Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0022116 (ischemia)
 91,303 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Peroxynitrite is a cytotoxic oxidant produced during shock, ischemia reperfusion, and inflammation. The cellular events mediating the cytotoxic effect of peroxynitrite include activation of poly(ADP-ribose) synthetase, inhibition of mitochondrial respiration, and activation of caspase-3. The aim of the present study was to investigate the role of intracellular calcium mobilization in the necrotic and apoptotic cell death induced by peroxynitrite. Peroxynitrite, in a low, pathophysiologically relevant concentration (20 microM), induces rapid (1 to 3 min) Ca(2+) mobilization in thymocytes. Inhibition of this early calcium signaling by cell-permeable Ca(2+) chelators [EGTA-acetoxymethyl ester (AM), 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N , N',N'-tetraacetic acid-tetra-AM] abolished cytotoxicity as measured by propidium iodide uptake. Intracellular Ca(2+) chelators also inhibited DNA single-strand breakage and activation of poly(ADP-ribose) synthase (PARS), which is a major mediator of cell necrosis in the current model. Intracellular Ca(2+) chelators also protected PARS-deficient thymocytes from peroxynitrite cytotoxicity, providing evidence for a PARS-independent, Ca(2+)-dependent cytotoxic pathway. Chelation of intracellular Ca(2+) blocked the peroxynitrite-induced decrease of mitochondrial membrane potential, secondary superoxide production, and mitochondrial membrane damage. Peroxynitrite-induced internucleosomal DNA cleavage was increased on BAPTA-AM pretreatment in the wild-type cells but decreased in the PARS-deficient cells. Two other apoptotic parameters (phosphatidylserine exposure and caspase 3 activation) were inhibited by BAPTA-AM in both the wild-type and the PARS-deficient thymocytes. Our findings provide evidence for the pivotal role of an early Ca(2+) signaling in peroxynitrite cytotoxicity.
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PMID:Requirement of intracellular calcium mobilization for peroxynitrite-induced poly(ADP-ribose) synthetase activation and cytotoxicity. 1049 67

We have previously shown that increased reactive oxygen species (ROS) generation occurs with ischemia in the oxygenated lung and have hypothesized that mechanotransduction is the initiating event. In the present study, we developed an in vitro model of oxygenated ischemia by interrupting medium flow to flow-adapted bovine pulmonary artery endothelial cells in an artificial capillary system. Cellular oxygenation during the "ischemic" period was maintained by perfusing medium over the abluminal surface of porous capillaries. Cells were assessed for ROS generation, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) binding activities, and DNA synthesis using dichlorofluorescein fluorescence by flow cytometry and spectrofluorometry, electrophoretic mobility shift assay of nuclear extracts with NF-kappaB-specific or AP-1-specific (32)P-labeled oligonucleotides, and (3)H-thymidine incorporation into DNA. Cells that were flow adapted for 2 to 7 days with 1 to 2 dyne/cm(2) shear stress exhibited a 1.6- to 1.9-fold increase in ROS generation during 1 hour of simulated ischemia compared with continuously perfused cells. This effect was abolished by diphenyleneiodonium chloride (DPI), indicating a role for a flavoprotein such as NADPH oxidase. The increase in ROS generation with ischemia was similar for cells from low and high passages. With ischemia, flow-adapted cells exhibited increases of 1.7-fold in nuclear NF-kappaB and 1.5-fold in nuclear AP-1; these changes were abolished by pretreatment with N-acetylcysteine or DPI. Ischemia for 24 hours resulted in a 1.8-fold increase of (3)H-thymidine incorporation into DNA and a significant increase of cells entering the cell cycle, as indicated by flow cytometry with propidium iodide. We conclude that flow-adapted endothelial cells generate ROS with ischemia that results in activation of NF-kappaB and AP-1 and an increase of DNA synthesis. This effect is not mediated by hypoxia, implicating a role for mechanotransduction in ischemia-mediated cell signaling.
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PMID:Simulated ischemia in flow-adapted endothelial cells leads to generation of reactive oxygen species and cell signaling. 1052 Dec 41

Extract from Scutellaria baicalensis Georgi Attenuates Oxidant Stress in Cardiomyocytes. Journal of Molecular and Cellular Cardiology (1999) 31, 1885-1895. Scutellaria baicalensis Georgi is a Chinese herbal medicine used to treat allergic and inflammatory diseases. The medicinal effects of S. baicalensis root may result, in part, from its constituent flavones reported to have antioxidant properties. Since oxidants play multiple roles in cells, we tested whether S. baicalensis could confer protection in a cardiomyocyte model of ischemia and reperfusion. The intracellular fluorescent probes 2',7'-dichlorofluorescin diacetate (DCFH-DA, sensitive to H(2)O(2) and hydroxyl radicals) and dihydroethidium (DHE, sensitive to superoxide) were used to assess intracellular reactive oxygen species (ROS), and propidium iodide (PI) was used to assess viability in cultured embryonic cardiomyocytes. S. baicalensis extract (SbE) quickly attenuated levels of oxidants generated during transient hypoxia and during exposure to the mitochondrial site III inhibitor antimycin A, as measured by DCFH oxidation or by DHE oxidation. These attenuated oxidant levels were associated with improved survival and function. Cell death after ischemia/reperfusion decreased from 47+/-3 % in untreated to 26+/-2 % in S. baicalensis treated cells (P<0.001). After antimycin A exposure, S. baicalensis decreased cell death from 49+/-6 % in untreated to 23+/-4 % in treated cells. Return of contraction occurred in S. baicalensis-treated cells but was not observed in control cells. Other in vitro studies revealed that baicalein, a major flavone component of SbE can directly scavenge superoxide, hydrogen peroxide, and hydroxyl radicals. Collectively, these findings indicate that SbE and its constituent flavones such as baicalein can attenuate oxidant stress and protect cells from lethal oxidant damage in an ischemia-reperfusion model.
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PMID:Extract from Scutellaria baicalensis Georgi attenuates oxidant stress in cardiomyocytes. 1052 26

Mitochondrial dysfunction has been implicated in a number of neurodegenerative diseases, such as ischemia and Parkinson's disease. We present here a method that allows the rapid quantification of interventions, aimed at inhibiting the effect of mitochondrial membrane potential uncouplers, based on the ratioing properties of the fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), by using currently available 96-well fluorescent plate readers. A method is presented for evaluation of cross-talk between the two excitation/emission channels. Further characterization of the probe shows that the effect of plasma membrane potential changes on JC-1 fluorescence ratio are negligible, but that the signal is very sensitive to pH. One of the most exciting applications is the possibility to perform end-point measurements, thanks to the ratioing properties of the probe. The system is tested in different culture types with different mitochondrial uncouplers. As an example of a quantitative evaluation, we show that flunarizine is able to inhibit, dose-dependently, FCCP mediated JC-1 signal increase. The procedure is simple and allows for the fast screening of mitochondria-protective compounds.
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PMID:A rapid method for the evaluation of compounds with mitochondria-protective properties. 1059 13

We examined the ability of ACh to mimic ischemic preconditioning in cardiomyocytes and the role of ATP-sensitive potassium (KATP) channels and mitochondrial reactive oxygen species (ROS) in mediating this effect. Chick embryonic ventricular myocytes were studied in a flow-through chamber while flow rate, pH, PO2, and PCO2 were controlled. Cell viability was quantified with propidium iodide (5 microM), and production of ROS was measured using 2', 7'-dichlorofluorescin diacetate. Data were expressed as means +/- SE. Preconditioning with 10 min of ischemia followed by 10 min of reoxygenation or 10 min of ACh (1 mM) followed by a drug-free period before 1 h of ischemia and 3 h of reoxygenation reduced cell death to the same extent [preconditioning 19 +/- 2% (n = 6, P < 0.05) ACh 21 +/- 5% (n = 6, P < 0.05) vs controls 42 +/- 5% (n = 9)]. Like preconditioning, ACh increased ROS production threefold before ischemia [0.60 +/- 0.16 (n = 7, P < 0.05) vs. controls, 0.16 +/- 0. 03 (n = 6); arbitrary units]. Protection and increased ROS production during ACh preconditioning were abolished with 5-hydroxydecanoate (5-HD, 100 microM), a selective mitochondrial K(ATP) channel antagonist, and the thiol reductant 2-mercaptopropionyl glycine (2-MPG, 1 mM), an antioxidant [cell death: 5-HD+ACh 37 +/- 7% (n = 5), 2-MPG+ACh 47 +/- 6% (n = 6); ROS signals: 5-HD+ACh 0.09 +/- 0.03 (n = 5), 2-MPG+ACh 0.01 +/- 0.04 (n = 4)]. In addition, ACh-induced ROS signaling was blocked by the mitochondrial site III electron transport inhibitor myxothiazol (0.02 +/- 0.07, n = 5). These results demonstrate that activation of mitochondrial K(ATP) channels and increased ROS production from mitochondria are important intracellular signals that participate in ACh-induced preconditioning in cardiomyocytes.
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PMID:Role of reactive oxygen species in acetylcholine-induced preconditioning in cardiomyocytes. 1060 Aug 75

Oxidative stress may cause apoptosis of cardiomyocytes in ischemic-reperfused myocardium. We investigated whether ischemia-reperfusion modifies the susceptibility of cardiomyocyte induction of apoptosis by oxidative stress. Ischemia was simulated by incubating isolated cardiomyocytes from adult rats in an anoxic, glucose-free medium, pH 6.4, for 3 h. Annexin V-fluorescein isothiocyanate/propidium iodide staining and the detection of DNA laddering were used as apoptotic markers. H(2)O(2) (7.5 micromol/l) induced apoptosis in 20.1 +/- 1.8% of cells under normoxic conditions but only 14.4 +/- 1.6% (n = 6, P < 0.05) after ischemia-reoxygenation. This partial protection of ischemic-reoxygenated cells was observed despite a reduction in their cellular glutathione content, from 11.4 +/- 1.9 in normoxic controls to 2.9 +/- 0.8 nmol/mg protein (n = 3, P < 0.05). Elevation of end-ischemic glutathione contents by pretreatment with 1 mmol/l N-acetylcysteine entirely protected ischemic-reoxygenated cells against induction of apoptosis by H(2)O(2). In conclusion, ischemia-reperfusion can protect cardiomyocytes against induction of apoptosis by exogenous oxidative stress. This endogenous protective effect is most clearly demonstrated when control and postischemic cardiomyocytes are compared at similar glutathione levels.
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PMID:Influence of simulated ischemia on apoptosis induction by oxidative stress in adult cardiomyocytes of rats. 1064 88

The relative contribution of xanthine oxidase (XO) and leukocytes to tissue injury after short-term ischemia is unknown. In this study, we subjected three groups of rat spinotrapezius muscles to 30-min ischemia and 1-h reperfusion: 1) ischemia-reperfusion (I/R) + 0.9% saline, 2) I/R + superoxide dismutase, and 3) I/R + oxypurinol. A fourth group served as nonischemic control. We quantified the increase in resistance (%DeltaR) caused by leukocyte-capillary plugging concurrently with myocyte uptake of propidium iodide (PI) [expressed as no. of PI spots per total volume of perfused tissue (N(PI)/V)] and performed assays to quantify XO activity, thiobarbituric acid-reactive substances (TBARS), and myeloperoxidase (MPO). Groups 2 and 3 exhibited significant decreases in N(PI)/V relative to group 1. MPO levels and TBARS were similar among all groups, and mean %DeltaR was significantly reduced in groups 2 and 3 relative to group 1. However, elevated XO was observed in groups 1 and 2 relative to group 3 and nonischemic controls. These data are consistent with the hypothesis that XO, rather than toxic species produced by plugging or venule-adherent leukocytes, is responsible for postischemic damage in this model.
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PMID:Role of leukocytes and tissue-derived oxidants in short-term skeletal muscle ischemia-reperfusion injury. 1066 73

There is recent evidence that angiotensin-converting enzyme (ACE) inhibition reduces postischemic injury and angiotensin II receptor inhibition may have similar effects. We therefore further characterized the role of ACE- vs. AT1-receptor inhibition on cell injury and temporal association of leukocyte endothelial interaction in response to ischemia-reperfusion. A combined in vivo and in vitro study comparing the ACE inhibitor enalapril and the AT1-receptor antagonist losartan was performed. The extent and temporal correlation of cellular damage (propidium-iodide staining), microvascular perfusion failure and leukocyte-endothelial interaction (leukocyte adherence) were investigated by means of intravital microscopy, after the application of hemodynamically ineffective doses of enalapril and losartan (5 mg/kg). A hamster dorsal skinfold model with a 4-h tourniquet ischemia was used. In vitro, the effect of enalapril and losartan on polymorphonuclear cell (PMN) adherence, as well as adhesion molecule expression (ICAM-1, VCAM-1), on hypoxia- or IL-1beta-stimulated endothelial cells (HUVEC) was assessed using a PMN-adhesion assay and flow cytometry, respectively. Ischemia-reperfusion responses revealed a biphasic pattern, comprised of an early phase (30 min) of acute cellular damage and microvascular perfusion failure, followed by a late increase (240 min) in leukocyte adherence in vivo. Enalapril significantly reduced early cellular damage, microvascular perfusion failure, and leukocyte adherence in response to ischemia-reperfusion. Conversely, AT1 receptor inhibition with losartan proved to be ineffective at attenuating postischemic microcirculatory disorders (leukocyte-endothelial interactions, microvascular perfusion failure) and aggravated cellular injury. In vitro, enalapril reduced PMN adherence and ICAM-1 and VCAM-1 expression, while losartan was ineffective in the same respect. Following ischemia-reperfusion injury, ACE- versus AT1-receptor inhibition induce differential effects concerning the extent and temporal association of cell injury and leukocyte-endothelial interaction. The use of enalapril combines the beneficial effects of preventing cell and vascular injury immediately after reperfusion, with a delayed inhibition of the inflammatory response. Since the AT1-receptor inhibitor losartan did not mimic effects obtained with ACE inhibition, it is conceivable that the responses in ischemia-reperfusion are mediated by a non-angiotensin II-AT1 receptor-dependent mechanism.
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PMID:Differential effects of short-term ace- and AT1-receptor inhibition on postischemic injury and leukocyte adherence in vivo and in vitro. 1071 75

Organotypic brain slice cultures have been used in a variety of studies on neurodegenerative processes [K.M. Abdel-Hamid, M. Tymianski, Mechanisms and effects of intracellular calcium buffering on neuronal survival in organotypic hippocampal cultures exposed to anoxia/aglycemia or to excitotoxins, J. Neurosci. 17, 1997, pp. 3538-3553; D.W. Newell, A. Barth, V. Papermaster, A.T. Malouf, Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures, J. Neurosci. 15, 1995, pp. 7702-7711; J.L. Perez Velazquez, M.V. Frantseva, P.L. Carlen, In vitro ischemia promotes glutamate mediated free radical generation and intracellular calcium accumulation in pyramidal neurons of cultured hippocampal slices, J. Neurosci. 23, 1997, pp. 9085-9094; L. Stoppini, L.A. Buchs, D. Muller, A simple method for organotypic cultures of nervous tissue, J. Neurosci. Methods 37, 1991, pp. 173-182; R.C. Tasker, J.T. Coyle, J.J. Vornov, The regional vulnerability to hypoglycemia induced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK 801, J. Neurosci. 12, 1992, pp. 4298-4308.]. We describe two methods to induce traumatic cell damage in hippocampal organotypic cultures. Primary trauma injury was achieved by rolling a stainless steel cylinder (0.9 g) on the organotypic slices. Secondary injury was followed after dropping a weight (0.137 g) on a localised area of the organotypic slice, from a height of 2 mm. The time course and extent of cell death were determined by measuring the fluorescence of the viability indicator propidium iodide (PI) at several time points after the injury. The initial localised impact damage spread 24 and 67 h after injury, cell death being 25% and 54%, respectively, when slices were kept at 37 degrees C. To validate these methods as models to assess neuroprotective strategies, similar insults were applied to slices at relatively low temperatures (30 degrees C), which is known to be neuroprotective [F.C. Barone, G.Z. Feuerstein, R.F. White, Brain cooling during transient focal ischaemia provides complete neuroprotection, Neurosci. Biobehav. Rev. 1, 1997, pp. 31-44; V.M. Bruno, M.P. Goldberg, L.L. Dugan, R.G. Giffard, D.W. Choi, Neuroprotective effect of hypothermia in cortical cultures exposed to oxygen glucose deprivation or excitatory aminoacids, J. Neurochem. 4, 1994, pp. 387-392; G.C. Newman, H. Qi, F.E. Hospod, K. Grundhmann, Preservation of hippocampal brain slices with in vivo or in vitro hypothermia, Brain Res. 1, 1992, pp. 159-163; J.Y. Yager, J. Asseline, Effect of mild hypothermia on cerebral energy metabolism during the evolution of hypoxic ischaemic brain damage in the immature rat, Stroke, 5, 1996, pp. 919-925.]. Low temperature incubation significantly reduced cell death, now being 9% at 24 h and 14% at 67 h. Our results show that these models of moderate mechanical trauma using organotypic slice cultures can be used to study neurodegeneration and neuroprotective strategies.
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PMID:Methods to induce primary and secondary traumatic damage in organotypic hippocampal slice cultures. 1077 35

The involvement of caspase-3 in cell death after hypoxia-ischemia (HI) was studied during brain maturation. Unilateral HI was produced in rats at postnatal day 7 (P7), 15 (P15), 26 (P26), and 60 (P60) by a combination of left carotid artery ligation and systemic hypoxia (8% O2). Activation of caspase-3 and cell death was examined in situ by high-resolution confocal microscopy with anti-active caspase-3 antibody and propidium iodide and by biochemical analysis. The active caspase-3 positive neurons were composed of more than 90% HI damaged striatal and neocortical neurons in P7 pups, but that number was reduced to approximately 65% in striatum and 34% in the neocortex of P15 pups, and approximately 26% in striatum and 2% in neocortex of P26 rats. In P60 rats, less than 4% of the damaged neurons in striatum and less than 1% in neocortex were positive for active caspase-3. Western blot analysis demonstrated that the level of inactive caspase-3 in normal forebrain tissue gradually declined from a high level in young pups to very low levels in adult rats. Concomitantly, HI-induced active caspase-3 was reduced from a relatively high level in P7, to moderate levels in P15 and P26, to a barely detectable level in P60 rats. The authors conclude that the involvement of caspase-3 in the pathogenesis of cell death after HI declines during neuronal maturation. The authors hypothesize that caspase-3 may play a major role in cell death in immature neurons but a minor role in cell death in mature neurons after brain injury.
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PMID:Involvement of caspase-3 in cell death after hypoxia-ischemia declines during brain maturation. 1099 50


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