Effects of the ascorbic acid supplementation on NADH-diaphorase myenteric neurons in the duodenum of diabetic rats

We assessed the ascorbic acid (AA) supplementation on the myenteric neurons in the duodenum of rats. Fifteen rats with 90 days of age were divided into three groups: control (C), diabetics (D) and ascorbic acid treated diabetics (DA). After 120 days of daily treatment with AA, the duodenum was submitted to the NADH-diaphorase (NADH-d) histochemical technique, which allowed us to evaluate theneuronal density in an area of 8.96 mm2 for each duodenum, and also to measure the cellular profile area of 500 neurons per group. The supplementation promoted an increase on AA levels. The neuronal density (p < 0.05) was higher in the group DA when compared to group D. There were no significant differences in the neuronal areas, when we compared groups C (204 +/- 16.5) and D (146.3 +/- 35.84) to groups D and DA (184.5 +/- 5.6 ) (p > 0.05). The AA-supplementation avoided the density reduction of the NADHd myenteric neurons in the duodenum of diabetic rats.

Effect of protein and vitamin B deficiency on the morpho-quantitative aspects of the myenteric plexus of the descending colon of adult rats

We carried out this work with the purpose of studying the effects of protein and vitamin B deficiency on the morphologic and quantitative aspects of the myenteric plexus of the descending colon of adult Rattus norvegicus. Twenty-eight rats were divided in two groups, one of them receiving chow with 22 percent protein level (control) and the other fed with chow having 8 percent protein level without vitamin B supplementation, during 120 days. Whole-mounts of the descending colon were prepared and stained with Giemsa, NADH-diaphorase and NADPH-diaphorase. The undernourished rats had a body weight 11.84 percent less than the control group. Relative to the controls, the experimental group had a colonic area 48 percent smaller, 51.9 percent less Giemsa-stained neurons, 28.3 percent less NADH-diaphorase positive neurons and 24.2 percent less NADPH-diaphorase positive neurons

Effects of protein and vitamin B deficiency on blood parameters and myenteric neurons of the colon of rats

The aims of this work were to evaluate the effects of the deficient ingestion of protein and vitamin B on the biochemical and hematologic parameters and on the NADH- and NADPH-diaphorase positive myenteric neurons. The control animals (n=10) received commercial chow and the experimental rats (n=10) received chow with protein level reduced to 8 percent during 120 days. At the time of killing blood was collected for assessment of the blood and hematologic parameters and the ascending colon for quantitative analysis of the neurons of the myenteric plexus. It was observed that the reduction of the protein level to 8 percent coupled to the reduction of the levels of vitamin B in adult rats neither led to qualitative or quantitative changes on red or white blood cells, nor decreased globulin levels, induced the formation of edema or gave rise to clinical signs typical of protein or vitamin B deficiency. On the other hand, the experimental protocol led to less weight gain, change on the body composition with fat deposition; decrease of the values of serum total protein and albumin; reduction of the area of colon and density of nitrergic and NADH-diaphorase myenteric neurons inferior to the expected

Dietary sodium intake induced myenteric neuron hypertrophy in Wistar rats

In the present study we investigated the effect of salt intake on myenteric neuron size of the colon of adult male Wistar rats. The animals were placed on either a high-salt (HS; 8 percent; 12 animals) or a low-salt diet (LS; 0.15 percent; 12 animals) for 15 or 52 weeks and blood pressure was measured. The sizes of myenteric neurons of the distal colon from both groups were measured. No difference in neuron size was observed between the HS and LS groups after 15 weeks. After 52 weeks on HS, neuron size was increased (P<0.005) when compared with the LS group. The rats also presented hypertension, which was significantly different at 52 weeks (142 +/- 11 vs 119 +/- 7 mmHg). These results suggest that a long time on an HS diet can significantly increase myenteric nerve cell size.

Number and size of myenteric neurons of the duodenum of adult rats with acute diabetes

This study had as its purpose to assess the effects of acute diabetes induced by streptozotocin (35 mg/kg body weight) on the number and size of the myenteric neurons of the duodenum of adult rats considering equally the antimesenteric and intermediate regions of the intestinal circunference. Experimental period extended for a week. Neuronal counts were carried out on the same number of fields of both regions of the duodenal circunference and measurements of neuronal and nuclear areas on equal numbers of cells. Number and size of the myenteric neurons stained with Giensa were not significantly different between groups. On the other hand, the proportion of NADH-positive neurons increased from 18.54 per cent on the controls to 39.33 per cent on the diabetics. The authors discuss that this increased reactivity probably results from a greater NADH/NAD* ratio, described in many tissues of diabetic animals, which has consequences on the modulation of the enzymes that use these cofactors and whose activity is detected by the NADH-diaphorase technique.

La mieloperoxidasa como fator de daño oxidativo del miocardio: inactivacion de la dihidrolipoamida deshidrogenasa / Inactivation of heart dihydrolipoamide dehydrogenase by leukocyte myeloperoxidase-dependent system. Prevention by captopril and other thiol compounds

La dihidrolipoamida deshidrogenasa de miocardio (LADH) es inactivada por incubación a 30 Graus Celssius con sistemas pro-oxidantes dependientes de la mieloperoxidasa (MPO) de leucocitos. La inactivación varía según la composición del sistema pro-oxidante y del tiempo de incubación con la LADH. Con el sistema MPO/H2O2/CINa las inactivaciones obtenidas fueron 64 y 87 por ciento a 30 y 60 min de incubación, respectivamente. En ausencia de CINa, la inactivación fue de 9 y 27 por ciento mientras que en ausencia de MPO fue de 4.0 y 11 por ciento, respectivamente. El CIONa, producto de la paroxidación del ión CL- por el sistema MPO/H2O2, inactivó la LADH 72-81 por ciento según el tiempo de incubación con la enzima, confirmando la función del ácido hipocloroso como agente del sistema MPO/H2O2/CINa. Los sistemas MPO/H2O2/IK, MPO/H2O2/SCNK y MPO/H2O2/NO2 Na también inactivaron la LADH, en grado dependiente de la naturaleza del anión. El anión ioduro fue el mas activo. El sistema MPO/NADH/IK, donde el NADH reemplazó al H2O2, inactivó la LADH. La catalasa previno esa inactivación, de acuerdo con la función del NADH como generador de H2O2. La MPO catalizó la inactivación de la LADH por los sistemas H2O2/Cloropromazina y H2O2/Paracetamol. Compuestos tiol (Captopril, penicilamina, GSH cisteína, N-acetilcisteína y mercaptopropioniglicina), la taurina y el ascorbato protegieron a la LADH frente a los sistemas dependientes de MPO; y frente al CIONa. Los resultados reseñados se discuten en relación a la función de la MPO como catalizador de la producción de oxidantes capaces de contribuir al daño del miocardio por isquemia-reperfusión, como consecuencia de la patología de la inflamación, o por acción de fármacos.

A rede testicular na cobaia: observaçöes morfológicas e histoquímicas / The rete testis in guinea pig: a morphological and histochemical examination

A rede testicular (RT) da cobaia tem características morfológicas de um tipo intermediário de RT. Esta rede foi caracterizada com cavitária, labiríntica e axial relativamente à disposiçäo de seus canais epiteliais interconectados. Morfologicamente consiste, predominantemente, de câmaras epiteliais interligadas que penetram no parênquima testicular, por uma pequena distância. Os canais e câmaras de todas as partes da rede säo revestidos por epitélio cúbico simples. Reatividades histoenzimáticas fortes foram observadas, predominantemente, na matriz da RT às enzimas fosfatases alcalina e ácida e m-ATPase. Ambas as fosfatases mostraram reatividades médias no epitélio da RT de cobaia, no qual a reatividade da m-ATPase foi fraca. Por outro lado, a NADH-d mostrou reatividade média, porém homogênea, na matriz e no epitélio da RT.

Localization of NADPH diaphorase activity in the human visual cortex

The present report describes the activity of NADPH-diaphorase (NADPHd) in area 17 of autopsied normal human visual cortex. Four human brains from autopsy tissue (4-8 h postmortem) were fixed by immersion in 4 per cent paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2-7.4, or in 10 per cent formalin for 24 h. NADPHd histochemistry was done using the malic enzyme indirect method. The neurpile pattern of enzyme activity presented a clear six layer appearance. Cell morphology and the laminar distribution of 73 NADPHd-positive neurons are descrived. All neurons found in area 17 of human cortex were sparsely spiny or smooth cells, located in all cortical layers exept layer 4c. Quantitative analysis of the branching pattern of the dendritic tree was carried out. A symmetrical pattern was observed with no particular dendritic bias except for a few white matter and layer 1 cells. Larger dendritic fields were found in white matter cells when compared to the other corical layers. Comparison of cell densities for gray and white matters showed that 85 per cent of the NADPHd-positive neurons were located in the white matter. NADPH was colocalized with nitric oxide synthase which produces nitric oxide, a short-life neuromediator implicated in synaptic plasticity, neuroprotection, and neurotoxicity. thus, the spatial distribution of the NADPHd cells is important for posterior functional studies of the neuromediators in the brain

Biochemical characterization of NADPH diaphorase in the chick embryo retina: stimulation by calcium ions and inhibition by arginine analogs

Nitric oxide is an important intercellular messenger in the central nervous system. NADPH-diaphorase, reported to be identical to nitric oxide synthase, is prsent in specific groups of cells in several neural tissues, including the retina. We determined NADPH-diaphorase activity in homogenates of the chick embryo retina. The enzyme activity was measured spectophotometrically at 585 nm after incubating retinal total homogenates (100-150 µg protein) with 1mMNADPH and 0.5 mM nitroblue tetrazolium in 50 mMTris buffer, pH8.1, at 37ºC. NADPH-diaphorse was detected in 14-day old retinas and 53-65 per cent of the enzyme activity was inhibited by 3 mM NG-nitro-L-arginine (NARG), the arginine analog. One mM L-N5-(1-iminoethyl)ornithine (NIO) was the most potent inhibitor (63 per cent inhibition) while 3 mM NG-nitro-L-arginine methyl ester (NAME) (33 per cent inhibition) and I mMNG-monomethyl-L-arginine acetate (NMMA) (14 per cent inhbition) were less effective. Enzyme activity was increased by 48 per cent by 2 mM calcium chloride, and effect reversed by 1 mMEGTA or EDTA. Basal enzyme levels were also partially inhibited by the chelators, indicating the presence of calcium-dependent and -independent isoforms of nitric oxide synthase in the retina. The results show that the NADPH-diaphorase assay is sample and sensitive and that the different isoforms of nitric oxide synthase expressed in chick retinal cells during development can be demonstrated

Inactivación de la lipoamida deshidrogenasa de miocardio por catecolaminas: protección por captopril y otros tioles / Inactivation of the myocardial lipoamide dehydrogenase by catecholamines: prevention by captopril and other thiol compounds

Inactivation of lipoamide dehydrogenase (LipDH) by the Cu(II)/H2O2 Fenton system (SF-Cu(II): (5.0 microM Cu(II), 3.0 mM H2O2) was enhanced by catecholamines (CAs), namely, epinephrine, levoDOPA (DOPA), DOPAMINE, 6-hydroxyDOPAMINE (OH-DOPAMINE) and related compounds (DOPAC, CATECHOL, etc.). After 5 min incubation with the Cu(II)/H2O2/CA system (0,4 mM CA), the enzyme activity decayed as indicated by the following percentage values (mean +/- S.D.; in parenthesis, number of determinations): SF-Cu(II) alone, 43 +/- 10 (18); SF-Cu(II) + epinephrine, 80 +/- 9 (5); SF-Cu(II)'+ DOPA, 78 +/- 2 (4); SF + Cu(II) + DOPAMINE, 88 +/- 7 (5); SF-Cu(II) + OH-DOPAMINE 87 +/- 6 (7); SF-Cu(II) + DOPAC, 88 +/- 3 (6); SF-Cu(II) + catechol, 85 +/- 6 (5). In all cases P < 0,05, with respect to the SF-Cu(II) control sample. CAs effect was concentration-dependent and at the 0-100 microM concentration range, it varied with the CA structure. Above the 100 MicroM concentration, CAs were equally effective and produced 90-100 percent enzyme, inactivation (Figure 2). In the absence of oxy-radical generation, the enzyme specific activity (mean + S.D.) was 149 +/- 10 (24) micromol NADH/min/mg protein. Assay of HO. production by the Cu(II)/H2O2/CA system in the presence of deoxyribose (TBA assay) yielded values much greater than those obtained omitting CA. Hydroxyl radical production depended on the presence of Cu(II) and H2O2, and significant HO. values were obtained with OH-DOPAMINE, DOPAC, epinephrine, DOPAMINE, DOPA and catecol supplemented systems (Table 2). LipDH (1.0 microM) inhibited 50-80 percent deoxyribose oxidation, the inhibition depending on the CA structure (Table 2). Native catalase (20 microg/ml) and bovine serum albumin (40 microg/ml) effectively prevented LipDH inactivation by the Cu(II)/H2O2/CA system, denaturated catalase, SOD, 0,3 M mannitol, 6,0 mM ethanol and 0,2 M benzoate were less effective or did not protect LipDH (Table 3). Incubation of CAs with the Cu(II)H2O2 system produced a time and Cu(II)-dependent destruction of CAs, the corresponding o-quinone, production as illustrated with epinephrine (figures 6 and 7), as illustrated with epinephrine and DOPAMINE (Table 4). These results support LipDH inactivation by (a) reduction of Cu(II) to Cu(I) by CAs followed by Cu-catalyzed production of HO. from H2O2; (b) CA oxidation followed by the corresponding o-quinone interaction with LipDH. CAPTOPRIL, N-acetylcysteine, mercaptopropionylglycine and penicillamine prevented to various degree LipDH inactivation by the Cu(II)/H2O2/CA systems (Table 1). The former was the most effective and 0,4 mM CAPTOPRIL prevented about 95-100 percent the effect of Cu(II)/H2O2/CA systems supplemented with epinephrine, DOPAMINE and OH-DOPAMINE (Figures 3 and Table 1). LipDH increased and CAPTOPRIL inhibited epinephrine oxidation by Cu(II)/H2O2 (Figures 4 and 5). Since un-physiological concentrations of CAs and Cu(II) may be released in the myocardium after ischemia-reperfusion, the summarized observations may contribute to explain myocardial damage in that condition.

Inactivacion de la dihidrolipoamida deshidrogenasa de miocardio por Cu(II) y peroxido de hidrogeno / Inactivation of myocardial dihydrolipoamide dehydrogenase by Cu(II) and hydrogen peroxide

Se estudió la inactivación de la dihidrolipoamida deshidrogenasa de corazón porcino (LipDH) por oxi-radicales, generados por Cu(II) suplementado o no con H2O2 (sistema SF-CU(II) o ácido ascórbico (sistema Cu(II)-Asc). As concentraciones utilizadas fueron: 2,5-10 µM Cu(II): 3,0 mM H2O2 y 0,5 mM ascorbato. Después de 5 minutos de incubación, la actividad lipoamida reductasa de la LipDH decayó de manera irrevedrsible: 83-98 por ciento con SF-Cu(II) y Cu(II)-Asc o 46-53 por ciento con Cu(II). La actividad diaforasa aumentó, demostrando un daño localizado de los grupos SH de la LipDH. NAD+, dihidrolipoamida, GSSG, CAPTOPRIL, complejantes de CU(II) (DL-histidina, batocuproína, EDTA y DETAPAG), la tripanotiona y el alopurinol, protegieron la LipDH frente al SF-Cu(II). El GSH, el ditiotretol, la N-acetilcisteína, la mercaptopropionilglicina, la DL-penicilamina y la L-cisteína, protegieron LipDH frente al CU(II) solamente. NADH, ADP (no ATP), OH-DOPAMINA, DOPA, DOPAC y catecol, aumentaron la inactivación de LipDH por el SF-Cu(II) mientras que OH-DOPAMINA aumentó el efecto del Cu(II). Se discute la acción de los oxi-radicales generados por Cu(II), como causa del daño miocárdico por la reoxigenación post-isquemia

Características histoquímicas e estruturais do testículo do morcego hematófago (desmodus rotundus rotundus, Geoffrey, 1810) / Histochemical and structural characteristics of the testis of the vampire bat (desmodus rotundus rotundus, Geoffrey, 1810)

O estroma testicular do morcego hematófago, incluindo a cápsula testicular e a lâmina própria dos túbulos seminíferos, mostrou forte reatividade ao PAS. Esta reatividade foi possivelmente decorrente da grande quantidade de mucossubstância neutras (glicogênio e outros glicoconjugados), presentes aos níveis dos elementos estruturais da túnica albugínea e lâmina própria tubular, ou seja das fibras musculares lisas; células contráteis alongadas (mióides) e arranjos colágenos, fibrilares ou lamelares. Ao nível da lâmina própria, as membranas basais dos túbulos seminíferos se mostraram intensamente PAS-positivas, comprovando sua composiçäo predominante por mucossubstâncias neutras, de estruturaçäo complexa, previamente reportada (Malmi et al., 1987). O revestimento epitelial do complexo cavitário e superficial da rede testicular mostrou baixas reatividades para mucossubstâncias; proteínas e lipídios totais e enzimas oxidativas estudadas. Contudo, foi observada granulaçäo epitelial apical fortemente PAS-positiva na rede testicular, com hipótese de secreçäo glicoprotéica a este nível. As reaçöes de PAS, Sudan Black B, NADH, MDH e LDH no tecido intersticial testicular parecem se relacionar ao metabolismo esteróide (biossíntese e secreçäo) ao nível das células de Leydig. O epitélio seminífero, de forma gerla, demonstrou baixas reaçöes a todos os métodos histoquímicos utilizados. No entanto, no epitélio adbasal as localizaqçöes histoquímicas da NADH e LDH observadas se relacionaram possivelmente...

Localizaçäo histoquímica da NADH-diaforase e succinato-desidrogenase no epidídimo do gato (Felis domestica, L.) / Histochemical localization of NADH-diaphorase and succinate dehydrogenase in the cat ( Felis domestica, L.)

Neste trabalho säo estudadas, histoquimicamente, as localizaçöes enzimáticas das enzímas NADH-diaforese e SHD no epitélio e estroma do edpidídimo do gato. Uma reaçäo forte e uniforme da NADH foi observada no epitélio do revestimento do corpo e da cauda do órgäo e uma reaçäo fraca, desta enzima, foi notada na cabeça epididimária. Quanto ao estroma ductular esta reatividade, à NADH, foi fraca ao nível da cabeça e média aos níveis do corpo e cauda epididimários. No concernente às reatividades da SDH, estas foram fracas no epitélio e muito fracas no estroma epididimário em todos os segmentos do ducto