjected to acute ischemia and reperfusion, NOS I/neuronal NOS mRNA and protein were elevated during reperfusion. NOS III/endothelial NOS was also upregulated at the protein level during reperfusion. No changes in NOS II/inducible NOS expression or NOS activity occurred. In conclusion, alterations in NOS I and III (neuronal NOS and endothelial NOS) at different levels occurred after acute ischemia and reperfusion in human skeletal muscle; however, this did not result in increased NOS activity. In the development of therapeutic agents based on manipulation of the NO pathway, targeting the appropriate NOS isoenzymes may be important.

Alterations of NOS, arginase, and DDAH protein expression in rabbit cavernous tissue after administration of cigarette smoke extract

Cigarette smoking is an independent risk factor for vasculogenic erectile dysfunction (ED). Nitric oxide (NO) has been demonstrated to be the principal mediator of cavernous smooth muscle relaxation and penile erection. Therefore, we examined whether or not enzyme activities and factors involved in the NO generation pathway are affected in rabbit corpus cavernosum after administration of nicotine- and tar-free cigarette smoke extract (CSE). CSE was prepared by bubbling a stream of cigarette smoke into phosphate-buffered saline. CSE was injected subcutaneously into adult male rabbits once a day for 5 wk. In the CSE group, significantly decreased cyclic GMP production as a marker of NO generation was associated with attenuated overall nitric oxide synthase (NOS) activity, enhanced arginase activity, accumulation of endogenous NOS inhibitors such as monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), and decreased dimethylarginine dimethylaminohydrolase (DDAH) activity as an metabolizing enzyme of endogenous NOS inhibitors. Neuronal NOS (nNOS) and DDAH I protein expression were decreased without altering endothelial NOS expression, while arginase I expression was upregulated. These results suggest that impaired NO production would result from blunted NOS activity, which is possibly brought about by the downregulation of nNOS protein, accumulation of endogenous NOS inhibitors, and enhanced arginase activity together with upregulation of arginase I protein in cavernous tissue. The impaired DDAH activity due to decreased expression of DDAH I protein would result in an accumulation of endogenous NOS inhibitors with CSE. These alterations may be relevant to induction of the erectile dysfunction following CSE.

Differential modulation of Rhodnius prolixus nitric oxide activities following challenge with Trypanosoma rangeli, T. cruzi and bacterial cell wall components

Nitric oxide (NO) is a key immune effector and signaling molecule in many organisms. However, the contribution NO makes towards insect immunity has received little attention, particularly in non-dipteran species. In this study, tissue- and time-specific alterations in NO synthase ( NOS) gene expression and NO production were documented in the hemipteran vector of Chagas’ disease, Rhodnius prolixus, following in vivo immune challenge by Trypanosoma cruzi, T. rangeli and crude bacterial lipopolysaccharide (LPS). The most pronounced reactions to crude LPS occurred in major immune tissues (fat body and blood cells), while tissues of the digestive tract were most responsive to infections by T. cruzi and T. rangeli. The data suggest the NO-mediated immune responses of R. prolixus are pathogen-specific and independently modified both at the transcriptional and enzyme levels.

Neuroprotective effect of arundic acid, an astrocyte-modulating agent, in mouse brain against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) neurotoxicity

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes the damage of dopaminergic neurons as seen in Parkinson's disease. Oxidative stress has been as one of several pathogenic hypotheses for Parkinson's disease. Here we investigated whether arundic acid, an astrocyte-modulating agent, can protect against alterations of nitric oxide synthase (NOS) and superoxide dismutase (SOD) expression on MPTP neurotoxicity in mice, utilizing an immunohistochemistry. For this purpose, anti-tyrosine hydroxylase (TH) antibody, anti-dopamine transporter (DAT) antibody, anti-Cu/Zn-SOD antibody, anti-Mn-SOD antibody, anti-nNOS antibody, anti-eNOS antibody and anti-iNOS antibody were used. The present study showed that the arundic acid had a protective effect against MPTP-induced neuronal damage in the striatum and substantia nigra of mice. The protective effect may be, at least in part, caused by the reductions of the levels of reactive nitrogen (RNS) and oxygen species (ROS) against MPTP neurotoxicity. These results suggest that the pharmacological modulation of astrocyte may offer a novel therapeutic strategy for the treatment of Parkinson's disease. Furthermore, our results provide further evidence that a combination of nNOS inhibitors, iNOS inhibitors and free radical scavengers may be effective in the treatment of neurodegenerative diseases. Thus our present results provide valuable information for the pathogenesis of degeneration of the nigrostriatal dopaminergic neuronal pathway.

Age-related alterations in NOS and oxidative stress in mesenteric arteries from male and female rats.

Epidemiological evidence suggests that advancing age affects the cardiovascular system of men and women differently. The purpose of this study was to determine whether the effects of aging on nitric oxide synthase (NOS), oxidative stress, and vascular function are different in males and females. Mesenteric arteries from young (3 mo) and old (24 mo) male and female Fischer 344/Brown Norway rats were studied. Western blot analysis and NOS activity were performed on the homogenized mesenteric arterial bed separated into cytosolic and membrane-associated fractions. Plasma 8-isoprostane measurements assessed oxidative stress. Vascular reactivity was determined by using a wire myograph in the absence and presence of a NOS inhibitor, N(omega)-nitro-l-arginine, to examine endothelial function and basal and stimulated nitric oxide release. In additional arteries, reactivity was performed in the presence of polyethylene glycol-SOD to assess the impact of superoxide on vascular function. Among females, aging was associated with a decline in membrane-associated NOS activity and membrane-associated NOS III protein expression. Advancing age in males was associated with increased cytosolic NOS III protein expression. Among both males and females, advancing age resulted in increased oxidative stress. Vascular function was maintained with age in arteries from both males and females, and there was no difference in either basal or stimulated nitric oxide release with age. Despite sex-specific effects of advancing age on the NOS system and increases in markers of oxidative stress, vascular function is maintained in mesenteric arteries from aged Fischer 344/Brown Norway rats. These data suggest that age-related alterations in the resistance vasculature are complex and likely involve multiple compensating vasoactive pathways.

Endothelial-dependent vasodilation is reduced in mesenteric arteries from superoxide dismutase knockout mice.

Oxidative stress has increasingly been implicated in the development and progression of many vascular diseases. Previous work from our laboratory indicated that peroxynitrite alters vasoactive pathways in endothelial cells, which could potentially reduce vascular relaxation. To test this hypothesis in vivo, we utilized an animal model of endogenous oxidative stress, the CuZn superoxide dismutase (SOD) knockout mouse, to assess vascular function. Vascular reactivity of mouse mesenteric arteries was assessed in the presence or absence of inhibitors to nitric oxide synthase (NOS) and/or prostaglandin H synthase (PGHS). Endothelial-dependent function was also measured after the addition of exogenous SOD. Peroxynitrite formation was detected by nitrotyrosine immunofluorescence in mesenteric arteries. Our data indicate that endothelial-dependent relaxation responses to methacholine are highly reduced in SOD-/- mice (P<0.01, ANOVA). In only the wild-type mice, NOS or PGHS inhibition significantly blunted relaxation, suggesting that vasodilators from these pathways are present only in the controls and not in SOD-/- mice. A combination of NOS and PGHS inhibitors reduced methacholine relaxation in both wild type and SOD-/- mice. This residual EDHF-like relaxation was not different between groups. After incubation with exogenous SOD, endothelial-dependent relaxation could be partially restored in SOD-/- mice, due to increased NOS-mediated vasodilation. In addition, peroxynitrite formation was significantly elevated in mesenteric arteries from SOD-/- mice. Our data suggest that in a novel animal model of oxidative stress, vessel function is compromised due to alterations in NOS and PGHS-dependent relaxation responses.