Nitric oxide synthase and NADPH-diaphorase after acute hypobaric hypoxia in the rat caudate putamen.

Changes in the production system of nitric oxide (NO), a multifunctional biological messenger known to participate in blood-flow regulation, neuromodulation, and neuroprotection or neurotoxicity, were investigated in the caudate putamen of adult rats submitted to hypobaric hypoxia. Employing immunohistochemistry, Western blotting, enzymatic assay, and NADPH-diaphorase staining, we demonstrate that neuronal nitric oxide synthase (nNOS) expression and constitutive nitric oxide synthase (cNOS) activity were transiently activated by 7 h of exposure to a simulated altitude of 8325 m (27,000 ft). In addition, endothelial nitric oxide synthase (eNOS) immunoreactivity and blood vessel NADPH-diaphorase staining peaked immediately after the hypoxic stimulus, whereas inducible nitric oxide synthase (iNOS) expression and activity remained unaltered. Nitrotyrosine formation, a marker of protein nitration, was evaluated by immunohistochemistry and Western blotting, and was found to increase parallel to nitric oxide synthesis. We conclude that the nitric oxide system undergoes significant transient alterations in the caudate putamen of adult rats submitted to acute hypobaric hypoxia.

Nitric oxide system and protein nitration are modified by an acute hypobaric hypoxia in the adult rat hippocampus.

Changes in the nitric oxide system of the hippocampus from rats submitted to hypobaric hypoxia were investigated. Adult rats were exposed to a simulated altitude of 8,325 m (27,000 ft) for 7 h and killed after 0 h, 1, 3, 5, 10 and 20 days of reoxygenation. The number of neuronal nitric oxide synthase immunoreactive neurons and their dendritic plexus, as well as neuronal nitric oxide synthase immunoblotting densitometry and calcium-dependent activity increased from 0 h to 3 days of reoxygenation. In addition, endothelial nitric oxide synthase immunoreactivity peaked after 7 h of hypobaric hypoxia. Nitrotyrosine immunoreactivity showed an increase in the pyramidal cells of CA2-CA3 and in glial cells surrounding the blood vessels after 0 h, 1 and 3 days of reoxygenation. Immunoblotting densitometry of 1 of the 2 nitrotyrosine-immunoreactive bands detected also increased after 0 h and 1 day of reoxygenation. Inducible nitric oxide synthase immunoreactivity was found only in some blood vessels after 0 h, 1 and 3 days of reoxygenation, but no changes in inducible nitric oxide synthase activity or immunoblotting were detected. We conclude that transient activation of the nitric oxide system constitutes a hippocampal response to hypobaric hypoxia.

Generation and role of angiostatin in human platelets.

Platelets regulate new blood vessel growth, because they contain a number of angiogenesis promoters and inhibitors. Additionally, platelets contain matrix metalloproteinases (MMPs), which when released mediate platelet adhesion and aggregation, and plasminogen, a fibrinolytic system enzyme that serves to limit blood clot formation. Enzymatic cleavage of plasminogen by MMPs generates angiostatin, an angiogenesis inhibitor. Therefore, we examined whether platelets generate angiostatin during aggregation in vitro. Platelets were isolated from healthy human donors and then aggregated with collagen, thrombin, or HT-1080 fibrosarcoma cells. Angiostatin was detected by Western blot analysis in the platelet releasates of all blood donors irrespective of the aggregating agent used. Platelet pellet homogenates showed the presence of angiostatin in all donors, which was released upon aggregation. Furthermore, platelet-derived angiostatin was isolated and purified by lysine-Sepharose affinity chromatography from collagen-aggregated platelet releasates. Bioassay of platelet-derived angiostatin showed that it inhibited the formation of capillary structures by human umbilical vein endothelial cells (HUV-EC-Cs) in an in vitro angiogenesis model. Inhibition of angiostatin in platelet releasates promoted the formation of capillary structures by HUV-EC-Cs. We conclude that healthy human platelets contain angiostatin, which is released in active form during platelet aggregation, and platelet-derived angiostatin has the capacity to inhibit angiogenesis.

Expression of nitrergic system and protein nitration in adult rat brains submitted to acute hypobaric hypoxia.

Changes in the nitric oxide (NO) system of the rat cerebral cortex were investigated by immunohistochemistry, immunoblotting, and NO synthase (NOS) activity assays in adult rats submitted for 30 min to hypoxia, in a hypobaric chamber at a simulated altitude of 38,000 ft (11000 m) (154.9 mm Hg). The cerebral cortex was studied after different survival times, 0 and 24 h, 5, 8, 15, and 30 days of reoxygenation. This situation led to morphological alterations in the large type I interneurons, as well as immunoreactive changes in the appearance and number of the small neurons (type II), both containing neuronal NOS (nNOS). Some of these small neurons showed immunoreactive cytoplasm and short processes; others, the more numerous during all reoxygenation periods, contained the immunoreactive product mainly related to a perinuclear ring. Ultrastructurally, these small neurons exhibited changes in nuclear structures as in the shape of the nuclear membrane, in the distribution of heterochromatin, and in the nucleolar morphology. The reaction product for nitrotyrosine, as a marker of protein nitration, showed modifications in distribution of the immunoreactive product. No expression was found for inducible NOS (iNOS). All these modifications were accompanied by increased nNOS and nitrotyrosine production as demonstrated by Western blotting and calcium-dependent activity, returning to control conditions after 30 days of reoxygenation, suggesting a reversible NO mechanism of action.

Hypobaric hypoxia modifies constitutive nitric oxide synthase activity and protein nitration in the rat cerebellum.

Ischemic hypoxia provokes alterations in the production system of nitric oxide in the cerebellum. We hypothesize that the nitric oxide system may undergo modifications due to hypobaric hypoxia and that may play a role in high altitude pathophysiology. Therefore, changes in the nitric oxide system of the cerebellum of rats submitted to acute hypobaric hypoxia were investigated. Adult rats were exposed for 7 h to a simulated altitude of 8235 m (27000 ft.) and then killed after 0 h or 1, 3, 5 and 10 days of reoxygenation. Nitric oxide synthase calcium-dependent and -independent activity, immunoblotting and immunohistochemistry of neuronal, endothelial, and inducible nitric oxide synthase, and nitrotyrosine were evaluated. Immunoreactivity for neuronal nitric oxide synthase slightly increased in the baskets of the Purkinje cell layer and in the granule cells, after 0 h of reoxygenation, although no changes in neuronal nitric oxide synthase immunoblotting densitometry were detected. Calcium-dependent activity significantly rose after 0 h of reoxygenation, reaching control levels in the following points, and being coincident with a peak of eNOS expression. Nitrotyrosine formation showed significant increments after 0 h and 1 day of reoxygenation. Nitrotyrosine immunoreactivity showed an intracellular location change in the neurons of the cerebellar nuclei and in addition, an appearance of nitration in the soma of the Purkinje cells was detected. No changes in inducible nitric oxide synthase activity, immunoblotting or immunohistochemistry were detected. We conclude that at least part of the nitric oxide system is involved in cerebellum responses to hypobaric hypoxia.

Postnatal changes in the nitric oxide system of the rat cerebral cortex after hypoxia during delivery.

The impact of hypoxia in utero during delivery was correlated with the immunocytochemistry, expression and activity of the neuronal (nNOS) and inducible (iNOS) isoforms of the nitric oxide synthase enzyme as well as with the reactivity and expression of nitrotyrosine as a marker of protein nitration during early postnatal development of the cortex. The expression of nNOS in both normal and hypoxic animals increased during the first few postnatal days, reaching a peak at day P5, but a higher expression was consistently found in hypoxic brain. This expression decreased progressively from P7 to P20, but was more prominent in the hypoxic group. Immunoreactivity for iNOS was also higher in the cortex of the hypoxic rats and was more evident between days P0 and P5, decreasing dramatically between P10 and P20 in both groups of rats. Two nitrated proteins of 52 and 38 kDa, were also identified. Nitration of the 52-kDa protein was more intense in the hypoxic animals than in the controls, increasing from P0 to P7 and then decreasing progressively to P20. The 38-kDa nitrated protein was seen only from P10 to P20, and its expression was more intense in control than in the hypoxic group. These results suggest that the NO system may be involved in neuronal maturation and cortical plasticity over postnatal development. Overproduction of NO in the brain of hypoxic animals may constitute an effort to re-establish normal blood flow and may also trigger a cascade of free-radical reactions, leading to modifications in the cortical plasticity.

Effects of oxygen and glucose deprivation on the expression and distribution of neuronal and inducible nitric oxide synthases and on protein nitration in rat cerebral cortex.

Changes in the nitric oxide (NO) system of the rat cerebral cortex were investigated by immunohistochemistry, immunoblotting, NO synthase (NOS) activity assay, and magnetic resonance imaging (MRI) in an experimental model of global cerebral ischemia and reperfusion. Brains were perfused transcardially with an oxygenated plasma substitute and subjected to 30 minutes of oxygen and glucose deprivation, followed by reperfusion for up to 12 hours with oxygenated medium containing glucose. A sham group was perfused without oxygen or glucose deprivation, and a further group was treated with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) before and during perfusion. Global ischemia led to cerebrocortical injury as shown by diffusion MRI. This was accompanied by increasing morphologic changes in the large type I interneurons expressing neuronal NOS (nNOS) and the appearance of nNOS immunoreactivity in small type II neurons. The nNOS-immunoreactive band and calcium-dependent NOS activity showed an initial increase, followed by a fall after 6 hours of reperfusion. Inducible NOS immunoreactivity appeared in neurons, especially pyramidal cells of layers IV-V, after 4 hours of reperfusion, with corresponding changes on immunoblotting and in calcium-independent NOS activity. Immunoreactive protein nitrotyrosine, present in the nuclear area of neurons in nonperfused controls and sham-perfused animals, showed changes in intensity and distribution, appearing in the neuronal processes during the reperfusion period. Prior and concurrent L-NAME administration blocked the changes on diffusion MRI and attenuated the morphologic changes, suggesting that NO and consequent peroxynitrite formation during ischemia-reperfusion contributes to cerebral injury.