Endothelial Function in Hypertension: Victim or Culprit?

Far from simply lining the inner surface of blood vessels, the cellular monolayer that comprises the endothelium is a highly active organ that regulates vascular tone. In health, the endothelium maintains the balance between opposing dilator and constrictor influences, while in disease, it is the common ground on which cardiovascular risk factors act to initiate the atherosclerotic process. As such, it is the site at which cardiovascular disease begins and consequently acts as a barometer of an individual's likely future cardiovascular health. The vascular endothelium is a very active organ responsible for the regulation of vascular tone through the effects of locally synthesized mediators, predominantly nitric oxide (NO), endothelial NO synthase (eNOS), and superoxide. NO is abundantly evident in normally functioning vasculature where it acts as a vasodilator, inhibits inflammation, and has an antiaggregant effect on platelets. Its depletion is both a sign and cause of endothelial dysfunction resulting from reduced activity of eNOS and amplified production of nicotinamide adenine dinucleotide oxidase, which, in turn, results in raised levels of reactive oxygen species. This cascade is the basis for reduced vascular compliance through an imbalanced regulation of tone with a predominance of vasoconstrictive elements. Further, structural changes in the microvasculature are a critical early step in the loss of normal function. This microvascular dysfunction is known to be highly predictive of future macrovascular events and is consequently a very attractive target for intervention in the hypertensive population in order to prevent cardiovascular events.

Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica

The inhalation of airborne pollutants, such as asbestos or silica, is linked to inflammation of the lung, fibrosis, and lung cancer. How the presence of pathogenic dust is recognized and how chronic inflammatory diseases are triggered are poorly understood. Here, we show that asbestos and silica are sensed by the Nalp3 inflammasome, whose subsequent activation leads to interleukin-1beta secretion. Inflammasome activation is triggered by reactive oxygen species, which are generated by a NADPH oxidase upon particle phagocytosis. (NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate.) In a model of asbestos inhalation, Nalp3-/- mice showed diminished recruitment of inflammatory cells to the lungs, paralleled by lower cytokine production. Our findings implicate the Nalp3 inflammasome in particulate matter-related pulmonary diseases and support its role as a major proinflammatory "danger" receptor

Association of NAD(P)H Oxidase with Glucose-Induced Insulin Secretion by Pancreatic beta-Cells

We previously described the presence of nicotinamide adenine dinucleotide phosphate reduced form [NAD(P)H] oxidase components in pancreatic beta-cells and its activation by glucose, palmitic acid, and proinflammatory cytokines. In the present study, the importance of the NAD(P)H oxidase complex for pancreatic beta-cell function was examined. Rat pancreatic islets were incubated in the presence of glucose plus diphenyleneiodonium, a NAD(P)H oxidase inhibitor, for 1 h or with the antisense oligonucleotide for p47(PHOX) during 24 h. Reactive oxygen species (ROS) production was determined by a fluorescence assay using 2,7-dichlorodihydrofluorescein diacetate. Insulin secretion, intracellular calcium responses, [U-(14)C] glucose oxidation, and expression of glucose transporter-2, glucokinase and insulin genes were examined. Antisense oligonucleotide reduced p47(PHOX) expression [an important NAD(P)H oxidase cytosolic subunit] and similarly to diphenyleneiodonium also blunted the enzyme activity as indicated by reduction of ROS production. Suppression of NAD(P)H oxidase activity had an inhibitory effect on intracellular calcium responses to glucose and glucose-stimulated insulin secretion by isolated islets. NAD(P)H oxidase inhibition also reduced glucose oxidation and gene expression of glucose transporter-2 and glucokinase. These findings indicate that NAD(P)H oxidase activation plays an important role for ROS production by pancreatic beta-cells during glucose-stimulated insulin secretion. The importance of this enzyme complex for the beta-cell metabolism and the machinery involved in insulin secretion were also shown. (Endocrinology 150: 2197-2201, 2009)

Alterations of NADPH Oxidase Activity in Rat Pancreatic Islets Induced by a High-Fat Diet

Objective: The aim of this study was to evaluate the effect of a high-fat diet (HFD) on nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in rat pancreatic islets. We investigated if changes in NADPH oxidase are connected to beta cell dysfunction reported in obese animals. Methods: Male Wistar rats were fed a HFD or control diet for 3 months. DNA fragmentation, insulin secretion, and [U-(14)C] glucose oxidation were examined in isolated pancreatic islets. The oxidative stress markers nitrotyrosine and 4-hydroxy-2-nonenal were assessed by immunohistochemistry. The protein content of gp91(phox) and p47(phox) was evaluated by Western blotting. Production of reactive oxygen species (ROS) was determined by a fluorescence assay using hydroethidine. Results: Occurrence of DNA fragmentation was reduced in pancreatic islets from HFD rats. There were no differences in oxidative stress markers between the groups. Glucose oxidation and insulin secretion were elevated due to high glucose in pancreatic islets from HFD rats. Protein concentrations of p47(phox) and gp91(phox) subunits were reduced and ROS production was diminished in pancreatic islets from HFD rats. Conclusions: The diminished content of NADPH oxidase subunits and ROS concentrations may be associated with increased glucose oxidation and insulin secretion in an attempt to compensate for the peripheral insulin resistance elicited by the HFD.

NAD(P)H Oxidase Participates in the Palmitate-Induced Superoxide Production and Insulin Secretion by Rat Pancreatic Islets

Nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase complex has been shown to be involved in the process of glucose-stimulated insulin secretion (GSIS). In this study, we examined the effect of palmitic acid on superoxide production and insulin secretion by rat pancreatic islets and the mechanism involved. Rat pancreatic islets were incubated during 1 h with 1 mM palmitate, 1% fatty acid free-albumin, 5.6 or 10 mM glucose and in the presence of inhibitors of NAD(P)H oxidase (DPI-diphenyleneiodonium), PKC (calphostin C) and carnitine palmitoyl transferase-I (CPT-I) (etomoxir). Superoxide content was determined by hydroethidine assays. Palmitate increased superoxide production in the presence of 5.6 and 10 mM glucose. This effect was dependent on activation of PKC and NAD(P)H oxidase. Palmitic acid oxidation was demonstrated to contribute for the fatty acid induction of superoxide production in the presence of 5.6 mM glucose. In fact, palmitate caused p47(PHOX) translocation to plasma membrane, as shown by immunohistochemistry. Exposure to palmitate for 1 h up-regulated the protein content of p47(PHOX) and the mRNA levels of p22(PHOX), gp91(PHOX), p47(PHOX), proinsulin and the G protein-coupled receptor 40 (GPR40). Fatty acid stimulation of insulin secretion in the presence of high glucose concentration was reduced by inhibition of NAD(P)H oxidase activity. In conclusion, NAD(P)H oxidase is an important source of superoxide in pancreatic islets and the activity of NAD(P)H oxidase is involved in the control of insulin secretion by palmitate. J. Cell. Physiol. 226: 1110-1117, 2011. (C) 2010 Wiley-Liss, Inc.

Upregulation of gp91(phox) Subunit of NAD(P)H Oxidase Contributes to Erectile Dysfunction Caused by Long-term Nitric Oxide Inhibition in Rats: Reversion by Regular Physical Training

OBJECTIVES To test the hypothesis that glyco protein 91phox (gp91(phox)) subunit of nicotinamide adenine dinucleotide phosphate [NAD(P) H] oxidase is a fundamental target for physical activity to ameliorate erectile dysfunction (ED). Vascular risk factors are reported to contribute to ED. Regular physical exercise prevents cardiovascular diseases by increasing nitric oxide (NO) production and/or decreasing NO inactivation. METHODS Male Wistar rats received the NO synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) for 4 weeks, after which animals were submitted to a run training program for another 4 weeks. Erectile functions were evaluated by in vitro cavernosal relaxations and intracavernous pressure measurements. Expressions of gp91(phox) subunit and neuronal nitric oxidase synthase in erectile tissue, as well as superoxide dismutase activity and nitrite/nitrate (NO(x)) levels were determined. RESULTS The in vitro acetylcholine-and electrical field stimulation-induced cavernosal relaxations, as well as the increases in intracavernous pressure were markedly reduced in sedentary rats treated with L-NAME. Run training significantly restored the impaired cavernosal relaxations. No alterations in the neuronal nitric oxidase synthase protein expression (and its variant penile neuronal nitric oxidase synthase) were detected. A reduction of NO(x) levels and superoxide dismutase activity was observed in L-NAME-treated animals, which was significantly reversed by physical training. Gene expression of subunit gp91(phox) was enhanced by approximately 2-fold in erectile tissue of L-NAME-treated rats, and that was restored to basal levels by run training. CONCLUSIONS Our study shows that ED seen after long-term L-NAME treatment is associated with gp91(phox) subunit upregulation and decreased NO bioavailability. Exercise training reverses the increased oxidative stress in NO-deficient rats, ameliorating the ED. UROLOGY 75: 961-967, 2010. (C) 2009 Elsevier Inc.

Serum amyloid A induces reactive oxygen species (ROS) production and proliferation of fibroblast

Serum amyloid A (SAA) levels are elevated highly in acute phase response and elevated slightly and persistently in chronic diseases such as rheumatoid arthritis and diabetes. Given that fibroblasts exert profound effects on progression of inflammatory chronic diseases, the aim of this study was to investigate the response of fibroblasts to SAA. A dose-dependent increase in O(2)(-) levels was observed by treatment of fibroblasts with SAA (r = 0.99 and P <= 0.001). In addition, the expression of p47-phox was up-regulated by SAA (P < 0.001) and diphenyliodonium (DPI), a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, reduced the release of O(2)(-) by 50%. Also, SAA raised fibroblast proliferation (P < 0.001) and this effect was completely abolished by the addition of anti-oxidants (P < 0.001). These findings support the notion that, in chronic inflammatory sites, SAA activated fibroblast proliferation and ROS production.

Links between pollen, atopy and the asthma epidemic

Pollen allergy has been found in 80–90% of childhood asthmatics and 40–50% of adult-onset asthmatics. Despite the high prevalence of atopy in asthmatics, a causal relationship between the allergic response and asthma has not been clearly established. Pollen grains are too large to penetrate the small airways where asthma occurs. Yet pollen cytoplasmic fragments are respirable and are likely correlated with the asthmatic response in allergic asthmatics. In this review, we outline the mechanism of pollen fragmentation and possible pathophysiology of pollen fragment-induced asthma. Pollen grains rupture within the male flowers and emit cytoplasmic debris when winds or other disturbances disperse the pollen. Peak levels of grass and birch pollen allergens in the atmosphere correlated with the occurrence of moist weather conditions during the flowering period. Thunderstorm asthma epidemics may be triggered by grass pollen rupture in the atmosphere and the entrainment of respirable-sized particles in the outflows of air masses at ground level. Pollen contains nicotinamide adenine dinucleotide phosphate (reduced) oxidases and bioactive lipid mediators which likely contribute to the inflammatory response. Several studies have examined synergistic effects and enhanced immune response from interaction in the atmosphere, or from co-deposition in the airways, of pollen allergens, endogenous pro-inflammatory agents, and the particulate and gaseous fraction of combustion products. Pollen and fungal fragments also contain compounds that can suppress reactive oxidants and quench free radicals. It is important to know more about how these substances interact to potentially enhance, or even ameliorate, allergic asthma.

Effects of nandrolone decanoate on the neuromuscular junction of rats submitted to swimming

This study addressed the effects of nandrolone decanoate (ND) on contractile properties and muscle fiber characteristics of rats submitted to swimming. Male Wistar rats were grouped in sedentary (S), swimming (Sw), sedentary+ND (SND), and swimming+ND (SwND), six animals per group. ND (3 mg/kg) was injected (subcutaneously) 5 days/week, for 4 weeks. Swimming consisted of 60-min sessions (load 2%), 5 days/week, for 4 weeks. After this period, the sciatic nerve extensor digitorum longus (EDL) muscle was isolated for myographic recordings. Fatigue resistance was assessed by the percent (%) decline of 180 direct tetanic contractions (30 Hz). Safety margin of synaptic transmission was determined from the resistance to the blockade of indirectly evoked twitches (0.5 Hz) induced by pancuronium (5 to 9 x 10(-7) M). EDL muscles were also submitted to histological and histochemical analysis (haematoxylin-eosin (HE); nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR)). Significant differences were detected by two-way ANOVA (p<0.05). ND did not change body mass, fatigue resistance or kinetic properties of indirect twitches in either sedentary or swimming rats. In contrast, ND reduced the safety margin of synaptic transmission in sedentary animals (SND=53.3+/-4.7% vs. S=75.7+/-2.0%), but did not affect the safety margin in the swimming rats (SwND=75.81+/-3.1% vs. Sw=71.0+/-4.0%). No significant difference in fiber type proportions or diameters was observed in EDL muscle of any experimental group. These results indicate that ND does not act as an ergogenic reinforcement in rats submitted to 4 weeks of swimming. on the other hand, this study revealed an important toxic effect of ND, that it reduces the safety margin of synaptic transmission in sedentary animals. Such an effect is masked when associated with physical exercise. (C) 2004 Elsevier B.V. All rights reserved.

Morphometric and histochemical study of the human vocal muscle

The present study was conducted on vocal muscles removed at autopsy Rom adult individuals (10 men and 8 women, aes ranging from 48 to 78 years) with no laryngeal disease. Histologic analysis was performed with hematoxylin and eosin staining, and histochemical analysis was performed by nicotinamide-adenine-dinucleotide tetrazolium reductase, succinate dehydrogenase, and acid and alkaline myofibrillar adenosine triphosphatase reactions. The histochemical reactions showed that the muscle consists of slow-twitch oxidative (SO), fast-twitch glycolytic (FG), and fast-twitch glycolytic oxidative (FOG) fibers distributed in mosaic form. The frequencies of SO, FOG, and FG fibers were 40.50%, 54.75%, and 4.75%, respectively. The higher frequency of SO and FOG oxidative fibers characterizes the muscle as having aerobic metabolism, resistance to fatigue, and fast contraction. The mean minimum diameters were 31.37 mu m for SO fibers and 36.46 mu m for FOG and FG fibers.

Upregulation of gp91(phox) Subunit of NAD(P)H Oxidase Contributes to Erectile Dysfunction Caused by Long-term Nitric Oxide Inhibition in Rats: Reversion by Regular Physical Training

OBJECTIVES To test the hypothesis that glyco protein 91phox (gp91(phox)) subunit of nicotinamide adenine dinucleotide phosphate [NAD(P) H] oxidase is a fundamental target for physical activity to ameliorate erectile dysfunction (ED). Vascular risk factors are reported to contribute to ED. Regular physical exercise prevents cardiovascular diseases by increasing nitric oxide (NO) production and/or decreasing NO inactivation.METHODS Male Wistar rats received the NO synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) for 4 weeks, after which animals were submitted to a run training program for another 4 weeks. Erectile functions were evaluated by in vitro cavernosal relaxations and intracavernous pressure measurements. Expressions of gp91(phox) subunit and neuronal nitric oxidase synthase in erectile tissue, as well as superoxide dismutase activity and nitrite/nitrate (NO(x)) levels were determined.RESULTS The in vitro acetylcholine-and electrical field stimulation-induced cavernosal relaxations, as well as the increases in intracavernous pressure were markedly reduced in sedentary rats treated with L-NAME. Run training significantly restored the impaired cavernosal relaxations. No alterations in the neuronal nitric oxidase synthase protein expression (and its variant penile neuronal nitric oxidase synthase) were detected. A reduction of NO(x) levels and superoxide dismutase activity was observed in L-NAME-treated animals, which was significantly reversed by physical training. Gene expression of subunit gp91(phox) was enhanced by approximately 2-fold in erectile tissue of L-NAME-treated rats, and that was restored to basal levels by run training.CONCLUSIONS Our study shows that ED seen after long-term L-NAME treatment is associated with gp91(phox) subunit upregulation and decreased NO bioavailability. Exercise training reverses the increased oxidative stress in NO-deficient rats, ameliorating the ED. UROLOGY 75: 961-967, 2010. (C) 2009 Elsevier B.V.

Morphological and histochemical analysis of the human vestibular fold

A morphological and histochemical study of the human vestibular fold was carried out using routine histological techniques. Seven μm-thick histological sections stained with hematoxylin-eosin (HE) and Calleja showed the presence of elastic collagen fibers and seromucous glands in the vestibular fold. Muscle fibers forming the ventricular muscle were also identified. Ultrastructural analyses of the epithelial layer by scanning electron microscopy (SEM) revealed ciliated cells and gland ducts opening on the epithelial surface. Histochemical analyses were performed on ventricular muscles submitted to nicotinamide-adenine-dinucleotide tetrazolium reductase (NADH-TR), succinate dehydrogenase (SDH), and myofibrillar adenosine triphosphatase (mATPase) reactions. Based on these reactions, it was observed that the muscle is formed by three types of muscle fibers: slow-twitch oxidative (SO), fast-twitch oxydative glycolytic (FOG) and fast-twitch glycolytic (FG) fibers distributed in a mosaic pattern. The fiber frequency was 22.7%, 69.9% and 7.4%, respectively. The higher frequency of SO and FOG fibers characterized the muscle as having aerobic metabolism and resistance to fatigue. The ventricular muscle was considered fast. The study of the neuromuscular junctions performed after nonspecific esterase reaction showed that they are of the en-plaque type and have multiple occurrences in the ventricular muscle.

Expression of the P2X(2) receptor in different classes of ileum myenteric neurons in the female obese ob/ob mouse

AIM: To examine whether the ob/ob mouse model of obesity is accompanied by enteric nervous system abnormalities such as altered motility. METHODS: The study examined the distribution of the P2X(2) receptor (P2X(2)R) in myenteric neurons of female ob/ob mice. Specifically, we used immunohistochemistry to analyze the co-expression of the P2X(2)R with neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and calretinin (CalR) in neurons of the small intestine myenteric plexus in ob/ob and control female mice. In these sections, we used scanning confocal microscopy to analyze the co-localization of these markers as well as the neuronal density (cm(2)) and area profile (mu m(2)) of P2X(2)R-positive neurons. In addition, enteric neurons were labeled using the nicotinamide adenine dinucleotide (NADH) diaphorase method and analyzed with light microscopy as an alternate means by which to analyze neuronal density and area. RESULTS: In the present study, we observed a 29.6% increase in the body weight of the ob/ob animals (OG) compared to the control group (CG). In addition, the average small intestine area was increased by approximately 29.6% in the OG compared to the CG. Immunoreactivity (IR) for the P2X(2)R, nNOS, ChAT and CaIR was detectable in the myenteric plexus, as well as in the smooth muscle, in both groups. This IR appeared to be mainly cytoplasmic and was also associated with the cell membrane of the myenteric plexus neurons, where it outlined the neuronal cell bodies and their processes. P2X(2)R-IR was observed to co-localize 100% with that for nNOS, ChAT and CaIR in neurons of both groups. In the ob/ob group, however, we observed that the neuronal density (neuron/cm(2)) of P2X(2)R-IR cells was increased by 62% compared to CG, while that of NOS-IR and ChAT-IR neurons was reduced by 49% and 57%, respectively, compared to control mice. The neuronal density of CaIR-IR neurons was not different between the groups. Morphometric studies further demonstrated that the cell body profile area (mu m(2)) of nNOS-IR, ChAT-IR and CaIR-IR neurons was increased by 34%, 20% and 55%, respectively, in the OG compared to controls. Staining for NADH diaphorase activity is widely used to detect alterations in the enteric nervous system; however, our qualitative examination of NADH-diaphorase positive neurons in the nnyenteric ganglia revealed an overall similarity between the two groups. CONCLUSION: We demonstrate increases in P2X(2)R expression and alterations in nNOS, ChAT and CaIR IR in ileal myenteric neurons of female ob/ob mice compared to wild-type controls. (c) 2012 Baishideng. All rights reserved.

Expression of NADPH Oxidase by Trophoblast Cells: Potential Implications for the Postimplanting Mouse Embryo

Cytochemical localization of hydrogen peroxide-generating sites suggests NADPH (nicotinamide adenine dinucleotide 3-phosphate [ reduced form]) oxidase expression at the maternal-fetal interface. To explore this possibility, we have characterized the expression and activity of the NADPH oxidase complex in trophoblast cells during the postimplantation period. Implantation sites and ectoplacental cones (EPCs) from 7.5-gestational day embryos from CD1 mice were used as a source for expression analyses of NADPH oxidase catalytic and regulatory subunits. EPCs grown in primary culture were used to investigate the production of superoxide anion through dihydroxyethidium oxidation in confocal microscopy and immunohistochemical assays. NADPH subunits Cybb (gp91phox), Cyba (p22phox), Ncf4 (p40phox), Ncf1 (p47phox), Ncf2 (p67phox), and Rac1 were expressed by trophoblast cells. The fundamental subunits of membrane CYBB and cytosolic NCF2 were markedly upregulated after phorbol-12-myristate-13-acetate (PMA) treatment, as detected by quantitative real-time PCR, Western blotting, and immunohistochemistry. Fluorescence microscopy imaging showed colocalization of cytosolic and plasma membrane NADPH oxidase subunits mainly after PMA treatment, suggesting assembly of the complex after enzyme activation. Cultured EPCs produced superoxide in a NADPH-dependent manner, associating the NADPH oxidase-mediated superoxide production with postimplantation trophoblast physiology. NADPH-oxidase cDNA subunit sequencing showed a high degree of homology between the trophoblast and neutrophil isoforms of the oxidase, emphasizing a putative role for reactive oxygen species production in phagocytic activity and innate immune responses.

Development of a fluorescent method for the detection in marine organisms of the respiratory electron transport system

Trabajo realizado por: Maldonado, F.; Packard, T.; Gómez, M.; Santana Rodríguez, J. J