Decreased Nitric Oxide Synthase Activity Research Articles

Nitrosative stress-based specific evaluation of creatine use in combination with aerobic running exercise at different speeds: a preclinical study in mice.

In acute and chronic aerobic exercise, skeletal muscle and liver are the main organs that adapt and regulate metabolic activity. The levels of nitrosative stress caused by exercise in these organs are extremely important in the continuity of exercise, its health-promoting benefits, and the evaluation of therapeutic efficacy. In this study, nitrosative stress levels were investigated in musculus quadriceps femoris tissue and liver tissue of mice that were given low and high-speed aerobic running exercise and also received Cr supplementation. In this study, nitrosative stress levels were investigated in the muscle/liver tissue of 42 BALB/c mice that were given low and high-speed aerobic running exercise and creatine monohydrate (Cr) (40 mg/kg of diet daily) supplementation with exercise. The study included six groups with and without Cr supplementation, low-speed aerobic running, high-speed aerobic running, and no exercise. The mice in groups with low-speed and high-speed aerobic exercise with and without Cr supplementation were run on the treadmill for 8 weeks. Then, nitric oxide (NO·), nitric oxide synthase (NOS), and peroxynitrite (ONOO-) levels in muscle/liver tissue were measured by spectrophotometric method. It was found that the nitrosative stress level in the groups that did low and high-speed aerobic running exercises increased compared to the group that did not exercise. It was found that NO· decreased NOS activity and ONOO- level increased in muscle tissues of low and high-speed aerobic exercise groups that received Cr supplementation compared to those that did not. However, NO· and ONOO- levels in liver tissue decreased while NOS activity did not change. The lowest level of nitrosative stress in both muscle and liver tissue was found in the low-speed exercise group receiving Cr supplementation. Although supplements in exercise are an important component, the simultaneously measured nitrosative stress level is critical in determining the optimal exercise.

Abstract 050: Endothelial Cell Mineralocorticoid Receptor Deletion Improves Endothelial Function, Blood Pressure And Fetal Growth In The RUPP Mouse Model Of Preeclampsia

Preeclampsia (PE) is a hypertensive disorder of pregnancy that causes adverse pregnancy outcomes. Progesterone in pregnancy increases endothelial mineralocorticoid receptor (ECMR) expression, which we showed mediates endothelial function in hypertensive female mice, however, whether ablating endothelial dysfunction in PE improves pregnancy outcomes is unknown. We hypothesized that improving endothelial function via ECMR deletion increases fetal growth and ablates hypertension in the reduced uterine perfusion pressure (RUPP) PE mouse. Pregnant ECMR-intact (WT) and -deficient (KO) mice underwent RUPP or sham surgery on GD13 and sacrificed on GD18 (WT Sham=9, WT RUPP=5, KO Sham=6, KO RUPP=6). Vascular function was measured via wire myography on 2 nd order mesenteric arteries and thoracic aorta. Systolic blood pressure (BP) was measured by radiotelemetry in a subset of mice. RUPP did not decrease maternal weights in either WT or KO mice (P>0.05) but decreased pup weight (726±20 WT+Sham vs 660±11mg WT+RUPP, *P<0.05) and fetal demise (16±4 vs 28±4% resoprtions/litter, *P<0.05) in WT pregnant mice, however, not in KO mice (710±15 KO+Sham vs 693±19 mg KO+RUPP, P>0.05, and 13±5 vs 28±12% resoprtions/litter, P>0.05), indicating KO protected fetal growth. RUPP reduced endothelial-dependent, acetylcholine-mediated vascular relaxation (10 -9 -3x10 -5 M) in WT, but not KO pregnant mice (*P<0.05, 2-way ANOVA w/ RM), but not aorta ACh-mediated relaxation (P>0.05). Nitric oxide synthase (NOS) inhibitor LNAME ablated differences in ACh-mediated relaxation between WT Sham and WT RUPP (P>0.05), with no differences observed in KO Sham or KO RUPP, in both aorta and mesenteric arteries, indicating RUPP impaired endothelial function via decreasing NOS activity. Preliminary data demonstrates RUPP increased BP on GD15-17 in WT (127±21 mmHg, N=3), however, not in KO pregnant mice (111±4, N=2). RUPP also ablated circadian rhythm in WT, but not KO, pregnant mice from GD15-18. Collectively, these data indicate that ECMR activation induces reduced pup weight, endothelial dysfunction and hypertension in the RUPP model which may lead to clinical studies for consideration of specific MR antagonists as therapeutic options for endothelial dysfunction in PE pregnancies.

Disorders of microcirculation in a comorbid patient

Background. Diabetes mellitus (DM) is a global epidemic. According to the experts, by 2045 the number of patients with diabetes will rise by 48 %. Patients with diabetes are characterized by the high cardiovascular mortality and a significant reduction in life expectancy. Almost half of the deaths associated with diabetes are due to cardiovascular diseases (CVD). Patients with diabetes often have concomitant pathology (hypertension, dyslipidemia, obesity), which increases the cardiovascular risk.
 Objective. To describe microcirculation disorders in patients with comorbid conditions.
 Materials and methods. Analysis of literature sources on this topic.
 Results and discussion. Type 2 diabetes (DM2) is a multifaceted disease that has a number of cardiovascular, metabolic and renal complications. The links of the pathogenesis of cardiovascular complications of DM2 include dyslipidemia, systemic inflammation, insulin resistance, autonomic imbalance, and endothelial dysfunction (ED). ED leads to vasoconstriction, increased chronic inflammation, increased vascular permeability and hypercoagulation, which ultimately causes micro- and macroangiopathy. Neurological complications of diabetes are also mediated by ED and microangiopathies, which lead to nerve hypoxia with a decrease in conductivity velocity. In recent years, the literature has been actively discussing the syndrome of early vascular aging – premature and accelerated development of structural and functional age-related changes in blood vessels. Microcirculation disorders that accompany diabetes, CVD and their complications are mediated by nitric oxide (NO) imbalance. NO reduces the contractility and proliferation of smooth muscle cells, platelet aggregation, endothelin production, adhesion of monocytes and platelets, and oxidation of low-density lipoproteins. In humans, NO is produced from L-arginine. Increased L-arginase activity and decreased NO-synthase activity lead to a decrease in NO content and to the development of ED, atherosclerosis, and decreased insulin sensitivity. L-arginine as a substrate of NO improves the functional state of the endothelium, reduces the manifestations of oxidative stress, reduces the level of pro-inflammatory cytokines and adhesion molecules, inhibits platelet aggregation, reduces insulin resistance. It should be noted that the administration of 4.2 g of L-arginine in DM is not enough. In clinical studies, doses of 6.4-9 g per day were found to be effective in diabetes and obesity. Therefore, it is advisable to use Tivortin-200 (“Yuria-Pharm”), which contains 8.4 g of L-arginine in one vial.
 Conclusions. 1. Patients with diabetes are characterized by the high cardiovascular mortality and a significant reduction in life expectancy. 2. The links of the pathogenesis of cardiovascular complications of DM2 include dyslipidemia, systemic inflammation, insulin resistance, autonomic imbalance, and ED. 3. Disorders of microcirculation that accompany diabetes, CVD and their complications, are mediated by NO imbalance. 4. L-arginine as a substrate of NO improves the functional state of the endothelium, reduces the manifestations of oxidative stress, reduces the level of pro-inflammatory cytokines and adhesion molecules, inhibits platelet aggregation, reduces insulin resistance. 5. In diabetes and obesity, it is advisable to use Tivortin-200, which contains 8.4 g of L-arginine in one vial.

Nitric Oxide Synthase activity in major depressive episodes before and after antidepressant treatment: Results of a large case-control treatment study.

Nitric oxide synthase (NOS) activity, an enzyme potentially involved in the major depressive episodes (MDE), could be indirectly measured by the L-Citrulline/L-Arginine ratio (L-Cit/L-Arg). The aim of this study was: (1) to compare the NOS activity of patients with a MDE to that of healthy controls (HC); (2) to assess its change after antidepressant treatment. A total of 460 patients with a current MDE in a context of major depressive disorder (MDD) were compared to 895 HC for NOS activity (L-Cit/L-Arg plasma ratio). L-Arg and L-Cit plasma levels were measured using a MS-based liquid chromatography method. Depressed patients were assessed at baseline, and after 3 and 6 months of antidepressant treatment for depression severity and clinical response. Depressed patients had a lower NOS activity than HC at baseline [0.31 ± 0.09 v. 0.38 ± 0.12; 95% confidence interval (CI) -0.084 to -0.062, p < 0.0001]. Lower NOS activity at baseline predicted a higher response rate [odds ratio (OR) = 29.20; 95% CI 1.58-536.37; p = 0.023]. NOS activity in depressed patients increased significantly up to 0.34 ± 0.08 after antidepressant treatment (Est = 0.0034; 95% CI 0.0002-0.0067; p = 0.03). Depressed patients have a decreased NOS activity that improves after antidepressant treatment and predicts drug response. NOS activity may be a promising biomarker for MDE in a context of MDD.

Effect of low dose L-NAME pretreatment on nitric oxide/reactive oxygen species balance and vasoactivity in L-NAME/salt-induced hypertensive rats.

Nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase-dependent reactive oxygen species (ROS) overproduction and decreased nitric oxide (NO) bioavailability lead to vascular dysfunction and development of hypertension. The goal of our study was to analyze an effect of salt diet and NO synthase (NOS) inhibition with NG-nitro-L-arginine methyl ester (L-NAME) on blood pressure (BP), arterial reactivity, NO production, as well as ROS level in adult rats pretreated with low dose of L-NAME (2 mg/kg/day) for three weeks. Higher dose of L-NAME (40 mg/kg/day), or salt diet (8% NaCl), or combination of both were applied for the following four weeks. The administration of L-NAME in low dose had no effect on BP but enhanced the expression of eNOS. Both higher dose of L-NAME and salt diet elevated BP, decreased NOS activity, and impaired the endothelium-dependent arterial relaxation. However, salt diet did not increase ROS production and sympathoadrenergic arterial contractions in low dose L-NAME-pretreated rats. Combination of salt diet with higher dose of L-NAME did not evoke additive decrease of NOS activity, but it caused elevation of conjugated dienes (CD) concentration and NADPH oxidase 2 (Nox-2) protein expression. In conclusion, these findings indicate that chronic low dose of L-NAME treatment has a potential to trigger adapting mechanisms to attenuate some cardiovascular disorders.

Different adaptive NO-dependent Mechanisms in Normal and Hypertensive Conditions.

Myocardial infarction (MI) remains the leading cause of death worldwide. We aimed to investigate the effect of NO deficiency on selective biochemical parameters within discreet myocardial zones after experimentally induced MI. To induce MI, the left descending coronary artery was ligated in two groups of 16-week-old WKY rats. In one group, NO production was inhibited by L-NAME (20 mg/kg/day) administration four weeks prior to ligation. Sham operations were performed on both groups as a control. Seven days after MI, we evaluated levels of nitric oxide synthase (NOS) activity, eNOS, iNOS, NFҡB/p65 and Nrf2 in ischemic, injured and non-ischemic zones of the heart. Levels of circulating TNF-α and IL-6 were evaluated in the plasma. MI led to increased NOS activity in all investigated zones of myocardium as well as circulating levels of TNF-α and IL-6. L-NAME treatment decreased NOS activity in the heart of sham operated animals. eNOS expression was increased in the injured zone and this could be a compensatory mechanism that improves the perfusion of the myocardium and cardiac dysfunction. Conversely, iNOS expression increased in the infarcted zone and may contribute to the inflammatory process and irreversible necrotic changes.

Nitric oxide modulates the responses of osteoclast formation to static magnetic fields

ABSTRACTNitric oxide (NO) is involved in osteoclast differentiation. Our previous studies showed that static magnetic fields (SMFs) could affect osteoclast differentiation. The inhibitory effects of 16 T of high SMF (HiMF) on osteoclast differentiation was correlated with increased production of NO. We raised the hypothesis that NO mediated the regulatory role of SMFs on osteoclast formation. In this study, 500 nT of hypomagnetic field (HyMF), 0.2 T of moderate SMF (MMF) and 16 T of high SMF (HiMF) were utilized as SMF treatment. Under 16 T, osteoclast formation was markedly decreased with enhanced NO synthase (NOS) activity, thus producing a high level of NO. When treated with NOS inhibitor N-Nitro-L-Arginine Methyl Ester (L-NAME), NO production could be inhibited, and osteoclast formation was restored to control group level in a concentration-dependent manner. However, 500 nT and 0.2 T increased osteoclast formation with decreased NOS activity and NO production. When treated with NOS substrate L-Arginine (L-Arg) or NO donor sodium nitroprusside (SNP), the NO level in the culture medium was obviously elevated, thus inhibiting osteoclast differentiation in a concentration-dependent manner under 500 nT or 0.2 T. Therefore, these findings indicate that NO mediates the regulatory role of SMF on osteoclast formation.

Renal response to L-arginine in diabetic rats. A possible link between nitric oxide system and aquaporin-2.

The aim of this study was to evaluate whether L-Arginine (L-Arg) supplementation modifies nitric oxide (NO) system and consequently aquaporin-2 (AQP2) expression in the renal outer medulla of streptozotocin-diabetic rats at an early time point after induction of diabetes. Male Wistar rats were divided in four groups: Control, Diabetic, Diabetic treated with L-Arginine and Control treated with L-Arginine. Nitric oxide synthase (NOS) activity was estimated by [14C] L-citrulline production in homogenates of the renal outer medulla and by NADPH-diaphorase staining in renal outer medullary tubules. Western blot was used to detect the expression of AQP2 and NOS types I and III; real time PCR was used to quantify AQP2 mRNA. The expression of both NOS isoforms, NOS I and NOS III, was decreased in the renal outer medulla of diabetic rats and L-Arg failed to prevent these decreases. However, L-Arg improved NO production, NADPH-diaphorase activity in collecting ducts and other tubular structures, and NOS activity in renal homogenates from diabetic rats. AQP2 protein and mRNA were decreased in the renal outer medulla of diabetic rats and L-Arg administration prevented these decreases. These results suggest that the decreased NOS activity in collecting ducts of the renal outer medulla may cause, at least in part, the decreased expression of AQP2 in this model of diabetes and constitute additional evidence supporting a role for NO in contributing to renal water reabsorption through the modulation of AQP2 expression in this pathological condition. However, we cannot discard that another pathway different from NOS also exists that links L-Arg to AQP2 expression.

Akita spontaneously type 1 diabetic mice exhibit elevated vascular arginase and impaired vascular endothelial and nitrergic function.

BackgroundElevated arginase (Arg) activity is reported to be involved in diabetes-induced vascular endothelial dysfunction. It can reduce L-arginine availability to nitric oxide (NO) synthase (NOS) and NO production. Akita mice, a genetic non-obese type 1 diabetes model, recapitulate human diabetes. We determined the role of Arg in a time-course of diabetes-associated endothelial dysfunction in aorta and corpora cavernosa (CC) from Akita mice.Methods and ResultsEndothelium-dependent relaxation, Arg and NOS activity, and protein expression levels of Arg and constitutive NOS were assessed in aortas and CC from Akita and non-diabetic wild type (WT) mice at 4, 12 and 24 wks of age. Systolic blood pressure (SBP) was assessed by tail cuff. In aorta and CC, Akita mice exhibited a progressive impairment of vascular endothelial and nitrergic function increased Arg activity and expression (Arg1 in aorta and both Arg1 and Arg2 in CC) compared with that of age-matched WT mice. Treatment of aorta and CC from Akita mice with an Arg inhibitor (BEC or ABH) reduced diabetes-induced elevation of Arg activity and restored endothelial and nitrergic function. Reduced levels of phospho-eNOS at Ser1177 (in aorta and CC) and nNOS expression (in CC) were observed in Akita mice at 12 and 24 wks. Akita mice also had decreased NOS activity in aorta and CC at 12 and 24 wks that was restored by BEC treatment. Further, Akita mice exhibited moderately increased SBP at 24 wks and increased sensitivity to PE-induced contractions in aorta and sympathetic nerve stimulation in CC at 12 and 24 wks.ConclusionsOver 24 wks of diabetes in Akita mice, both aortic and cavernosal tissues exhibited increased Arg activity/expression, contributing to impaired endothelial and nitrergic function and reduced NO production. Our findings demonstrate involvement of Arg activity in diabetes-induced impairment of vascular function in Akita mouse.

The relationship between enzyme activity of gastric mucosa with microcirculation and acid output

The aim of this study is to reveal the relationship between gastric blood flow with acid output and enzymatic systems of the gastric mucosa (GM). We examined 14 healthy young individuals and 20 healthy elderly patients without atrophic changes in GM. Blood flow in GM microvessels was assessed using Doppler laser flowmetry, acid output was assessed by 24-h pH monitoring, and H+-K+-ATPase and nitric oxide synthase activity was assessed by standard biochemical techniques. A statistically significant decreased blood flow level in microvessels of the GM secretory zone (large curvature, front and back walls in the stomach body) was revealed for healthy individuals, which was accompanied by decreased NO synthase activity. Acid output and H+-K+-ATPase activity of parietal cells in this zone do not decrease with age. Nevertheless, in elderly patients, a higher level of blood flow in the secretory zone was associated with a higher acid output level, which points to an increase in the blood flow level values for hydrochloric acid secretion in elderly individuals.

Serum nitric oxide synthase activity is a novel predictor of impaired vasorelaxation in rats

1. It is well documented that both acetylcholine (ACh)-evoked arterial relaxation and brachial artery flow-mediated vasodilatation are blunted in hypercholesterolaemic patients. However, there are no simple diagnostic methods to detect the pathology of blood vessels of patients. 2. To establish the use of serum nitric oxide synthase (NOS) activity as a diagnostic parameter for impaired vasorelaxation, animals with different levels of vascular healthiness were made by feeding Sprague-Dawley rats a normal diet, a high-cholesterol diet (HCD) or an HCD supplemented with 10 mg/kg per day, p.o., simvastatin, a cholesterol-lowering drug, for 30 days. Serum total cholesterol levels, serum NOS activity and ACh-induced vasorelaxation of the isolated aorta were determined at the end of the experiment. 3. Consumption of HCD for 30 days resulted in an increase in serum total cholesterol, attenuated ACh-induced nitric oxide/endothelium-dependent aortic relaxation and decreased NOS activity. Concomitant administration of simvastatin lowered the elevated blood cholesterol levels with complete reversal of the attenuated ACh-induced aortic relaxation and serum NOS activity. An attempt was made to correlate serum NOS activity and the magnitude of ACh-elicited vascular relaxation among the different groups. A positive correlation (r = 0.8329; P < 0.001; n = 30) was found between serum NOS activity and vascular relaxation. 4. This finding is a good foundation for the development of a simple and low-cost alternative for diagnosing vascular diseases and evaluating the effectiveness of drugs on the vascular system in patients.

Nitric oxide synthase activity in the abdominal aorta of rats is decreased after 4 weeks of simulated microgravity

1. The aim of the present study was to investigate the effects of simulated microgravity on the arterial dilatory responsiveness and L-arginine (L-Arg)-nitric oxide (NO)-cGMP pathway in the abdominal aorta of rats. 2. Twenty healthy male Sprague-Dawley were randomly divided into control and simulated microgravity groups. Rats in the simulated microgravity group were subjected to hindlimb unweighting (HU). After 4 weeks, arterial dilatory responsiveness was examined in vitro in isolated abdominal aortic rings. Western blotting was used to measure endothelial (e) and inducible (i) NO synthase (NOS) protein content. Total concentrations of nitrate and nitrite (NO(x)), the stable metabolites of NO, were determined by the chemiluminescence method. Nitric oxide synthase activity in the abdominal aorta was determined through the conversion of [(3)H]-L-Arg to [(3)H]-L-citrulline. 3. The data showed that the dilatory responses of the arterial rings to L-Arg and acetylcholine decreased in rats exposed to simulated microgravity, but the dilatory responses to sodium nitroprusside and 8-bromo-cGMP were similar in both simulated microgravity and control rats. The expression of eNOS and iNOS did not differ significantly between the two groups. The NO(x) concentration in the abdominal aorta of HU rats was significantly less than that in control rats. Nitric oxide synthase activity in the aorta decreased after 4 weeks of HU. 4. The data indicate that endothelium-dependent vasorelaxation in the abdominal aorta decreased due to 4 weeks of simulated microgravity in rats and that this impaired dilatory responsiveness may result from decreased NOS activity.

Apocynin prevents decrease of nitric oxide synthase activity due to the L-NAME treatment in SHR

The aim of our study was to analyse the effect of apocynin, NADPH-oxidase inhibitor, on blood pressure (BP) and total nitric oxide synthase (NOS) activity of spontaneously hypertensive rats (SHR) treated with NOS inhibitor-NG-nitro-l-arginine methyl ester (L-NAME). Young 6-week-old male SHR were treated with L-NAME (2 mg/kg/day) or apocynin (30 mg/kg/day) or L-NAME along with apocynin for 6 weeks. At the end of the treatment, apocynin had no significant effect on both BP and NOS activity while L-NAME potentiated BP increase by 10% and decreased NOS activity by 45% in the heart and by 53% in the aorta.

Regression of L-NAME–Induced Hypertension: The Role of Nitric Oxide and Endothelium-Derived Constricting Factor

N(G)-Nitro-L-arginine-methyl ester (L-NAME)-induced hypertension is a well established model of experimental hypertension. Although regression experiments are effective at approximating a clinical setting the reversal of already established L-NAME hypertension has not been intensively researched. We investigated whether spontaneous regression of L-NAME hypertension after discontinuing the drug administration was associated with recovery of endothelial dysfunction. Special attention was devoted to NO signaling and endothelium-derived constricting factor (EDCF) formation in various parts of the vascular tree. Male adult Wistar rats were divided into 4 groups: an L-NAME (5 weeks), a spontaneous recovery (5 weeks L-NAME + 3 weeks of recovery) and two age-matched control groups (a 5- and 8-week control group). The NO-mediated and EDCF-mediated components of acetylcholine-induced responses were evaluated in preconstricted small mesenteric and femoral arteries. The activity, mRNA and protein expression of NO synthase together with the mRNA expression of cyclooxygenase were determined in the aorta. L-NAME administration caused hypertension, impaired NO signaling (as indicated by the reduced NO component of acetylcholine-induced relaxation and decreased NO synthase activity) in all arteries investigated and reduced the inner diameter of the femoral artery. Moreover, we observed enhanced cyclooxygenase-dependent EDCF formation in the femoral arteries and enhanced cyclooxygenase-2 expression in the aortas of L-NAME-treated rats. During spontaneous recovery a functional restoration of NO signaling took place in all parts of the vascular tree. However, the increases in systolic blood pressure, EDCF formation, and cyclooxygenase expression and the reduction in femoral artery diameter were not completely restored. We conclude that impaired NO signaling was improved after the cessation of L-NAME administration. However, persisting arterial structural alterations and enhanced EDCF formation may decelerate blood pressure reduction even after the restoration of NO synthase activity.

Nitric oxide synthase expression and activity in the tight-skin mouse model of fibrosis

Nitric oxide (*NO) is an important physiological signalling molecule and a potent vasodilator. We have previously demonstrated abnormal *NO metabolism in the plasma of patients with systemic sclerosis (SSc; scleroderma), a disease that features vascular dysfunction as well as collagen overproduction and fibrosis. The aim of the present study was to examine nitric oxide synthase (NOS) expression and activity and assess the potential role of antioxidants in the scleroderma-like syndrome of the tight-skin 1 (TSK-1/+) mouse, an experimental animal model for fibrosis. Skin, lung or plasma was taken from TSK-1/+ (n = 15) and wild-type (WT; n = 12) littermate mice. Type 1 collagen, endothelial NOS (eNOS), haemoxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein and gene expression were determined by western blot and reverse transcriptase-polymerase chain reaction. eNOS expression was further determined by immunohistochemistry. NOS activity was evaluated by conversion of [14C] L-arginine to [14C] L-citrulline. Levels of circulating plasma nitrite/nitrate (NO(x)) were also measured. Total antioxidant activity was evaluated by ABTS+ production (ABTS = 2,2'-azino-bis-[3-ethylbenz-thiazoline-6-sulphonic acid). In the skin, eNOS was present in the epidermal layer, hair follicles and also in the endothelial cells lining the blood vessels. Expression of both the eNOS protein and gene was significantly reduced in TSK-1/+ skin tissue, while type 1 collagen protein was elevated compared with WT. Furthermore, there was decreased NOS activity in TSK-1/+ skin tissue; however, there was no measurable difference in plasma NO(x). Correspondingly, the protective antioxidant enzyme HO-1 and the associated transcription factor Nrf2 showed reduced protein and gene expression levels in TSK-1/+ skin, while there was also less total antioxidant activity. In TSK-1/+ lung tissue, however, we observed no difference in collagen protein expression, *NO metabolism or HO-1 expression and total antioxidant activity compared with WT. The findings suggest that there is also abnormal *NO metabolism in the TSK-1/+ mouse model of fibrosis, particularly in the skin, while expression and activity of protective antioxidants are reduced. The TSK-1/+ mouse may also be useful for testing treatments that target vascular endothelial cell function in patients with SSc.

Nitric oxide synthase activity in rat gastric mucosa contributes to mucin synthesis elicited by calcitonin gene-related peptide

The majority of research for the calcitonin gene-related peptide (CGRP) in the stomach has been devoted to the submucosal blood flow, and only slight attention has been paid to its involvement in the gastric epithelial function. In this study, we examined the age-related change in the CGRP-containing nerves and its effects on the mucus metabolism. We compared the immunoreactivity for CGRP in the gastric mucosa of 7-week-old rats (young) to that of 52-week-old animals (middle-aged). The effects of CGRP on the mucin biosynthesis were compared using the stomachs from both young and middle-aged rats. The nitric oxide synthase (NOS) activity was measured in the surface and deep mucosa of the gastric corpus. The density of the CGRP nerve fibers was reduced in both the lamina propria and submucosa of the middle-aged rats compared to the young rats. CGRP stimulated the mucin biosynthesis in the cultured corpus mucosa from the 7-week-old rats, but not from the 52-week-old rats. The total NOS activity of the surface layer in the corpus mucosa was markedly reduced in the middle-aged rats compared to the young rats. These findings demonstrate the age-dependent reduction in the CGRP-induced mucin biosynthesis, as well as in the density of the CGRP fibers in the rat stomach. The decreased NOS activity in the surface layer of the oxyntic mucosa in the aged rats may also be a principal cause for the lack of regulation of the mucin biosynthesis by CGRP.

Dysregulation of Dimethylarginine Dimethylaminohydrolase Causes Elevation of Asymmetric Dimethylarginine in a Rat Model of Vasculogenic Erectile Dysfunction

Purpose: Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) and the major metabolic pathway of ADMA is enzymatic degradation via dimethylarginine dimethylaminohydrolasease (DDAH). In this study, we hypothesized that an elevated cavernosal ADMA level might result from poor DDAH activity in the corpus cavernosum. We examined whether ADMA was accumulated in our atherosclerotic rat model of vasculogenic erectile dysfunction (VED). Materials and Methods: Twelve 12-wk-old Sprague-Dawley rats were grouped in either the atherosclerosis group (AS, n=6) or the control (n=6) group. The AS group received a 1% cholesterol diet for 6 weeks and the rats were also treated with NG-nitro-L-arginine methyl ester (3mg/ml) for the initial 2 weeks. The control group received a normal diet. Six weeks later, all the rats were anesthetized with urethane (1.6g/kg) and cavernous electrostimulation was done under continuous arterial and cavernosal pressure monitoring (6V, 0.5ms, 20Hz, 50sec). The methylarginine level in both the AS group and the control group was measured respectively. Also, the NOS activity and DDAH activity in the corpus cavernosum were evaluated. Results: Upon cavernous electostimulation, the peak intracavernosal pressure (ICP) of the control group was 88.5±5.5mmHg (n=6). In contrast, the peak ICP level was markedly reduced in the atherosclerotic group to 54.2±4.8mmHg (n=6, p<0.001). The cavernosal level of ADMA in the control group was 320.5±23.6μM and it was 860.7±34.7μM in the AS group. The constitutive NOS activity in the rat corpus cavernosum of the AS group was markedly reduced compared to the control group. Also, the cavernosal DDAH activity was reduced in the AS rats and the activity showed significant negative correlation with the cavernosal ADMA level. Conclusions: In this study, we have demonstrated that the dysregulation of DDAH activity may be one of the causes of decreased NOS activity in atherosclerotic erectile dysfunction. (Korean J Urol 2006;47:1079-1085)

Inhaled nitric oxide induced NOS inhibition and rebound pulmonary hypertension: a role for superoxide and peroxynitrite in the intact lamb

Previous in vivo studies indicate that inhaled nitric oxide (NO) decreases nitric oxide synthase (NOS) activity and that this decrease is associated with significant increases in pulmonary vascular resistance (PVR) upon the acute withdrawal of inhaled NO (rebound pulmonary hypertension). In vitro studies suggest that superoxide and peroxynitrite production during inhaled NO therapy may mediate these effects, but in vivo data are lacking. The objective of this study was to determine the role of superoxide in the decrease in NOS activity and rebound pulmonary hypertension associated with inhaled NO therapy in vivo. In control lambs, 24 h of inhaled NO (40 ppm) decreased NOS activity by 40% (P<0.05) and increased endothelin-1 levels by 64% (P<0.05). Withdrawal of NO resulted in an acute increase in PVR (60.7%, P<0.05). Associated with these changes, superoxide and peroxynitrite levels increased more than twofold (P<0.05) following 24 h of inhaled NO therapy. However, in lambs treated with polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) during inhaled NO therapy, there was no change in NOS activity, no increase in superoxide or peroxynitrite levels, and no increase in PVR upon the withdrawal of inhaled NO. In addition, endothelial NOS nitration was 18-fold higher (P<0.05) in control lambs than in PEG-SOD-treated lambs following 24 h of inhaled NO. These data suggest that superoxide and peroxynitrite participate in the decrease in NOS activity and rebound pulmonary hypertension associated with inhaled NO therapy. Reactive oxygen species scavenging may be a useful therapeutic strategy to ameliorate alterations in endogenous NO signaling during inhaled NO therapy.

Impaired Male Sexual Behavior in Activin Receptor Type II Knockout Mice1

Integration of multiple hormonal and neuronal signaling pathways in the medial preoptic area (mPOA) is required for elicitation of male sexual behavior in most vertebrates. Perturbation of nitric oxide synthase (NOS) activity in the mPOA causes significant defects in male sexual behavior. Although activins and their signaling components are highly expressed throughout the brain, including the mPOA, their functional significance in the central nervous system (CNS) is unknown. Here, we demonstrate a neurophysiologic role for activin signaling in male reproductive behavior. Adult activin receptor type II null (Acvr2-/-) male mice display multiple reproductive behavioral deficits, including delayed initiation of copulation, reduced mount, and intromission frequencies, and increased mount, intromission, and ejaculation latencies. These behavioral defects in the adult mice are independent of gonadotropin-releasing hormone (GnRH) homeostasis or mating-induced changes in luteinizing hormone (LH) and testosterone levels. The impairment in behavior can be correlated to the nitric oxide content in the CNS because Acvr2-/- males have decreased NOS activity in the mPOA but not the rest of the hypothalamus or cortex. Olfactory acuity tests confirmed that Acvr2-/- mice have no defects in general odor or pheromone recognition. In addition, motor functions are not impaired and the mutants demonstrate normal neuromuscular coordination and balance. Furthermore, the penile histology in mutant mice appears normal, with no significant differences in the expression of penile differentiation marker genes compared with controls, suggesting the observed behavioral phenotypes are not due to structural defects in the penis. Our studies identify a previously unrecognized role of activin signaling in male sexual behavior and suggest that activins and/or related family members are upstream regulators of NOS activity within the mPOA of the forebrain.

L-Arginine attenuates cardiovascular impairment in DOCA-salt hypertensive rats

Nitric oxide (NO) is essential for normal function of the cardiovascular system. This study has determined whether chronic administration of l-arginine, the biological precursor of NO, attenuates the development of structural and functional changes in hearts and blood vessels of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Uninephrectomized rats treated with DOCA (25 mg every 4th day sc) and 1% NaCl in the drinking water for 4 wk were treated with l-arginine (5% in food, 3.4 +/- 0.3 g x kg body wt(-1) x day(-1)). Changes in cardiovascular structure and function were determined by echocardiography, microelectrode studies, histology, and studies in isolated hearts and thoracic aortic rings. DOCA-salt hypertensive rats developed hypertension, left ventricular hypertrophy with increased left ventricular wall thickness and decreased ventricular internal diameter, increased inflammatory cell infiltration, increased ventricular interstitial and perivascular collagen deposition, increased passive diastolic stiffness, prolonged action potential duration, increased oxidative stress, and inability to increase purine efflux in response to an increased workload. l-Arginine markedly attenuated or prevented these changes and also normalized the reduced efficacy of norepinephrine and acetylcholine in isolated thoracic aortic rings of DOCA-salt hypertensive rats. This study suggests that a functional NO deficit in blood vessels and heart due to decreased NO synthase activity or increased release of reactive oxygen species such as superoxide may be a key change initiating many aspects of the cardiovascular impairment observed in DOCA-salt hypertensive rats. These changes can be prevented or attenuated by administration of l-arginine.