Posthypoxic Reoxygenation Research Articles

Effect of catecholamines on activity of Na +, K +-ATPase in neonatal piglet brain during posthypoxic reoxygenation

The present study examined the possible role of dopamine on the response of Na +, K +-ATPase activity in the striatum of newborn piglets to 1 h of bilateral carotid ligation with hemorrhage and 2 h of recovery. Newborn piglets, 2–4 days of age and with and without prior treatment with α-methyl- p-tyrosine (AMT), an inhibitor of catecholamines synthesis, were used for the study. The oxygen pressure in the microvasculature of the cortex (PcO 2) was measured by oxygen dependent quenching of the phosphorescence. In sham-operated animals the PcO 2 was 50±3 torr. Following ligation and hemorrhage the PcO 2 decreased to 8±0.5 torr. After release of ligation and reperfusion PcO 2 increased to 45±4 torr, a value not significantly different from controls, in approximately 30 min. There were no significant differences in PcO 2 between AMT treated and untreated animals. In sham-operated animals striatal Na +,K +-ATPase was 29.1±3 μmol/mg protein per h and decreased by 25% after 2 h of recovery. Depleting the brain of catecholamines prior to ligation and hemorrhage abolished this decrease. It is postulated that the decrease in the level of dopamine in the brain prior to ligation and hemorrhage can be at least partly responsible for the observed decrease in activity of Na +, K +-ATPase in the striatum of newborn piglets.

Operation Everest III (Comex'97): the effect of simulated severe hypobaric hypoxia on lipid peroxidation and antioxidant defence systems in human blood at rest and after maximal exercise

Eight subjects were placed in a decompression chamber for 31 days at pressures from sea level (SL) to 8848 m altitude equivalent. Whole blood lipid peroxidation (LP) was increased at 6000 m by a mean of 23% ( P<0.05), at 8000 m by 79% ( P<0.01) and at 8848 m by 94% ( P<0.01). (All figures are means.) Two days after return to sea level (RSL), it remained high, by 81% ( P<0.01), while corresponding erythrocyte GSH/GSSG ratios decreased by 31, 46, 49, 48%, respectively (each P<0.01). Erythrocyte SOD and plasma ascorbate did not change significantly. At sea level, maximal exercise induced a 49% increase in LP ( P<0.01), and a 27% decrease in erythrocyte GSH/GSSG ratio relative to resting values ( P<0.05). At 6000 m, the LP was enhanced further from 23 ( P<0.05) to 66% ( P<0.01), and after RSL from 81 ( P<0.01) to 232% ( P<0.01), while pre-exercise GSH/GSSG ratios did not change significantly. Exercise did not change plasma ascorbate relative to sea level or to 6000 m, but decreased after RSL by 32% ( P<0.01). These findings suggest that oxidative stress is induced by prolonged hypobaric hypoxia, and is maintained by rapid return to sea level, similar to the post-hypoxic re-oxygenation process. It is increased by physical exercise.

Hypoxia/Reoxygenation Stimulates Intracellular Calcium Oscillations in Human Aortic Endothelial Cells

We have previously shown that hydrogen peroxide stimulates endothelial [Ca(2+)](i) oscillations. This study was performed to determine whether posthypoxic reoxygenation stimulates [Ca(2+)](i) oscillations in vascular endothelial cells. Hypoxia (glucose-free 95% N(2)/5% CO(2) bicarbonate buffer for 60 minutes) stimulated an increase in [Ca(2+)](i) from 111.9+/-7. 9 to 161.7+/-17.7 nmol/L (n=12, P:<0.01) in indo 1-loaded human aortic endothelial cells. On reoxygenation (glucose-containing 95% air/5% CO(2) bicarbonate buffer), 13 of 16 cells responded with repetitive [Ca(2+)](i) oscillations with an average amplitude of 570. 6+/-59.3 nmol/L, occurring at a mean interval of 0.28+/-0.04/min and persisting for >/=60 minutes. [Ca(2+)](i) oscillations were still observed in 4 of 7 cells studied in Ca(2+)-free buffer but did not occur when the intracellular Ca(2+) store was first depleted during hypoxia by either 1 micromol/L thapsigargin or by 10 mmol/L caffeine (n=6 for each). Reoxygenation-induced [Ca(2+)](i) oscillations were abolished by 10 micromol/L diphenyleneiodonium, an inhibitor of NAD(P)H oxidase (n=7), and by polyethylene glycol (PEG)-catalase (5000 U/mL, n=4) but were not prevented by inhibitors of xanthine oxidase (n=5), cyclooxygenase (n=4), nitric oxide synthase (n=5), the mitochondrial electron transport chain (n=4), or by PEG-superoxide dismutase (n=5). Posthypoxic reoxygenation stimulates repetitive [Ca(2+)](i) oscillations that are dependent on Ca(2+) release from an intracellular pool and require extracellular Ca(2+) to be maintained. These oscillations may be initiated by NAD(P)H oxidase-derived hydrogen peroxide and may play a role in signal transduction during ischemia/reperfusion in vivo.

Alterations in relaxation to lactate and H(2)O(2) in human placental vessels from gestational diabetic pregnancies.

We determined whether alterations in the mechanism of relaxation to H(2)O(2) potentially contribute to the enhanced prostaglandin-mediated contractile response to H(2)O(2) and posthypoxic reoxygenation seen in human placental vessels of pregnancies with gestational diabetes mellitus (GDM). Isolated placental arteries and veins from GDM and uncomplicated full-term pregnancies were precontracted with prostaglandin F(2alpha) (PO(2) 35-38 Torr) and then exposed to lactate (1-10 mM), arachidonic acid (0.01-10 microM), nitroglycerin (1 nM-1 microM), forskolin (0.01-10 microM), or H(2)O(2) (1 microM-1 mM + 10 microM indomethacin). The rates of tissue H(2)O(2) metabolism by catalase and nitrite production were measured. The relaxation to lactate was reduced in GDM placental arteries and veins by 54-85 and 66-80%, and the relaxation to H(2)O(2) was inhibited by 80-94% in GDM placental veins compared with vessels from uncomplicated full-term pregnancies. H(2)O(2) caused only minimal relaxation of placental arteries. Responses to other relaxing agents were not altered in the GDM placental vessels. Diabetic vessels showed rates of nitrite production that were increased by 113-195% and rates of H(2)O(2) metabolism by catalase that were decreased by 44-61%. The loss of relaxation to H(2)O(2) and lactate (mediated via H(2)O(2)), perhaps as a result of the inhibition of catalase by nitric oxide, may explain the previously reported enhancement of prostaglandin-mediated contractile responses to H(2)O(2) and posthypoxic reoxygenation seen in GDM placental vessels.

Cross-influence of membrane polyunsaturated fatty acids and hypoxia-reoxygenation on alpha- and beta-adrenergic function of rat cardiomyocytes.

The purpose of the present investigation was to determine whether the beneficial effects of polyunsaturated fatty acids (PUFA) may influence ischemia-reperfusion-induced alterations of myocardial alpha- and beta-adrenoceptor (alpha-AR, beta-AR) responsiveness. This study was carried out using monolayer cultures of neonatal rat ventricular myocytes in a substrate-free, hypoxia-reoxygenation model of ischemia. The cardiomyocytes (CM) were incubated during 4 days in media enriched either with n-6 PUFA (arachidonic acid, AA) or with n-3 PUFA (eicosapentaenoic acid, EPA, and docosahexaenoic acid, DHA). The n-6/n-3 ratio in n-3 CM was close to 1.2, compared to 20.1 in n-6 CM. The contractile parameters of n-6 CM and n-3 CM were similar in basal conditions as well as during hypoxia and reoxygenation. In basal conditions, the phospholipid (PL) enrichment with long chain n-3 PUFA resulted in an increased chronotropic response to isoproterenol (ISO) and to phenylephrine (PHE). After posthypoxic reoxygenation, the chronotropic response to beta-AR activation in n-6 CM was significantly enhanced as compared with the control response in normoxia. In opposition, the ISO-induced rise in frequency in n-3 CM in control normoxia and after reoxygenation was similar. In these n-3 CM, the changes in contractile parameters, which accompanied the chronotropic response, were also similar in reoxygenation and in normoxic periods, although the rise in shortening velocity was slightly increased after reoxygenation. In response to PHE addition, only the chronotropic effect of n-6 CM appeared significantly enhanced after hypoxic treatment. These results suggested that increasing n-3 PUFA in PL reduced the increase in alpha- and beta-AR functional responses observed after hypoxia-reoxygenation. This effect may partly account for the assumed cardiac protective effect of n-3 PUFA, through the attenuation of the functional response to catecholamines in the ischemic myocardium.

Altered ligand binding of D1 and D2 dopamine receptors in response to hypoxia and posthypoxic reoxygenation.

The physiological effects of dopamine, a major striatal neurotransmitter, are mediated by several distinct receptor subtypes. On the basis of structural, pharmacological, functional, and distributional similarities, these subtypes fall into one of two groups, the D1-like receptor family and the D2-like receptor family. The D1-like receptors are linked to stimulation of adenylate cyclase via Gs proteins whereas the D2-like receptors inhibit adenylate cyclase through a Gi/o protein.1 The D1-like receptors are also associated with inositol phosphate production and calcium mobilization while the D2-like receptors stimulate potassium efflux and indirectly inhibit inositol phosphate production. Thus, changes in the properties of these dopaminergic receptors could have a major impact on cellular metabolism. Several studies have examined the effect of ischemia-hypoxia on ligand binding properties of the striatal dopaminergic receptors, particularly on adult animals, and the authors suggested that the changes in the D1 and/or D2 receptors play a key role in the pathogenesis of damage to the striatum induced by ischemia. For example, Bonfenati et al.4 proposed that reduction in the number of D1 receptors plays a key role in the pathogenesis of ischemic striatal damage. The purpose of the present study was to determine whether arterial hypoxia is associated with alterations in the affinities (Kd) and numbers (Bmax) of the dopaminergic receptor subtypes, D1 and D2, in the striatum of newborn piglets.KeywordsDopamine ReceptorDopaminergic ReceptorNewborn PigletInositol Phosphate ProductionReoxygenation PeriodThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Influence of glutathione peroxidase on coronary artery responses to alterations in PO2 and H2O2.

Our previous work suggests that relaxation of endothelium-removed bovine coronary arteries (BCA) to posthypoxic reoxygenation is mediated by NADH oxidase-dependent superoxide anion-derived H2O2 and cGMP. The purpose of this study was to investigate if altering BCA GSH peroxidase activity by enhancing its activity with a GSH peroxidase-mimetic (0.1 mM Ebselen) or by inhibiting its activity with an inhibitor of GSH peroxidase [10 mM mercaptosuccinic acid (MS)] causes a selective modulation of responses to exogenously (1 microM-1 mM H2O2) and endogenously generated (reoxygenation and 1-10 mM lactate) H2O2. Ebselen inhibited and MS enhanced all of the responses that are thought to be mediated by H2O2, without having significant effects on relaxation to hypoxia or a nitric oxide donor [1 nM-10 microM S-nitroso-N-acetylpenicillamine (SNAP)]. Thus enhancement of BCA GSH peroxidase activity with Ebselen inhibits relaxation to reoxygenation, lactate, and H2O2, whereas inhibition of GSH peroxidase with MS causes potentiation of responses thought to be mediated by H2O2 in BCA. Inactivation of catalase by pretreatment of BCA with 3-amino-1,2,4-triazole (50 mM, 30 min) inhibited relaxation to H2O2 and the potentiation by MS. Whereas the actions of these probes are not consistent with a role for oxidation of GSH in the relaxation to H2O2, their effects are potentially a result of modulating the metabolism of H2O2 by endogenous catalase, which is thought to mediate the stimulation of the cytosolic or soluble form of guanylate cyclase.

Inhibition of rat cardiac muscle contraction and mitochondrial respiration by endogenous peroxynitrite formation during posthypoxic reoxygenation.

This study was designed to investigate the potential role of endogenous peroxynitrite (ONOO-) formation in the inhibition of cardiac muscle contractility and mitochondrial respiration during posthypoxic reoxygenation. Isometric contraction of isolated rat left ventricular posterior papillary muscle was virtually eliminated at the end of an exposure to 15 minutes of hypoxia and remained 40+/-5% depressed an hour after the reintroduction of O2. O2 uptake by rat left ventricular cardiac muscle, measured by a Clark-type O2 electrode, was also inhibited by 24+/-2% at 10 minutes after reoxygenation. The inhibition of contractility and respiration during posthypoxic reoxygenation was markedly attenuated by the NO synthase inhibitor nitro-L-arginine, exogenous superoxide dismutase, and the ONOO- scavenger urate but not by the hydroxyl radical scavenger mannitol. Generation of ONOO- with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) plus the superoxide-releasing agent pyrogallol caused an irreversible inhibition of cardiac contractile and respiratory function. Unlike ONOO-, exogenous (SNAP) and endogenous (bradykinin) sources of NO inhibited contractility in a reversible manner. Under conditions of comparable amounts of respiratory inhibition in unstimulated incubated muscle, the NO-dependent agents and the mitochondrial antagonist NaCN produced a smaller degree of suppression of contractility compared with ONOO- and posthypoxic reoxygenation. These results are consistent with a contributing role for endogenous ONOO- formation in the inhibition of cardiac muscle contractility and mitochondrial respiration during posthypoxic reoxygenation.

Lactate and PO2 modulate superoxide anion production in bovine cardiac myocytes: potential role of NADH oxidase.

Lactate increases lucigenin chemiluminescence (CL)-detectable superoxide anion (O2.-) generation in bovine vascular smooth muscle and endothelium, and a microsomal flavoprotein-containing NADH oxidase whose activity is regulated by PO2 and cytosolic NAD(H) redox appears to be the detected source of O2.- production. Little is known about the importance of this O2.(-)-producing system in cardiac myocytes. In isolated bovine cardiac myocytes, lactate (10 mmol/L) increased lucigenin-detectable O2.- levels to approximately 1.8 times baseline, whereas pyruvate (10 mmol/L) and mitochondrial probes did not increase the detection of O2.-. A nonmitochondrial NADH oxidase activity, found in microsomes containing a cytochrome b558, was a major source of O2.- production in the homogenate of myocytes, because NADH (0.1 mmol/L) increased basal lucigenin CL >100-fold. NADPH oxidases, mitochondria, and xanthine oxidase were minor sources of detectable O2.- production. However, mitochondria released H2O2 in the presence of 5 mmol/L succinate and 30 micromol/L antimycin, based on its detection as catalase-inhibitable luminol (+horseradish peroxidase)-elicited CL. Diphenyliodonium (DPI), an inhibitor of flavoprotein-containing oxidases, significantly attenuated basal, lactate, and NADH-elicited lucigenin CL. Hypoxia eliminated myocyte lucigenin CL, and posthypoxic reoxygenation caused an 8.6-fold increase in the detection of O2.- that was potentiated by lactate and inhibited by DPI. NADH oxidase activity linked to cytosolic NAD(H) redox appears to be a key source of O2.- production in cardiac myocytes that could contribute to oxidant signaling mechanisms and injury upon exposure to changes in PO2 and metabolites produced under hypoxia, such as lactate. These processes could contribute to the previously observed potentiation of injury caused by lactate in cardiac ischemia/reperfusion.

Effect of Post-Hypoxic Reoxygenation with Room Air or 100% Oxygen on Cerebral Cortical Function in Newborn Piglets. † 800

Effect of Post-Hypoxic Reoxygenation with Room Air or 100% Oxygen on Cerebral Cortical Function in Newborn Piglets. † 800

Evaluation of purine nucleotide loss, lipid peroxidation and ultrastructural alterations in post-hypoxic hepatocytes.

1. Hypoxic alterations in isolated rat hepatocytes were demonstrated by a 90% ATP loss during 60 min of ischaemia and temporary increases of nucleotide degradation products. 2. The oxidative stress during reoxygenation was demonstrated in these cells by a decrease in reduced glutathione (GSH) concentration (30%) and a threefold increase in lipid peroxidation products such as 4-hydroxynonenal and thiobarbituric acid-reactive substances (TBA-RSs). The tremendous GSH loss could not be balanced by the slight oxidized glutathione (GSSG) increase during reoxygenation. 3. For the first time the involvement of free radicals was directly demonstrated using electron spin resonance (ESR) spectroscopy in reoxygenated liver cells. Using the spin trap 5,5-dimethylpyrroline-1-oxide (DMPO), a carbon-centred radical and the adduct of the hydroxyl radical could be detected during early reoxygenation. 4. Morphological alteration of cells was observed, beginning during hypoxia and increasing during post-hypoxic reoxygenation. Electron microscopic findings of hypoxic and post-hypoxic cell damage included pyknosis of nuclei, spherical transformation of mitochondria and increased number of vesicles.

Oxygen-elicited responses in calf coronary arteries: role of H2O2 production via NADH-derived superoxide.

Our previous studies in isolated endothelium-removed calf pulmonary arteries suggest that PO2-elicited responses are primarily mediated through modulation of guanosine 3',5'-cyclic monophosphate via changes in the generation of H2O2 originating from superoxide anion (O2-.) produced by NADH oxidase activity. In the present study we examined the importance of this mechanism in PO2-elicited responses of endothelium-removed calf coronary arteries. NADH oxidase activity was found to be the major source of O2-. in the homogenate of endothelium-removed calf coronary arteries detected by lucigenin-elicited chemiluminescence. Precontracted endothelium-removed calf coronary arteries show a relaxation to hypoxia, and reoxygenation causes a transient additional relaxation before the recovery of normoxic levels of force. Under these conditions the detection of O2-. was decreased by hypoxia and a transient overproduction was observed during reoxygenation. The relaxation to reoxygenation, but not to hypoxia, was significantly inhibited by a scavenger of O2-. that prevents the formation of H2O2 (nitro blue tetrazolium), an inhibitor of NAD(P)H oxidases and other O2(-.)-generating flavoproteins (diphenyliodonium), and inhibition of the stimulation of soluble guanylate cyclase (LY-83583). A scavenger of O2-. that promotes H2O2 formation (Tiron) did not inhibit the PO2-elicited responses examined. Hypoxia and diphenyliodonium (but not Tiron) decreased the metabolism of endogenous H2O2 by catalase (as measured by the H2O2-dependent co-oxidation of methanol to formaldehyde by catalase), and reoxygenation caused a stimulation of H2O2 metabolism by catalase. The presence of endothelium resulted in minor modifications of the PO2 responses, which were partially mediated via prostaglandins and nitric oxide on the basis of the effects of indomethacin and nitro-L-arginine, respectively. These results suggest that in calf coronary arteries the stimulation of guanylate cyclase via H2O2 originating from NADH-derived O2-(.) production contributes to the transient relaxation to posthypoxic reoxygenation, but not the response to hypoxia.

Uncoupling of mitochondrial oxidative phosphorylation alters lipid peroxidation-derived free radical production but not recovery of postischemic rat hearts and post-hypoxic endothelial cells.

The contribution of mitochondrial free radical production towards the initiation of lipid peroxidation (LPO) and functional injury in the post-ischemic heart is unclear. Using the isolated rat heart model, the effects of the uncoupler of mitochondrial oxidative phosphorylation dinitrophenol (DNP, 50 microM final) on post-ischemic lipid peroxidation-derived free radical production and functional recovery were assessed. Hearts were subjected to 30 min total global ischemia followed by 15 min of reperfusion in the presence of DNP. As expected, DNP enhanced oxygen consumption before (11.3 +/- 0.9 mumol/min, p < 0.001) and during reperfusion (at 10 min: 7.9 +/- 0.7 mu umol/min), compared to the heart with control treatment (8.2 +/- 0.5 and 6.7 +/- 0.3, respectively). This effect was only associated with a higher incidence of ventricular tachycardia during reperfusion (80 vs. 50% for control treatment, p < 0.05). Electron spin resonance spectroscopy (ESR) and spin trapping with alpha-phenyl-tert-butylnitrone PBN-radical adducts (untreated: 6.4 +/- 1.0 nM, at 10 min) decreased in the presence of DNP (1.7 +/- 0.4 nM, p < 0.01). The radical concentration inversely correlated with myocardial oxygen consumption. Total liberation of free radical adducts during the initial 10 min of reperfusion was reduced by DNP (0.59 +/- 0.09 nmol, p < 0.01) compared to the respective control treatment (1.26 +/- 0.16 nmol). Similar effects, prevention of PBN adduct formation and unchanged viability in the presence of DNP, were obtained with endothelial cells during post-hypoxic reoxygenation. Since inhibition of mitochondrial phosphorylation can inhibit the formation of LPO-derived free radicals after an ischemic/hypoxic interval, mitochondria may represent an important source of free radicals capable of initiating lipid peroxidative injury during reperfusion/reoxygenation.

Effects of hypoxia, simulated ischemia and reoxygenation on the contractile function of human atrial trabeculae.

Hypoxia, ischemia and reoxygenation cause contractile dysfunction which will be characterized by the time course of isometric contraction of human atrial trabeculae. Post-rest potentiation (PRP) and postextrastimulatory potentiation (PEP) were elicited to obtain indirect information about the the role of the sarcoplasmic reticulum (SR) in excitation-contraction coupling. As lipid peroxidation could cause SR dysfunction, thiobarbituric acid reactive substances (TBARS) were measured. After 30 min of hypoxia (H) or simulated ischemia (H combined with acidosis-SI), contractile force decreased to 15% and 6%, respectively, of control (p < or = 0.05), whereas the normalized rate of both contraction and relaxation increased. In group H, rapid reoxygenation produced a recovery of contractile force to about 60%. After post-hypoxic reoxygenation the TBARS concentration was increased. In group SI, rapid reoxygenation and a rather gradual correction of acidosis produced complete recovery of contractile force. PRP and PEP were maintained during H and SI. Particularly post-ischemic reoxygenation caused a marked depression of PRP and partly of PEP. Thus, alteration of SR Ca2+ handling occurs predominantly during reoxygenation rather than during H or SI, probably associated with the damaging effect of increased oxygen radicals. The depression of potentiation occurred along with delayed relaxation, temporary increased resting force, mechanical alternans, and spontaneous activity which are further characteristics for SR dysfunction. Thus, for a possibly beneficial effect of low pH during SI combined with its gradual correction during reoxygenation on the recovery of contractile function, developed force should not be the only index.

Relationships of dopamine, cortical oxygen pressure, and hydroxyl radicals in brain of newborn piglets during hypoxia and posthypoxic recovery.

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 (FiO2) from 22% (control) to 7% for 1 h. The FiO2 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 FiO2 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Eicosanoid synthesis in cardiomyocytes: influence of hypoxia, reoxygenation, and polyunsaturated fatty acids

The synthesis of eicosanoids was investigated in cultured rat ventricular myocytes. Under normoxia, the cardiomyocytes released 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) and prostaglandin (PG) E2 and smaller amounts of PGF2 alpha and thromboxane B2. Hypoxia enhanced the production of PGE2 and PGF2 alpha, whereas the synthesis of 6-keto-PGF1 alpha was not affected. Conversely, posthypoxic reoxygenation greatly increased the synthesis of 6-keto-PGF1 alpha, whereas the synthesis of PGF2 alpha, was not affected and that of PGE2 was reduced. The cardiomyocyte polyunsaturated fatty acid (PUFA) profile was altered by arachidonic acid or eicosapentaenoic acid and docosahexaenoic acid. Under normoxia, the eicosanoid production appeared to be roughly related to the cell phospholipid arachidonic acid content. Conversely, during posthypoxic reoxygenation, the production of eicosanoids was related to the cell phospholipid n-3 PUFA content, with the n-3-rich cells displaying a marked inhibition of the synthesis. This inhibition was mainly attributed to eicosapentaenoic acid and/or docosapentaenoic acid. Whether this inhibition occurs in vivo during postischemic reperfusion, it may contribute to the beneficial effect of n-3 PUFA on the heart.

Lipophilic antioxidant U-18 and superoxide dismutase prevent cultured hippocampal neurons from destruction during hypoxia and in the posthypoxic period

The lipophilic antioxidant U-18 from the class of hindered phenols prevents the destruction of cultured hippocampal neurons during hypoxia and also in the posthypoxic reoxygenation period, apparently by being stably incorporated into their phospholipid membranes and by safeguarding these from free-radical damage in the course of reoxygenation. On the other hand, the protection afforded to the cultured hippocampal neurons by superoxide dismutase is probably due to its ability to interfere with the posthypoxic neuron-degrading processes mediated through hyperproduction of superoxide radicals in the neuronal cytoplasm.

Energetic status and viability of porcine kidney cells after hypoxic, hypothermic preservation in UW solution and subsequent reoxygenation: the influence of taurine

Taurine (2-aminoethane sulfonic acid) is a physiologic compound with osmoregulative properties and presumbly an inhibitor of lipid peroxidation. The present study evaluates the effects on the viability of isolated kidney cells of the addition of various doses of taurine to the UW preservation solution. Porcine tubular cell monolayers were incubated at 4°C for 9 h in UW solution under hypoxic conditions, and subsequently reoxygenated for 30 min in Hank's balanced salt solution equilibrated with room air. Taurine was added to the preservation solution at final concentrations of 0, 1, 10 ot 20 mmol/1. Cel viability was assessed by exclusion on Trypan blue and by determination of the energetic status reoxygenation. While tuarine had no influence on the post-hypoxic vaibility according to Trypan blue exclusion, a dose-related increases in cellular content of high energy phosphates after post-hypoxic reoxygenation was observed after addition of taurine to the preservation medium. It is concluded that taurine is able to reduce tissue alterations during hypoxia and reoxygenation and that the determination of post-hypoxic recovery of energy metabolism may be a more sensitive indicator of cell viability than the morphological Trypan blue exclusion test.

Some Biochemical Aspects of the Protective Effect of Trimetazidine on Rat Cardiomyocytes During Hypoxia and Reoxygenation

This study was undertaken to evaluate the direct cardioprotective effect of trimetazidine (TMZ), an anti-anginal drug devoid of haemodynamic action, on isolated myocytes. Cultured rat ventricular myocytes were treated with the drug 16 h and 1 h before the experiments. The drug-treated cells and control cells were placed in a substrate free medium and submitted in a specially designed device to either normoxia (N4), or hypoxia (150 min. H2.5, or 240 min, H4), or 150 min hypoxia followed by 90 min reoxygenation (HR). The treatment of the cells with TMZ (5 × 10-4 M) resulted in a significant decrease of lactate dehydrogenase (LDH) leakage (-58% in H2.5, -36% in H4 and -37% in HR). The LDH release provoked by oxidizing agents, H2O2 and 13-s-HpOTrE (13(S)-hydroperoxyoctadecatrienoic acid) during post-hypoxic reoxygenation was also lowered by TMZ. However, this effect reflected the beneficial action of TMZ during hypoxia since the drug was not efficient in altering the LDH leakage induced by the oxidizing agents in normal conditions. Moreover, the hypoxia-induced decreased of ATP content was not affected by TMZ, and resynthesis of ATP during substrate-free reoxygenation was similar in TMZ-treated and control cells. The respiration parameters have been studied in rat heart mitochondria isolated from control and TMZ-treated rats, in the presence or absence of TMZ in the respiration medium (10-4 M). The main result was a rapid and potent inhibition of palmitoylcarnitine oxidation, when TMZ was added to the respiration medium. The chronic treatment only resulted in a slight alternation of pyruvate oxidation. In conclusion, a pre-treatment of ventricular myocytes with TMZ resulted in an increased cell resistance to hypoxic stress, as evidenced by LDH leakage. This cytoprotective effect to TMZ should not be mediated through an antioxidant activity, but could be related to a modification of lipid metabolism.

Endogenous angiotensin II inhibits production of cerebrospinal fluid during posthypoxemic reoxygenation in the rabbit.

The choroid plexus, the major source of cerebrospinal fluid (CSF), contains receptors for angiotensin II and a very high concentration of angiotensin converting enzyme. Circulating angiotensin II decreases blood flow to the choroid plexus and the production of CSF. During recovery from hypoxia, marked increases in circulating angiotensin II have been described in some studies. We tested the hypothesis that CSF production decreases during posthypoxemic reoxygenation and examined related changes in plasma concentrations of angiotensin II. We also determined whether effect of posthypoxic reoxygenation on production of CSF is due to endogenous release of angiotensin II. We measured production of CSF in chloralose-anesthetized rabbits using ventriculocisternal perfusion of artificial CSF containing blue dextran. After control measurements, rabbits were subjected to one of the following interventions: (1) 30 minutes of hypoxia (PaO2 = 36 +/- 1 mmHg [mean +/- SE]) followed by 90 minutes of reoxygenation; (2) 30 minutes of hypoxia (PaO2 = 37 +/- 2 mmHg) followed by 90 minutes of reoxygenation in the presence of the angiotensin II antagonist saralasin; (3) hypoxia for 120 minutes (PaO2 = 35 +/- 1 mmHg); and (4) infusion of vehicle under normoxic conditions for 120 minutes (time control). Plasma concentrations of angiotensin II were also measured (radioimmunoassay) under control conditions, during hypoxia, and during posthypoxic reoxygenation (first intervention) and at corresponding time intervals in time control animals (fourth intervention). Under control conditions, the rate of production of CSF averaged 6.7 +/- 0.1 microL.min-1. During posthypoxemic reoxygenation, production of CSF decreased by 31 +/- 4% (P < .05). In the presence of sarlasin, CSF production did not change significantly during posthypoxemic reoxygenation (-12 +/- 6%, P > .05). In time control animals and during prolonged hypoxia, CSF production did not change significantly (-12 +/- 5% [P > .05] and 9 +/- 7% [P > .05], respectively). Plasma concentrations of angiotensin were below the threshold of sensitivity of the assay under control conditions and during interventions in animals that were made hypoxic and then reoxygenated and in time control animals. CSF production decreases during posthpoxemic reoxygenation. Since plasma concentrations of angiotensin II did not change during posthypoxic reoxygenation, this effect does not appear to be mediated by increases in circulating angiotensin II. We speculate that endogenous release of angiotensin II, perhaps in the choroid plexus epithelium, decreases production of CSF after hypoxic brain injury.