Lipid free radical generation and brain cell membrane alteration following nitric oxide synthase inhibition during cerebral hypoxia in the newborn piglet

Nitric oxide (NO) is reported to cause neuronal damage through various mechanisms. The present study tests the hypothesis that NO synthase inhibition by N(omega)-nitro-L-arginine (NNLA) will result in decreased oxygen-derived free radical production leading to the preservation of cell membrane structure and function during cerebral hypoxia. Ten newborn piglets were pretreated with NNLA (40 mg/kg); five were subjected to hypoxia, whereas the other five were maintained with normoxia. An additional 10 piglets without NNLA treatment underwent the same conditions. Hypoxia was induced with a lowered FiO2 and documented biochemically by decreased cerebral ATP and phosphocreatine levels. Free radicals were detected by using electron spin resonance spectroscopy with a spin trapping technique. Results demonstrated that free radicals, corresponding to alkoxyl radicals, were induced by hypoxia but were inhibited by pretreatment with NNLA before inducing hypoxia. NNLA also inhibited hypoxia-induced generation of conjugated dienes, products of lipid peroxidation. Na+,K+-ATPase activity, an index of cellular membrane function, decreased following hypoxia but was preserved by pretreatment with NNLA. These data demonstrate that during hypoxia NO generates free radicals via peroxynitrite production, presumably causing lipid peroxidation and membrane dysfunction. These results suggest that NO is a potentially limiting factor in the peroxynitrite-mediated lipid peroxidation resulting in membrane injury.     

Oxygen free radical generation during in-utero hypoxia in the fetal guinea pig brain: the effects of maturity and of magnesium sulfate administration

Previous studies have shown, employing direct measurements with electron spin resonance (ESR) spectroscopy, that hypoxia induces an increased production of oxygen free radicals (OFR) in the brain of the guinea pig fetus. The present study using the same approach, investigated the effects of maturity and Mg2+-pretreatment on hypoxia-induced OFR formation in the guinea pig fetal brain. The normoxic and the hypoxic groups were exposed for 60 min to 21% or 7% oxygen, respectively. The control group consisted of term fetuses exposed to normoxia (n=7) and hypoxia (n=7). The experimental groups consisted of the following: (a) for the investigation on maturity effect, preterm fetuses (40 days) exposed to normoxia (n=6) or hypoxia (n=6); and (b) for the Mg2+-pretreatment investigation, term fetuses (60 days) exposed to normoxia (n=6) or hypoxia (n=6) following maternal pretreatment with Mg2+ which consisted of an initial bolus of MgSO4 (600 mg/kg, i.p.) 1 h prior to hypoxia followed by a second dose (300 mg/kg, i.p.). Oxygen free radicals were measured by ESR spectroscopy in the fetal cerebral cortical tissue utilizing phenyl-N-tert-butylnitrone (PBN) spin trapping. Fetal brain tissue hypoxia was documented biochemically by decreased tissue levels of ATP and phosphocreatine. In the control group of term fetuses, the cortical tissue from hypoxic fetuses showed a significant increase in spin adducts (71% increase, p<0.01). In the preterm group, the cortical tissue from hypoxic fetuses showed a 33% increase in spin adducts (p<0.001). The baseline free radical generation during normoxia was 22.5% higher at preterm than at term (41.4+/-3.5 units/g issue vs. 33.8+/-9.3 units/g tissue, p<0.05). In Mg2+-treated groups, spin adduct levels in cortical tissue from hypoxic fetuses did not significantly differ from those of the normoxic group (30.2+/-9.9 units/g tissue, normoxic-Mg2+ vs. 30. 6+/-8.1 units/g tissue, hypoxic-Mg2+). The results indicate that the fetal brain at term may be more susceptible to hypoxia-induced free radical damage than at preterm and that Mg2+ administration significantly decreased the hypoxia-induced increase in oxygen free radical generation in the term fetal guinea pig brain in comparison with non-treated hypoxic group.     

Effect of propentofylline on free radical generation during cerebral hypoxia in the newborn piglet

The present study tests the hypothesis that propentofylline, an adenosine re-uptake inhibitor, will reduce free radical generation during cerebral hypoxia. Ten newborn piglets were pretreated with propentofylline (10 mg/kg), five of which were subjected to hypoxia, while the other five were maintained at normoxia. Five untreated control piglets underwent the same conditions. Hypoxia was induced through a decrease in FiO2 to 0.11 and documented biochemically by a decrease in ATP and phosphocreatine levels. Free radical formation in the cortex was detected directly using electron spin resonance spectroscopy with a spin trap technique. Results demonstrate that free radicals, corresponding to the alkoxyl radical, increased significantly following hypoxia, and that this increase was inhibited by pretreatment with propentofylline. Conjugated dienes, a lipid peroxidation product, also increased following hypoxia and were subsequently inhibited by propentofylline. The administration of propentofylline also significantly limited the hypoxia-induced decrease in tissue levels of ATP and phosphocreatine. These data demonstrate that pretreatment with propentofylline decreased free radical generation and lipid peroxidation as well as preserved high energy phosphates during cerebral hypoxia.     

Effect of hyperglycemia on brain cell membrane function and energy metabolism during hypoxia-ischemia in newborn piglets

The purpose of this study was to test the hypothesis that hyperglycemia ameliorates changes in brain cell membrane function and preserves cerebral high energy phosphates during hypoxia-ischemia in newborn piglets. A total of 42 ventilated piglets were divided into 4 groups, normoglycemic/normoxic(group 1, n=9), hyperglycemic/normoxic(group 2, n=8), normoglycemic/hypoxic-ischemic(group 3, n=13) and hyperglycemic/hypoxic-ischemic(group 4, n=12) group. Cerebral hypoxia-ischemia was induced by occlusion of bilateral common carotid arteries and simultaneous breathing with 8% oxygen for 30 min. Hyperglycemia (blood glucose 350-400 mg/dl) was maintained for 90 min before and throughout hypoxia-ischemia using modified glucose clamp technique. Changes in cytochrome aa3 were continuously monitored using near infrared spectroscopy. Blood and CSF glucose and lactate were monitored. Na+, K+-ATPase activity, lipid peroxidation products (conjugated dienes), tissue high energy phosphates (ATP and phosphocreatine) levels and brain glucose and lactate levels were determined biochemically in the cerebral cortex. During hypoxia-ischemia, glucose levels in blood and CSF were significantly elevated in hyperglycemic/hypoxic-ischemic group compared with normoglycemic/hypoxic-ischemic group, but lactate levels in blood and CSF were not different between two groups. At the end of hypoxia-ischemia of group 3 and 4, triangle up Cyt aa3, Na+, K+-ATPase activity, ATP and phosphocreatine values in brain were significantly decreased compared with normoxic groups 1 and 2, but were not different between groups 3 and 4. Levels of conjugated dienes and brain lactate were significantly increased in groups 3 and 4 compared with groups 1 and 2, and were significantly elevated in group 4 than in group 3 (0.30+/-0.11 vs. 0.09+/-0.02 micromol g-1 protein, 26.4+/-7.6 vs. 13.1+/-2.6 mmol kg-1, p<0.05). These findings suggest that hyperglycemia does not reduce the changes in brain cell membrane function and does not preserve cerebral high energy phosphates during hypoxia-ischemia in newborn piglets. We speculate that hyperglycemia may be harmful during hypoxia-ischemia due to increased levels of lipid peroxidation in newborn piglet.     

The antioxidant action of taurine in acute hypoxic hypoxia

It was found that preliminary treatment by amino acid taurine protected rats from lipid peroxidation intensification (expressed in terms of malondialdehyde and conjugated dienes contents) in the liver, brain and heart under acute severe normobaric hypoxic hypoxia. The mechanisms of the antioxidant action of taurine are connected to the prevention of lactate accumulation in tissues and cell membrane structure disorders (expressed in a decrease of membrane Na+, K(+)-ATPase activity). It was also shown that taurine reduced significantly a decrease of glutathione antioxidant system activity protecting tissues against reduced glutathione pool depletion and preventing a decrease of glutathione reductase and glutathione peroxidase activities in acute severe hypoxia.     

Modulation of mouse cerebral Na+,K(+)-ATPase activity by oxygen free radicals

There is increasing evidence that oxygen free radicals (OFR) are involved in cerebral ischaemia-reperfusion injury, possibly via a modulation of Na+,K(+)-ATPase activity, one of the major membrane pumps responsible for ionic homeostasis. We measured OFR-mediated modulation of this enzymatic activity and examined the roles of lipid and/or protein alterations. Using mouse brain microsomes exposed to UV-C irradiation, our results show a good correlation between activity inhibition and lipoperoxidation estimated by PUFA loss as well as malondialdehyde production. The protective effect of thiourea (OH scavenger) and the lack of effect noted with DTT (thiol protector) suggest that the functionality of the Na+,K(+)-ATPase is altered by perturbation of membrane integrity rather than by a structural alteration of the protein itself.     

The role of sports training in the efficiency of oxygen regimens in children in the North]

The comparative study of the sensitivity of Na+, K(+)-ATPase isozymes from cerebral cortex to ascorbate-dependent membrane peroxidation was conducted. With highly inactivated Na+, K(+)-ATPase the degree of inactivation of the SH-dependent ouabain-sensitive forms alpha+ (alpha 2 and alpha 3) is higher than glycoside-resistant isoform alpha 1. The process is accompanied by simultaneous lipid peroxidation and decrease of SH-groups amount in enzyme preparations. The combined nature of the oxidative Na+, K(+)-ATPase inactivation, accompanied by the direct oxidation of enzyme SH-groups and modification of lipid environment is supposed.     

8%-9% and 12%-13% hypoxic gas induced free radicals generation in rat's left and right myocardium

Alpha-phenyl-tert-butylnitrone (PBN) was administered intravenously to capture free radicals of rat's myocardium. Rats were exposed to hypoxic gas (8%-9% O2 in N2) for 15 min. The ESR (electron spin resonance) signal intensity of PBN-spin adduct in the left myocardium increased significantly as compared with the normoxia group (n = 5, P < 0.05), but in the right myocardium there was no significant changes between hypoxia and normoxia. Rats exposed to hypoxic gas (12%-13% O2 in N2) were divided into four groups: I (hypoxia for 15 min), II (hypoxia for 60 min), III (hypoxia for 30 min/normoxia for 15 min/hypoxia for 30 min) and IV (injected MPEG- SOD intravenously before hypoxia for 60 min). The ESR signal intensity of PBN-spin adduct of left and right myocardium in group II increased significantly as compared with normoxia group (n = 5, P < 0.01), but the ESR signal intensity of group I didn't show obvious change as compared with normoxia group (n = 5, P > 0.05). In the right myocardium of group III the ESR signal intensity of PBN-spin adduct decreased significantly as compared with group II (n = 5, P < 0.05) and in the left myocardium did not decrease evidently. In the left myocardium of group IV the ESR signal intensity of PBN-spin adduct decreased evidently as compared with group II (n = 5, P < 0.05) and that in the right myocardium did not decrease evidently. When the rats were exposed to 8%-9% hypoxic gas for 15 min and 12%-13% hypoxic gas for 60 min, the SOD (superoxide dismutase, EC 1.11.1.9) activity of myocardium decreased and the content of MDA (malondialdehyde) increased significantly (n = 8, P < 0.05 or P < 0.01). The above results suggested that one way of myocardium free radical gereration may be relevant to decrease of SOD activity. The generation of free radicals pertained chiefly to superoxide free radical in the left myocardium and the membrane structure of myocardium cells might have been damaged largely during hypoxia.     

Basic FGF attenuates amyloid beta-peptide-induced oxidative stress, mitochondrial dysfunction, and impairment of Na+/K+-ATPase activity in hippocampal neurons

Basic fibroblast growth factor (bFGF) exhibits trophic activity for many populations of neurons in the brain, and can protect those neurons against excitotoxic, metabolic and oxidative insults. In Alzheimer's disease (AD), amyloid beta-peptide (A beta) fibrils accumulate in plaques which are associated with degenerating neurons. A beta can be neurotoxic by a mechanism that appears to involve induction of oxidative stress and disruption of calcium homeostasis. Plaques in AD brain contain high levels of bFGF suggesting a possible modulatory role for bFGF in the neurodegenerative process. We now report that bFGF can protect cultured hippocampal neurons against A beta25-35 toxicity by a mechanism that involves suppression of reactive oxygen species (ROS) accumulation and maintenance of Na+/K+-ATPase activity. A beta25-35 induced lipid peroxidation, accumulation of H2O2, mitochondrial ROS accumulation, and a decrease in mitochondrial transmembrane potential; each of these effects of A beta25-35 was abrogated in cultures pre-treated with bFGF. Na+/K+-ATPase activity was significantly reduced following exposure to A beta25-35 in control cultures, but not in cultures pre-treated with bFGF. bFGF did not protect neurons from death induced by ouabain (a specific inhibitor of the Na+/K+-ATPase) or 4-hydroxynonenal (an aldehydic product of lipid peroxidation) consistent with a site of action of bFGF prior to induction of oxidative stress and impairment of ion-motive ATPases. By suppressing accumulation of oxyradicals, bFGF may slow A beta-induced neurodegenerative cascades.     

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.     

Inhibition of membrane lipid peroxidation by a radical scavenging mechanism: a novel function for hydroxyl-containing ionophores

In the present study we show that K+/H+ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe2+-citrate and 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-induced lipid peroxidation in intact rat liver mitochondria and in egg phosphatidylcholine (PC) liposomes containing negatively charged lipids--dicetyl phosphate (DCP) or cardiolipin (CL)--and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-containing ionophore with higher affinity for Na+ than for K+, promotes a similar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-insensitive O2 consumption, formation of thiobarbituric acid-reactive substances (TBARS), conjugated dienes, and electron paramagnetic resonance (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid peroxidation induced by both inductor systems in liposomes. Nitroxide destruction is inhibited by butylated hydroxytoluene, a known antioxidant, and by the hydroxyl-containing ionophores. In contrast, valinomycin (VAL), which does not possess alcoholic groups, does not display this protective effect. Effective order parameters (Seff), determined from the spectra of an incorporated spin label are larger in the presence of salt and display a small increase upon addition of the ionophores, as a result of the increase of counter ion concentration at the negatively charged bilayer surface. This condition leads to increased formation of the ion-ionophore complex, the membrane binding (uncharged) species. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution (in the absence of membranes) showed that NIG and LAS, but not VAL, decrease the Fe2+-citrate-induced production of radicals derived from piperazine-based buffers, demonstrating their property as radical scavengers. Both Fe2+-citrate and ABAP promote a much more pronounced decrease of LAS fluorescence in PC/CL liposomes than in dimyristoyl phosphatidylcholine (DMPC, saturated phospholipid)-DCP liposomes, indicating that the ionophore also scavenges lipid peroxyl radicals. A slow decrease of fluorescence is observed in the latter system, for all lipid compositions in sucrose medium, and in the absence of membranes, indicating that the primary radicals stemming from both inductors also attack the ionophore. Altogether, the data lead to the conclusion that the membrane-incorporated cation complexes of NIG, LAS and MON inhibit lipid peroxidation by blocking initiation and propagation reactions in the lipid phase via a free radical scavenging mechanism, very likely due to the presence of alcoholic hydroxyl groups in all three molecules and to the attack of the aromatic moiety of LAS.     

Changes in cytosol Ca(2+)-, Mg(2+)-, H(+)-, N(+)-, and K(+)-concentrations in cultivated hepatocytes in hypoxic conditions

AIM: It is assumed that disturbances of cellular ion homeostasis, especially an increase in the cytosolic Ca2+ concentration, are of decisive importance for hypoxic cell injury. The aim of this study is the determination of alterations in the cytosolic Ca2+, Mg2+, H+, Na+ and K+ concentration in cultured hepatocytes during hypoxia. METHODS: The alterations of ion homeostasis under hypoxic conditions were studied in primary cultures of isolated rat hepatocytes by using fluorescence microscopy. RESULTS: The measurements of cytosolic Ca2+ concentration showed no alterations during the first 3-4 h of hypoxia. About 1-2 h before cell injury became evident Ca2+ increased from 147 +/- 28 to 385 +/- 31 nM. Similarly the cytosolic Mg2+ concentration increased from 0.63 +/- 0.05 to 1.42 +/- 0.11 mM in a late stage of hypoxia. In contrast, the cytosolic Na+ concentration increased continuously from 16 +/- 2 mM at start to 76 +/- 9 mM after 5 h of hypoxic conditions. The cytosolic K+ concentration remained constant for 2 h (129 +/- 7 mM) but then decreased down to 31 +/- 18 mM. The intracellular H+ concentration increased slightly under hypoxic conditions, the pH decreased from 7.35 +/- 0.02 to 7.19 +/- 0.04. CONCLUSION: The results indicate that cytosolic Ca2+ plays only a minor role in the pathomechanism of hypoxic hepatocellular injury but suggest an important role of the cytosolic Na+ concentration in this process.     

A role for 4-hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion homeostasis and neuronal death induced by amyloid beta-peptide

Peroxidation of membrane lipids results in release of the aldehyde 4-hydroxynonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. Because accumulating data indicate that free radicals mediate injury and death of neurons in Alzheimer's disease (AD) and because amyloid beta-peptide (A beta) can promote free radical production, we tested the hypothesis that HNE mediates A beta 25-35-induced disruption of neuronal ion homeostasis and cell death. A beta induced large increases in levels of free and protein-bound HNE in cultured hippocampal cells. HNE was neurotoxic in a time- and concentration-dependent manner, and this toxicity was specific in that other aldehydic lipid peroxidation products were not neurotoxic. HNE impaired Na+, K(+)-ATPase activity and induced an increase of neuronal intracellular free Ca2+ concentration. HNE increased neuronal vulnerability to glutamate toxicity, and HNE toxicity was partially attenuated by NMDA receptor antagonists, suggesting an excitotoxic component to HNE neurotoxicity. Glutathione, which was previously shown to play a key role in HNE metabolism in nonneuronal cells, attenuated the neurotoxicities of both A beta and HNE. The antioxidant propyl gallate protected neurons against A beta toxicity but was less effective in protecting against HNE toxicity. Collectively, the data suggest that HNE mediates A beta-induced oxidative damage to neuronal membrane proteins, which, in turn, leads to disruption of ion homeostasis and cell degeneration.     

Oxidative stress following traumatic brain injury in rats

BACKGROUND: Free radicals may be involved in the pathophysiology of traumatic brain injury (TBI) through oxidative damage of neurovascular structures. Endogenous antioxidants, such as ascorbate and alpha-tocopherol, may play a critical role in combating these oxidative reactions and their oxidized products can serve as an important index of oxidative stress. METHODS: We used electron spin resonance (ESR) spectroscopy and in vivo spin trapping (reaction of an organic compound with free radical species) to detect the possible generation of free radicals after TBI. Injury was inflicted by a weight drop technique over the head (5.7 kg-cm). Rats were intravenously infused with either 1 mL, 0.1 M of the spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN), or an equivalent volume of saline immediately before TBI or sham-injury. Animals were divided into four groups: (1) Group I: PBN-infused sham-injured, (2) Group II: PBN-infused injured, (3) Group III: saline-infused sham-injured, and (4) Group IV: saline-infused injured. Additional groups of saline-infused uninjured, saline-infused, and PBN-infused injured animals were used for histopathology. Sixty minutes after TBI or sham-injury, rats were again anesthetized and decapitated. The brains were removed within 1 minute, homogenized, and extracted for lipids. The extracts were analyzed by ESR spectroscopy. Brain ascorbic acid (AA) concentration was determined spectrophotometrically, using the ascorbate oxidase assay. RESULTS: No PBN spin adduct signals (indicating trapped free radical species) were visible 60 minutes after TBI. All groups of rats showed an ascorbyl free radical signal. The ascorbyl signal intensity (AI) was, however, significantly higher in the injured rats, while the brain (AA) was significantly reduced. In addition, the ratio of AI/AA, which eliminates the effect of variable ascorbate concentrations in the brain, was also significantly higher in the injured animals. CONCLUSIONS: We conclude that 60 minutes following TBI there was a significantly increased level of oxidative stress in the brain. This may reflect formation of free radical species with subsequent interaction with ascorbate (antioxidant) during the 60 minute period. The lack of PBN spin adduct signals 1 hour after TBI may indicate that free radical generation is time dependent and might be detectable earlier or later than the 60 minute period.     

Charge translocation by the Na+/K+-ATPase investigated on solid supported membranes: cytoplasmic cation binding and release

In the preceding publication (. Biophys. J. 76:000-000) a new technique was described that was able to produce concentration jumps of arbitrary ion species at the surface of a solid supported membrane (SSM). This technique can be used to investigate the kinetics of ion translocating proteins adsorbed to the SSM. Charge translocation of the Na+/K+-ATPase in the presence of ATP was investigated. Here we describe experiments carried out with membrane fragments containing Na+/K+-ATPase from pig kidney and in the absence of ATP. Electrical currents are measured after rapid addition of Na+. We demonstrate that these currents can be explained only by a cation binding process on the cytoplasmic side, most probably to the cytoplasmic cation binding site of the Na+/K+-ATPase. An electrogenic reaction of the protein was observed only with Na+, but not with other monovalent cations (K+, Li+, Rb+, Cs+). Using Na+ activation of the enzyme after preincubation with K+ we also investigated the K+-dependent half-cycle of the Na+/K+-ATPase. A rate constant for K+ translocation in the absence of ATP of 0.2-0.3 s-1 was determined. In addition, these experiments show that K+ deocclusion, and cytoplasmic K+ release are electroneutral.     

Characterization of the radical trapping activity of a novel series of cyclic nitrone spin traps

alpha-Phenyl-tert-butyl nitrone (PBN) is a nitrone spin trap, which has shown efficacy in animal models of oxidative stress, including stroke, aging, sepsis, and myocardial ischemia/reperfusion injury. We have prepared a series of novel cyclic variants of PBN and evaluated them for radical trapping activity in vitro. Specifically, their ability to inhibit iron-induced lipid peroxidation in liposomes was assessed, as well as superoxide anion (O2(-.)) and hydroxyl radical ((.)OH) trapping activity as determined biochemically and using electron spin resonance (ESR) spectroscopy. All cyclic nitrones tested were much more potent as inhibitors of lipid peroxidation than was PBN. The unsubstituted cyclic variant MDL 101,002 was approximately 8-fold more potent than PBN. An analysis of the analogs of MDL 101,002 revealed a direct correlation of activity with lipophilicity. However, lipophilicity does not solely account for the difference between MDL 101,002 and PBN, inasmuch as the calculated octanol/water partition coefficient for MDL 101,002 is 1.01 as compared to 1.23 for PBN. This indicated the cyclic nitrones are inherently more effective radical traps than PBN in a membrane system. The most active compound was a dichloro analog in the seven-membered ring series (MDL 104,342), which had an IC50 of 26 mum, which was 550-fold better than that of PBN. The cyclic nitrones were shown to trap (.)OH with MDL 101,002 being 20 25 times more active than PBN as assessed using 2-deoxyribose and p-nitrosodimethylaniline as substrates, respectively. Trapping of (.)OH by MDL 101,002 was also examined by using ESR spectroscopy. When Fenton's reagent was used, the (.)OH adduct of MDL 101,002 yielded a six-line spectrum with hyperfine coupling constants distinct from that of PBN. Importantly, the half-life of the adduct was nearly 5 min, while that of PBN is less than 1 min at physiologic pH. MDL 101,002 also trapped the O2(-.) radical to yield a six-line spectrum with coupling constants very distinct from that of the (.)OH adduct. In mice, the cyclic nitrones ameliorated the damaging effects of oxidative stress induced by ferrous iron injection into brain tissue. Similar protection was not afforded by the lipid peroxidation inhibitor U74006F, thus implicating radical trapping as a unique feature in the prevention of cell injury. Together, the in vivo activity, the stability of the nitroxide adducts, and the ability to distinguish between trapping of (.)OH and O2(-.) suggest the cyclic nitrones to be ideal reagents for the study of oxidative cell injury.     

A hydroxylated analog of the beta-adrenoceptor antagonist, carvedilol, affords exceptional antioxidant protection to postischemic rat hearts

The antioxidant and cardioprotective effects of the beta-adrenoceptor antagonist, carvedilol, and its hydroxylated analog. BM-910228, were compared using the postischemic rat heart model. Hearts were infused with either agent (0.01, 0.10, or 10 nM final, or drug-free infusate) for 10 min prior to 30 min global ischemia, and also during the initial 15 min of reperfusion. Recovery of postischemic hemodynamic parameters (left ventricular systolic and developed pressures, mean diastolic pressure, cardiac output, coronary flow rate, and cardiac pressure-volume work), and the extent of postischemic tissue lactate dehydrogenase (LDH) loss, lipid hydroperoxide (LOOH) formation, and lipid peroxidation (LPO)-derived free radical production were assessed and compared among the treatment groups. The depressive pharmacological properties (beta- and alpha-blockade) of both agents masked the extent of postischemic hemodynamic recovery, except at the lowest dose (10 pM) of the analog, which provided significant improvements in systolic and developed pressures, and cardiac work. Treatment with both agents provided significant dose-dependent reductions in postischemic LOOH formation and lipid alkoxyl radical production, as determined by electron spin resonance spectroscopy and alpha-phenyl-tert-butylnitrone. (PBN) spin trapping (PBN/alkoxyl adduct hyperfine splitting alpha N = 13.63 G and alpha H = 1.93 G). Although both agents reduced oxidative injury, the hydroxylated analog was clearly the superior antioxidant (equipotent at doses two to three orders of magnitude lower) compared to the parent compound. This was also reflected with respect to three orders of magnitude lower) compared to the parent compound. This was also reflected with respect to drug-mediated improvement in myocardial preservation (reduced LDH release), which paralleled the antioxidant protective effects. Because neither agent displayed significant primary radical scavenging ability at doses (< or = 10 nM), which did provide substantial inhibition of postischemic LOOH and alkoxyl formation, our data suggest that the antioxidant properties of carvedilol and its analog are mediated primarily through a LPO chair-breaking mechanism. Moreover, the significant antioxidant protection afforded by the analog BM-910228 at subnanomolar levels places this agent into an exclusive category reserved for exceptionally potent antioxidants.     

Functional activity of Na+,K(+)-pump in normal and pathological tissues

Na+,K(+)-ATPase, supporting the ionic homeostasis of the cell, is under control of Na+, K+, Mg2+, and ATP. The regulating effect of Mg2+ is rather unclear, whereas the Na+/K+ ratio in the cytoplasm is a potent regulatory factor, especially for osmotic balance in excitable cells. We have demonstrated two possibilities for regulation of ion pumping activity: First, via the number of Na+,K(+)-ATPase molecules under operation, and second, via changes in the turnover rate of the active molecules. In the presence of low ATP concentration, which is typical for cells with membrane damage (ischemic cardiac myocytes, tumor cells, fatigued muscles) Na+,K(+)-ATPase is transformed to a regime of the decreased efficiency. Radiation inactivation study demonstrates the weakening of the interprotein interactions in the enzyme complexes during ATP deficiency. Thus, measurements of ATPase activity of the purified enzyme under optimal conditions in vitro may be useless for the discrimination of pathological from normal tissues. In such a case, the estimation of lipid composition and microviscosity of the membranes under study could be important. This review briefly discusses several basic mechanisms of the regulation of Na+,K(+)-ATPase--an integral protein of the outer cell membranes.     

Detection of free radicals in blood by electron spin resonance in a model of respiratory failure in the rat

Previous work has suggested that a free radical mechanism is involved in some types of muscle fatigue and that there can be free radicals released extracellularly. Because muscle fatigue may be an important factor in respiratory failure, the authors tested the hypothesis that increased concentrations of free radicals could be detected in the blood of animals undergoing severe resistive loading to respiratory failure. An ex vivo spin trapping technique with alpha-phenyl-N-tert-butylnitrone (PBN) was used to investigate the possible formation of free radicals in systemic blood samples by electron spin resonance (ESR) spectrometry. After 2.5-3 h of severe inspiratory resistive loading with 70% supplemental inspired oxygen, free radical levels in the form of PBN-adducts were found to rise significantly over the control group breathing room air and the control group breathing 70% oxygen (p < 0.05, N = 8). There were no significant differences between control groups breathing room air and control groups breathing 70% oxygen. This study presents direct evidence that free radicals are produced ex vivo and that they can be detected in the systemic circulation due to excessive resistive loading of the respiratory muscles.