Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation

The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of this study was to determine the role of PKC activation in BBB paracellular permeability changes induced by hypoxia and posthypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% O2-99% N2; 24 h), a significant increase in total PKC activity was observed, and this was reduced by posthypoxic reoxygenation (95% room air-5% CO2) for 2 h. The expression of PKC-betaII, PKC-gamma, PKC-eta, PKC-mu, and PKC-lambda also increased following hypoxia (1% O2-99% N2; 24 h), and these protein levels remained elevated following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Increases in the expression of PKC-epsilon and PKC-zeta were also observed following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 microM) attenuated the hypoxia-induced increases in [14C]sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC-gamma and PKC-theta in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the 11 PKC isozymes.     

Acetaminophen in the hypoxic and reoxygenated guinea pig myocardium

We investigated the effects of 0.35-mM acetaminophen and its vehicle on isolated, perfused guinea pig hearts made hypoxic and subsequently reoxygenated. Hearts were allowed 30 min postinstrumentation to reach baseline, steady-state values, and then were exposed to 6 min of hypoxia (5% O(2), 5% CO(2), balance N(2)) followed by 36 min of reoxygenation (95% O(2), 5% CO(2)). We recorded hemodynamic, metabolic, and mechanical data in addition to assessing ultrastructure and the capacity of coronary venous effluent to reduce reactive oxygen species. We found that acetaminophen-treated hearts retained a greater fraction of mechanical function during hypoxia and reoxygenation. For example, the average percentage change from baseline of left ventricular developed pressure in acetaminophen- and vehicle-treated hearts at 6 min reoxygenation was 9 +/- 2% and -8 +/- 5% (P < 0.05), respectively. In addition, electron micrographs revealed greater preservation of myofibrillar ultrastructure in acetaminophen-treated hearts. Biochemical analyses revealed the potential of coronary effluent from acetaminophen-treated hearts to significantly neutralize peroxynitrite-dependent chemiluminescence in all recorded time periods. During early reoxygenation, the percentage inhibition of peroxynitrite-mediated chemiluminescence was 56 +/- 10% in vehicle-treated hearts and 99 +/- 1% in acetaminophen-treated hearts (P < 0.05). We conclude that acetaminophen has previously unreported cardioprotective properties in the nonischemic, hypoxic, and reoxygenated myocardium mediated through the reduction of reactive oxygen species.     

Hypoxia/reoxygenation alters endothelial prostacyclin synthesis--protection by superoxide dismutase

An in vitro model was designed to study the role of ischemia/reperfusion and oxygen free radicals on vascular prostacyclin (PGI2) synthesis and protection provided by superoxide dismutase (SOD). Cultured bovine aortic endothelial cells (BAEC) were subjected to various times of hypoxia (30 min to 5 h) followed by 30 min reoxygenation. An increase or a decrease in PGI2 synthesis capacity was then observed according to the duration of hypoxia. Inhibition of PGI2 synthesis after 5 h hypoxia/30 min reoxygenation was accompanied by a rise in lipoperoxidation products and a slight cytotoxicity. Superoxide anion could be implicated in these cellular alterations as SOD efficiently prevented these effects. Incubation of normoxic or H/R-treated BAEC with SOD led to an increase in cellular SOD activity as compared to controls. This increase, inhibited by incubation at 4 degrees C but not by addition of cycloheximide, strongly suggested endocytosis of SOD. This study emphasizes the role of endothelium as a source and target of free radicals and provides a new insight into the mechanism of protection by SOD in ischemia-related vascular pathology.     

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.     

Hypoxia affects the physiological behavior of rat cortical synaptosomes

Nerve cells, especially synaptosomes, are very susceptible to hypoxia and the subsequent oxidative stress. In this paper, we examined the effects of hypoxia (93% N(2):2% O(2):5% CO(2), v/v/v) on rat cortical synaptosomes by evaluating modifications of synaptosomal mitochondrial respiration rate and ATP production, membrane potential, intrasynaptosomal mitochondrial Ca(2+) concentration ([Ca(2+)](i)), and desferoxamine-chelatable free iron and esterified F2-isoprostane levels after different periods of hypoxia and after 30 min of reoxygenation. Oxygen consumption decreased significantly during 120 min of hypoxia and was restored after reoxygenation. At the same time, ATP production decreased and remained significantly lower even after reoxygenation. This involved a depolarization of the synaptosomal mitochondrial membrane, although the [Ca(2+)](i) remained practically unchanged. Indeed, iron and F2-isoprostane levels, representing useful prediction markers for neurodevelopmental outcome, increased significantly after hypoxia, and there was a strong correlation between the two variables. On the whole our results indicate that synaptosomal mitochondria undergo mitoptosis after 2 h of hypoxia.     

L-arginine protects human heart cells from low-volume anoxia and reoxygenation

Protective effects of L-arginine were evaluated in a human ventricular heart cell model of low-volume anoxia and reoxygenation independent of alternate cell types. Cell cultures were subjected to 90 min of low-volume anoxia and 30 min of reoxygenation. L-Arginine (0-5.0 mM) was administered during the preanoxic period or the reoxygenation phase. Nitric oxide (NO) production, NO synthase (NOS) activity, cGMP levels, and cellular injury were assessed. To evaluate the effects of the L-arginine on cell signaling, the effects of the NOS antagonist N(G)-nitro-L-arginine methyl ester, NO donor S-nitroso-N-acetyl-penicillamine, guanylate cyclase inhibitor methylene blue, cGMP analog 8-bromo-cGMP, and ATP-sensitive K+ channel antagonist glibenclamide were examined. Our data indicate that low-volume anoxia and reoxygenation increased NOS activity and facilitated the conversion of L-arginine to NO, which provided protection against cellular injury in a dose-dependent fashion. In addition, L-arginine cardioprotection was achieved by the activation of guanylate cyclase, leading to increased cGMP levels in human heart cells. This action involves a glibenclamide-sensitive, NO-cGMP-dependent pathway.     

Changes in membrane properties during energy depletion-induced cell injury studied with fluorescence microscopy.

The changes in membrane structural properties occurring during the process of ATP depletion-induced cell injury in adherent human astrocytoma cells (UC-11 MG) were studied with two epifluorescence techniques: 1) steady-state fluorescence anisotropy (r) to examine microstructural changes in the membrane phospholipids and 2) fluorescence redistribution after photobleaching (FRAP) to examine membrane fluidity changes. A new method for r measurement was established that provides the unique advantage of simultaneously monitoring both vertical and horizontal polarized fluorescence emissions needed for the calculation of r. In this study, r in the astrocytoma cells labeled with 1-(4-trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate was shown to remain stable for up to 90 min. However, when the cells were treated with 75 microM iodoacetic acid (IAA), a metabolic inhibitor that induces rapid depletion of cellular ATP, r continually decreased, indicating a decrease in membrane lipid order and perturbation of the bilayer structure. This decrease in r could be prevented by the pretreatment of cells with lipophilic antioxidants such as tirilazad or gossypol. Tirilazad itself caused a significant increase in r, suggesting that tirilazad intercalates into the membrane bilayer and profoundly increases the lipid order in uninjured cells. Gossypol, however, did not exhibit this property. Further investigations into these phenomena with FRAP confirmed the r results and indicated that membrane fluidity increased while its structure became less rigid during the process of ATP-induced cell injury. In addition, lipophilic antioxidants prevented the membrane structural aberrations induced by IAA. Experimental results suggest that different mechanisms of cytoprotective action may exist for tirilazad and the antioxidant gossypol. Gossypol appears to prevent or delay the observed cell injury entirely because of its antioxidant action, whereas tirilazad's protection is mediated not only via its antioxidant activity, but also by its ability to increase cell membrane lipid order.     

Hypothermia preserves myocardial function and mitochondrial protein gene expression during hypoxia

Hypothermia before and/or during no-flow ischemia promotes cardiac functional recovery and maintains mRNA expression for stress proteins and mitochondrial membrane proteins (MMP) during reperfusion. Adaptation and protection may occur through cold-induced change in anaerobic metabolism. Accordingly, the principal objective of this study was to test the hypothesis that hypothermia preserves myocardial function during hypoxia and reoxygenation. Hypoxic conditions in these experiments were created by reducing O2 concentration in perfusate, thereby maintaining or elevating coronary flow (CF). Isolated Langendorff-perfused rabbit hearts were subjected to perfusate (Po2 = 38 mmHg) with glucose (11.5 mM) and perfusion pressure (90 mmHg). The control (C) group was at 37 degrees C for 30 min before and 45 min during hypoxia, whereas the hypothermia (H) group was at 29.5 degrees C for 30 min before and 45 min during hypoxia. Reoxygenation occurred at 37 degrees C for 45 min for both groups. CF increased during hypoxia. The H group markedly improved functional recovery during reoxygenation, including left ventricular developed pressure (DP), the product of DP and heart rate, dP/dtmax, and O2 consumption (MVo2) (P < 0.05 vs. control). MVo2 decreased during hypothermia. Lactate and CO2 gradients across the coronary bed were the same in C and H groups during hypoxia, implying similar anaerobic metabolic rates. Hypothermia preserved MMP betaF1-ATPase mRNA levels but did not alter adenine nucleotide translocator-1 or heat shock protein-70 mRNA levels. In conclusion, hypothermia preserves cardiac function after hypoxia in the hypoxic high-CF model. Thus hypothermic protection does not occur exclusively through cold-induced alterations in anaerobic metabolism.     

The relationship between cell membrane damage and lipid peroxidation under the condition of hypoxia‐reoxygenation: analysis of the mechanism using antioxidants and electron transport inhibitors

We consecutively observed lipid peroxidation and cell membrane damage under the condition of hypoxia‐reoxygenation (H/R) in cells and analyzed their mechanisms by using electron transport inhibitors and an antioxidant. In H/R experiments, lipid peroxidation and cell membrane damage were observed during the hypoxia phase. In the reoxygenation phase, lipid peroxidation stopped, while cell membrane damage did not. An antioxidant, n‐acetylcystein (NAC), and potassium cyanide (KCN) inhibited lipid peroxidation and cell membrane damage, while rotenone did not inhibit either of them. Although antimycin A did not inhibit lipid peroxidation, it inhibited cell membrane damage during the hypoxia phase but not during the reoxygenation phase. These results suggested that lipid peroxidation can affect cell membrane damage as a trigger during the hypoxia phase and the generation of oxidative stress can vary depending on the inhibition locations in the electron transport system. Copyright © 2009 John Wiley & Sons, Ltd.     

培养大鼠搏动心肌细胞在缺氧和复氧时的电生理观察

应用细胞内微电极技术记录到37个培养大鼠搏动心肌细胞充氮前后和复氧后的电活动参数。结果提示:充氮10min后,最大舒张电位(MDP),最大除极速度(V_(max)),动作电位振幅(APA)和动作电位时程(APD)等参数明显降低;自发节律增快,并出现多种形式的节律失常。83.8％细胞在充氮后30min内停搏,16.2％在50min左右停搏。复氧后,86.5％细胞在5min内复跳,13.5％未能复跳;12.5％复跳细胞在复跳10min内再次停搏。复跳细胞的各项电活动参数在30min内未能恢复到充氮前水平(p<0.05),且呈现不同程度的各类异常电活动。本结果对进一步研究心肌细胞缺氧和复氧损伤有一定意义。     

七氟醚预处理对大鼠海马脑片缺氧无糖损伤的保护作用:线粒体KATP通道的作用

利用离体海马脑片缺氧无糖(oxygen-glucose deprivation,OGD)损伤模型,探讨七氟醚预处理对神经细胞的保护作用及该作用与线粒体内膜ATP敏感钾通道(mitochondrial ATP-sensitive potassium channels,mitoKATPchannels)的关系,随机将脑片用2%、4%、6%七氟醚,以及6%七氟醚复合mitoKATP通道阻滞剂5-羟基奎酸盐(5-hydroxydecanoic acid,5-HD)预处理30 min,观察OGD损伤14 min复氧1 h期间顺向群峰电位(orthodromic population spike,OPS)的变化,并应用透射电镜观察细胞超微结构的改变.结果表明,与单纯OGD组相比,七氟醚预处理可使海马脑片OPS消失时间明显延长(P＜0.01),使OPS明显恢复,其中4%、6%七氟醚组的恢复率均为71.4%(P＜0.05 vs OGD),相应恢复程度为(61.0±42.3)%和(78.7±21.1)%(P＜0.01),而且6%七氟醚的保护作用可被5-HD取消.OGD组的海马CA1区锥体细胞明显水肿,核膜皱缩、破裂,染色质聚集,线粒体肿胀畸形,嵴断裂或消失,而4%和6%七氟醚组仅见海马CA1区锥体细胞轻度水肿,核膜皱缩不明显,染色质均匀,线粒体轻度肿胀.结果提示,七氟醚预处理对大鼠海马脑片OGD损伤有一定的保护作用,且七氟醚对神经细胞的保护作用与激活mitoKATP通道有关.     

Bovine pituitary extract provides remarkable protection against oxidative stress in human prostate epithelial cells

Bovine pituitary extract (BPE) is routinely used as a mitogenic supplement in serum-free growth medium. In addition to its mitogenic activity, BPE contains a variety of growth factors and hormones with reported antioxidant activity. This study examines the antioxidant potential of BPE in nontumorigenic human prostate epithelial cells (RWPE-1). Treatment of RWPE-1 cells with BPE (50 microg/ml) provided significant protection against H(2)O(2)-induced cell death, deoxyribonucleic acid fragmentation, protein oxidation, and membrane damage. Treatment with heat (71 degrees C, 10 min) and proteolytic enzymes reduced the antioxidant activity of BPE, suggesting that proteins present in BPE may be responsible for the antioxidant activity. Residual catalase activity present in BPE was responsible for a portion (30%) of the antioxidant activity. Interestingly, RWPE-1 cells treated with BPE and H(2)O(2) rapidly accumulated intracellular reactive oxygen species (ROS) to a greater extent than cells receiving only H(2)O(2). Pretreatment of RWPE-1 cells with tyrosine kinase inhibitors (genistein, tyrphostin 47, and AG-1296) before the addition of H(2)O(2) diminished BPE protection against H(2)O(2)-induced cell death, whereas treatment with purified mitogens commonly found in BPE, growth hormone and basic fibroblast growth factor, did not protect against oxidative damage. Taken together, these data suggest that BPE contains proteins or protein complexes with remarkable antioxidant activity. These yet unidentified compounds appear to confer protection against H(2)O(2)-induced cell death by tyrosine kinase-dependent pathways that increase intracellular ROS generation. The antioxidant activity of BPE may represent a confounding variable when studying oxidative stress in cells maintained in BPE-supplemented serum-free medium.     

Role of catalase on the hypoxia/reoxygenation stress in the hypoxia-tolerant Nile tilapia

The specific contribution of each antioxidant enzyme to protection against the reoxygenation-associated oxidative stress after periods of hypoxia is not well understood. We assessed the physiological role of catalase during posthypoxic reoxygenation by the combination of two approaches. First, catalase activity of Nile tilapias (Oreochromis niloticus) was 90% suppressed by intraperitoneal injection of 3-amino-1,2,4-triazole (ATZ, 1g/kg). In ATZ-injected fish, liver GSH levels, oxidative stress markers, and activities of other antioxidant enzymes remained unchanged. Second, animals with depleted catalase activity (or those saline-injected) were subjected to a cycle of severe hypoxia (dissolved O(2) = 0.28 mg/l for 3 h) followed by reoxygenation (0.5 to 24 h). Hypoxia did not induce changes in the above-mentioned parameters, either in saline- or in ATZ-injected animals. Reoxygenation increased superoxide dismutase activity in saline-injected fish, whose levels were similar to ATZ-injected animals. The activities of glutathione S-transferase, selenium-dependent glutathione peroxidase, and total-GPX and the levels of GSH-eq, GSSG, and thiobarbituric acid reactive substances remained unchanged during reoxygenation in both saline- and ATZ-injected fish. The GSSG/GSH-eq ratio in ATZ-injected fish increased at 30 min of reoxygenation compared with saline-injected ones. Reoxygenation also increased carbonyl protein levels in saline-injected fish, whose levels were similar to the ATZ-injected group. Our work shows that inhibition of liver tilapia catalase causes a redox imbalance during reoxygenation, which is insufficient to induce further oxidative stress. This indicates the relevance of hepatic catalase for hypoxia/reoxygenation stress in tilapia fish.     

Anoxia reversibly suppresses neuronal calcium currents in rat hippocampal slices

Intracellular recording from CA1 neurons confirmed that short periods of anoxia (95% N2 + 5% CO2 for 2-4 min) have a hyperpolarizing action, caused by a rise in K conductance. After blockage of K channels with extracellular Cs+ and tetraethylammonium (or intracellular Cs+), large inward currents of Ca were evoked by depolarizing pulses: transient currents at a holding potential near -70 mV, and more sustained ones near -50 mV. Both types of Ca current were much reduced or fully suppressed after 1-3 min of anoxia, but they largely (or fully) recovered within 1-10 min of starting reoxygenation.     

Effect of ischemia reperfusion or hypoxia reoxygenation on lung vascular permeability and resistance

The effect of ischemia reperfusion or hypoxia reoxygenation on pulmonary vascular permeability and resistance was studied in 25 isolated blood-perfused dog lungs. Vascular permeability, assessed by determining filtration coefficient (Kf), and vascular resistances were measured at the beginning and end of the experiment. Ischemia reperfusion was produced by occluding blood flow to the lung for 3 h and reperfusing for 1 h, whereas hypoxia reoxygenation was obtained by ventilating the lung with 95% N2-5% CO2 for 3 h and then ventilating with 95% O2-5% CO2 for 1 h with no interruption of perfusion. There was a significant increase in Kf in both ischemia reperfusion and hypoxia reoxygenation groups (51 and 85%, respectively), and total vascular resistance increased greatly in both groups (386 and 532%, respectively). Two additional groups were also studied in which the ischemia reperfusion or hypoxia reoxygenation lungs were pretreated with allopurinol (20 micrograms/ml). The Kf did not significantly increase in either the allopurinol ischemia reperfusion or the allopurinol hypoxia reoxygenation groups (22 and 6%, respectively). However, total vascular resistance significantly increased in both groups (239 and 224%, respectively). Although vascular permeability is modestly increased by both ischemia reperfusion and hypoxia reoxygenation, the predominant change in these conditions is the increased vascular resistance, which predominantly affects the postcapillary resistance and would result in a greater tendency for edema to develop in these slightly damaged lungs. Allopurinol, which inhibits xanthine oxidase, attenuated the permeability changes in both groups and may be useful in preventing ischemia reperfusion injury in certain conditions.     

Signal transduction of flumazenil-induced preconditioning in myocytes

The objective of this study was to examine the role of oxygen radicals, protein kinase C (PKC), and ATP-sensitive K(+) (K(ATP)) channels in mediating flumazenil-produced preconditioning. Chick cardiomyocyte death was quantified using propidium iodide, and oxygen radical generation was assessed using 2',7'-dichlorofluorescin oxidation. Preconditioning was initiated with 10 min of ischemia followed by 10 min of reoxygenation. Alternatively, flumazenil was infused for 10 min and removed 10 min before ischemia. Flumazenil (10 microM) and preconditioning increased oxygen radicals [1,693 +/- 101 (n = 3) and 1,567 +/- 98 (n = 3), respectively, vs. 345 +/- 53 (n = 3) in control] and reduced cell death similarly [22 +/- 3% (n = 5) and 18 +/- 2% (n = 6), respectively, vs. controls 49 +/- 5% (n = 8)]. Protection and increased oxygen radicals by flumazenil were abolished by pretreatment with the antioxidant thiol reductant 2-mercaptopropionyl glycine (800 microM; 52 +/- 10%, n = 6). Specific PKC inhibitors Go-6976 (0.1 microM) and chelerythrine (2 microM), given during ischemia and reoxygenation, blocked flumazenil-produced protection (47 +/- 5%, n = 6). The PKC activator phorbol 12-myristate 13-acetate (0.2 microM), given during ischemia and reoxygenation, reduced cell death similarly to that with flumazenil [17 +/- 4% (n = 6) and 22 +/- 3% (n = 5)]. Finally, 5-hydroxydecanoate (1 mM), a selective mitochondrial K(ATP) channel antagonist given during ischemia and reoxygenation, abolished the protection of flumazenil and phorbol 12-myristate 13-acetate. Thus flumazenil mimics preconditioning to reduce cell death in cardiomyocytes. Oxygen radicals activate mitochondrial K(ATP) channels via PKC during the process.     

Oxygen radicals generated during anoxia followed by reoxygenation reduce the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in human endothelial cell culture

The effect of anoxia and reoxygenation on the synthesis and secretion of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) was studied in primary cultures of human umbilical vein endothelial cells. Sublethal anoxia, determined by trypan blue dye exclusion and lactate dehydrogenase release, was produced by cell culture under a 95% N2, 5% CO2 atmosphere for 2-24 h and was followed by reoxygenation with 95% air, 5% CO2 for 24 or 48 h. Anoxia did not alter the levels of mRNA for t-PA or PAI-1 in the cells or the secretion of t-PA or PAI-1 into the medium. At 24 h, t-PA secreted into conditioned medium was 7.0 +/- 1.4 ng/2 x 10(6) cells (n = 9) and PAI-1 was 300 +/- 13 IU/2 x 10(6) cells (n = 9), whereas the content of t-PA mRNA was 2.2 pg/micrograms of RNA and PAI-1 mRNA was 180 pg/micrograms of RNA. During reoxygenation, however, t-PA antigen and PAI-1 activity as well as mRNA for PAI-1 decreased proportionally to the duration of anoxia, to reach 27 +/- 1.0, 49 +/- 2.0, and 47 +/- 14% of control values, respectively, within 24 h of anoxia. t-PA mRNA also decreased significantly during reoxygenation following anoxia, but the extent could not be accurately quantitated. Addition, during anoxia, of a 200 micrograms/ml concentration of the superoxide anion radical scavenger superoxide dismutase or of a 5 mM concentration of the iron chelator deferoxamine mesylate prevented the subsequent decrease of t-PA antigen during reoxygenation; addition of these compounds during reoxygenation had no effect. Superoxide dismutase, but not deferoxamine mesylate, when added during anoxia prevented the subsequent decrease in PAI-1 activity. These studies suggest that the marked alteration of endothelial cell fibrinolysis during anoxia followed by reoxygenation is most likely mediated by a mechanism dependent on oxygen radicals. Impaired endothelial cell fibrinolysis may contribute to the pathophysiology of ischemia/reperfusion injury.     

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.     

Effects of prostaglandins on rat renal adenylate cyclase-cyclic AMP systems.

The effects of prostaglandin (PG) E1, E2, A1, F1alpha, F2alpha or D2 on the rat renal cortical, outer medullary and inner medullary adenylate cyclase-cyclic AMP systems were examined. While high concentrations (8X10-4M) of each prostaglandin stimulated adenylate cyclase activity in each area of the kidney, PGE1 was the only prostaglandin to stimulate at 10-7M. PGA's were the only prostaglandins tested besides PGE's which stimulated adenylate cyclase at less than 10-4M. This effect of PGA's was limited to the outer medulla. PGD2 was the least stimulatory. Observations with renal slices yielded qualitatively similar results. The PGE's were the most potent in each area with PGA's only stimulatory in the outer medulla. O2 deprivation (5% O2) lowered the slice cyclic AMP content in each area of the kidney. In the cortex and outer medulla, prostaglandin mediated increases in cyclic AMP content were either lower or absent at 5% O2 compared to 95% O2. However, in the inner medulla PGE stimulation was observed only at 5% O2 and not 95% O2. No other prostaglandins were found to increase inner medullary cyclic AMP content at 95% or 5% O2. These results illustrate that the adenylate cyclase-cyclic AMP system responds uniquely to prostaglandins in each area of the kidney. Consideration of these results along with correlative observations suggests that inner medullary produced PGE's may act as local modulators of inner medullary adenylate cyclase.     

Fullerene derivative attenuates ischemia-reperfusion-induced lung injury

Reactive oxygen species are the major contributing factors to lung ischemia-reperfusion (IR) injury. In this study, we tested whether a water soluble antioxidant fullerene derivative [C(60)(ONO(2))(7 +/- 2)] attenuates IR lung injury. Young Wistar rats were divided into two groups: control and C(60)(ONO(2))(7 +/- 2). Under ventilation with 95% air-5% CO(2) gas mixture and a 2.5 cm H(2)O end-expiratory pressure, the isolated lungs were perfused with a physiological solution. The experimental protocol included three periods: baseline (10 min), ischemia (45 min) and reperfusion (60 min, ventilated with 95% O(2)-5% CO(2) gas mixture). Before and after ischemia, we measured pulmonary arterial pressure (Ppa), pulmonary venous pressure and lung weight (W). Then, pulmonary capillary pressure and filtration coefficient (K(fc)) were calculated. Ischemia caused increases in Ppa, W and K(fc) in the control group. For most cases, the above ischemia-induced increases were attenuated by the C(60)(ONO(2))(7 +/- 2) pretreatment. Our results suggest that the antioxidant C(60)(ONO(2))(7 +/- 2) attenuates IR-induced lung injury.