Metabolic and Biosynthetic Alterations in Cultured Astrocytes Exposed to Hypoxia/Reoxygenation

To investigate the astrocyte response to hypoxia/reoxygenation, as a model relevant to the pathogenesis of ischemic injury, cultured rat astrocytes were exposed to hypoxia. On restoration of astrocytes to normoxia, there was a dramatic increase in protein synthesis within 3 h, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocyte lysates showed multiple induced bands on fluorograms. Levels of cellular ATP declined during the first 3 h of reoxygenation and the concentration of AMP increased to ± 3.6 nmol/mg of protein within 1 h of reoxygenation. Reoxygenated astrocytes generated oxygen free radicals early after replacement into ambient air, and addition of diphenyliodonium, an NADPH oxidase inhibitor, diminished the generation of free radicals as well as the induction of several bands on fluorogram. Although addition of cycloheximide on reoxygenation resulted in inhibition of both astrocyte protein synthesis and accumulation of cellular AMP, it caused cell death within 6 h, suggesting the importance of protein synthesis in adaptation of hypoxic astrocytes to reoxygenation. Potential physiologic significance of biosynthetic products of astrocytes in hypoxia/reoxygenation was suggested by the recovery of glutamate uptake. These results indicate that the astrocyte response to hypoxia/reoxygenation includes generation of oxygen free radicals and de novo synthesis of products that influence cell viability and function in ischemia.     

Hypoxia/reoxygenation stimulates endothelium to promote neutrophil adhesion.

An in vitro model was designed to study the role of ischemia/reperfusion and endothelium-derived oxygen free radicals on neutrophil adhesion, with particular interest in the endothelial adhesion molecules involved. Human umbilical vein endothelial cells were submitted to 5 h hypoxia followed by various times (20 min to 24 h) of reoxygenation. Human resting neutrophils were added to monolayers for the last 15 min of reoxygenation. Adherence was evaluated by myeloperoxidase assay. Under these conditions, we found an increased adhesion of neutrophils with two peaks after 20 min and 4 h reoxygenation. This was correlated with the respective expression of the preformed granule membrane protein 140 (GMP-140) and of the de novo synthesized endothelial leukocyte adhesion molecule 1 (ELAM-1) on endothelial surface. Superoxide dismutase and/or catalase, or oxypurinol added to cultures before hypoxia efficiently prevented neutrophil adhesion. These results underline the crucial role played by endothelial oxy radicals at reoxygenation in adhesion of leukocytes, which could lead to an amplification of the oxidative stress injury. The protection offered by free radical scavengers emphasizes the potential therapeutic use of antioxidants in postischemic vascular disorders.     

Antioxidant changes in heart hypertrophy: significance during hypoxia-reoxygenation injury.

Because hypertrophied rat hearts display an increase in antioxidant enzyme activities and because hypoxia-reoxygenation injury is known to involve free radicals, we tested the hypothesis that the hypertrophied heart may be more resistant to this type of injury. Hypertrophied rat hearts after 10 weeks of chronic pressure overload showed elevated superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) activities and a decrease in lipid peroxidation as indicated by malondialdehyde (MDA) content. Glucose-free hypoxia for 15 min resulted in a complete failure of developed tension and about 200% increase in resting tension in both hypertrophied and sham control groups (p < 0.05). Upon reoxygenation for up to 30 min, hypertrophied hearts recovered developed tension to 60% and resting tension was higher by only 80% of prehypoxic values. In contrast, sham hearts showed only a 25% recovery of developed tension, whereas resting tension remained 130% higher than prehypoxic control values. During hypoxia, the SOD activity was significantly reduced in both sham and hypertrophied groups, whereas GSHPx was reduced only in the sham group. Upon reoxygenation there was no further change in these enzyme activities. Both the SOD and GSHPx activities in the hypertrophied group remained significantly higher than the corresponding reoxygenated sham hearts. During hypoxia, there was no apparent change in MDA content in either the sham or hypertrophied hearts. However, reoxygenation resulted in a significant increase in MDA content in both sham and hypertrophied hearts, but the MDA content was significantly less in the hypertrophied group (p < 0.05). It is suggested that maintenance of an adequate endogenous antioxidant reserve during hypoxia may be important in recovery upon reoxygenation.     

Enzyme release and mitochondrial activity in reoxygenated cardiac muscle: relationship with oxygen-induced lipid peroxidation

The aim of this work was to precisely determine the sites of the peroxidative action on unsatured lipids by oxygen-derived free radicals and the lytic cell damage on reoxygenated perfused hearts. The cellular load of lipid peroxidation products (malondialdehyde) during the reoxygenation was dependent on PO2. This unfavorable biochemical response was linked to creatine kinase leakage, alteration of coronary flow and mitochondrial injury. When an enzymatic (superoxide dismutase, 290 IU/minute) or tripeptide scavenger of oxygen radicals (reduced glutathione, 0.5 mmol/l) was administered at the end of hypoxia and during reoxygenation, the abnormal intolerance of hypoxic heart to molecular oxygen was significantly weakened; the load of lipid peroxides load, enzyme release, and vascular alteration were all reduced. Moreover, mitochondrial activity was enhanced and the oxygen-induced uncoupling of mitochondrial remained limited: both the respiratory control ratio (RCR) and the ADP/O ratio were higher than in control reoxygenated hearts. The inhibition by rotenone (100 mumol/l) of reoxidation of electron chain transfer during oxygen readmission also reduced the unfavorable cardiac accumulation of lipid peroxidation products and the release of creatine kinase. These data demonstrate that in the oxygen paradox, the peroxidative attack on lipids plays an important role in inducing alterations of sarcolemmal permeability and mitochondrial activity. An uncontrolled reactivation of oxidative function of mitochondria during reoxygenation enhances the synthesis of oxygen-derived free radicals and triggers the peroxidation of cardiac lipids resulting in irreversible injury to cellular and intracellular membranes.     

Vascular endothelial growth factor (VEGF)-A165-induced prostacyclin synthesis requires the activation of VEGF receptor-1 and -2 heterodimer

We previously reported that vascular endothelial growth factor (VEGF)-A(165) inflammatory effect is mediated by acute platelet-activating factor synthesis from endothelial cells upon the activation of VEGF receptor-2 (VEGFR-2) and its coreceptor, neuropilin-1 (NRP-1). In addition, VEGF-A(165) promotes the release of other endothelial mediators including nitric oxide and prostacyclin (PGI(2)). However, it is unknown whether VEGF-A(165) is mediating PGI(2) synthesis through VEGF receptor-1 (VEGFR-1) and/or VEGF receptor-2 (VEGFR-2) activation and whether the coreceptor NRP-1 potentiates VEGF-A(165) activity. In this study, PGI(2) synthesis in bovine aortic endothelial cells (BAEC) was assessed by quantifying its stable metabolite (6-keto prostaglandin F(1alpha), 6-keto PGF(1alpha)) by enzyme-linked immunosorbent assay. Treatment of BAEC with VEGF analogs, VEGF-A(165) (VEGFR-1, VEGFR-2 and NRP-1 agonist) and VEGF-A(121) (VEGFR-1 and VEGFR-2 agonist) (up to 10(-9) m), increased PGI(2) synthesis by 70- and 40-fold within 15 min. Treatment with VEGFR-1 (placental growth factor and VEGF-B) or VEGFR-2 (VEGF-C) agonist did not increase PGI(2) synthesis. The combination of VEGFR-1 and VEGFR-2 agonists did not increase PGI(2) release. Pretreatment with a VEGFR-2 inhibitor abrogated PGI(2) release mediated by VEGF-A(165) and VEGF-A(121), and pretreatment of BAEC with antisense oligomers targeting VEGFR-1 or VEGFR-2 mRNA reduced PGI(2) synthesis mediated by VEGF-A(165) and VEGF-A(121) up to 79%. In summary, our data demonstrate that the activation of VEGFR-1 and VEGFR-2 heterodimer (VEGFR-1/R-2) is essential for PGI(2) synthesis mediated by VEGF-A(165) and VEGF-A(121), which cannot be reproduced by the parallel activation of VEGFR-1 and VEGFR-2 homodimers with corresponding agonists. In addition, the binding of VEGF-A(165) to NRP-1 potentiates its capacity to promote PGI(2) synthesis.     

Regulation of endothelial heme oxygenase activity during hypoxia is dependent on chelatable iron

The regulation of heme oxygenase (HO) activity and its dependence on iron was studied in bovine aortic endothelial cells (BAEC) subjected to hypoxia-reoxygenation (H/R). HO activity was induced by hypoxia (10 h) and continued to increase during the reoxygenation phase. HO-1 protein levels were strongly induced by hypoxia from undetectable levels and remained elevated at least 8 h postreoxygenation. Addition of the Fe(3+) chelator desferrioxamine mesylate (DFO) or the Fe(2+) chelator o-phenanthroline during hypoxia alone or during the entire H/R period inhibited the induction of HO activity and HO-1 protein levels. However, DFO had no effect and o-phenanthroline had a partial inhibitory effect on HO activity and protein levels when added only during reoxygenation. Loading of BAEC with Fe(3+) enhanced the activation of the HO-1 gene by H/R, whereas loading with L-aminolevulinic acid, which stimulates heme synthesis, had little effect. These results suggest that chelatable iron participates in regulating HO expression during hypoxia.     

超氧化物歧化酶对内皮细胞缺氧复氧损伤的防护作用

体外培养扔兔胸主动脉内皮细胞缺氧３０ｍｉｎ后复氧１０ｍｉｎ,可以发现缺氧后复氧可引起细胞乳酸脱氢酶释放量,细胞悬液丙二醛含量增加,谷胱甘肽过氧化酶活性降低,细胞合成释放一氧化氮减少,细胞内钙离子浓度明显升高;ＥＣ的这些损伤在缺氧期间即有表现,复氧后更为加剧。而在缺氧前预先加入终浓度为２００Ｕ／ｍｌ的超氧化物歧化酶可改善细胞的抗氧化能力,减轻缺氧复氧对ＥＣ的损伤。     

Enhancement of thrombin- and ionomycin-stimulated prostacyclin and platelet-activating factor production in cultured endothelial cells by a tumor-promoting phorbol ester

Tumor-promoting phorbol esters such as 4 beta-phorbol 12-myristate 13-acetate (PMA) have been shown to act synergistically with Ca2+ ionophores in cell activation, including stimulation of arachidonic acid metabolism. The effects of PMA on unstimulated and Ca2+ ionophore- or thrombin-stimulated PGI2 and platelet-activating factor (PAF) production in cultured bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were investigated. Incubation of BAEC or HUVEC for 5-10 min with 100 nM PMA alone slightly increased basal PGI2 production. PGI2 production was rapidly stimulated in BAEC and HUVEC treated with the Ca2+ ionophore ionomycin. Preincubation of BAEC or HUVEC with 100 nM PMA for 5-10 min followed by ionomycin for up to 60 min enhanced PGI2 production up to 2.5-fold. Pretreatment with 100 nM PMA for 5 min also caused a 2-fold enhancement of thrombin-stimulated (1 U/ml) PGI2 production in HUVEC. The production of other prostaglandins, PGF2 alpha, PGE2, and PGD2, was also enhanced. In contrast, PMA had no effect on PGI2 synthesized directly from exogenous arachidonic acid or PGH2. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect. Since the biosyntheses of both PGI2 and PAF share a common first step, the hydrolysis of their respective phospholipid precursors by phospholipase A2, we investigated whether PMA preincubation could also enhance PAF biosynthesis. Incubation of HUVEC with 100 nM PMA alone had a negligible effect on PAF production. However, thrombin-stimulated (1 U/ml) PAF production was enhanced 2.6-fold by preincubation with 100 nM PMA. The protein kinase C inhibitors H-7 and staurosporine ablated the enhancing effect of PMA on thrombin-stimulated PGI2 and PAF biosynthesis. These results demonstrate that PMA can significantly alter the production of PGI2 and PAF in vascular endothelial cells, and suggest that protein kinase C activation modulates phospholipase A2 activity in this cell type.     

Propranolol preserves ultrastructure in adult cardiocytes exposed to anoxia/reoxygenation: a morphometric analysis

The protective effect of d,l-propranolol was studied using freshly isolated canine ventricular cardiocytes (1.5 x 10(6)/mL) exposed to 30 min anoxia (95% N2/5% CO2) and 0, 3, 20, and 45 min of reoxygenation (95% O2/5% CO2). In addition to preventing lipid peroxide formation, propranolol maintained cellular viability, and minimized ultrastructural alterations. In the absence of propranolol, the outer mitochondria become swollen and rounded up within the first few minutes of reoxygenation. The perinuclear mitochondrial area increased only slightly. We observed that the cellular injury process proceeded differentially from the exterior to the interior, with a mitochondrial area increase and outer membrane rupture. Sarcolemmal damage was also observed with prevalent blebbing and membrane loss. The Z-lines became wider and more diffuse with reoxygenation. Injury to the nuclear double membrane was observed. Incubation with propranolol showed significant protection during postanoxia reoxygenation. In contrast, the more water soluble beta-blocker atenolol only exhibited slight protection. In addition, d-propranolol (the non beta-blocking isomer) and the antioxidant enzymes, SOD and catalase, showed significant protection. These data support previous findings concerning the antioxidant properties of propranolol which appear to be independent of beta-receptor blockade.     

Xanthine Oxidase is Not Responsible for Reoxygenation Injury in Isolated-Perfused Rat Heart

The massive leakage of intracellular enzymes which occurs during reoxygenation of heart tissue after hypoxic or ischemic episodes has been suggested to result from the formation of oxygen radicals. One purported source of such radicals is the xanthine oxidase-mediated metabolism of hypoxanthine and xanthine. Xanthine oxidase (O form) has been suggested to be formed in vivo by limited proteolysis of xanthine dehydrogenase (D form) during the hypoxic period (Granger el ai. Gastroenterology 81, 22 (1981)). We measured the activities of xanthine oxidase in both fresh and isolated-perfused (Langendorff) rat heart tissue. Approximately 32% of the total xanthine oxidase was in the O form in fresh and isolated-perfused rat heart. This value was unchanged following 60min of hypoxia and 30 minutes of reoxygenation. The infusion of 250/JM allopurinol throughout the perfusion completely inhibited xanthine oxidase activity but had no effect on the massive release of lactate dehydrogenase (LDH) into the coronary effluent upon reoxygenation of heart tissue subjected to 30 or 60min of hypoxia. Protection from 30min of hypoxia was also not obtained when rats were pretreated for 48 h with allopurinol at a dose of 30mg/kg/day and perfused with allopurinol containing medium. Superoxide dismutase (50 units/ml), catalase (200 units/ml), or the antioxidant cyanidanol (100μM) also had no effect on LDH release upon reoxygenation after 60 min of hypoxia. Xanthine oxidase activity was detected in a preparation enriched in cardiac endothelial cells while no allupurinol-inhibitable activity could be measured in purified isolated cardiomyocytes. It is concluded that xanthine dehydrogenase is not converted to xanthine oxidase in hypoxic tissue of the isolated perfused rat heart, and that the release of intracellular enzymes upon reoxygenation in this experimental model is mediated by factors other than reactive oxygen generated by xanthine oxidase.     

Superoxide Disrnutase Does Protect the Cdtured Rat Cardiac Myocytes Against Hypoxia/Reoxygenation Injury

The effect of superoxide dismutase (SOD) on membrane integrity and fluidity of the cultured neonatal rat cardiac myocytes in vitro was investigated under the condition of hypoxia and hypoxia/reoxygenation. Lactate dehydrogenase (LDH) concentration was used as the biochemical indicator for the loss of cell membrane integrity. Fluorescence polarization (FP), average microviscosity (N) and anisotropy (Ast), which are inversely proportional to the fluidity of cell membrane, were assayed. Cells were respectively exposed to hypoxia or hypoxia/reoxygenation for different periods of time in the absence or presence of SOD at various concentrations. Hypoxia alone or hypoxia/ reoxygenation brought injury to the cultured myocytes. This was demonstrated by changes in LDH and membrane fluidity. In the former LDH concentration gradually increased in a time-dependent manner and the values of FP, N and Asf were significantly increased. The changes in membrane integrity and fluidity induced by hypoxia or hypoxia/reoxygenation could be prevented by adding SOD to the culture medium. The results provide a direct evidence that SOD (740 u.ml^-1, the effective dose) was effective in protecting cultured myocytes against the injury as well as an indirect evidence of free radical generation. Based on the results obtained from this study and the establishment of concept of optimally effective dose by Bernier and Omar et al, it was suggested that some previous reports, in which no evidence was found both in protective effect of SOD and in free radical generation by using only one dose in hypoxia/reoxygenation model, should be interpreted with caution.     

Extracellular iron chelators protect kidney cells from hypoxia/reoxygenation

Iron is an important contributor to reoxygenation injury because of its ability to promote hydroxyl radical formation. In previous in vivo studies, we demonstrated that iron chelators that underwent glomerular filtration provided significant protection against postischemic renal injury. An in vitro system was employed to further characterize the protection provided by extracellular iron chelators. Primary cultures of rat proximal tubular epithelial cells were subjected to 60 min hypoxia and 30 min reoxygenation (H/R). During H/R, there was a 67% increase in ferrozine-detectable iron in cell homogenates and increased release of iron into the extracellular space. Cells pretreated with either deferoxamine (DFO) or hydroxyethyl starch-conjugated deferoxamine (HES-DFO), an iron chelator predicted to be confined to the extracellular space, were greatly protected against lethal cell injury. To further localize the site of action of DFO and HES-DFO, tracer quantities of 59Fe were added to DFO or HES-DFO, and their distribution after 2 h was quantitated. Less than 0.1% of DFO entered the cells, whereas essentially none of the HES-DFO was cell-associated. These findings suggest that iron was released during hypoxia/reoxygenation and caused lethal cell injury. Iron chelators confined to the extracellular space provided substantial protection against injury.     

Preconditioning improves function and recovery of single muscle fibers during severe hypoxia and reoxygenation

Reperfusion following prolonged ischemia induces cellular damage in whole skeletal muscle models. Ischemic preconditioning attenuates the deleterious effects. We tested whether individual skeletal muscle fibers would be similarly affected by severe hypoxia and reoxygenation (H/R) in the absence of extracellular factors and whether cellular damage could be alleviated by hypoxic preconditioning. Force and free cytosolic Ca2+ ([Ca2+]c) were monitored in Xenopus single muscle fibers (n = 24) contracting tetanically at 0.2 Hz during 5 min of severe hypoxia and 5 min of reoxygenation. Twelve cells were preconditioned by a shorter bout of H/R 1 h before the experimental trial. In preconditioned cells, force relative to initial maximal values (P/P(o)) and relative peak [Ca2+]c fell (P < 0.05) during 5 min of hypoxia and recovered during reoxygenation. In contrast, P/P(o) and relative peak [Ca2+]c fell more during hypoxia (P < 0.05) and recovered less during reoxygenation (P < 0.05) in control cells. The ratio of force to [Ca2+]c was significantly higher in the preconditioned cells during severe hypoxia, suggesting that changes in [Ca2+]c were not solely responsible for the loss in force. We conclude that 1) isolated skeletal muscle fibers contracting in the absence of extracellular factors are susceptible to H/R injury associated with changes in Ca2+ handling; and 2) hypoxic preconditioning improves contractility, Ca2+ handling, and cell recovery during subsequent hypoxic insult.     

Xanthine Oxidase is Not Responsible for Reoxygenation Injury in Isolated-Perfused Rat Heart

The massive leakage of intracellular enzymes which occurs during reoxygenation of heart tissue after hypoxic or ischemic episodes has been suggested to result from the formation of oxygen radicals. One purported source of such radicals is the xanthine oxidase-mediated metabolism of hypoxanthine and xanthine. Xanthine oxidase (O form) has been suggested to be formed in vivo by limited proteolysis of xanthine dehydrogenase (D form) during the hypoxic period (Granger el ai. Gastroenterology 81, 22 (1981)). We measured the activities of xanthine oxidase in both fresh and isolated-perfused (Langendorff) rat heart tissue. Approximately 32% of the total xanthine oxidase was in the O form in fresh and isolated-perfused rat heart. This value was unchanged following 60min of hypoxia and 30 minutes of reoxygenation. The infusion of 250/JM allopurinol throughout the perfusion completely inhibited xanthine oxidase activity but had no effect on the massive release of lactate dehydrogenase (LDH) into the coronary effluent upon reoxygenation of heart tissue subjected to 30 or 60min of hypoxia. Protection from 30min of hypoxia was also not obtained when rats were pretreated for 48 h with allopurinol at a dose of 30mg/kg/day and perfused with allopurinol containing medium. Superoxide dismutase (50 units/ml), catalase (200 units/ml), or the antioxidant cyanidanol (100μM) also had no effect on LDH release upon reoxygenation after 60 min of hypoxia. Xanthine oxidase activity was detected in a preparation enriched in cardiac endothelial cells while no allupurinol-inhibitable activity could be measured in purified isolated cardiomyocytes. It is concluded that xanthine dehydrogenase is not converted to xanthine oxidase in hypoxic tissue of the isolated perfused rat heart, and that the release of intracellular enzymes upon reoxygenation in this experimental model is mediated by factors other than reactive oxygen generated by xanthine oxidase.     

Interrelationships between the development of the gastric cytoprotective effects of prostacyclin, atropine, cimetidine and the gastric mucosal superoxide dismutase activity in rats

Gastric mucosal damage was produced by the intragastric administration of 96% ethanol or 0.6 M HCl. The cytoprotective doses of prostacyclin (PGI2) (5 micrograms/kg), atropine (0.025 mg/kg) or cimetidine (2.5 mg/kg) were given intraperitoneally 30 min before the administration of the necrotizing agents. The animals were killed 1 hr later. The number and severity of gastric mucosal lesions (ulcer) were recorded. At the time of the sacrifice of the animals, superoxide dismutase (SOD) was prepared from the gastric fundic mucosa and its activity was measured. It was found that PGI2 (5 micrograms/kg), atropine (0.025 mg/kg) and cimetidine (2.5 mg/kg) significantly decreased the number and severity of gastric mucosal lesions (ulcers) produced by the intragastric administration of 96% ethanol a 0.6 M HCl, PGI2, atropine, cimetidine, given in cytoprotective doses, significantly mounted the ethanol-induced increase of gastric mucosal SOD activity; PGI2, atropine, cimetidine, given them in cytoprotective doses significantly shunted the HCl-induced decrease of gastric mucosal SOD activity. It has been concluded that; chemically different cytoprotective agents (PGI2, atropine, cimetidine) give rise to similar tendencies in the changes of gastric mucosal SOD activity; both the significant decrease (in the ethanol-model) and the significant increase (in the HCl-model) of this enzyme seem to be involved in the development of gastric mucosal protection by PGI2, atropine and cimetidine.     

Chronic and intermittent hypoxia induce different degrees of myocardial tolerance to hypoxia-induced dysfunction

Chronic hypoxia (CH) is believed to induce myocardial protection, but this is in contrast with clinical evidence. Here, we test the hypothesis that repeated brief reoxygenation episodes during prolonged CH improve myocardial tolerance to hypoxia-induced dysfunction. Male 5-week-old Sprague-Dawley rats (n = 7-9/group) were exposed for 2 weeks to CH (F(I)O(2) = 0.10), intermittent hypoxia (IH, same as CH, but 1 hr/day exposure to room air), or normoxia (N, F(I)O(2) = 0.21). Hearts were isolated, Langendorff perfused for 30 min with hypoxic medium (Krebs-Henseleit, PO(2) = 67 mmHg), and exposed to hyperoxia (PO(2) = 670 mm Hg). CH hearts displayed higher end-diastolic pressure, lower rate x pressure product, and higher vascular resistance than IH. During hypoxic perfusion, anaerobic mechanisms recruitment was similar in CH and IH hearts, but less than in N. Thus, despite differing only for 1 hr daily exposure to room air, CH and IH induced different responses in animal homeostasis, markers of oxidative stress, and myocardial tolerance to reoxygenation. We conclude that the protection in animals exposed to CH appears conferred by the hypoxic preconditioning due to the reoxygenation rather than by hypoxia per se.     

Reoxygenation-induced constriction in murine coronary arteries: the role of endothelial NADPH oxidase (gp91phox) and intracellular superoxide

Previous work suggests that superoxide mediates hypoxia/reoxygenation (H/R)-induced constriction of isolated mouse coronary arteries (CA). To determine the source of superoxide overproduction during H/R we studied CA obtained from transgenic (Tg) mice overexpressing human CuZn-superoxide dismutase (SOD) and mice lacking gp91(phox) using an in vitro vascular ring bioassay. We found that under normoxic conditions CA isolated from wild type (wt) mice, CuZn-SOD Tg mice and gp91(phox) knock-out mice had similar contractile responses to U46619 and hypoxia and similar dilation responses to acetylcholine. In wt CA, 30 min of hypoxia (1% O(2)) followed by reoxygenation (16% O(2)) resulted in further coronary vasoconstriction (internal diameter from 105 +/- 11 to 84.5 +/- 17.9 microm), whereas this response was completely blocked in both CuZn-SOD Tg and gp91(phox) knock-out CA (104.3 +/- 10.5 to 120.7 +/- 14 microm and 143.3 +/- 15.3 to 172.7 +/- 12.5 microm, respectively, p < 0.01). Furthermore, we show that H/R enhances the generation of superoxide radicals in wt CA (25.8 +/- 0.7 relative light units per second (RLU/s)), whereas CuZn-SOD Tg CA (12.2 +/- 0.8 RLU/s, p < 0.01) and gp91(phox) CA (12.5 +/- 0.9 RLU/s, p < 0.01) show reduced levels. These results demonstrate that H/R-induced vasoconstriction is mediated by intracellular superoxide overproduction via endothelial NADPH oxidase gp91(phox). Therefore, increasing endogenous levels of CuZn-SOD in CA may provide a novel cardioprotective strategy for maintaining coronary perfusion under conditions of H/R.     

Hypoxia/reoxygenation stimulates Ca2+-dependent ICAM-1 mRNA expression in human aortic endothelial cells

Increased endothelial ICAM-1 expression is found in normal aging and in atherosclerosis and is related to the chronic effects of oxidative stress. We examined the Ca(2+)-dependence of ICAM-1 mRNA expression in human aortic endothelial cells (HAEC) exposed to hypoxia/reoxygenation (H/R) as a model of oxidative stress. HAEC were exposed to glucose-free hypoxia (95% N(2)/5% CO(2)) for 60 min and were then reoxygenated (21% O(2)/5% CO(2)) and observed for up to 6h. Reactive oxygen species (ROS) generation was measured by dichlorofluorescein fluorescence and ICAM-1 mRNA was assessed by Northern blot. Upon reoxygenation after hypoxia, ROS production occurred in HAEC and was inhibited by diphenyleneiodonium and by polyethylene glycol-catalase, suggesting the involvement of NADPH oxidase-derived hydrogen peroxide. Hypoxia alone did not increase either ROS production or ICAM-1 mRNA levels, but a 2.5-fold increase in ICAM-1 mRNA was noted by 30 min of reoxygenation. This was not observed in Ca(2+)-free buffer or in cells treated with diphenyleneiodonium. Thus, H/R upregulates ICAM-1 mRNA in HAEC by a Ca(2+)- and ROS-dependent mechanism. Characterizing the signaling pathways involved in H/R-induced adhesion molecule expression may result in a better understanding of the vascular biology of normal aging and the pathobiology of atherosclerosis.     

Effect of short-term hypoxia and re-oxygenation on mice peritoneal macrophages in vitro

Microfluorimetry of single cells could help to analyze their morphology and function state during changes of gas environment. It is very important to have a possibility of the cell visual control during hypoxia and collection of dynamic fluorimetric data in digital form. The effects of short-term pO2 decrease were studied. For estimating the effects of hypoxia and reoxygenation we used the mice peritoneal macrophages, which are very sensitive to physical, chemical and regulatory stimuli. A special small chamber for fluorimetric measurements during pO2 changes, was developed. The level of active oxygen forms, intracellular pH, and cell membrane instability were investigated during replacement of air by nitrogen or argon (of the basal level decreased to 20% of basic level) and in subsequent reoxygenation. The increase of active oxygen forms was shown during 30 min of hypoxia and their level continued to rise immediately after reoxygenation. A short-term decrease and subsequent increase of pO2 in the medium led to an increase of intracellular pH level. The shifts of measured cell indices were stabilized after 30-40 min of pO2 changes thus suggesting a fast comprehension of countermeasure cell mechanisms. No macrophages with membrane disorders were found despite the rise of the active oxygen forms level during hypoxia and reoxygenation in vitro. There were no significant differences between nitrogen and argon used for replacement of air in the medium. The data obtained suggest a high resistance of macrophages against pO2 changes and an involvement of the antioxidative mechanisms for cell protection especially during reoxygenation period.     

Relationships of Dopamine, Cortical Oxygen Pressure, and Hydroxyl Radicals in Brain of Newborn Piglets During Hypoxia and Posthypoxic Recovery

Abstract: The present study describes the relationships of extracellular striatal dopamine, cortical oxygen pressure, and striatal hydroxyl radicals in brain of newborn piglets during hypoxia and posthypoxic reoxygenation. Hypoxia was induced by reducing the fraction of inspired oxygen (F_iO₂) from 22% (control) to 7% for 1 h. The F_iO₂ was then returned to the control value and measurements were continued for 2 h. Cerebral oxygen pressure was measured by the oxygen dependent quenching of phosphorescence and extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and hydroxy radicals in the striatum were determined by in vivo microdialysis. Hypoxia decreased the cortical oxygen pressure from 47 ± 2 to 9 ± 1.3 torr (p < 0.001); the levels of extracellular dopamine in the striatum increased to 16,000 ± 3,270% of control (p < 0.01), whereas the levels of DOPAC and HVA decreased to 25.3 ± 6% (p < 0.001) and 36 ± 5% (p < 0.01) of control, respectively. Compared with control, the hydroxyl radical levels at each time point were not significantly increased during hypoxia, although the sum of the measured values was significantly increased (p < 0.05). During the first 5 min after F_iO₂ was returned to 22%, the cortical oxygen pressure increased to control values and stayed at this level for the remainder of the measurement period. The extracellular level of dopamine declined to values not statistically different from control during 40 min of reoxygenation. During the first 10 min of reoxygenation, DOPAC and HVA further decreased and then began to slowly increase. By 70 min of reoxygenation, the values were not significantly different from control. Hydroxyl radicals were above control during the entire period of reoxygenation, with maximal values observed after 100 min of reoxygenation. This increase was largely abolished by injecting the animals with α-methyl-p-tyrosine 5 h before hypoxia, a procedure that depleted the brain of dopamine. Our results suggest that oxidation of striatal dopamine during posthypoxic reoxygenation is at least partly responsible for the observed increase in striatal level of hydroxyl radicals that may exacerbate posthypoxic cerebral injury.