The rs2682826 Polymorphism of the NOS1 Gene Is Associated with the Degree of Disability of Erectile Dysfunction.

Erectile dysfunction (ED) is a common male disorder, often associated with cardiovascular disease and ageing. The Sildenafil, a PDE5 inhibitor, can improve the erectile function by prolonging the nitric oxide (NO) downstream effect. NO is a molecule of pivotal importance in erection physiology and is mainly produced by neuronal nitric oxide synthase (nNOS) and endothelial NO synthase (eNOS). While it has been shown that eNOS and nNOS genetic polymorphisms could be associated with Sildenafil responsiveness in ED, no study so far has assessed whether nNOS polymorphisms and PDE5A polymorphism could be associated with increased risk to ED or with intensity of symptoms. A total of 119 ED patients and 114 controls were studied, with evaluation of the clinical disability by the International Index for Erectile Function instrument, plasma assessment of nitrite levels and genomic DNA analysis regarding the rs41279104 and rs2682826 polymorphisms of the NOS1 gene and the rs2389866, rs3733526 and rs13124532 polymorphisms of the PDE5A gene. We have found a significant association of the rs2682826 with lower IIEF scores in the clinical ED group. While this result should be confirmed in other populations, it may be helpful in establishing a genetic panel to better assess disease risk and prognosis on ED therapy.

The protective role of neuronal nitric oxide synthase in endothelial vasodilation in chronic ß-adrenoceptor overstimulation.

AIMS: Nitric oxide synthases (NOSs) are key enzymes regulating vascular function. Previously, we reported that ß-adrenergic (ß-AR) overstimulation, a common feature of cardiovascular diseases, did not impair endothelium-dependent vasodilation, although it resulted in endothelial NOS (eNOS) uncoupling and reduced NO bioavailability. In addition to NO, neuronal NOS (nNOS) produces H2O2, which contributes to vasodilation. However, there is limited information regarding vascular ß-AR signaling and nNOS. In the present study, we assessed the possible role of nNOS-derived H2O2 and caveolins on endothelial vasodilation function following ß-AR overstimulation. MAIN METHODS: Male C57BL/6 wild-type and nNOS knockout mice (nNOS-/-) were treated with the ß-AR agonist isoproterenol (ISO, 15 mg·kg-1·day-1, s.c.) or vehicle (VHE) for seven days. Relaxation responses of aortic rings were evaluated using wire myograph and H2O2 by Amplex Red. KEY FINDINGS: Acetylcholine- or calcium ionophore A23187-induced endothelium-dependent relaxation was similar in aortic rings from VHE and ISO. However, this relaxation was significantly reduced in aortas from ISO compared to VHE when (1) caveolae were disrupted, (2) nNOS was pharmacologically inhibited or genetically suppressed and (3) H2O2 was scavenged. NOS-derived H2O2 production was higher in the aortas of ISO mice than in those of VHE mice. Aortas from ISO-treated mice showed increased expression of caveolin-1, nNOS and catalase, while caveolin-3 expression did not change. SIGNIFICANCE: The results suggest a role of caveolin-1 and the nNOS/H2O2 vasodilatory pathway in endothelium-dependent relaxation following ß-AR overstimulation and reinforce the protective role of nNOS in cardiovascular diseases associated with high adrenergic tone.

Decreased expression of neuronal nitric oxide synthase contributes to the endothelial dysfunction associated with cigarette smoking in human.

Endothelial nitric oxide synthase (eNOS) malfunctioning has been proposed to contribute to the endothelial damage produced by cigarette. Besides eNOS, neuronal NOS (nNOS) is also expressed in most vascular tissues and plays an important role in the endothelium-dependent vascular relaxation. We hypothesize that nNOS may contribute to the endothelium dysfunction produced by cigarette in smokers. Vascular function was assessed in human resistance mesenteric arteries using a wire myograph, the level of protein expression by Western blot, eNOS and nNOS localization by immunofluorescence. Measurement of NO was assessed by fluorescence microscopy. Arteries of smokers showed impaired endothelium-dependent vascular relaxation in response to acetylcholine. Pharmacological nonselective blockade of NOS with l-NAME and selective nNOS blockade with inhibitor 1 reduced the relaxation of the mesenteric artery of both smokers and nonsmokers. Interestingly, the inhibitory effect of NOS inhibitors was greater in nonsmokers than in smokers. The expression of total nNOS and eNOS and the level of phosphorylation at eNOS-pSer1177 were reduced in arteries of smokers as compared with nonsmokers. No differences between groups were observed in the expression of total COX-1, COX-2, catalase and SOD-1. Immunofluorescence analysis showed the presence of nNOS in the vascular endothelium in both groups. Acetylcholine-induced NO production was impaired in arteries from smokers as compared to nonsmokers. Selective inhibition of nNOS caused a decreased in NO production, which was greater in nonsmokers than in smokers. Our data show that a decrease in nNOS expression contributes to the endothelial dysfunction caused by cigarette smoking in human.

Cognitive interference as a possible therapeutic strategy to prevent expression of benzodiazepine withdrawal.

Benzodiazepines are usually prescribed for anxiety and sleep disorders in long-term schedules that may cause drug dependence. Discontinuation after prolonged administration may lead to withdrawal expression, being anxiety the most predominant sign. The context-dependent associative learning process that underlies diazepam dependence can be interfered by pre-exposure to the drug administration context, an effect known as latent inhibition. Considering this background, the primary aim of the present investigation is to develop a therapeutic strategy to prevent diazepam withdrawal in male Wistar rats by interfering with this learning process. Nitric oxide is a crucial player in learning and memory, hippocampal synaptic transmission and in diazepam withdrawal. Then, a secondary goal is to determine how latent inhibition could alter functional plasticity and neuronal nitric oxide synthase enzyme (NOS-1) expression within the hippocampus, by using multi-unitary cell recordings and Western blot, respectively. Our results indicate that chronic diazepam treated animals under latent inhibition did not show anxiety, or changes in hippocampal synaptic transmission, but a significant reduction in NOS-1 expression was observed. Accordingly, pharmacological NOS-1 inhibition resembles behavioral and electrophysiological changes induced by latent inhibition. Contrary, diazepam treated animals under Control protocol expressed anxiety and evidenced an increased hippocampal-plasticity, without alterations in NOS-1 expression. In conclusion, manipulation of the contextual cues presented during diazepam administration may be considered as an effective non-pharmacological tool to prevent the withdrawal syndrome. This behavioral strategy may influence hippocampal synaptic transmission, probably by alterations in nitric oxide signaling pathways in this structure.

Autonomic dysregulation at multiple sites is implicated in age-associated underactive bladder in female mice.

AIMS: To evaluate the functional and molecular alterations of contractile and relaxant machinery in the bladder and urethra that lead to the underactive bladder (UAB) in old female mice. METHODS: Female young (3-months) and old (18-months) C57BL/6 mice were used. Urodynamic was assessed in awake and anaesthetized mice. Electrical-field stimulation (EFS) and concentration-response curves to contractile and relaxing agents in isolated bladders and urethras were performed. Messenger RNA (mRNA) expressions of muscarinic, adrenergic, and transient receptor potential vanilloid-4 (TRPV4), and of the enzymes tyrosine hydroxylase and neuronal nitric oxide synthase (nNOS) were determined. Bladder cyclic adenosine monophosphate (cAMP) levels were measured. RESULTS: Cystometry in old mice showed incapacity to produce bladder emptying. On filter paper, old mice showed reduced urinary spots. Compared to the young group, bladder contractions induced by EFS and carbachol were lower in old mice. The ß3 -adrenoceptor agonist mirabegron promoted higher bladder relaxation and elevation of cAMP levels in old mice. In old mice urethras, the α1a -adrenoceptor agonist phenylephrine produced higher contractions, but no differences were found for the NO donor sodium nitroprusside-induced relaxations. In old mice, increased mRNA expressions of ß3 - and α1a -adrenoceptors in bladder and urethra were found, respectively, whereas the muscarinic M2 and M3 receptors and ß2 -adrenoceptors did not change between groups. Reduced mRNA expressions of tyrosine hydroxylase and nNOS were found in old mouse urethras. Additionally, TRPV4 expression was reduced in bladder urothelium from old mice. CONCLUSION: Age-associated mouse UAB is the result of autonomic dysfunction at multiple levels leading to the less sensitive and overrelaxed bladder, along with urethral hypercontractility.

Striatal Protection in nNOS Knock-Out Mice After Quinolinic Acid-Induced Oxidative Damage.

Under pathological conditions, nitric oxide can become a mediator of oxidative cellular damage, generating an unbalance between oxidant and antioxidant systems. The participation of neuronal nitric oxide synthase (nNOS) in the neurodegeneration mechanism has been reported; the activation of N-methyl-D-aspartate (NMDA) receptors by agonist quinolinic acid (QUIN) triggers an increase in nNOS function and promotes oxidative stress. The aim of the present work was to elucidate the participation of nNOS in QUIN-induced oxidative stress in knock-out mice (nNOS-/-). To do so, we microinjected saline solution or QUIN in the striatum of wild-type (nNOS +/+), heterozygote (nNOS+/-), and knock-out (nNOS-/-) mice, and measured circling behavior, GABA content levels, oxidative stress, and NOS expression and activity. We found that the absence of nNOS provides a protection against striatal oxidative damage induced by QUIN, resulting in decreased circling behavior, oxidative stress, and a partial protection reflected in GABA depletion. We have shown that nNOS-derived NO is involved in neurological damage induced by oxidative stress in a QUIN-excitotoxic model.

Neuronal nitric oxide synthase contributes to the normalization of blood pressure in medicated hypertensive patients.

Neuronal nitric oxide synthase (nNOS) is expressed in the cardiovascular system and besides NO, generates H2O2. nNOS has been proposed to contribute to the control of blood pressure in healthy humans. The aim of this study was to verify the hypothesis that nNOS can contribute to the control of vascular relaxation and blood pressure in hypertensive patients undergoing drug treatment. The study was conducted in resistance mesenteric arteries from 63 individuals, as follows: 1) normotensive patients; 2) controlled hypertensive patients (patients on antihypertensive treatment with blood pressure normalized); 3) uncontrolled hypertensive patients (patients on antihypertensive treatment that remained hypertensive). Only mesenteric arteries from uncontrolled hypertensive patients showed impaired endothelium-dependent vasorelaxation in response to acetylcholine (ACh). Selective nNOS blockade with inhibitor 1 and catalase, which decomposes H2O2, decreased vasorelaxation in the three groups. However, the inhibitory effect was greater in controlled hypertensive patients. Decreased eNOS expression was detected in both uncontrolled and controlled hypertensive groups. Interestingly nNOS expression and ACh-stimulated H2O2 production were greater in controlled hypertensive patients, than in the other groups. ACh-stimulated NO production was lower in controlled hypertensive when compared to normotensive patients, while uncontrolled hypertensive patients showed the lowest levels. Catalase and nNOS blockade inhibited ACh-induced H2O2 production. In conclusion, nNOS-derived H2O2 contributes to the endothelium-dependent vascular relaxation in human resistance mesenteric arteries. The endothelial dysfunction observed in uncontrolled hypertensive patients involves decreased eNOS expression and NO production. The normalization of vascular relaxation and blood pressure in controlled hypertensive patients involves increased nNOS-derived H2O2 and NO production.

The essential role of hypothalamic paraventricular nucleus nNOS in the modulation of autonomic control in exercised rats.

Nitric oxide (NO), an intercellular signaling molecule is relevant for circulatory autonomic control. Brain NO synthase (NOS) and NO levels were downregulated in pathological conditions, but rescued after exercise training. We hypothesized that exercise training was also able to improve NO modulation within the hypothalamic paraventricular nucleus (PVN) of healthy rats. Male Wistar rats were submitted to two 4-weeks protocols: i) swimming training (T) or kept sedentary (S), ii) l-arginine (62,5 mg/mL, 1 mL/day p. o.) or vehicle supplementation. Rats underwent stereotaxic surgery (PVN bilateral guide cannulas) and chronic catheterization of artery/vein. Arterial pressure (AP), heart rate (HR) and baroreflex sensitivity were recorded in conscious rats at rest and following a selective nNOS inhibitor (Nw-Propyl-l-Arginine, 4 nmol/100 nL) within the PVN. Rats were deeply anesthetized for brain perfusion/harvesting after respiratory arrest. In separate groups (T and S, l-arginine and Vehicle supplemented) not submitted to PVN cannulation, fresh and fixed brains were obtained for gene and protein nNOS expression (qPCR and immunohistochemistry) and nitrite levels (Griess reaction). T and l-arginine treatment were accompanied by resting bradycardia, augmented parasympathetic and reduced sympathetic activity to heart and vessels (power spectral analysis) and increased baroreflex sensitivity (†P < 0.05). In contrast, PVN nNOS inhibition blocked/attenuated these effects in addition to significantly increase in resting MAP and HR (with larger effects in T and l-arginine treated rats vs. respective controls, †P < 0.05). T increased nNOS gene and protein expression within the ventromedial and posterior PVN nuclei (†P < 0.05). PVN nitirite levels were also increased in T and l-arginine groups (†P < 0.05). Data strongly suggest that training by increasing NO availability within PVN preautonomic nuclei favors both the slow down of sympathetic and the augmentation of parasympathetic activity and facilitates baroreflex control, therefore improving autonomic regulation of the heart in healthy rats.

Retinal exposure to high glucose condition modifies the GABAergic system: Regulation by nitric oxide.

Diabetic retinopathy is a severe retinal complication that diabetic patients are susceptible to present. Although this disease is currently characterized as a microvascular disease, there is growing evidence that neural changes occur and maybe precede vascular impairments. Using chicken retina, an avascular tissue with no direct contact with blood vessels and neural retina, this study aimed to evaluate the influence of acute exposure to high glucose concentration in the retinal GABAergic system, and the role of nitric oxide (NO) in this modulation. Therefore, in ex vivo experiments, retinas were incubated in control (10 mM glucose) or high glucose condition (35 mM) for 30 min. By using DAF-FM to evaluate NO production, it was possible to show that high glucose (HG) significantly increased NO levels in the outer nuclear layer, inner nuclear layer (outer and inner portion), and inner plexiform layer. It was also observed that HG increased GABA immunoreactivity (IR) in amacrine and horizontal cells. HG did not change glutamic acid decarboxylase-IR, whereas it decreased GABA Transporter (GAT) 1-IR and increased GAT-3-IR. The co-treatment with 7-NI, an inhibitor of neuronal nitric oxide synthase (nNOS), blocked all changes stimulated by HG exposure. The concomitant exposure with SNAP-5114, a GAT-2/3 inhibitor, blocked the increase in GABA-IR caused by HG incubation. Therefore, our data suggest that hyperglycemia induces GABA accumulation in the cytosol by modulating GABA transporters. This response is dependent on NO production and signaling.

Fish oil prevents rodent anxious states comorbid with diabetes: A putative involvement of nitric oxide modulation.

There is an urgent need to understand the pathophysiological mechanisms related to anxiety associated with diabetes, seeking more effective alternative treatments to treat it. For that, the effect of a preventive and prolonged treatment with fish oil (FO), a source of omega-3 polyunsaturated fatty acid, was tested in streptozotocin-diabetic (DBT) rats submitted to the anxiety tests. Additionally, an immunohistochemistry for neuronal NO synthase (nNOS) was performed in brain areas related to anxiety, such as lateral amygdala (AMY), hippocampus (HIP) and dorsolateral periaqueductal gray (dlPAG). Lastly, the effect of NO precursor L-arginine (L-Arg) or nNOS inhibitor 7-nitroindazole (7-NI) was tested in DBT animals treated with vehicle (VEH) or FO. Our data demonstrated that vehicle-treated DBT animals exhibited a more pronounced anxiogenic-like response and also presented high nNOS levels in the AMY, HIP and rostral dlPAG, what were both significantly prevented by FO treatment. This treatment was able to prevent the impairment in locomotor activity besides improving the high glycemic levels in DBT rats. Interestingly, while injection of 7-NI or L-Arg in VEH-treated DBT animals induced an anxiogenic-like and anxiolytic-like effect, respectively; the previous treatment with both L-Arg and 7-NI in FO-DBT animals abolished the anxiolytic-like effect induced by FO treatment. Altogether, our data support the hypothesis that a dysregulation in the NO production in brain areas as AMY, HIP and dlPAG may contribute to the mechanisms that link anxiety and diabetes, and the prevention of nNOS brain expression changes induced by a prolonged treatment with FO may be an important mechanism related to its anxiolytic-like effect.

The synthetic peptide PnPP-19 induces peripheral antinociception via activation of NO/cGMP/KATP pathway: Role of eNOS and nNOS.

BACKGROUND: and purpose: The peptide PnPP-19, derived from the spider toxin PnTx2-6 (renamed as δ-CNTX-Pn1c), potentiates erectile function by activating the nitrergic system. Since NO has been studied as an antinociceptive molecule and PnPP-19 is known to induce peripheral antinociception, we intended to evaluate whether PnPP-19 could induce peripheral antinociception through activation of this pathway. EXPERIMENTAL APPROACH: Nociceptive thresholds were measured by paw pressure test. PGE2 (2 µg/paw) was administered intraplantarly together with PnPP-19 and inhibitors/blockers of NOS, guanylyl cyclase and KATP channels. The nitrite concentration was accessed by Griess test. The expression and phosphorylation of eNOS and nNOS were determined by western blot. KEY RESULTS: PnPP-19 (5, 10 and 20 µg/paw) induced peripheral antinociception in rats. Administration of NOS inhibitor (L-NOarg), selective nNOS inhibitor (L-NPA), guanylyl cyclase inhibitor (ODQ) and the blocker of KATP (glibenclamide) partially inhibited the antinociceptive effect of PnPP-19 (10 µg/paw). Tissue nitrite concentration increased after PnPP-19 (10 µg/paw) administration. Expression of eNOS and nNOS remained the same in all tested groups, however the phosphorylation of nNOS Ser852 (inactivation site) increased and phosphorylation of eNOS Ser1177 (activation site) decreased after PGE2 injection. Administration of PnPP-19 reverted this PGE2-induced effect. CONCLUSIONS AND IMPLICATIONS: The peripheral antinociceptive effect induced by PnPP-19 is resulting from activation of NO-cGMP-KATP pathway. Activation of eNOS and nNOS might be required for such effect. Our results suggest PnPP-19 as a new drug candidate to treat pain and reinforce the importance of nNOS and eNOS activation, as well as endogenous NO release, for induction of peripheral antinociception.

Neuronal Nitric Oxide Synthase in Vascular Physiology and Diseases.

The family of nitric oxide synthases (NOS) has significant importance in various physiological mechanisms and is also involved in many pathological processes. Three NOS isoforms have been identified: neuronal NOS (nNOS or NOS 1), endothelial NOS (eNOS or NOS 3), and an inducible NOS (iNOS or NOS 2). Both nNOS and eNOS are constitutively expressed. Classically, eNOS is considered the main isoform involved in the control of the vascular function. However, more recent studies have shown that nNOS is present in the vascular endothelium and importantly contributes to the maintenance of the homeostasis of the cardiovascular system. In physiological conditions, besides nitric oxide (NO), nNOS also produces hydrogen peroxide (H2O2) and superoxide ([Formula: see text]) considered as key mediators in non-neuronal cells signaling. This mini-review highlights recent scientific releases on the role of nNOS in vascular homeostasis and cardiovascular disorders such as hypertension and atherosclerosis.

Endothelial dysfunction in DOCA-salt-hypertensive mice: role of neuronal nitric oxide synthase-derived hydrogen peroxide.

Endothelial dysfunction is a common problem associated with hypertension and is considered a precursor to the development of micro- and macro-vascular complications. The present study investigated the involvement of nNOS (neuronal nitric oxide synthase) and H2O2 (hydrogen peroxide) in the impaired endothelium-dependent vasodilation of the mesenteric arteries of DOCA (deoxycorticosterone acetate)-salt-hypertensive mice. Myograph studies were used to investigate the endothelium-dependent vasodilator effect of ACh (acetylcholine). The expression and phosphorylation of nNOS and eNOS (endothelial nitric oxide synthase) were studied by Western blot analysis. Immunofluorescence was used to examine the localization of nNOS and eNOS in the endothelial layer of the mesenteric artery. The vasodilator effect of ACh is strongly impaired in mesenteric arteries of DOCA-salt-hypertensive mice. Non-selective inhibition of NOS sharply reduced the effect of ACh in both DOCA-salt-hypertensive and sham mice. Selective inhibition of nNOS and catalase led to a higher reduction in the effect of ACh in sham than in DOCA-salt-hypertensive mice. Production of H2O2 induced by ACh was significantly reduced in vessels from DOCA-salt-hypertensive mice, and it was blunted after nNOS inhibition. The expression of both eNOS and nNOS was considerably lower in DOCA-salt-hypertensive mice, whereas phosphorylation of their inhibitory sites was increased. The presence of nNOS was confirmed in the endothelial layer of mesenteric arteries from both sham and DOCA-salt-hypertensive mice. These results demonstrate that endothelial dysfunction in the mesenteric arteries of DOCA-salt-hypertensive mice is associated with reduced expression and functioning of nNOS and impaired production of nNOS-derived H2O2 Such findings offer a new perspective for the understanding of endothelial dysfunction in hypertension.

Control of the neurovascular coupling by nitric oxide-dependent regulation of astrocytic Ca(2+) signaling.

Neuronal activity must be tightly coordinated with blood flow to keep proper brain function, which is achieved by a mechanism known as neurovascular coupling. Then, an increase in synaptic activity leads to a dilation of local parenchymal arterioles that matches the enhanced metabolic demand. Neurovascular coupling is orchestrated by astrocytes. These glial cells are located between neurons and the microvasculature, with the astrocytic endfeet ensheathing the vessels, which allows fine intercellular communication. The neurotransmitters released during neuronal activity reach astrocytic receptors and trigger a Ca(2+) signaling that propagates to the endfeet, activating the release of vasoactive factors and arteriolar dilation. The astrocyte Ca(2+) signaling is coordinated by gap junction channels and hemichannels formed by connexins (Cx43 and Cx30) and channels formed by pannexins (Panx-1). The neuronal activity-initiated Ca(2+) waves are propagated among neighboring astrocytes directly via gap junctions or through ATP release via connexin hemichannels or pannexin channels. In addition, Ca(2+) entry via connexin hemichannels or pannexin channels may participate in the regulation of the astrocyte signaling-mediated neurovascular coupling. Interestingly, nitric oxide (NO) can activate connexin hemichannel by S-nitrosylation and the Ca(2+)-dependent NO-synthesizing enzymes endothelial NO synthase (eNOS) and neuronal NOS (nNOS) are expressed in astrocytes. Therefore, the astrocytic Ca(2+) signaling triggered in neurovascular coupling may activate NO production, which, in turn, may lead to Ca(2+) influx through hemichannel activation. Furthermore, NO release from the hemichannels located at astrocytic endfeet may contribute to the vasodilation of parenchymal arterioles. In this review, we discuss the mechanisms involved in the regulation of the astrocytic Ca(2+) signaling that mediates neurovascular coupling, with a special emphasis in the possible participation of NO in this process.

Involvement of dorsal hippocampus glutamatergic and nitrergic neurotransmission in autonomic responses evoked by acute restraint stress in rats.

The dorsal hippocampus (DH) is a structure of the limbic system that is involved in emotional, learning and memory processes. There is evidence indicating that the DH modulates cardiovascular correlates of behavioral responses to stressful stimuli. Acute restraint stress (RS) is an unavoidable stress situation that evokes marked and sustained autonomic changes, which are characterized by elevated blood pressure (BP), intense heart rate (HR) increase and a decrease in cutaneous temperature. In the present study, we investigated the involvement of an N-methyl-D-aspartate (NMDA) glutamate receptor/nitric oxide (NO) pathway of the DH in the modulation of autonomic (arterial BP, HR and tail skin temperature) responses evoked by RS in rats. Bilateral microinjection of the NMDA receptor antagonist AP-7 (10 nmol/500 nL) into the DH attenuated RS-evoked autonomic responses. Moreover, RS evoked an increase in the content of NO2/NO3 in the DH, which are products of the spontaneous oxidation of NO under physiological conditions that can provide an indirect measurement of NO production. Bilateral microinjection of N-propyl-L-arginine (0.1 nmol/500 nL; N-propyl, a neuronal NO synthase (nNOS) inhibitor) or carboxy-PTIO (2 nmol/500 nL; c-PTIO, an NO scavenger) into the DH also attenuated autonomic responses evoked by RS. Therefore, our findings suggest that a glutamatergic system present in the DH is involved in the autonomic modulation during RS, acting via NMDA receptors and nNOS activation. Furthermore, the present results suggest that NMDA receptor/nNO activation has a facilitatory influence on RS-evoked autonomic responses.

Inhibition of nitric oxide is a good therapeutic target for bladder tumors that express iNOS.

Bladder cancer is the second cause of death for urological tumors in man. When the tumor is nonmuscle invasive, transurethral resection is curative. On the other hand, radical cystectomy is the treatment chosen for patients with invasive tumors, but still under treatment, these patients have high risk of dying, by the development of metastatic disease within 5 years. It is therefore important to identify a new therapeutic target to avoid tumor recurrences and tumor progression. Nitric oxide (NO) is an important biological messenger known to influence several types of cancers. In bladder cancer, production of NO and expression and activity of inducible NO synthase was associated to recurrence and progression. The objective of this work was to analyze if inhibition of nitric oxide production could be considered a therapeutic target for bladder tumors expressing iNOS. Using a bladder cancer murine model with different invasiveness grade we have demonstrated that NO inhibition was able to inhibit growth of bladder tumors expressing iNOS. Furthermore, invasive properties of MB49-I orthotopic growth was inhibited using NO inhibitors. This paper also shows that levels of NO in urine can be correlated with tumor size. In conclusion, inhibition of NO could be considered as a therapeutic target that prevents tumor growth and progression. Also, urine NO levels may be useful for measuring tumor growth.

Diabetic neuropathy: an evaluation of the use of quercetin in the cecum of rats.

AIM: To investigate the effect of quercetin supplementation on the myenteric neurons and glia in the cecum of diabetic rats. METHODS: Total preparations of the muscular tunic were prepared from the ceca of twenty-four rats divided into the following groups: control (C), control supplemented with quercetin (200 mg/kg quercetin body weight) (CQ), diabetic (D) and diabetic supplemented with quercetin (DQ). Immunohistochemical double staining technique was performed with HuC/D (general population)/nitric oxide synthase (nNOS), HuC-D/S-100 and VIP. Density analysis of the general neuronal population HuC/D-IR, the nNOS-IR (nitrergic subpopulation) and the enteric glial cells (S-100) was performed, and the morphometry and the reduction in varicosity population (VIP-IR) in these populations were analyzed. RESULTS: Diabetes promoted a significant reduction (25%) in the neuronal density of the HuC/D-IR (general population) and the nNOS-IR (nitrergic subpopulation) compared with the C group. Diabetes also significantly increased the areas of neurons, glial cells and VIP-IR varicosities. Supplementation with quercetin in the DQ group prevented neuronal loss in the general population and increased its area (P < 0.001) and the area of nitrergic subpopulation (P < 0.001), when compared to C group. Quercetin induced a VIP-IR and glial cells areas (P < 0.001) in DQ group when compared to C, CQ and D groups. CONCLUSION: In diabetes, quercetin exhibited a neuroprotective effect by maintaining the density of the general neuronal population but did not affect the density of the nNOS subpopulation.

Mitochondrial dysfunction in the hippocampus of rats caused by chronic oxidative stress.

The aim of this study was to analyze the effects of chronic oxidative stress on mitochondrial function and its relationship to progressive neurodegeneration in the hippocampus of rats chronically exposed to ozone. Animals were exposed to 0.25 ppm ozone for 7, 15, 30, or 60 days. Each group was tested for (1) protein oxidation and, manganese superoxide dismutase (Mn-SOD), glutathione peroxidase (GPx) and succinate dehydrogenase (SDH) activity using spectrophotometric techniques, (2) oxygen consumption, (3) cytochrome c, inducible nitric oxide synthase (iNOS), peroxisome proliferator-activated receptor γ Co-activator 1α (PGC-1α), B-cell lymphoma (Bcl-2), and Bax expression using Western blotting, (4) histology using hematoxylin and eosin staining, and (5) mitochondrial structure using electron microscopy. Our results showed increased levels of carbonyl protein and Mn-SOD activity after 30 days of ozone exposure and decreased GPx activity. The SDH activity decreased from 7 to 60 days of exposure. The oxygen consumption decreased at 60 days. Western blotting showed an increase in cytochrome c at 60 days of ozone exposure and an increase in iNOS up to 60 days of ozone exposure. The expression of PGC-1α was decreased after 15, 30, and 60 days compared to the earlier time Bcl-2 was increased at 60 days compared to earlier time points, and Bax was increased after 30 and 60 days of exposure compared to earlier time points. We observed cellular damage, and mitochondrial swelling with a loss of mitochondrial cristae after 60 days of exposure. These changes suggest that low doses of ozone caused mitochondrial abnormalities that may lead to cell damage.

Antidepressant-like effect of α-tocopherol in a mouse model of depressive-like behavior induced by TNF-α.

Taking into account that pro-inflammatory cytokines and oxidative and nitrosative stress are implicated in the pathogenesis of depression and that α-tocopherol has antidepressant, anti-inflammatory and antioxidant properties, this study investigated the ability of α-tocopherol to abolish the depressive-like behavior induced by i.c.v. administration of TNF-α in the mouse TST. Additionally, we investigated the occurrence of changes in the levels of Bcl2 and Bax and phosphorylation of GSK-3ß (Ser9) in the hippocampus of mice. The administration of TNF-α (0.001fg/site, i.c.v.) increased the immobility time in the TST, which was prevented by the administration of α-tocopherol at the doses of 10, 30 and 100mg/kg (p.o.). Subeffective doses of α-tocopherol (10mg/kg, p.o.) and/or the antidepressants fluoxetine (5mg/kg, p.o.), imipramine (0.1mg/kg, p.o.) and bupropion (1mg/kg, p.o.), the NMDA receptor antagonist MK-801 (0.001mg/kg, p.o.) or the neuronal nitric oxide synthase inhibitor 7-nitroindazole (25mg/kg, i.p.) prevented the depressive-like effect induced by TNF-α. None of the treatments altered the locomotor activity of mice. Treatment with TNF-α and/or α-tocopherol did not alter the levels of Bax and Bcl2 or the phosphorylation of GSK-3ß in the hippocampus of mice. Together, our results show a synergistic antidepressant-like effect of α-tocopherol with antidepressants against the depressive-like behavior induced by an inflammatory insult, suggesting that this vitamin may be useful to optimize conventional pharmacotherapy of depression, including depressive states associated with inflammatory conditions.

Nitric oxide and AQP2 in hypothyroid rats: a link between aging and water homeostasis.

OBJECTIVE: Hypothyroid state and aging are associated with impairment in water reabsorption and changes in aquaporin water channel type 2 (AQP2). Nitric oxide (NO) is involved in AQP2 trafficking to the apical plasma membrane in medullary collecting duct cells. The purpose of this study was to investigate whether aging and hypothyroidism alter renal function, and whether medullary NO and AQP2 are implicated in maintaining water homeostasis. MATERIALS/METHODS: Sprague-Dawley rats aged 2 and 18months old were treated with 0.02% methimazole (w/v) during 28days. Renal function was examined and NO synthase (NOS) activity ([(14)C (U)]-L-arginine to [(14)C (U)]-L-citrulline assays), NOS, caveolin-1 and -3 and AQP2 protein levels were determined in medullary tissue (Western blot). Plasma membrane fraction and intracellular vesicle fraction of AQP2 were evaluated by Western blot and immunohistochemistry. RESULTS: A divergent response was observed in hypothyroid rats: while young rats exhibited polyuria with decreased medullary NOS activity, adult rats exhibited a decrease in urine output with increased NOS activity. AQP2 was increased with hypothyroidism, but while young rats exhibited increased AQP2 in plasma membrane, adult rats did so in the cytosolic site. CONCLUSIONS: Hypothyroidism contributes in a differential way to aging-induced changes in renal function, and medullary NO and AQP2 would be implicated in maintaining water homeostasis.