Biomedical subjects

Luiz R G Britto

Publications and source records attributed to Luiz R G Britto.

At least 19 recordsLinked to original sources

PubMed2006

Production of nitric oxide by airways neutrophils in the initial phase of murine asthma.

In experimental models of asthma, nitric oxide (NO) is produced and contributes to the physiopathology of the disease. Neutrophil is the first cell to infiltrate the lung in response to antigen stimulation, it has the capacity to produce NO but a clear demonstration that neutrophils contribute to NO production in asthma is lacking. This was the aim of the present study. At weekly intervals C57Bl/6 mice were sensitized twice with ovalbumin-alumen and challenged twice with ovalbumin aerosol. The peak of neutrophil infiltration in the bronchoalveolar lavage fluid (BALF) was 12 h after challenge, when neutrophils constituted 70% of the cell population and eosinophils only 1.5%. BALF cell preparations were stained with a NO-sensitive fluorescent dye (DAF-2) and with a nucleus marker (DAPI). Most DAF-2 stained cells could be identified as polymorphonuclear leukocytes, by the co-localization of both DAF-2 and DAPI staining. Cells from animals treated with l-NAME, were not stained for DAF-2 confirming the specificity of DAF-2 staining for NO. Moreover, the peak expression of inducible nitric oxide synthase (NOS2), in BALF cells and lung homogenates, was coincident with the peak of BALF neutrophil influx. NOS2 protein expression (arbitrary units) was detected 6 h after challenge (17.8+/-9.1 in BALF cells; 47.5+/-7.7 in lung homogenates), peak expression was at 12 h (54.5+/-8.7 and 133.7+/-10), decreasing thereafter, being no longer detected after 24 h. Thus, the neutrophils infiltrating the lung in the initial phase of murine asthma are producing NO via NOS2.

Animals↗

PubMed2006

Differential regulation of the neuronal isoform of nitric oxide synthase in the superior colliculus and dorsal lateral geniculate nucleus of the adult rat brain following eye enucleation.

Nitric oxide has been shown to play various physiological and pathological roles in the visual system. We studied here the expression of the neuronal isoform of nitric oxide synthase in the rat superior colliculus and in the dorsal lateral geniculate nucleus after unilateral enucleation, by means of immunohistochemistry, immunoblotting, and real-time PCR. Immunohistochemistry revealed an increase of nitric oxide synthase-positive neurons in specific layers of the superior colliculus and in the dorsal lateral geniculate nucleus between 1 and 30 days post-lesion. Immunoblotting analyses confirmed that the neuronal isoform of nitric oxide synthase is upregulated in the superior colliculus and in the dorsal lateral geniculate nucleus after retinal removal. Diaminofluorescein histochemistry suggested that nitric oxide production was increased in both deafferented retinorecipient areas. Our real-time PCR results indicated that nitric oxide synthase transcript levels in the superior colliculus were not significantly altered after monocular enucleation, although an upregulation of the enzyme transcription was detected into the deafferented dorsal lateral geniculate nucleus. These findings indicated that neuronal nitric oxide synthase may undergo different forms of regulation in the adult deafferented visual system.

Analysis of Variance↗

PubMed2006

Down-regulation of ABCB1 transporter by atorvastatin in a human hepatoma cell line and in human peripheral blood mononuclear cells.

PURPOSE: The effect of atorvastatin, an HMG-CoA reductase inhibitor, on expression and activity of the drug transporter ABCB1 in HepG2 cells and peripheral blood mononuclear cells (PBMCs) was examined. METHODS: Localization and expression of ABCB1 in hepatocytes was examined by indirect immunofluorescence. Expression of ABCB1 mRNA and ABCB1 activity were examined in atorvastatin-treated and control cells and PBMCs using real-time PCR and Rhodamine 123 efflux assay. RESULTS: Immunohistochemical analysis revealed that ABCB1 is located at the apical membrane of the bile canaliculi. Atorvastatin at 10 and 20 microM up-regulated ABCB1 expression resulting in a significant 1.4-fold increase of the protein levels. Treatment of HepG2 cells with 20 microM atorvastatin caused a 60% reduction on mRNA expression (p<0.05) and a 41% decrease in ABCB1-mediated efflux of Rhodamine123 (p<0.01) by flow cytometry. Correlation was found between ABCB1 mRNA levels and creatine kinase (r=0.30; p=0.014) and total cholesterol (r=-0.31; p=0.010). CONCLUSIONS. Atorvastatin leads to decreased ABCB1 function and modulates ABCB1 synthesis in HepG2 cells and in PBMCs. ABCB1 plays a role in cellular protection as well as in secretion and/or disposition, therefore, inhibition of ABCB1 synthesis may increase the atorvastatin efficacy, leading to a more pronounced reduction of plasma cholesterol.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2006

Ontogeny of subunits 2 and 3 of the AMPA-type glutamate receptors in Purkinje cells of the developing chick cerebellum.

Several molecules, involved in cellular communication in the mature nervous system, appear to play important roles during neural development. These roles include neuronal growth, morphological changes of neurites, and neuronal survival. Such plasticity processes seem to be in part the result of activation of different receptor subtypes, which could cause Ca(2+) influx, a major candidate to be an outgrowth promoter. In this context, we performed immunohistochemical and in situ hybridization experiments to examine the following aspects of the development of chick cerebellum Purkinje cells: (i) expression of AMPA-type glutamate receptor GluR2/3 proteins; (ii) the levels of mRNAs coding for the GluR2 and GluR3 flip/flop isoforms; and (iii) expression of calbindin (CB) and parvalbumin (PV). Expression of GluR2/3 proteins, CB, PV, and the mRNAs coding for GluR2 and GluR3 splice variants all revealed a differential expression during development in chick Purkinje cells. GluR2/3 proteins and the GluR3 flop variant start to be expressed at E10, while the expression of CB, PV, the GluR3 flip isoform and the splice variants of GluR2 all started around E12-E14. All proteins showed an increasing expression from embryonic stages into the posthatching period. These results reveal a developmentally regulated expression of GluR2/3 proteins, including their splice variants, and of CB and PV in Purkinje cells. These findings may suggest a relationship between these proteins and specific cerebellar developmental processes.

Animals↗

PubMed2006

5-Aminolevulinate and 4, 5-dioxovalerate ions decrease GABA(A) receptor density in neuronal cells, synaptosomes and rat brain.

Porphyrias are heme-associated metabolic disorders such as intermittent acute porphyria (IAP) and lead poisoning, where 5-aminolevulinate (ALA) accumulates. Effects of ALA on the CNS have been explained by ALA binding to GABA(A) receptors, followed by receptor lesions from oxyradicals and 4, 5-dioxovalerate (DOVA) generated from metal-catalyzed ALA oxidation by oxygen. We have characterized the effects of ALA and DOVA on GABA(A) receptor density in synaptosomes and neurons in vitro and also in brains of rats treated with ALA or succinylacetone methyl ester (SAME), a tyrosine catabolite derivative able to induce ALA accumulation. Radiolabeling assays revealed that following exposure to DOVA the concentration of synaptosomal GABAergic sites decreased by approximately 50%. Pretreatment with DOVA resulted in less GABA(A) receptor density in P19 and WERI cells and altered cell morphology. Furthermore, exposure to DOVA also induced a 5-fold increase in WERI cell mortality rate. Treatment with ALA resulted in loss of neuronal morphology and decrease of GABA(A) density in P19 neuronal cells. ALA and SAME treatment diminished the density of GABAergic receptors in the habenular complex and the parabigeminal nucleus of rat brain as studied by immunohistochemical procedures. Our results strongly suggest that ALA- and DOVA-promoted damage to GABA(A) receptors may contribute to the neurological manifestations of AIP and plumbism.

Aminolevulinic Acid↗

PubMed2006

Correction of endothelial dysfunction in diabetic female rats by tetrahydrobiopterin and chronic insulin.

Diabetes-induced vascular dysfunction has mainly been studied in males. However, the mechanisms involved may not correspond to those in females. Here we analyzed the effects of tetrahydrobiopterin (BH(4)) and chronic insulin on the physiology of mesenteric arterioles of alloxan-diabetic female rats. The parameters studied were the mesenteric arteriolar reactivity (intravital microscopy), nitric oxide synthase (NOS) activity (conversion of L-arginine to L-citrulline), eNOS gene expression (RT-PCR), NO production (diaminofluorescein), reactive oxygen species (ROS) generation (intravital fluorescence microscopy) and Cu/Zn superoxide dismutase (SOD) activity (spectrophotometry) and gene expression (RT-PCR). The reduced endothelium-dependent vasodilation of diabetic females was corrected by both BH(4) and insulin. NOS activity was decreased by diabetes, but insulin did not correct it. However, NOS expression was not modified by either diabetes or insulin. Arterioles of diabetic rats exhibited lower NO production, which was fully corrected by BH(4) and only partially by insulin. ROS generation was increased in diabetic rats, and both BH(4) and insulin normalized it. Diabetes did not change SOD activity and gene expression. However, insulin increased SOD activity but not its expression. Our data suggest that, similarly to males, endothelial dysfunction in female diabetic rats involves an altered ROS/NO imbalance. In contrast to males, however, insulin does not regulate NOS in the microcirculation of diabetic females.

Acetylcholine↗

PubMed2005

Dehydroepiandrosterone increases beta-cell mass and improves the glucose-induced insulin secretion by pancreatic islets from aged rats.

The effect of dehydroepiandrosterone (DHEA) on pancreatic islet function of aged rats, an animal model with impaired glucose-induced insulin secretion, was investigated. The following parameters were examined: morphological analysis of endocrine pancreata by immunohistochemistry; protein levels of insulin receptor, IRS-1, IRS-2, PI 3-kinase, Akt-1, and Akt-2; and static insulin secretion in isolated pancreatic islets. Pancreatic islets from DHEA-treated rats showed an increased beta-cell mass accompanied by increased Akt-1 protein level but reduced IR, IRS-1, and IRS-2 protein levels and enhanced glucose-stimulated insulin secretion. The present results suggest that DHEA may be a promising drug to prevent diabetes during aging.

Adjuvants, Immunologic↗

PubMed2005

NF-kappaB, MEF2A, MEF2D and HIF1-a involvement on insulin- and contraction-induced regulation of GLUT4 gene expression in soleus muscle.

The GLUT4 gene transcriptional activity has a profound impact on the insulin-mediated glucose disposal and it is, therefore, important to understand the mechanisms underlying it. Insulin and exercise modulate GLUT4 expression in vivo, but the net control and involved mechanisms of each one have not been established yet. This paper sought to discriminate, in soleus muscle, the effects of insulin and muscle contraction on GLUT4 gene expression, and the involvement of transcriptional factors: myocite enhancer factor 2 (MEF2 A/C/D), hypoxia inducible factor 1-a (HIF1-a) and nuclear factor-kappa B (NF-kappaB). The GLUT4 mRNA was reduced by fasting (40%), and increased by in vitro incubation with insulin (25%) or insulin plus glucose (40%), which was accompanied by opposite regulations of NF-kappaB mRNA. Differently, in vitro, muscle contraction led to a rapid increase (35-80%) in GLUT4, MEF2A, MEF2D and HIF1-a mRNAs. Additionally, electrophoretic mobility shift assay confirmed changes in the binding activity of nuclear proteins to consensus NF-kappaB, GLUT4-Ebox and GLUT4-AT-rich element probes, parallel to the mRNA changes of their respective transcriptional factors NF-kappaB, HIF1-a and MEF2s. Concluding, insulin- and contraction-induced regulation of GLUT4 expression involves distinct transcriptional factors.

Animals↗

PubMed2005

Developmental expression of cannabinoid receptors in the chick retinotectal system.

The cannabinoid system has been suggested to participate in processes such as antinociception, cognition, motor control, and, more recently, development of the nervous system. This study describes the expression of the CB1 cannabinoid receptor in the developing chick retina and optic tectum by means of conventional immunoperoxidase protocols. CB1 immunoreactivity was initially detected around the embryonic day 4 (E4) in both the retina and tectum. In the retina, CB1 immunoreactivity was first observed in presumptive ganglion cells and, subsequently, in the inner plexiform layer and two populations of neurons of the inner nuclear layer. The post-hatched chick exhibited a pattern of staining that included four sublayers of the inner plexiform layer, a few stained cells in the ganglion cell layer, and labeled neurons both in the inner and central parts of the inner nuclear layer. The latter two types of neurons appear to be amacrine and bipolar cells, respectively. In the tectum, CB1 first appeared in its most superficial zone and later in several tectal laminae, including a white matter layer (stratum album centrale; Cajal's layer 14). There was a remarkable and transient increase of labeling at E10, followed by a continuous reduction of staining until E18. In the post-hatched chick, tectal staining was mostly confined to layers 2-3 and 5-6. Stained perikarya were seldom observed in the tectum at any stage. These data are in agreement with a possible developmental function of CB1, as it is expressed several days before synaptogenesis ensues and exhibits transient expression in the optic tectum.

Animals↗

PubMed2005

Retinal projections to the midline and intralaminar thalamic nuclei in the common marmoset (Callithrix jacchus).

In this study, we report the identification of a hitherto not reported direct retinal projection to midline and intralaminar thalamic nuclei in the marmoset brain. After unilateral intravitreal injections of cholera toxin subunit B (CTb), anterogradely transported CTb-immunoreactive fibers and presumptive terminals were seen in the following thalamic midline nuclei: paraventricular, rhomboid, interanteromedial, and reuniens, and thalamic intralaminar nuclei: central medial, central lateral, central dorsal, and parafascicular. Studies employing sensitive tracers in other primate species are needed in order to verify the possible universality of these projections. Some of the possible functional correlates of the present data are briefly discussed. The present results may contribute to the elucidation of the anatomical substrate of the functionally demonstrated involvement of this midline/intralaminar thalamic nuclear complex in several domains that include arousal and awareness, besides specific cognitive, sensory, and motor functions.

Animals↗

PubMed2004

Increased expression of nitric oxide synthase in visual structures of the chick brain after retinal removal.

Nitric oxide (NO) seems to act as a retrograde messenger in the establishment and refinement of synaptic connections during development and in neural plasticity processes in adult life. Previous studies have shown that the expression of NO synthase (NOS) in the optic tectum of developing chicks is regulated by the retinal innervation. The aim of this study was to observe the effects of unilateral retinal lesions upon the expression of NOS in central visual areas of the adult chick brain. After different survival times (1-30 days), the chick brains were submitted to immunohistochemical, immunoblotting, and NO imaging procedures to evaluate NOS expression and activity. Our results indicate that NOS expression in visual areas is also regulated by retinal innervation in the adult chick. However, differently from the case in the developing animal, the deafferentation seems to generate an increase of the NOS expression in retinorecipient visual areas. Our results suggest that NOS expression in visual structures of the adult chick brain may be down-regulated by the retinal innervation. Alternatively, the increase of NOS expression observed after retinal removal could be an indicative of a role of the NO system in plasticity processes.

Animals↗

PubMed2004

Snake venom components enhance pain upon subcutaneous injection: an initial examination of spinal cord mediators.

Snakebites are a relevant public health problem in Central and South America. Snake bite envenomations cause intense pain, not relieved by anti-venom. The fangs of many species are short, causing subcutaneous injection. Fangs of larger species inflict subcutaneous or intramuscular envenomation. To understand pain induced by subcutaneous venom, this study examined spinal mechanisms involved in pain-enhancing effects of subcutaneous Lys49 and Asp49 secretory phospholipase-A(2) (sPLA2), two components of Bothrops asper snake venom showing highly different enzymatic activities. Unilateral intraplantar sPLA2-Lys49 (catalytically inactive) or sPLA2-Asp49 (catalytically active) into rat hindpaws each induced mechanical hyperalgesia (Randall-Selitto test), whereas only catalytically active sPLA2-Asp49 caused mechanical allodynia (von Frey test). Effects induced by both sPLA2s were inhibited by intrathecal fluorocitrate, a reversible glial metabolic inhibitor. In support, immunohistochemical analysis revealed activation of dorsal horn astrocytes and microglia after intraplantar injection of either sPLA2. Spinal proinflammatory cytokines, nitric oxide, and prostanoids each appear to be involved in the pain-enhancing effects of these sPLA2s. Blockade of interleukin-1 (IL1) inhibited hyperalgesia induced by both sPLA2s, while leaving allodynia unaffected. Blockade of tumor necrosis factor reduced responses to sPLA2-Asp49. An inhibitor of neuronal nitric oxide synthase, 7-nitroindazole (7-NI), inhibited hyperalgesia induced by both sPLA2s, without interfering with allodynia induced by sPLA2-Asp49. On the other hand, L-N(6)-(1-iminoethyl)lysine (L-NI), an inhibitor of the inducible nitric oxide synthase, did not alter any sPLA2-induced effect. Lastly, celecoxib, an inhibitor of cyclooxygenase-2, attenuated sPLA2 actions. These data provide the first evidence of spinal mediators involved in pain facilitation induced by subcutaneous venoms.

Animals↗

PubMed2003

Nicotine and alpha-bungarotoxin modify the dendritic growth of cholinoceptive neurons in the developing chick tectum.

Nicotinic acetylcholine receptors have been suggested to participate in morphogenetic processes during development of the nervous system. In this study, nicotine applied both in ovo and in vitro produced a reduction of the neuritic length of cholinoceptive neurons of the developing chick tectum, whereas alpha-bungarotoxin produced the opposite effect. Taken together with previous data, our results are indicative of a role of the alpha-bungarotoxin-sensitive nicotinic receptors in neural development.

Animals↗

PubMed2003

Static, but not optokinetic visual stimuli induce Fos expression in the retina and brain of retinal degeneration mice.

The stimulated expression of the c-fos gene was used in this study to characterize the residual vision of retinal degeneration (rd) mice. Conventional immunohistochemistry was employed to detect Fos-positive nuclei in the rd mouse retina and brain after monocular application of static and dynamic photic stimuli. Diffuse light induced Fos-positive nuclei in the inner nuclear and ganglion cell layers of the stimulated retina and in the contralateral pretectal olivary nucleus. On the other hand, Fos-positive nuclei were rarely detected in the rd mouse retina and brain after stimulation with a slowly displaced random-dot pattern. These results suggest that the residual vision of the rd mouse involves mainly the circuits that are related to the processing of static photic stimuli.

Animals↗

PubMed2003

Differential effects of aging on the distribution of calcium-binding proteins in a pretectal nucleus of the chicken brain.

The nucleus pretectalis (PT) of birds is an ovoid-shaped visuomotor cell group of the pretectum that receives tectal input and projects back to the optic tectum. We performed immunohistochemical single- and double-labeling to determine the distribution and abundance of neurons containing three calcium-binding proteins, parvalbumin (PV), calretinin (CR), and calbindin (CB), in the PT in chickens at three ages. We found that PV-positive and CR-positive cells co-localize and are largely found in the outer part of PT at all ages. The GluR4 subunit of the AMPA-type glutamate receptor was selectively localized to these neurons. CB-positive neurons, however, were largely absent from the PT in young and adult chickens. The abundance of PV-positive and CR-positive neurons in PT in old birds was indistinguishable from that in the younger birds, but CB-positive perikarya were 10-20-fold more common than in young birds, and were again mainly found in the outer part of PT. The overall abundance of neurons in PT was reduced to about 50% of its former abundance in the old birds, with this loss restricted to the central part of the nucleus. These data indicate that a cell loss process develops in PT as birds age, that parvalbuminergic and calretinergic neurons resist this process, and that this process is associated with increased expression of CB.

Aging↗

PubMed2003

The C-type natriuretic peptide precursor of snake brain contains highly specific inhibitors of the angiotensin-converting enzyme.

The bradykinin-potentiating peptides from Bothrops jararaca venom are the most potent natural inhibitors of the angiotensin-converting enzyme. The biochemical and biological features of these peptides were crucial to demonstrate the pivotal role of the angiotensin-converting enzyme in blood pressure regulation. In the present study, seven bradykinin-potentiating peptides were identified within the C-type natriuretic peptide precursor cloned from snake brain. The bradykinin-potentiating peptides deduced from the B. jararaca brain precursor are strong in vitro inhibitors of the angiotensin-converting enzyme (nanomolar range), and also potentiate the bradykinin effects in ex vivo and in vivo experiments. Two of these peptides are novel bradykinin-potentiating peptides, one of which displays high specificity toward the N-domain active site of the somatic angiotensin-converting enzyme. In situ hybridization studies revealed the presence of the bradykinin-potentiating peptides precursor mRNAs in distinct regions of the B. jararaca brain, such as the ventromedial hypothalamus, the paraventricular nuclei, the paraventricular organ, and the subcommissural organ. The biochemical and pharmacological properties of the brain bradykinin-potentiating peptides, their presence within the neuroendocrine regulator C-type natriuretic peptide precursor, and their expression in regions of the snake brain correlated to neuroendocrine functions, strongly suggest that these peptides belong to a novel class of endogenous vasoactive peptides.

Amino Acid Sequence↗

PubMed2003

Pancreatic beta-cells express phagocyte-like NAD(P)H oxidase.

The presence of a phagocyte-like NAD(P)H oxidase in pancreatic beta-cells was investigated. Three NAD(P)H oxidase components were found in pancreatic islets by RT-PCR: gp91(PHOX), p22(PHOX), and p47(PHOX). The components p67(PHOX) and p47(PHOX) were also demonstrated by Western blotting. Through immunohistochemistry, p47(PHOX) was mainly found in the central area of the islet, confirming the expression of this component by insulin-producing cells. Activation of NAD(P)H oxidase complex in the beta-cells was also examined by immunohistochemistry. The pancreatic islets presented slower kinetics of superoxide production than HIT-T15 cells, neutrophils, and macrophages, but they reached 66% that of the neutrophil nitroblue tetrazolium (NBT) reduction after 2 h of incubation. Glucose (5.6 mmol/l) increased NBT reduction by 75% when compared with control. The involvement of protein kinase C (PKC) in the stimulatory effect of glucose was confirmed by incubation of islets with phorbol myristate acetate (a PKC activator) and bysindoylmaleimide (GF109203X) (a PKC-specific inhibitor). Diphenylene iodonium [an NAD(P)H oxidase inhibitor] abolished the increase of NBT reduction induced by glucose, confirming the NAD(P)H oxidase activity in pancreatic islets. Because reactive oxygen species are involved in intracellular signaling, the phagocyte-like NAD(P)H oxidase activation by glucose may play an important role for beta-cell functioning.

Animals↗

PubMed2002

Interspecific differences in the expression of the AMPA-type glutamate receptors and parvalbumin in the nucleus of Edinger-Westphal of chicks and pigeons.

The distribution of AMPA-type glutamate receptor (GluR) subunits was studied in the Edinger-Westphal nucleus (EW) of chicks and pigeons. GluR1, GluR2, GluR3 and GluR4 subunits appeared to be present in EW neurons of both species, but interspecific differences were observed in the abundance of the different types of subunits found in EW neurons. Of particular note, GluR2 immunoreactivity was present in the vast majority (ca. 80%) of neurons of pigeon EW but was found in only a small fraction (ca. 15%) of chick EW neurons. Scarcity of the GluR2 subunit in chick EW was confirmed by in situ hybridization. Because of the tendency for parvalbumin to be localized to neurons that are selectively deficient in GluR2, we also studied the localization of parvalbumin, as well as other calcium-binding proteins, in EW of chick and pigeon. Parvalbumin was found in more than 50% of chick EW neurons but was not detected in pigeon EW neurons. Our results suggest that there are major glutamatergic inputs to EW neurons in both pigeons and chicks. Furthermore, there are likely to be more AMPA-type calcium-permeable glutamate receptors in EW neurons of chick than in pigeon, since it is known that the subtype containing the edited GluR2 subunit is not calcium permeable.

Animals↗