Santos Blanco

Age-related changes of the nitric oxide system in the rat brain

This work examines the age-related changes of the NO pathway in the central nervous system (CNS), analyzing nitric oxide synthase (NOS) isoform expression, the level of nitrotyrosine-modified proteins, and the NOS activity in the cerebral cortex, decorticated brain (basal ganglia, thalamus, hypothalamus, tegtum and tegmentum) and cerebellum of young, adult and aged rats. Our data demonstrate that the different NOS isoforms are not uniformly expressed across the CNS. In this sense, the nNOS and eNOS isoenzymes are expressed mainly in the cerebellum and decorticated brain, respectively, while the iNOS isoenzyme shows the highest level in cerebellum. Concerning age, in the cerebral cortex nNOS significantly increased its expression only in adult animals; meanwhile, in the cerebellum the eNOS expression decreased whereas iNOS increased in adult and aged rats. No age-related changes in any isoform were found in decorticated brain. NOS activity, determined by nitrate plus nitrite quantification, registered the highest levels in the cerebellum, where the significant increase detected with aging was probably related to iNOS activity. The number of nitrotyrosine-modified immunoreactive bands differed among regions; thus, the highest number was detected in the decorticated brain while the cerebellum showed the least number of bands. Finally, bulk protein nitration increased in cerebral cortex only in adult animal. No changes were found in the decorticated brain, and the decrease detected in the cerebellum of aged animals was not significant. According to these results, the NO pathway is differently modified with age in the three CNS regions analyzed.

Glutathione S-transferase isoenzymatic response to aging in rat cerebral cortex and cerebellum

Aging is associated with increased oxidant generation. One mechanism involved in the defense of oxidative products is the family of glutathione transferases (GST). We have analyzed the activity, distribution and expression of GSTP1 and GSTA4 isoenzymes in the cerebral cortex and cerebellum of young, adult and aged rats. The total GST activity, measured with the universal substrate 1-chloro-2,4-dinitrobenzene (CDNB), increased only with the maturation process; however GSTA4 activity, using the specific substrate 4-hydroxynonenal (HNE), did show an age-dependent increase in both brain regions. Cellular location of GSTA4 in astrocytes was not changed except for young cerebral cortex and adult/aged cerebellum that also showed immunoreactivity in layer III pyramidal neurons and Bergman radial glia, respectively. Distribution of GSTP1 was similar among groups and only an increased number of positive oligodendrocytes was found in the Purkinje and granular layer of adult/aged cerebellum. The GSTA4 and GSTP1 expression increased from young to adult/aged brain and GSTA4 even augmented in the aged cerebral cortex. These results suggest a GST isoenzymatic response with aging, but above all with the maturation process.

Upregulation of endothelial nitric oxide synthase maintains nitric oxide production in the cerebellum of thioacetamide cirrhotic rats

This study examines the expression and cellular distribution pattern of nitric oxide synthase (NOS) isoforms, nitrotyrosine-derived complexes, and the nitric oxide (NO) production in the cerebellum of rats with cirrhosis induced by thioacetamide (TAA). The results showed local changes in the tissue distribution pattern of the NOS isoforms and nitrated proteins in the cerebellum of these animals. Particularly, eNOS immunoreactivity in perivascular glial cells of the white matter was detected only in TAA-treated animals. In addition, although neither neuronal NOS (nNOS) nor inducible NOS (iNOS) cerebellar protein levels appeared to be affected, the endothelial NOS (eNOS) isoform significantly increased its expression, and NO production slightly augmented in TAA-treated rats. These NOS/NO changes may contribute differently to the evolution of the hepatic disease either by maintaining the guanosine monophosphate-NO signal transduction pathways and the physiological cerebellar functions or by inducing oxidative stress and cell damage. This model gives rise to the hypothesis that the upregulation of the eNOS maintains the physiological production of NO, while the iNOS is silenced and the nNOS remains unchanged. The differential NOS-distribution and expression pattern may be one of the mechanisms involved to balance cerebellar NO production in order to minimize TAA toxic injury. These data help elucidate the role of the NOS/NO system in the development and progress of hepatic encephalopathy associated with TAA cirrhosis.

Immunohistochemical localisation of neuronal nitric oxide synthase in the rainbow trout kidney.

The distribution of nitrergic nervous structures in the trout kidney was studied by peroxidase-linked ABC immunostaining procedures using a polyclonal antibody raised against the neuronal isoform of nitric oxide synthase. The nitrergic plexus reaches the kidney along the vasculature, mainly running with the postcardinal vein where nitrergic fibres, microganglia like cellular clusters and isolated neurones were detected. The atubular head-kidney only showed isolated nitrergic fibres close to the larger arteries. On the other hand, the collecting tubules, collecting ducts, large arteries and glomerular arterioles of the tubular middle and posterior trunks were innervated by nitrergic fibres even though immunoreactive neurones were also observed in close apposition to some tubular elements and large arteries. These results suggest that, according to morphofunctional differences between the fish and mammalian kidneys, nitrergic neural structures may be involved in the control of particular renal functions in the rainbow trout.

Study of the nitric oxide system in the rat cerebellum during aging

BackgroundThe cerebellum is the neural structure with the highest levels of nitric oxide, a neurotransmitter that has been proposed to play a key role in the brain aging, although knowledge concerning its contribution to cerebellar senescence is still unclear, due mainly to absence of integrative studies that jointly evaluate the main factors involved in its cell production and function. Consequently, in the present study, we investigate the expression, location, and activity of nitric oxide synthase isoenzymes; the protein nitration; and the production of nitric oxide in the cerebellum of adult and old rats.ResultsOur results show no variation in the expression of nitric oxide synthase isoforms with aging, although, we have detected some changes in the cellular distribution pattern of the inducible isoform particularly in the cerebellar nuclei. There is also an increase in nitric oxide synthase activity, as well as greater protein-nitration levels, and maintenance of nitrogen oxides (NOx) levels in the senescent cerebellum.ConclusionsThe nitric oxide/nitric oxide syntahses system suffers from a number of changes, mainly in the inducible nitric oxide synthase distribution and in overall nitric oxide synthases activity in the senescent cerebellum, which result in an increase of the protein nitration. These changes might be related to the oxidative damage detected with aging in the cerebellum.

Constitutive nitric oxide synthases are responsible for the nitric oxide production in the ischemic aged cerebral cortex

Aged brain shows reduced biological plasticity to meet emergency conditions such as ischemia, a process in which nitric oxide (NO) and apoptosis have been shown to play important roles. Using a model of transient global ischemia, we have analyzed the NO system and the p53, bax and bcl-2 response in the cerebral cortex of aged rats. Although immediately after ischemia the NO level is maintained, the reperfusion period increases NO concentrations together with the following: (i) greater bulk-protein nitration mainly due to a 50-kDa immunoreactive band; (ii) an increase in p53 protein; and (iii) an up-regulation of Bax together with a down-regulation of Bcl-2. These results match up with induced endothelial nitric oxide synthase expression immediately after ischemia and in neuronal nitric oxide synthase with the reperfusion. However, inducible nitric oxide synthase was not altered with ischemia/reperfusion. Altogether, these data suggest that NO production in cerebral cortex of aged ischemic animals is due to the constitutive NO synthase isoforms. This response is accompanied by the increased expression of pro-apoptotic proteins.

Age modulates the nitric oxide system response in the ischemic cerebellum.

To determine whether age influences the nitric oxide system response to ischemia in the cerebellum, we have analyzed the levels of nitrogen oxides (NOx) and the expression of the different nitric oxide synthase isoforms (NOS) in mature adult (4-5 months old) and aged rats (24-27 months old) subjected to a transient global ischemia/reperfusion (I/R) model. We also analyzed the nitrated proteins and the glial fibrillary acidic protein (GFAP) expression. NOx concentration in adult rats, which more than doubled the values found in the aged rats, decreased after the ischemia and reperfusion. However, in the aged animals, these NOx levels did not significantly change after I/R. Constitutive isoforms were first down-regulated in the ischemic period, in both adult and aged animals. However, after 6 h of reperfusion, these isoforms were up-regulated, but only in aged rats. After I/R, iNOS was up-regulated in adults but down-regulated in the aged rats. Hence, after an episode of transient global ischemia and reperfusion, the aged cerebellum maintains a balanced NO production, silencing the iNOS isoform and inducing a weak expression of nNOS and eNOS; this allows NO physiological functions while avoiding possible undesirable effects such as the nitrative damage or astrocyte activation.

Melatonin influences NO/NOS pathway and reduces oxidative and nitrosative stress in a model of hypoxic-ischemic brain damage

In this work, using a rat model combining ischemia and hypobaric hypoxia (IH), we evaluate the relationships between the antioxidant melatonin and the cerebral nitric oxide/nitric oxide synthase (NO/NOS) system seeking to ascertain whether melatonin exerts its antioxidant protective action by balancing this key pathway, which is highly involved in the cerebral oxidative and nitrosative damage underlying these pathologies. The application of the IH model increases the expression of the three nitric oxide synthase (NOS) isoforms, as well as nitrogen oxide (NOx) levels and nitrotyrosine (n-Tyr) impacts on the cerebral cortex. However, melatonin administration before IH makes nNOS expression response earlier and stronger, but diminishes iNOS and n-Tyr expression, while both eNOS and NOx remain unchanged. These results were corroborated by nicotine adenine dinucleotide phosphate diaphorase (NADPH-d) staining, as indicative of in situ NOS activity. In addition, the rats previously treated with melatonin exhibited a reduction in the oxidative impact evaluated by thiobarbituric acid reactive substances (TBARS). Finally, IH also intensified glial fibrillary acidic protein (GFAP) expression, reduced hypoxia-inducible factor-1alpha (HIF-1α), but did not change nuclear factor kappa B (NF-κB); meanwhile, melatonin did not significantly affect any of these patterns after the application of the IH model. The antioxidant melatonin acts on the NO/NOS system after IH injury balancing the release of NO, reducing peroxynitrite formation and protecting from nitrosative/oxidative damage. In addition, this paper raises questions concerning the classical role of some controversial molecules such as NO, which are of great consequence in the final fate of hypoxic neurons. We conclude that melatonin protects the brain from hypoxic/ischemic-derived damage in the first steps of the ischemic cascade, influencing the NO/NOS pathway and reducing oxidative and nitrosative stress.

Proteomic characterization of nitrated cell targets after hypobaric hypoxia and reoxygenation in rat brain.

UNLABELLED This study analyzes the nitrated protein profile of the rat-brain cortex in a model of hypoxia/reoxygenation, identifying the nitrated proteins and assessing spot changes. The proteins identified were grouped into categories, according to their function: 1) metabolism: pyruvate kinase (PK), α-enolase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphoglycerate mutase 1 (PGAM1), and glutamine synthetase (GS); 2) cytoskeletal proteins: α-tubulin, β-tubulin, γ-actin, and glial fibrillary acidic protein (GFAP); 3) chaperones: heat-shock protein 71kDa (HSP71); and 4) carrier proteins: voltage-dependent anion-selective channel protein 1 (VDAC-1) and Atp6v1a. PK, α-enolase, and GS nitration rates were upregulated, increasing progressively during reoxygenation and peaking at 24h. GAPDH and PGAM1 nitration levels fell after hypoxia/reoxygenation. α-Tubulin, β-tubulin, γ-actin, and GFAP nitration rates augmented at 24h, but diminished at 5d. HSP71 suffered from nitration immediately after hypoxia, but not during reoxygenation. VDAC-1 tyrosine nitration was identified only in the control group, whereas detectable Atp6v1a nitration levels were observed only immediately after hypoxia. The data have been deposited to the ProteomeXchange with identifier PXD001049. Our findings suggest a hypothetically crucial linkage between nitration-related protein modifications and metabolic and cell-structure alterations. These changes are probably needed for the remodeling and plasticity processes activated by the hypoxic brain response. BIOLOGICAL SIGNIFICANCE For the first time the spectrum of nitrated proteins in the hypoxic brain as well as its changes during reoxygenation are described. Our findings suggest a hypothetically crucial linkage between nitration-related protein modifications and metabolic and cell-structure alterations. These changes are probably needed for the remodeling and plasticity processes activated by the hypoxic brain response. The biological relevance of these findings is linked to the important role developed by the signaling molecule NO in the hypoxic brain, and could be interpreted in two different but complementary ways: first, as a mechanism of damage due to nitration impacts over some key proteins affecting its structure and function; and second, as a regulation mechanism involved in the hypoxic response. Hence, based on the modified proteins identified and their functions, it would be possible to design new tools and therapies to prevent brain damage in low-oxygen-pressure atmospheres.

Colorimetric quantification and in situ detection of collagen

A simple multidisciplinary and inexpensive laboratory exercise is proposed, in which the undergraduate student may correlate biochemical and anatomical findings. The entire practical session can be completed in one 2.5–3 hour laboratory period, and consists of the quantification of collagen and total protein content from tissue sections — without previous homogenisation — by a colorimetric assay, and a staining method for in situ collagen detection carried out on the same tissue samples using the same chemicals. Because collagen accumulation is a common characteristic of hepatic diseases such as fibrosis and cirrhosis, the experiment may also serve as the basis for discussing the importance of collagen detection and quantification in estimating the degree of fibrosis from subjects with liver disease.