Viera Mézešová

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.

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 protects rats under global ischemia

Rat brain subjected to 45-min global ischemia is characterized by decreased activity of K-p-nitrophenyl phosphatase and monoamine oxidase B and a disordering of the membrane bilayer by reactive oxygen species attack, the latter being monitored by the fluorescence of the membrane fluorescent probe, 1-anilino, 8-naphtalene sulphonate (ANS). Ischemic injury resulted in 67% mortality of the animals. In the group of animals pre-treated with the neuropeptide carnosine the mortality was only 30%. At the same time, carnosine protected both the activity of the above-mentioned enzymes and the brain membrane disordering, which was also tested by ANS fluorescence. The conclusion was made that carnosine protects the brain against oxidative injury and thereby increases the survival of the animals.

Lipid peroxidation both inhibits Ca2 ‐ATPase and increases Ca2 permeability of endoplasmic reticulum membrane

Incubation of reticular membranes with Fe2+‐EDTA and H2O2 plus Fe2+‐EDTA at 37 °C for 30 min. led to the loss of membranes efficiency to sequester Ca2+ to 21.8 % and 3.6 % of control values, respectively. The incubation of microsomes with Fe2+‐EDTA and H2O2 plus Fe2+‐EDTA also caused decrease of Ca2+‐ATPase activity; to 44.9 % and 44.4 % (measured under the same conditions as Ca2+‐uptake) or to 79.6 % and 62.1 % (uncoupled from Ca2+ transport by detergent). In addition, incubation of membranes with Fe2+‐EDTA and H2O2 plus Fe2+‐EDTA at 37 °C for 30 min. led to the increase of Ca2+ permeability to 125.1 % and 124.2 %, respectively. Preincubation of membranes with membrane‐soluble antioxidants (U‐74500A, U‐83836E, t‐butyl hydroxytoluene and stobadine) protected the reticular membranes against depression of Ca2+ uptake values and Ca2+‐ATPase inhibition in a dose and an antioxidant nature dependent manner. Our results indicate that both processes, Ca2+‐ATPase inhibition and increase of endoplasmic reticulum membrane Ca2+ permeability, participate in the lipid peroxidation induced loss of membranes efficiency to sequester Ca2+.

Distribution of plasma membrane Ca2+ pump (PMCA) isoforms in the gerbil brain: effect of ischemia-reperfusion injury.

Non-species isoform-specific antibodies against three isoforms of the plasma membrane Ca2+ pump (PMCA) were used for immuno-localization of PMCA by Western blot analysis in membrane preparations isolated from different regions of gerbil brain. All three gene products were detected in the membranes from hippocampus, cerebral cortex and cerebellum. However, they showed a distinct distribution pattern. Two proteins were revealed in the case of PMCA1 with molecular masses 129 and 135 kDa. The antibody against PMCA2 recognized three proteins of about 130-137 kDa. Only one protein was detected with the anti-PMCA3 antibody. Levels of immuno-signal for the PMCA isoforms varied significantly among the different brain regions. The PMCA1 is the most abundant in the cerebro-cortical and hippocampal membrane preparations. The PMCA2 was detected in a lesser amount comparing to PMCA1 and was highest in the membrane preparations from cerebellum and in a slightly lesser amount from cerebral cortex. Anti-PMCA3 antibody stained weakly and was localized in the cerebellar and hippocampal membrane preparations. Transient forebrain ischemia (10 min) and reperfusion (for a prolonged period up to 10 d) leads to a significant decrease of PMCA immuno-signal. This decrease could be ascribed to the loss of PMCA1 signal, especially in hippocampal membrane preparations.

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.

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.

Fe2+-induced inhibition of gerbil forebrain microsomal Ca2+-ATPase: Effect of stobadine, glutathione and combination of both antioxidants

The incubation of the gerbil forebrain microsomes in the presence of ferrous sulphate and EDTA for either 30 min or for 60 min at a temperature of 37 degrees C led to the inhibition of Ca2+-ATPase in both a concentration- and time-dependent manner. The concentrations of Fe2+ which led to the inhibition of 50% of the Ca2+-ATPase activity (IC50-value) at these times were 0.59 mM and 0.07 mM, respectively. The preincubation of microsomes with 0.1 mM of stobadine prevented the inhibition of Ca2+-ATPase, however, the effectivity of prevention was dependent on the Fe2+ concentration. The net effect of stobadine was an increase in IC50-value to 0.76 mM. Unlike stobadine, reduced glutathione is a naturally occurring water soluble antioxidant. Glutathione at the concentration of 0.1 mM had no significant protective effect on the inhibition of Ca2+-ATPase. The protective effect of a stobadine-glutathione mixture was also investigated; 0.1 mM of stobadine in combination with 0.1 mM of glutathione was more potent in prevention of Fe2+-induced inhibition of Ca2+-ATPase than stobadine alone (IC50=1. 31 mM). In addition, we have investigated the effect of various stobadine-glutathione molar ratios (the total concentration of both antioxidants being 0.2 mM) on Fe2+-induced inhibition of Ca2+-ATPase. The results indicated that the best stobadine-glutathione ratio was close to 1 : 1. The effect of 0.04 mM stobadine in combination with 0.16 mM glutathione was comparable to the effect of 0.2 mM of stobadine alone, whereas 0.2 mM glutathione was almost ineffective. These results may suggest a possible role of membrane in Fe2+-induced inhibition of Ca2+-ATPase.

Ischemia-Reperfusion Decreases Protein Levels of InsP3 Receptor and PMCA but not Organellar Ca2+ Pump and Calreticulin in Gerbil Forebrain

Intracellular Ca2+ overload, excitotoxic activity of glutamate and concomitant generation of free radicals are thought to play the prominent role as triggering factors in the pathogenesis of brain ischemic-reperfusion injury (IRI) (1). While calcium acts as an important second messenger in the regulation of various neural activities (2), it also acts as an essential mediator of neuronal cell damage in IRI by stimulating of Ca2+ -dependent degradative processes and disaggregation of cytoskeletal components (3). Post-ischemic accumulation of Ca2+ is thought to be responsible for delayed neuronal death of selective regions of hippocampus, but it is not yet clear which sources of Ca2+ and which pathways are involved (4).

Transport mechanism of L-[14C]glutamate in cortical slices and synaptosomes of rabbits exposed to brain ischemia and reperfusion

Changes in the functioning of the glutamatergic system in rabbit brain were studied after partial brain ischemia and reperfusion. In vitro studies were conducted relating to the release of L-[14C]glutamate from cortical brain slices, L-[14C]glutamate uptake in synaptosomes, and 45Ca uptake in synaptosomes. It was found that basal release of L-[14C]glutamate from rabbit brain cortical slices after 30 min of partial ischemia and 1 d of reperfusion was essentially without change compared to the control values. After 3 d of reperfusion, there was an increase in basal release of L-[14C]glutamate from rabbit brain cortical slices. K+ stimulated release of L-[14C]glutamate in normal Krebs-Ringer medium was essentially the same in the control group and in the experimental group after 30 min of ischemia. The K+ stimulated release of L-[14C]glutamate independent of calcium was increased to 145% after 30 min of ischemia and 1 d of reperfusion. The decreased Km value at the glutamate transporter may have contributed to this difference. Kinetic parameters of the L-[14C]glutamate uptake (Km and Vmax) in synaptosomes from rabbit brain were significantly lower after 30 min of ischemia. The authors discovered that during the reperfusion period, Vmax was almost the same as in the control group. The activity of the Na+/Ca2+ exchanger in synaptosomes of rat brain was about 70% of the control values after 30 min of ischemia and 72 h of reperfusion. According to our results, increased L-[14C]glutamate release after 30 min of ischemia appears to be the result of higher intracellular calcium concentration and possibly also of a higher uptake of glutamate.