M Matejovicova

Membrane ion transport systems during oxidative stress in rodent brain: protective effect of stobadine and other antioxidants.

The effect of oxidative stress in vitro induced by radical generating systems (RGS) (Fe2+-EDTA and Fe2+-EDTA plus H2O2) on synaptosomal and microsomal ion transport systems as well as on the membrane fluidity was investigated. Oxidative insult reduced Na+, K+-ATPase activity by 50.7% and Na+-dependent Ca2+ uptake measured in choline media by 46.7%. Membrane fluidity was also significantly reduced as observed with the fluorescent probe. Stobadine (ST) prevented the decrease in membrane fluidity and Na+-dependent Ca2+ uptake, however Na+, K+-ATPase activity was only partially protected, indicating a more complex mechanism of inhibition. Incubation of microsomes with RGS led to the loss of ability of membranes to sequester Ca2+, as well as to the decrease of Ca2+-ATPase activity and to the increase of Ca2+ permeability to 125.1%. The relative potency of the two RGS to decrease membrane fluidity correlated well with the systems potencies to induce lipid peroxidation. The extent of protection against depression of Ca2+ uptake values and Ca2+-ATPase activity by membrane soluble antioxidants (U-74500A, U-83836E, t-butylated hydroxytoluene-BHT and ST) was dependent on the experimental conditions and on the dose and nature of antioxidant used. ST seems to be at least as affective as BHT and 21-aminosteroids, and more potent than tocopherol acetate. Water soluble glutathione had no significant effect on the RGS induced inhibition of Ca2+-ATPase activity. Combination of ST with glutathione enhanced ST antioxidant efficacy, so drug combination might be beneficial therapeutically.

Na/K-ATPase under Oxidative Stress: Molecular Mechanisms of Injury

Abstract1. The authors compare oxidative injury to brain and kidney Na/K-ATPase using in vitro and in vivo approaches. The substrate dependence of dog kidney Na/K-ATPase was examined both before and after partial hydrogen peroxide modification. A computer simulation model was used for calculating kinetic parameters.2. The substrate dependence curve for the unmodified endogenous enzyme displayed a typical curve with an intermediate plateau, adequately described by the sum of hyperbolic and sigmoidal components.3. The modified enzyme demonstrated a dependent curve that closely approximates normal hyperbola. The estimated ATP Km value for the endogenous enzyme was about 85 μM; the Kh was equal to 800 μM. The maximal number of protomers interacting was 8. Following oxidative modification, the enzyme substrate dependence curve did not show a significant change in the maximal protomer rate Vm, while the Km was increased slightly and interprotomer interaction was abolished.4. Na/K-ATPase from an ischemic gerbil brain showed a 22% decrease in specific activity. The maximal rate of ATP hydrolysis by an enzyme protomer changed slightly, but the sigmoidal component, characterizing the enzymes ability to form oligomers was abolished completely. The Km value was almost unchanged, but the Hill coefficient fell to 1. These data show that Na/K-ATPase molecules isolated from the ischemic brain have lost the ability to interact with one another.5. We suggest that the most important consequence of oxidative modification is Na/K-ATPase oligomeric structure formation and subsequent hydrolysis rate suppression.

Kinetic parameters of Na/K-ATPase modified by free radicals in vitro and in vivo.

NdK-ATPase is characterized by complex kinetic behavior reflected in abnormal substrate dependence and described as a curve with an intermediary plateau.’ This feature does not depend on the source of the enzyme studied,2 but is closely connected to interprotomer interaction of the enzyme and the modulating effect of ATP on the activity. SoIubilization of the enzyme into a monomeric state by nonionic detergents results in transformation of the complex substrate dependence curve into one resembling a hyperb~la.’.~ From these observations we concluded that ATP stimulates enzyme activity modifying interprotomer interactions within oligomeric ensembles of NdKATPase. NdK-ATPase is a key enzyme regulating ionic homeostasis of the cell. Unfavorable conditions such as oxidative stress accompanied by increased generation of reactive oxygen species result in inhibition of NdK-ATPase in V ~ V O . ~ The same result can be achieved using different oxidants in vitro.6 In our experiments we compared the kinetic properties of Na/K-ATPase after oxidative modification by hydrogen peroxide or hypochlorous anion in vitro and after experimental brain ischemia in vivo. Hydrogen peroxide inhibited NdK-ATPase very slowly; 5 mM H202 led to 25-30% inhibition after 30 minutes of preincubation. Hypochlorous anion provided the same inhibiting effect much faster and at as low a concentration as 5 pM. Experimental brain ishemia for 15 minutes in rats or gerbils was also accompanied by pronounced (24-28%) inhibition of brain NdK-ATPase. To elucidate the mechanism of inhibition, we compared the kinetic properties of highly purified membrane-bound enzyme2 before and after oxidative attack. Kinetic

CARNOSINE : AN ENDOGENOUS NEUROPROTECTOR IN THE ISCHEMIC BRAIN

Abstract1. The biological effects of carnosine, a natural hydrophilic neuropeptide, on the reactive oxygen species (ROS) pathological generation are reviewed.2. We describe direct antioxidant action observed in the in vitro experiments.3. Carnosine was found to effect metabolism indirectly. These effects are reflected in ROS turnover regulation and lipid peroxidation (LPO) processes.4. During brain ischemia carnosine acts as a neuroprotector, contributing to better cerebral blood flow restoration, electroencephalography (EEG) normalization, decreased lactate accumulation, and enyzmatic protection against ROS.5. The data presented demonstrate that carnosine is a specific regulator of essential metabolic pathways in neurons supporting brain homeostasis under unfavorable conditions.

Iron-induced inhibition of Na+, K(+)-ATPase and Na+/Ca2+ exchanger in synaptosomes: protection by the pyridoindole stobadine.

The effect of oxidative stress, induced by Fe2+-EDTA system, on Na+,K+-ATPase, Na+/Ca2+ exchanger and membrane fluidity of synaptosomes was investigated. Synaptosomes isolated from gerbil whole forebrain were incubated in the presence of 200 μM FeSO4-EDTA per mg of protein at 37°C for 30 min. The oxidative insult reduced Na+,K+-ATPase activity by 50.7 ± 5.0 % and Na+/Ca2+ exchanger activity measured in potassium and choline media by 47.1 ± 7.2 % and 46.7 ± 8.6 %, respectively. Membrane fluidity was also significantly reduced as observed with the 1,6-diphenyl-1,3,5-hexatriene probe. Stobadine, a pyridoindole derivative, prevented the decrease in membrane fluidity and in Na+/Ca2+ exchanger activity. The Na+,K+-ATPase activity was only partially protected by this lipid antioxidant, indicating a more complex mechanism of inhibition of this protein. The results of the present study suggest that the Na+/Ca2+ exchanger and the Na+,K+-ATPase are involved in oxidation stress-mediated disturbances of intracellular ion homeostasis and may contribute to cell injury.

Effect of free radical scavengers on myocardial function and Na+, K+-ATPase activity in stunned rabbit myocardium

Objectives. The role of reactive oxygen species (ROS) in the mechanism of myocardial stunning was investigated. Material and methods. Isolated Langendorff-perfused rabbit hearts were subjected to 15 min normothermic ischemia followed by 10 min reperfusion with Krebs-Henseleit solution±mannitol or histidine. Results. In hearts reperfused without free radical scavenger the left ventricular developed pressure as well as its maximal positive and negative first derivatives (+dP/dt, −dP/dt) was significantly depressed, whereas end diastolic pressure (LVEDP) increased when compared to preischemic values. Treatment with mannitol had little protective effects, whereas singlet oxygen scavenger histidine significantly improved the recovery of LVEDP and −dP/dt. Sarcolemmal Na+, K+-ATPase activity (control, 400±41 nmol Pi.min−1.mg−1) was depressed in untreated stunned hearts (260±27 nmol Pi.min−1.mg−1), but was almost completely recovered in hearts pretreated with histidine (364±27 nmol Pi.min−1.mg−1). The inhibition of Na+, K+-ATPase was only slightly prevented by mannitol (302±29 nmol Pi.min−1.mg−1l). Conclusions. The results suggest that ROS-induced inhibition of Na+, K+-ATPase activity is involved in the mechanism of postischemic contractile dysfunction and support the view that singlet oxygen may be one of the major causes of oxidative injury during ischemia and reperfusion.

Synaptosomal Na, K-ATPase during forebrain ischemia in Mongolian gerbils

We studied the activity and kinetic parameters of synaptosomal Na, K-ATPase during 15 min of forebrain ischemia and following 60 min of reperfusion produced by reversible common carotid occlusion in Mongolian gerbils. A synaptosomal fraction was obtained by both differential centrifugation of brain tissue homogenate and centrifugation of crude mitochondrial fraction at a discontinual sucrose density gradient. We found two components of ATP concentration dependence of ATP hydrolysis that represent two types of ATP-binding sites: high affinity and low affinity. Neither ischemia nor reperfusion affected kinetic parameters of a high-affinity site. However, low-affinity site parameters were affected by both ischemia and ischemia followed by reperfusion. Maximal velocity (Vmax) decreased by 43 and 42% after ischemia and after ischemia/reperfusion, respectively. The apparent Km for ATP decreased by 52% after ischemia and by 47% after ischemia/reperfusion. The apparent affinities for K+ and Na+ were determined from the ATP hydrolysis rate as a function of Na+ and K+ concentrations. We found the half-maximal activation constant for K+ (KaK+) increased by 60% after ischemia and by 146% after ischemia/reperfusion. On the other hand, we found that KaNa+ decreased significantly after ischemia/reperfusion (16%). We concluded that it is the dephosphorylation step of the ATPase reaction cycle that is primarily affected by both ischemia and ischemia/reperfusion. This might be caused by alteration of the protein molecule and/or its surroundings subsequent to ischemia.

Effect of myocardial stunning on thiol status, myofibrillar ATPase and troponin I proteolysis

To investigate the mechanism underlying postischemic contractile dysfunction (myocardial stunning) we examined myocardial sulfhydryl group content, myofibrillar Ca2+-dependent Mg2+-ATPase activity and protein profile after global ischemia and reperfusion. The Langerdorff-perfused rabbit hearts were subjected to 15 min normothermic ischemia followed by 10 min reperfusion and myofibrils were isolated from homogenates of left ventricular tissues. Depressed contractile function during reperfusion was accompanied by a decrease in total sulfhydryl group content. However, myofibrillar protein profile was unchanged and Western immunoblotting analysis showed no significant differences in troponin I immunoreactive bands between control and stunned hearts. Likewise, myofibrillar Mg2+-ATPase activity was unaltered after ischemia and reperfusion. We conclude that myocardial stunning is not caused by altered myofibrillar function and protein degradation but may be partly due to the oxidative modification of as yet undefined proteins.

Protein kinase C expression and subcellular distribution in chronic myocardial ischemia. Comparison of two different canine models

We studied protein kinase C (PKC) isozyme expression and activity distribution in two models of chronically ischemic canine myocardium: (1) single vessel obstruction (SVO), produced by tight stenosis of LAD followed by preconditioning and acute ischemia (40 min); (2) three vessel obstruction (3VO), produced by LAD-stenosis and gradual occlusion of right coronary artery and left circumflex. In both models after 8 weeks of chronic ischemia the dogs were either sacrificed or had PTCA of the LAD with a follow up of another 4 weeks. Control dogs were sham operated. PKC activity was measured in subcellular fractions of tissue samples from anterior and posterior regions in the presence of histone and γ-[32P]-ATP. PKC isozymes were detected by Western blotting. All regions perfused by the obstructed coronaries were dysfunctional at 8 weeks when compared to baseline, with improvement of anterior wall function after PTCA of LAD. PKC activity was elevated in the membrane fraction of SVO, but unchanged in the 3VO model. PKCs α, ε, and ζ prevailed in cytosol fraction of the controls (cytosol/membrane ratios were ± 3.34, 1.38 and 4.56 for α, ε and ζ, respectively), consistent with PKC activity distribution, while δ was not detected. There was no significant difference between the groups concerning the relative membrane amount of the isozymes. PKCs α and ε were decreased in the cytosol fraction of both models at 8 weeks (for anterior region, by 56 and 57% in SVO and by 49 and 46% in 3VO, respectively) without there being any differences between anterior and posterior regions, and were low also in the PTCA group. PKC ζ distribution however varied between the two models. The amount of PKC ζ isozyme was downregulated by 45% after 8 weeks of chronic ischemia and returned towards the control values after PTCA in the anterior region of SVO, while it did not change in anterior wall after 8 weeks in 3VO but was significantly decreased (by 47%) in posterior region after PTCA. In conclusion, our results suggest modified PKC signalling in chronically ischemic canine myocardium.