Adventitia-derived nitric oxide in rat aortas exposed to endotoxin: cell origin and functional consequences.
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The role of adventitial cells in bacterial lipopolysaccharide (LPS)-induced vascular nitric oxide (NO) overproduction has been largely ignored. In rat aortas exposed to LPS in vitro or in vivo, it was found that adventitia contained the major part of NO synthase (NOS)-2 protein (Western blot and immunohistochemistry) and generated the largest amount of NO (electron paramagnetic resonance spin trapping). NOS-2 immunoreactive cells were mainly resident macrophages at an early stage (5 h, in vitro or in vivo) and fibroblasts at a later stage (20 h, in vitro). Adventitial NOS-2 activity largely accounted for 1) the relaxing effect of L-arginine in rings exposed to LPS in vivo, 2) generation of an "NO store" revealed by N-acetylcysteine-induced relaxation, and 3) formation of protein-bound dinitrosyl iron complexes in the medial layer of aortic rings exposed to LPS in vitro. In conclusion, the adventitia is a powerful source of NO triggered by LPS in the rat aorta. This novel source of NO has an important impact on smooth muscle function and might be implicated in various inflammatory diseases. (+info)
Normoxic stabilization of hypoxia-inducible factor-1 expression and activity: redox-dependent effect of nitrogen oxides.
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Hypoxia-inducible factor-1 (HIF-1) is an essential transcription factor involved in the oxygen-dependent regulation of gene expression. Thiol groups in HIF-1 or in proteins that modify HIF-1 are conventional targets for regulation by nitric oxide (NO). Moreover, NO delivery to tissue by hemoglobin appears to be oxygen dependent. Therefore, the role NO plays in regulating HIF-1 activity and expression was examined. The 1-substituted diazen-1-ium-1, 2-diolate NOC-18 induced HIF-1 DNA-binding activity in normoxic bovine pulmonary artery endothelial cells and rat aortic smooth muscle cells in a time- and dose-dependent manner. Induction of HIF-1-binding activity was consistent with an increased expression of HIF-1 subunit proteins HIF-1alpha and HIF-1beta. The effect of NOC-18 on HIF-1 activity was blocked by cycloheximide, consistent with a post-transcriptional effect. NOC-18 induction of HIF-1 DNA-binding activity was not blocked with oxyhemoglobin, nor was it related to the rate of NO evolution, arguing against NO-mediation of the effect. Additionally, the effect of NOC-18 could not be mimicked by Angeli's salt, arguing against nitroxyl mediation. However, the NOC-18 effect could be reproduced by S-nitrosoglutathione (GSNO), an endogenous nitrosonium donor formed in the presence of deoxyhemoglobin. Furthermore, the GSNO effect could be reversed by dithiothreitol as well as acivicin, an inhibitor of GSNO bioactivation. Taken together, these results suggest that an S-nitrosylation reaction stabilizes HIF-1 protein expression and activity. We speculate that one signaling mechanism by which deoxyhemoglobin may activate HIF-1 involves NO. (+info)
Development of separable electron spin resonance-computed tomography imaging for multiple radical species: an application to .OH and .NO.
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A method of separable ESR-CT (electron spin resonance-computed tomography) imaging for multiple radical species was developed and applied to imaging of .OH and .NO. The algorithm was improved by combining filtered back-projection with a modified algebraic reconstruction technique to enhance accuracy and shorten calculation time. With this algorithm, spectral-spatial images of the phantom consisting of 3-carbamoyl-2,2,5,5,-tetramethylpyrrolidine-N-oxyl and 2-phenyl-4,4,5,5,-tetramethylimidazoline-3-oxide-1-oxyl could be obtained in different directions by rotating the spatial axis. The spatial function of individual radicals was extracted by each of the two methods from each spectral-spatial image. The separative 2D images of each radical were individually constructed using the spatial function obtained with the two methods. By comparing the separative images with the phantom sample, the algorithm for separable ESR-CT imaging was established. This ESR-CT technique was combined with L-band ESR spectroscopy and applied to the separative imaging of .OH and .NO, which were spin trapped with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) and Fe(2+)-N-methyl-D-glucamine dithiocarbamate complex, respectively. The ESR signal of DMPO-OH decreased gradually during data acquisition, and the decrease was calibrated by extrapolating the signal intensity to the beginning of data sampling. Both the position and size of the individual images for .OH and .NO were in very good agreement with the findings for the sample. (+info)
The free radical scavenger alpha-phenyl-tert-butyl nitrone aggravates hippocampal apoptosis and learning deficits in experimental pneumococcal meningitis.
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The effect of adjuvant therapy with the radical scavenger alpha-phenyl-tert-butyl nitrone (PBN; 100 mg/kg given intraperitoneally every 8 h for 5 days) on brain injury and learning function was evaluated in an infant rat model of pneumococcal meningitis. Meningitis led to cortical necrotic injury (median, 3.97% [range, 0%-38.9%] of the cortex), which was reduced to a median of 0% (range, 0%-30.9%) of the cortex (P<.001) by PBN. However, neuronal apoptosis in the hippocampal dentate gyrus was increased by PBN, compared with that by saline (median score, 1.15 [range, 0.04-1.73] vs. 0.31 [range, 0-0.92]; P<.001). Learning function 3 weeks after cured infection, as assessed by the Morris water maze, was decreased, compared with that in uninfected control animals (P<.001). Parallel to the increase in hippocampal apoptosis, PBN further impaired learning in infected animals, compared with that in saline-treated animals (P<.02). These results contrast with those of an earlier study, in which PBN reduced cortical and hippocampal neuronal injury in group B streptococcal meningitis. Thus, in pneumococcal meningitis, antioxidant therapy with PBN aggravates hippocampal injury and learning deficits. (+info)
Novel activation of non-selective cationic channels by dinitrosyl iron-thiosulfate in PC12 cells.
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Low molecular mass dinitrosyl iron complexes (DNICs) are nitrosating agents and it is known that the dinitrosyl iron moiety can be transferred to proteins. The aim of the present study was to determine if the formation of protein-bound dinitrosyl iron can modulate ionic channel activity. In PC12 cells, dinitrosyl iron-thiosulfate (50 microM) caused irreversible activation of a depolarizing inward current (IDNIC). IDNIC was partially inhibited by the metal chelator diethyldithiocarbamate (DETC, 1 mM), but not by the reducing/denitrosylating agent dithiothreitol (DTT, 5 mM). The activation of IDNIC was not reproduced by application of nitric oxide (NO., 100 microM), S-nitrocysteine (200 microM) or ferrous iron-thiosulfate (50 microM), and was not prevented by the irreversible guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ, 1 microM). Similarly, intracellular perfusion of dinitrosyl iron-thiosulfate (100 microM) did not result in activation of IDNIC. Ion replacement experiments show that the DETC-sensitive component of IDNIC is a non-selective cationic current. In accordance, IDNIC was blocked by antagonists of receptor-operated calcium entry, gadolinium (25 microM) and SK&F 96365 (25 microM). Single-channel measurements from outside-out patches reveal that the DETC-sensitive component of IDNIC is an inward current carried by a cationic channel having a conductance of 50 pS. The present observations suggest that the formation of ion channel-bound dinitrosyl iron represents another mechanism of regulation of ion channel activity by NO.-related species, which may be particularly important in pathophysiological processes where NO. is overproduced. (+info)
Nitric oxide modulation of interleukin-1[beta]-evoked intracellular Ca2+ release in human astrocytoma U-373 MG cells and brain striatal slices.
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Intracellular Ca(2+) mobilization and release into mammal CSF plays a fundamental role in the etiogenesis of fever induced by the proinflammatory cytokine interleukin-1beta (IL-1beta) and other pyrogens. The source and mechanism of IL-1beta-induced intracellular Ca(2+) mobilization was investigated using two experimental models. IL-1beta (10 ng/ml) treatment of rat striatal slices preloaded with (45)Ca(2+) elicited a delayed (30 min) and sustained increase (125-150%) in spontaneous (45)Ca(2+) release that was potentiated by l-arginine (300 microm) and counteracted by N-omega-nitro-l-arginine methyl ester (l-NAME) (1 and 3 mm). The nitric oxide (NO) donors diethylamine/NO complex (sodium salt) (0.3 and 1 mm) and spermine/NO (0.1 and 0.3 mm) mimicked the effect of IL-1beta on Ca(2+) release. IL-1beta stimulated tissue cGMP concentration, and dibutyryl cGMP enhanced Ca(2+) release. The guanyl cyclase inhibitors 1H-[1,2, 4]oxadiazole[4,3-a] quinoxalin-1-one (100 microm) and 6-[phenylamino]-5,8 quinolinedione (50 microm) counteracted Ca(2+) release induced by 2.5 but not 10 ng/ml IL-1beta. Ruthenium red (50 microm) and, to a lesser extent, heparin (3 mg/ml) antagonized IL-1beta-induced Ca(2+) release, and both compounds administered together completely abolished this response. Similar results were obtained in human astrocytoma cells in which IL-1beta elicited a delayed (30 min) increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) (402 +/- 71.2% of baseline), which was abolished by 1 mm l-NAME. These data indicate that the NO/cGMP-signaling pathway is part of the intracellular mechanism transducing IL-1beta-evoked Ca(2+) mobilization in glial and striatal cells and that the ryanodine and the inositol-(1,4,5)-trisphosphate-sensitive Ca(2+) stores are involved. (+info)
Estradiol-induced attenuation of pulmonary hypertension is not associated with altered eNOS expression.
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Female rats develop less severe pulmonary hypertension (PH) in response to chronic hypoxia compared with males, thus implicating a potential role for ovarian hormones in mediating this gender difference. Considering that estrogen upregulates endothelial nitric oxide (NO) synthase (eNOS) in systemic vascular tissue, we hypothesized that estrogen inhibits hypoxic PH by increasing eNOS expression and activity. To test this hypothesis, we examined responses to the endothelium-derived NO-dependent dilator ionomycin and the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate in U-46619-constricted, isolated, saline-perfused lungs from the following groups: 1) normoxic rats with intact ovaries, 2) chronic hypoxic (CH) rats with intact ovaries, 3) CH ovariectomized rats given 17 beta-estradiol (E(2)beta), and 4) CH ovariectomized rats given vehicle. Additional experiments assessed pulmonary eNOS levels in each group by Western blotting. Our findings indicate that E(2)beta attenuated chronic hypoxia-induced right ventricular hypertrophy, pulmonary arterial remodeling, and polycythemia. Furthermore, although CH augmented vasodilatory responsiveness to ionomycin and increased pulmonary eNOS expression, these responses were not potentiated by E(2)beta. Finally, responses to S-nitroso-N-acetylpenicillamine and spermine NONOate were similarly attenuated in all CH groups compared with normoxic control groups. We conclude that the inhibitory influence of E(2)beta on chronic hypoxia-induced PH is not associated with increased eNOS expression or activity. (+info)
Pharmacological effects of the spin trap agents N-t-butyl-phenylnitrone (PBN) and 2,2,6, 6-tetramethylpiperidine-N-oxyl (TEMPO) in a rabbit thromboembolic stroke model: combination studies with the thrombolytic tissue plasminogen activator.
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BACKGROUND AND PURPOSE: It has been proposed that spin trap agents such as N:-t-butyl-phenylnitrone (PBN) may be useful as neuroprotective agents in the treatment of ischemia and stroke. However, to date, there is little information concerning the effectiveness of spin trap agents when administered in combination with the only Food and Drug Administration-approved pharmacological agent for the treatment of stroke, the thrombolytic tissue plasminogen activator (tPA). Thus, we determined the effects of PBN when administered before tPA on hemorrhage and infarct rate and volume. We also compared the effects of PBN with those of 2,2,6, 6-tetramethylpiperidine-N:-oxyl (TEMPO), another spin trap agent that has a different chemical structure and trapping profile, on the incidence of infarcts and hemorrhage. METHODS: One hundred sixty-five male New Zealand White rabbits were embolized by injecting a blood clot into the middle cerebral artery via a catheter. Five minutes after embolization, PBN or TEMPO (100 mg/kg) was infused intravenously. Control rabbits received saline, the vehicle required to solubilize the spin traps. In tPA studies, rabbits were given intravenous tPA starting 60 minutes after embolization. Postmortem analysis included assessment of hemorrhage, infarct size and location, and clot lysis. RESULTS: In the control group, the hemorrhage rate after a thromboembolic stroke was 24%. The amount of hemorrhage was significantly increased to 77% if the thrombolytic tPA was administered. The rabbits treated with PBN in the absence of tPA had a 91% incidence of hemorrhage compared with 33% for the TEMPO-treated group. In the combination drug-treated groups, the PBN/tPA group had a 44% incidence of hemorrhage, and the TEMPO/tPA group had a 42% incidence of hemorrhage. tPA, PBN/tPA, and TEMPO/tPA were similarly effective at lysing clots (49%, 44%, and 33%, respectively) compared with the 5% rate of lysis in the control group. There was no significant effect of drug combinations on the rate or volume of infarcts. CONCLUSIONS: This study suggests that certain spin trap agents may have deleterious effects when administered after an embolic stroke. However, spin trap agents such as PBN or TEMPO, when administered in combination with tPA, may improve the safety of tPA by reducing the incidence of tPA-induced hemorrhage. Overall, the therapeutic benefit of spin trap agents for the treatment of ischemic stroke requires additional scrutiny before they can be considered "safe" therapeutics. (+info)