Increased Nitric Oxide Synthesis Research Articles

In vivo arginine production and nitric oxide synthesis in pregnant Indian women with normal and low body mass indices

Background/Objectives:Nitric oxide (NO) has been proposed as a mediator of vascular expansion during pregnancy. Inability to increase NO synthesis and/or production of its precursor, arginine, may be a contributor to pregnancy-induced hypertension or preeclampsia. Because maternal weight is associated with blood pressure and risk of preeclampsia during pregnancy, it may also influence arginine and/or NO production. The purpose of this study was to determine the in vivo arginine production and NO synthesis rate in pregnant women with normal (n=10) and low (n=10) body mass indices (BMIs).Subjects/Methods:Arginine flux and NO synthesis rate were measured in the postabsorptive state with constant infusions of 15N2-arginine and 13C,2H4-citrulline. Plasma concentrations of arginine and NO metabolites were also measured. Kinetic parameters were correlated to maternal variables, gestational age, birth weight and blood pressure.Results:Endogenous arginine flux was significantly faster in the low-BMI compared with normal-BMI women in the first trimester (63.1±3.4 vs 50.2±2.0 μmol/kg per h, P<0.01), but not in the second. Plasma NO concentration was higher (44.7±5.3 vs 30.4±1.9 μmol/l, P=0.03) and its rate of synthesis trended faster in the low-BMI compared with normal-BMI group in the second trimester. Maternal weight and BMI were negatively correlated with arginine flux in both trimesters and NO synthesis in the second trimester.Conclusions:These findings suggest, but do not prove, that maternal BMI may be a factor in the ability to produce NO during pregnancy and may be one way by which BMI influences blood pressure during pregnancy.

L-arginine supplementation normalizes bone turnover and preserves bone mass in streptozotocin-induced diabetic rats

Osteopenia, an important complication of diabetes mellitus, is responsible of an increase in bone fracture and of a delay in fracture healing. The pathogenesis of this complication is unclear, however decreased availability and synthesis of nitric oxide (NO) may be regarded as a possible cause of disregulation of bone turnover. The aim of our study was to evaluate the effect of streptozotocin (STZ)-induced diabetes in the rat on bone mineral density (BMD) and bone turnover. We also examined whether supplementation of L-arginine (which acts as a NO substrate) could be beneficial for bone. After 6 weeks of STZ treatment, diabetic rats showed a significant decrease of BMD in the whole body, at the spine, at the pelvis, and at the femur. Bone turnover evaluation revealed a significant decrease in the serum levels of osteocalcin (a marker of bone formation), and an increase of the serum levels of the C-terminal telopeptide of type I collagen (RatLaps; a marker of bone resorption). L-arginine supplementation prevented the diabetes-induced reduction of BMD and osteocalcin, and the increase of RatLaps. These pharmacological actions of L-arginine produce a new suggestion that increase of NO synthesis and availability is potentially useful for effective prevention and treatment of osteopenia associated with diabetes.

Expression of inducible nitric oxide synthase by Corydalis turtschaninovii on interferon-γ stimulated macrophages

Corydalis turtschaninovii (CT) has been used for tumor therapy. However, it is still unclear how this herb prevents the diseases in experimental models. Nitric oxide (NO) as a potent macrophage-derived effector molecule against a variety of tumors has received increasing attention. In this study, using mouse peritoneal macrophages, we have examined the mechanism by which CT regulates NO production. When CT was used in combination with recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production. However, CT had no effect on NO production by itself. The increase in NO synthesis was reflected as an increased amount of inducible NO synthase (iNOS) protein. The increased production of NO from rIFN-gamma plus CT-stimulated peritoneal macrophages was decreased by the treatment with N(G)-monomethyl-L-arginine or N(alpha)-Tosyl-Phe Chloromethyl Ketone, iNOS inhibitor. The increased production of NO from rIFN-gamma plus CT-stimulated cells was almost completely inhibited by pre-treatment with pyrrolidine dithiocarbamate, an inhibitor of nuclear factor kappa B (NF-kappaB). However, treatment of peritoneal macrophages with rIFN-gamma plus CT had no effect on the increase in tumor necrosis factor-alpha (TNF-alpha) production. Our findings demonstrate that CT increases the production of NO and TNF-alpha by rIFN-gamma-primed macrophages and suggest that NF-kappaB plays a critical role in mediating these effects of CT.

Role of glutamate NMDA receptors and nitric oxide located within the periaqueductal gray on defensive behaviors in mice confronted by predator

The midbrain periaqueductal gray (PAG) is part of the brain system involved in active defense reactions to threatening stimuli. Glutamate N-methyl-D: -aspartate (NMDA) receptor activation within the dorsal column of the PAG (dPAG) leads to autonomic and behavioral responses characterized as the fear reaction. Nitric oxide (NO) has been proposed to be a mediator of the aversive action of glutamate, since the activation of NMDA receptors in the brain increases NO synthesis. We investigated the effects of intra-dPAG infusions of NMDA on defensive behaviors in mice pretreated with a neuronal nitric oxide synthase (nNOS) inhibitor [Nomega-propyl-L: -arginine (NPLA)], in the same midbrain site, during a confrontation with a predator in the rat exposure test (RET). Male Swiss mice received intra-dPAG injections of NPLA (0.1 or 0.4 nmol/0.1 microl), and 10 min later, they were infused with NMDA (0.04 nmol/0.1 microl) into the dPAG. After 10 min, each mouse was placed in the RET. NMDA treatment enhanced avoidance behavior from the predator and markedly increased freezing behavior. These proaversive effects of NMDA were prevented by prior injection of NPLA. Furthermore, defensive behaviors (e.g., avoidance, risk assessment, freezing) were consistently reduced by the highest dose of NPLA alone, suggesting an intrinsic effect of nitric oxide on defensive behavior in mice exposed to the RET. These findings suggest a potential role of glutamate NMDA receptors and NO in the dPAG in the regulation of defensive behaviors in mice during a confrontation with a predator in the RET.

TGF-β1 inhibits expression and activity of hENT1 in a nitric oxide-dependent manner in human umbilical vein endothelium

We studied whether transforming growth factor beta1 (TGF-beta1) modulates human equilibrative nucleoside transporters 1 (hENT1) expression and activity in human umbilical vein endothelial cells (HUVECs). hENT1-mediated adenosine transport and expression are reduced in gestational diabetes and hyperglycaemia, conditions associated with increased synthesis and release of nitric oxide (NO) and TGF-beta1 in this cell type. TGF-beta1 increases NO synthesis via activation of TGF-beta receptor type II (TbetaRII), and NO inhibits hENT1 expression and activity in HUVECs. HUVECs (passage 2) were used for experiments. Total and hENT1-mediated adenosine transport was measured in the absence or presence of TGF-beta1, NG-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor), S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor), and/or KT-5823 (protein kinase G inhibitor) in control cells and cells expressing a truncated form of TGF-beta1 receptor type II (TTbetaRII). Western blot and real-time PCR were used to determine hENT1 protein abundance and mRNA expression. SLC29A1 gene promoter and specific protein 1 (Sp1) transcription factor activity was assayed. Vascular reactivity was assayed in endothelium-intact or -denuded umbilical vein rings. TGF-beta1 reduced hENT1-mediated adenosine transport, hENT1 protein abundance, hENT1 mRNA expression, and SLC29A1 gene promoter activity, but increased Sp1 binding to DNA. TGF-beta1 effect was blocked by L-NAME and KT-5823 and mimicked by SNAP in control cells. However, TGF-beta1 was ineffective in cells expressing TTbetaRII or a mutated Sp1 consensus sequence. Vasodilatation in response to TGF-beta1 and S-(4-nitrobenzyl)-6-thio-inosine (an ENT inhibitor) was endothelium-dependent and blocked by KT-5823 and ZM-241385. hENT1 is down-regulated by activation of TbetaRII by TGF-beta1 in HUVECs, a phenomenon where NO and Sp1 play key roles. These findings comprise physiological mechanisms that could be important in diseases where TGF-beta1 plasma level is increased as in gestational diabetic mothers or patients with diabetes mellitus.

Nitric oxide producing effect of six extracts from Harungana madagascariensis Lam. ex Poiret (Guttiferae) stem bark

Preliminary pharmacological screening of the aqueous extract from the leaves, stem and root of Harungana madagascariensis Lam. ex Poiret (Guttiferae) are reported to have effect on some cardiovascular diseases like bleeding, cadiopathy, hematuria, and other complications. However, the mechanism underlying its therapeutic effect is not known. The effects of six extracts from the stem bark of H. madagascariensis on equine platelets and chicken hepatocyte cells were explored to examine the involvement of nitric oxide (NO) in platelet aggregation and cell viability capacity. Extracts exhibited concentration dependent platelet aggregation, and synergistically increased NO synthesis in platelets pre-treated with NO and prostaglandin (PG) inhibitors (L-NAME and ASA). The ED50 values observed in the extracts of H. madagascariensis extracts in this study were potentiated and the graphs were shifted to the left in the presence of both inhibitors. However, acontrary effect was observed in berberine, in which its ED50 was shifted to the right non-competitively by the inhibitors. These results demonstrate that extracts from the stem of H. madagascariensis stimulates NO release and this may be a mechanism whereby the constituents of the plant elicit its therapeutic effects in herbal medicine. This study may have relevance in hemostasis, thrombosis and cancer chemotherapy. Key words: Harungana madagascariensis, nitric oxide, equnine platelets, chicken hepatocyte cells, cytotoxicity.

EFFECTS OF HAEMODIALYSIS AND CONTINUOUS AMBULATORY PERITONEAL DIALYSIS ON NITRIC OXIDE SERUM CONCENTRATION IN PATIENTS WITH CHRONIC RENAL FAILURE

Introduction: Nitric oxide (NO) plays an important role in a wide range of physiologic and pathophysiological processes. A major mediator of endothelial function, NO regulates vasodilatory and antithrombotic actions in the vasculature and plays a role in reproductive functions, bronchodilation, bone formation, memory, insulin sensitivity, and gastrointestinal relaxation. Impaired NO bioactivity is strongly associated with endothelial dysfunction. NO, an L-arginine derivative, also exerts a variety of renal and extrarenal physiological and pathophysiological effects. It seems that NO synthetic pathway could have a key role in mediating the complex hemodynamic and hemostatic disorders associated to the progression of renal disease. It remains unclear whether endogenous NO production is increased or decreased in patients with chronic renal failure. The objective of this study was to present the effect of different dialysis treatment on NO serum concentration in patients with chronic renal failure.Patients and Methods: To evaluate endogenous NO production in these patients we studied plasma NO2 and NO3 levels (determined with the Griess method) in patients who underwent regular continuous ambulatory peritoneal dialysis or repeated haemodialysis and in healthy subjects. The study included 51 patients suffering from chronic renal failure and 30 healthy subjects.Results: Our results show that patients with chronic renal failure had a significantly higher NO serum concentration than controls. These values did not differ between patients on haemodialysis and those on continuous ambulatory peritoneal dialysis. NO serum concentration did not differ between female and male independently of the patient’s treatment.Discussion and Conclusion: From obtained results we can concluded that uremia is associated with excessive systemic NO release independently of the patient’s treatment. Alter (increase) NO synthesis may help to explain some pathological changes seen in uraemia such as bleeding tendency, a well-known complication of uremia and hemodialysis hypotension.

Abstract 1618: Role of Nitric Oxide in Regulation of Myocardial Glucose Metabolism; A Potential Mechanism of Cardioprotection

Ischemia induces a switch in myocardial substrate utilization from fatty acids to glucose and stimulates nitric oxide (NO) production. Although increased NO synthesis can ameliorate ischemic injury, mechanism by which NO confers cardioprotection remains unclear. Based on our recent observation that increased NO production contributes to ischemic activation of aldose reductase (AR), we tested the hypothesis that NO prevents ischemic injury by increasing reduction of glucose to sorbitol via polyol pathway over its oxidation to lactate in glycolysis, that could prevent acidosis. Our data show that in isolated mouse hearts subjected to 30 min of ischemia, sorbitol levels increased from 43.2 ± 2.4 to 68.7 ± 4.6 nmol/mg (n=6; P &lt; 0.05) and returned to pre-ischemic levels after 30 min of reperfusion. Treatment with eNOS agonist, bradykinin (10 μM), prior to ischemia, increased AR activity from 27.5 ± 0.7 to 35.9 ± 1.4 mU/mg (n=6; P &lt; 0.01) and decreased lactate levels from 3.7 ± 0.6 to 1.5 ± 0.4 mM (n=6; P &lt; 0.05) upon reperfusion. Bradykinin also decreased creatine kinase (CK) and lactate dehydrogenase (LDH) release by 65 ± 7% and 85 ± 9%, respectively. In contrast, NOS inhibitor, L-NAME (0.1 mM) decreased AR activity (22.4 ± 2.0 mU/mg), but increased the activity of glyceraldehyde-3-phosphate dehydrogenase (from 2.4 ± 0.1 to 3.5 ± 0.2 U/mg) and lactate production (5.8 ± 0.9 mM). AR inhibitor, sorbinil (0.1 mM) increased lactate production (5.9 ± 0.1 mM) and exacerbated ischemic injury. Pretreatment with bradykinin normalized lactate levels and reduced injury caused by sorbinil. To examine whether upregulation of AR is sufficient to confer cardioprotection, cardio-specific AR transgenic mice were generated. Echocardiography confirmed, that these mice had normal myocardial function and when subjected to 30 min of coronary occlusion followed by 24 h of reperfusion displayed reduced myocardial infarct size (49.6 ± 4.1 vs 64.6 ± 3.5 % of the area at risk; transgenic vs wild type; n=7; P &lt; 0.01). Collectively, these results show that NO mediates cardioprotection by increasing glucose flux via polyol pathway, which may represent an adaptive mechanism protecting the heart from ischemic injury. This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania &amp; West Virginia).

Brief femoral artery ischaemia provides protection against myocardial ischaemia–reperfusion injury in rats: the possible mechanisms

The present study was conducted to examine the role of nitric oxide (NO), mitochondrial ATP-sensitive K(+) channels (mito K(+)(ATP) channels) and reactive oxygen species (ROS) and their interdependence in brief femoral artery ischaemia-induced myocardial preconditioning. To assess myocardial injury, myocardial infarction was induced by occlusion followed by reperfusion of the left anterior descending (LAD) coronary artery in anaesthetized rats and was assessed by triphenyl tetrazolium chloride (TTC) staining. Left ventricular function was assessed by left ventricular end-diastolic pressure (LVEDP) and the maximal rate of rise of left ventricular pressure [LV(dP/dt)(max)]. Serum creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) were determined by colorimetric kits. Remote preconditioning (RPC) was induced by 15 min occlusion of femoral arteries followed by 10 min of reperfusion just before LAD coronary artery occlusion. Brief femoral artery ischaemia led to a 61% reduction in myocardial infarct size, 57% reduction in elevated serum LDH and 72% reduction in elevated CK-MB activities, and a significant improvement in LVEDP and LV(dP/dt)(max) compared with control animals. Pretreatment with 5-hydroxydecanoate (5-HD) or l-NAME or N-acetylcystein (NAC) blocked this protective effect of femoral artery ischaemia. Moreover, infusion of l-arginine or diazoxide before coronary artery occlusion markedly reduced the myocardial infarction and improved the left ventricular function. This effect of l-arginine was found to be abolished by the blockade of mito K(+)(ATP) channels with 5-HD and, similarly, the effect of diazoxide was blocked in the presence of a ROS scavenger, NAC. The results suggest that brief femoral artery ischaemia-induced RPC is mediated by a combination of increased NO synthesis, opening of mito K(+)(ATP) channels and increased ROS production. Moreover, it appears that NO is working upstream and acts via activation of mito K(+)(ATP) channels, which subsequently increases the production of ROS.

Prostaglandins inhibit cytochrome P450 4A activity and contribute to endotoxin-induced hypotension in rats via nitric oxide production

Increased production of nitric oxide (NO) and prostaglandins contribute to development of hypotension during endotoxemia. We have previously demonstrated that endotoxemia-induced increase in NO production suppresses renal cytochrome P450 (CYP) 4A expression and activity, and that selective inhibition of inducible NO synthase (iNOS) with 1,3-PBIT restores renal CYP 4A protein and activity and mean arterial pressure (MAP). By using cyclooxygenase (COX) inhibitor indomethacin, we investigated herein whether prostaglandins, via NO production, inhibit renal CYP 4A1 protein expression and CYP 4A activity and contribute to the endotoxin-induced hypotension. In conscious male Sprague-Dawley rats, endotoxin (10 mg/kg, intraperitoneal (i.p.)) reduced MAP, increased serum nitrite and bicyclo PGE2 levels, renal nitrite production and iNOS protein expression, and decreased renal CYP 4A1 protein expression and CYP 4A activity after 4 h injection. All of the endotoxin-induced changes, except for increase in renal nitrite production, were prevented by indomethacin (5 mg/kg, i.p. 1 h after endotoxin). The effects of indomethacin on the endotoxin-induced decrease in MAP, CYP 4A1 protein expression and CYP 4A activity were minimized by the CYP 4A inhibitor, aminobenzotriazole (50 mg/kg, i.p. 1 h after endotoxin). These data suggest that prostaglandins produced during endotoxemia increase iNOS protein expression and NO synthesis, and decrease CYP 4A protein expression and CYP 4A activity and that inhibition of iNOS or COX restores renal CYP 4A protein level and CYP 4A activity and MAP presumably due to increased production of arachidonic acid metabolites derived from CYP 4A.

Urinary excretion of nitrite and nitrate in experimental glomerulonephritis reflects systemic immune activation and not glomerular synthesis.

In immune-induced glomerulonephritis (gn), glomeruli (gl) synthesize nitric oxide (NO), and urinary nitrite (NO2-) excretion is increased. In mammals on a low nitrate (NO3-) diet, urinary NO3- is a measure of endogenous NO3- synthesis. Excretion is increased after administration of macrophage activators, reflecting induction of NO production. To determine whether increased urinary NO2- gn is due to glomerular synthesis we studied urinary NO2-/NO3- in accelerated nephrotoxic nephritis induced by preimmunization with rabbit immunoglobulin G (IgG), followed by rabbit anti-rat nephrotoxic globulin, and in control rats similarly preimmunized with rabbit IgG, but followed by normal rabbit serum. Both urinary NO2- and NO3- were increased by i.p. preimmunization with rabbit IgG (peak 463 +/- 171 nmol NO2-/60.3 +/- 9.4 nmol NO3-/24 h, P < 0.001 for both NO2- and NO3- compared with preimmunization levels). Repeat immunization with i.v. rabbit anti-rat nephrotoxic globulin (nephritic rats) or normal rabbit globulin (control rats) again increased urinary NO2- and NO3-. There was no statistically significant difference in urinary NO2- and NO3- levels between nephritic rats where globulin had nephrotoxic activity and the control rats injected with normal rabbit globulin, despite increased NO2- synthesis in ex vivo nephritic glomeruli after nephrotoxic globulin (7.9 +/- 1.9 nmol/2000 gl/48 h; controls 3.2 +/- 1.0 nmol/2000 gl/48 h). Thus neither urinary NO2- nor NO3- levels reflect local activation of the NO pathway in glomeruli. As reported for other stimulants, we show here that systemic stimulation with foreign antigen increased NO synthesis.

Nitric oxide production of T lymphocytes is increased in rheumatoid arthritis

Experimental and clinical evidence for T cell involvement in the pathology of rheumatoid arthritis (RA) is compelling, and points to a local dysregulation of T cell function in the inflamed joint. Nitric oxide (NO) has been shown to regulate T cell function under physiological conditions, but overproduction of NO may contribute to lymphocyte dysfunction characteristic of RA. Several investigations in patients with RA have documented evidence of increased NO synthesis, but these studies have focused largely on macrophage-derived NO and its impact on innate immune and inflammatory responses. In this study, we set out to explore the contribution that T cells make to NO production. We find that T cells from RA patients produce >2.5 times more NO than healthy donor T cells (p<0.001). Although NO is an important physiological mediator of mitochondrial biogenesis, mitochondrial mass is similar in RA and control T cells. In contrast, increased NO production is associated with increased cytoplasmic Ca(2+) concentrations in RA T cells (p<0.001). In vitro treatment of human peripheral blood lymphocytes, or Jurkat cells with TNF increases NO production (p=0.006 and p=0.001, respectively), whilst infliximab treatment in RA patients decreases T cell derived NO production within 6 weeks of the first infusion (p=0.005). Together, these data indicate that TNF induced NO production in T lymphocytes may contribute to perturbations of immune homeostasis in RA.

Ferroportin Modulates Macrophage‐Mediated Immune Responses

The iron exporter ferroportin (FPN) plays a key role in iron homeostasis. Infection and inflammation down‐regulate FPN to limit iron efflux from macrophages. Whether FPN contributes to other macrophage immune responses is unknown. In particular, the intersection of iron metabolism and nitric oxide (NO) production by macrophage inducible nitric oxide synthase (iNOS) has been the subject of much debate. Induction of NO synthesis by macrophage iNOS is a key inflammatory response mechanism triggered by cytokines such as INF‐γ, TNF‐α, and IL‐1 and microbial lipopolysaccharide (LPS). While heme iron is required for iNOS dimerization, it remains unclear whether iron increases or decreases NO production. J774 murine macrophages over‐expressing FPN were used to investigate the influence of iron efflux on iNOS activity. When challenged with LPS or Mycobacterium tuberculosis (Mtb), control J774 cells increase NO synthesis as measured by the Greiss reaction. J774 cells over‐expressing FPN have significantly impaired NO induction in response to LPS or Mtb. Western blot analysis shows that increased NO synthesis in control J774 cells is accompanied by increased iNOS expression, but that upregulation of iNOS protein is markedly reduced when J744 cells over‐expressing FPN are challenged with LPS or Mtb. These results suggest that iron efflux by FPN can limit macrophage‐mediated immune responses.

Effect of Telmisartan on Nitric Oxide–Asymmetrical Dimethylarginine System

Telmisartan, in addition to blocking angiotensin (Ang) II type 1 receptor (AT(1)R), activates peroxisome proliferator activated receptor gamma (PPARgamma) signaling that interferes with nitric oxide (NO) system. Because aging of endothelial cells (ECs) is hallmarked by a reduction in NO synthesis, we hypothesized that telmisartan increases NO formation by regulated asymmetrical dimethylarginine (ADMA)-dimethylarginine dimethylaminohydrolase (DDAH)-system through blocking AT(1)R and activating PPARgamma signaling. To test this hypothesis, ECs were cultured with telmisartan, eprosartan, Ang II, and GW9662 (PPARgamma antagonist) until the twelfth passage. During the process of aging, PPARgamma protein expression decreased significantly, whereas the expression of AT(1)R increased. Telmisartan reversed these effects and dose-dependently decreased reactive oxygen species and 8-iso-prostaglandin (PG) F(2alpha) formation. This effect was associated with an upregulated activity and protein expression of DDAH, accompanied by a decrease in ADMA concentration, an increase in NO metabolites, and delayed senescence. Blockade of PPARgamma signaling by GW9662 or PPARgamma small-interference RNA prevented the effect of telmisartan on ADMA-DDAH-NO system. Coincubation with Ang II did not affect the effect of telmisartan-delayed senescence, whereas Ang II itself accelerated endothelial aging. Moreover, AT(1)R blocker eprosartan that did not influence PPARgamma protein expression had no effect on ADMA system and senescence. We have demonstrated that telmisartan mainly by activating PPARgamma signaling can alter the catabolism and release of ADMA as an important cardiovascular risk factor. We therefore propose that telmisartan translationally and posttranslationally upregulated DDAH expression via activation of PPARgamma signaling, causing ADMA to diminish and increase NO synthesis sufficient to delay senescence.

Dimethylarginine Dimethylaminohydrolase Overexpression Enhances Insulin Sensitivity

Previous studies suggest that nitric oxide (NO) may modulate insulin-induced uptake of glucose in insulin-sensitive tissues. Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase (NOS). We hypothesized that a reduction in endogenous ADMA would increase NO synthesis and thereby enhance insulin sensitivity. To test this hypothesis we used a transgenic mouse in which we overexpressed human dimethylarginine dimethylaminohydrolase (DDAH-I). The DDAH-I mice had lower plasma ADMA at all ages (22 to 70 wk) by comparison to wild-type (WT) littermates. With a glucose challenge, WT mice showed a prompt increase in ADMA, whereas DDAH-I mice had a blunted response. Furthermore, DDAH-I mice had a blunted increase in plasma insulin and glucose levels after glucose challenge, with a 50% reduction in the insulin resistance index, consistent with enhanced sensitivity to insulin. In liver, we observed an increased Akt phosphorylation in the DDAH-I mice after i.p. glucose challenge. Incubation of skeletal muscle from WT mice ex vivo with ADMA (2 mumol/L) markedly suppressed insulin-induced glycogen synthesis in fast-twitch but not slow-twitch muscle. These findings suggest that the endogenous NOS inhibitor ADMA reduces insulin sensitivity, consistent with previous observations that NO plays a role in insulin sensitivity.

Oral Sorbent AST-120 Increases Renal NO Synthesis in Uremic Rats

The urine level of nitric oxide (NO) metabolites, i.e., nitrates/nitrites (NOx), in chronic renal failure (CRF) is decreased because of reduced renal synthesis of NO. We determined whether the administration of an oral sorbent, AST-120, increases the urine level of NOx and the renal expression of nitric oxide synthase (NOS) isoforms in CRF rats. Chronic renal failure rats were produced by 4/5 nephrectomy. Rats were randomized into two groups: CRF control rats, and AST-120-treated CRF rats. The AST-120 was administered to the rats at a dose of 4 g/kg with powder chow for 16 weeks, whereas powder chow alone was administered to control rats. The urine levels of NOx were measured by using a NOx colorimetric assay kit. The expression of endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) in the kidney was determined by immunohistochemistry. Serum and urine levels of indoxyl sulfate were determined by high-performance liquid chromatography. Urine levels of NOx and the expression of glomerular eNOS and tubulointerstitial nNOS were significantly decreased in CRF rats compared with normal rats. The administration of AST-120 to CRF rats significantly increased urine levels of NOx and the expression of glomerular eNOS and tubulointerstitial nNOS. The administration of AST-120 to CRF rats significantly decreased urine and serum levels of indoxyl sulfate. The oral sorbent AST-120 increases NO synthesis in the kidneys of uremic rats by increasing the renal expression of eNOS and nNOS, through alleviation of indoxyl sulfate overload on the kidney.

Abnormally increased nitric oxide synthesis and increased endothelin‐1 in plasma in patients with obstructive sleep apnoea

Objective. Obstructive sleep apnoea (OSA) is a risk factor for cardiovascular morbidity and mortality. The mechanism is unknown, but endothelial dysfunction might contribute. We tested the hypothesis that patients with OSA have abnormal nitric oxide (NO) synthesis, which results in an abnormal response in blood pressure, renal hemodynamics, renal tubular function and vasoactive hormones in response to inhibition of the NO synthesis with l‐NMMA. Material and methods. A randomized, placebo‐controlled, single‐blinded, crossover study was done in 13 OSA patients and 14 controls. We measured changes in systolic and diastolic blood pressure (SBP and DBP), pulse rate, glomerular filtration rate, renal plasma flow, fractional excretion of sodium and lithium and plasma concentrations of vasoactive hormones after injection of l‐NMMA and placebo. Results.l‐NMMA induced a significant increase in SBP and DBP in both groups. The placebo‐corrected increase in DBP was more pronounced in patients with OSA than in controls (9 (5–11) versus 3 (1–6) mmHg; p = 0.01). Endothelin‐1 (ET‐1) was significantly higher in patients compared with controls (1.1 (1.0–1.3) versus 0.8 (0.7–0.9) pg/mL; p<0.01). In controls, l‐NMMA induced a small increase in ET‐1, while no changes were seen in patients. The placebo‐corrected change in ET‐1 was lower in patients compared to controls (−01 (−0.3–0.0) versus 0.1 (0.0–0.1) pg/mL; p = 0.04). Conclusions. Patients with OSA have abnormally increased NO synthesis and an increased unresponsive level of ET‐1 in plasma. This might be due to an imbalance between NO synthesis and ET‐1 production.

The NADPH oxidase inhibitor apocynin induces nitric oxide synthesis via oxidative stress

We have recently shown that apocynin elicits an oxidative stress in N11 mouse glial cells and other cell types. Here we report that apocynin increased the accumulation of nitrite, the stable derivative of nitric oxide (NO), in the extracellular medium of N11 cell cultures, and the NO synthase (NOS) activity in cell lysates. The increased synthesis of NO was associated with increased expression of inducible NOS (iNOS) mRNA, increased nuclear translocation of the redox-sensitive transcription factor NF-kappa B and decreased intracellular level of its inhibitor IkB alpha. These effects, accompanied by increased production of H2O2, were very similar to those observed after incubation with bacterial lipopolysaccharide (LPS) and were inhibited by catalase. These results suggest that apocynin, similarly to LPS, induces increased NO synthesis by eliciting a generation of reactive oxygen species (ROS), which in turn causes NF-kappa B activation and increased expression of iNOS. Therefore, the increased bioavailability of NO reported in the literature after in vivo or in vitro treatments with apocynin might depend, at least partly, on the drug-elicited induction of iNOS, and not only on the inhibition of NADPH oxidase and the subsequent decreased scavenging of NO by oxidase-derived ROS, as it is often supposed.

Nitric Oxide-Dependent Neovascularization Role in the Lower Extremity Disease

Peripheral arterial occlusive disease (PAD) describes vascular disorders associated with ischemia and PAD affects about 8 million people in the United States. Moreover, PAD's prevalence can increase dramatically if cardiovascular disease is present. In healthy individuals reducing blood flow through the lower extremity is followed by a physiological process to limit ischemia in the distal tissue. This process is called revascularization and impairing revascularization results in PAD. Studies suggest nitric oxide (NO) maybe involved in the ischemia-dependent hindlimb revascularization process. NO is increased in the ischemic hindlimb and eliminating NO impairs the revascularization process. Moreover, restoring NO improves hindlimb revascularization. NO may be acting through its effects on vascular tone, cell migration, or extracellular matrix degradation. The present review illustrates nitric oxide's critical role in the ischemia-induced hindlimb revascularization. Thus, restoring normal NO levels in diseased arteries may represent a viable therapeutic avenue by supplementing exogenous NO or employing therapeutic strategies to either increase NO synthesis and its messengers or decrease NO catabolism.

The direct action of estrone on vascular tissue involves genomic and non-genomic actions

A two step model mechanism of steroid action has been recently postulated. In this study, we test the hypothesis that, the biochemical action of estrone (E(1)) on vascular tissue could be performed via genomic and non-genomic actions. Rat aortic rings or vascular smooth muscle cell cultures (VSMC) were used to test the effect of the hormone on nitric oxide (NO) production, protein kinases activities and cell proliferation. Our data showed that estrone increased NO synthesis between 30 s and 20 min treatment, and this stimulatory effect was dependent on MAPK cascade activation, since it was prevented in the presence of a MAPK inhibitor (PD98059). Using a phosphorylation assay, we also showed that E(1) significantly increased MAPK activity. The effect of the hormone on PKC activity was measured in concentrations and time course studies. Direct treatment of rat aortic homogenates with E(1) significantly enhanced PKC activity (1-10 fold increase, p<0.01) at all concentrations (1; 10; 50 nM) and time tested (1-10 min). We demonstrated that 24 h of E(1) treatment markedly increased VSMC proliferation (53% above control), and this effect was suppressed by a PKC inhibitor. The rapid and the long term effects of the hormone were completely suppressed in the presence of an estradiol receptor antagonist (ICI 182780). In summary, we provided evidence that, the steroid exerts both non-genomic and genomic actions, the former associated with MAPK kinase dependent on NO production, and the latter related with induction of VSMC proliferation involving PKC pathway activation.