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.

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 system's 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.

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.

[The effect of ischemia and ischemia-reperfusion on ion transport systems]. / Vplyv ischémie a ischémie-reperfúzie na iónové transportné systémy.

Interruption of cerebral blood flow leads to dissipation of ionic gradients as the consequence of ionic channel overstimulation and ionic pump failure. The aim of this work was to study the possible effects of ischaemia and ischaemia followed by reperfusion on biochemical properties of endoplasmic calcium pump and synaptosomal sodium pump and sodium/calcium exchanger. The results presented in this study showed that 15 minute ischaemia led to the inhibition of all three ionic transport systems, however in different degrees. 60 minute reperfusion following 15 minute ischaemia led to partial recovery of calcium pump and sodium/calcium exchanger. The activity of sodium pump was still significantly depressed. Ischaemia and ischemia followed by reperfusion did not affect kinetic parameters of calcium pump. On the other side, both ischaemia and ischaemia-reperfusion led to an increase of sodium pump affinity to ATP and a decrease of the enzyme affinity to potassium. The possible causes of the changes, as the alteration of membrane structure or altered enzymes phosphorylation are discussed in the study. In addition to the inhibitory effect of ischaemia-reperfusion injury, intracellular water accumulation, as the possible consequence of altered ion homeostasis, is documented by nuclear magnetic resonance (imaging).

[Neurochemical changes associated with ischemic-reperfusion injury of the CNS]. / Neurochemické zmeny spojené s ischemicko-reperfúznym poskodením CNS.

Ischaemia-reperfusion injury of the central nervous system is the third leading cause of death in the European countries. Since nerve cells are exclusively dependent on glucose oxidation, reduction of the glucose and oxygen delivery to the neurons affect all cellular metabolic pathways. Decrease of ATP production and its concentration as well as activation of ionic gradients, seems to be a primary process. Several degrading cellular enzymes, as lipases, phospholipases, proteases and endonucleases, are activated, in contrast to the severe inhibition of protein and phospholipid synthesis. Signal transduction pathways, playing a role in the regulation of cellular metabolism, are depressed due to the energy deprivation. After certain time of ischaemia, reperfusion does not lead to restoration of normal cell metabolism and intracellular situation could be even worsen, depending on the neuron type and duration of ischaemic period. Inhibition of glucose oxidation, decrease of ATP concentration, inhibition of proteosynthesis and signal transduction disturbances were observed during the period of reperfusion. Despite of proteosynthesis inhibition, several proteins, especially transcription and growth factors are overproduced. The loss of intracellular homeostasis, as the consequence of the above mentioned processes, could lead to neuronal death.

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.

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 Fe(2+)-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 microM FeSO4-EDTA per mg of protein at 37 degrees 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.

Lipid peroxidation both inhibits Ca(2+)-ATPase and increases Ca2+ permeability of endoplasmic reticulum membrane.

Incubation of reticular membranes with Fe(2+)-EDTA and H2O2 plus Fe(2+)-EDTA at 37 degrees C for 30 min led to the loss of membrane's efficiency to sequester Ca2+ to 21.8% and 3.6% of control values, respectively. The incubation of microsomes with Fe(2+)-EDTA and H2O2 plus Fe(2+)-EDTA also caused decrease of Ca(2+)-ATPase activity; to 44.9% and 44.4% (measured under the same conditions as Ca(2+)-uptake) or to 79.6% and 62.1% (uncoupled from Ca2+ transport by detergent). In addition, incubation of membranes with Fe(2+)-EDTA and H2O2 plus Fe(2+)-EDTA at 37 degrees 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 Ca(2+)-ATPase inhibition in a dose and an antioxidant nature dependent manner. Our results indicate that both processes, Ca(2+)-ATPase inhibition and increase of endoplasmic reticulum membrane Ca2+ permeability, participate in the lipid peroxidation induced loss of membrane's efficiency to sequester Ca2+.

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.

Rabbit brain endoplasmic reticulum membranes as target for free radicals. Changes in Ca(2+)-transport and protection by stobadine.

Incubation of rabbit brain endoplasmic reticulum membranes with either ferrous sulfate/EDTA or ferrous sulfate/EDTA and hydrogen peroxide led to the loss of efficiency of membranes to sequester Ca2+, which did not correlate with changes in conjugated diene formation. The production of practically non-detectable amount of conjugated dienes that occurs during the period of incubation of microsomes with lipid peroxidation initiators represents lipid peroxidation that is enough to produce changes in membrane permeability towards Ca2+. Addition of stobadine was able to prevent Ca2+ transport damage in a dose-dependent manner and drug concentrations higher than 200 microM were able in our model system to confer the defense against free radical and heavy metal initiated lipid peroxidation. The EC50 values for microsomes treated with Fe2+ and Fe2+/H2O2 were 12 microM and 25 microM, respectively. In our model system stobadine seems to be at least as effective as butylated hydroxytoluene, which is considered to be a good chain-breaking antioxidant. In contrast to stobadine alpha-tocopherole acetate was less potent; the effect of 1 mM alpha-tocopherole acetate being comparable to the effect of 20 microM stobadine.

Change in fluidity of brain endoplasmic reticulum membranes by oxygen free radicals: a protective effect of stobadine, alpha-tocopherol acetate, and butylated hydroxytoluene.

Effect of various oxygen free radical generating systems and an oxidant H2O2 on brain endoplasmic reticulum (ER) membrane fluidity was examined using fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene, DPH. The relative potency of free radical generating systems to decrease membrane fluidity increased in this order: FeCl3-EDTA, FeSO4-EDTA, FeSO4-EDTA/hydrogen peroxide. Potency to decrease membrane fluidity correlated well with these systems' potencies to induce lipid peroxidation, as detected by conjugated diene formation. Treatment of ER membranes with H2O2 had no effect on fluidity or conjugated diene formation. Using the two most potent free radical generating systems, FeSO4-EDTA and FeSO4-EDTA/hydrogen peroxide, a protective effect of the novel antihypoxic and antiarrhytmic drug stobadine was tested. Stobadine and two well-known antioxidants, alpha-tocopherol acetate and butylated hydroxytoluene, demonstrated the ability to prevent free radical induced alterations in ER membrane fluidity. These results provide new evidence of stobadine's protective effect on membranes attacked by oxygen free radicals.

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.

Relative resistance of rabbits to MPTP neurotoxicity.

The neurotoxic actions of MPTP and its 4-(O-tolyl) analog (2'-Me-MPTP) on two breeds of rabbits were investigated. MPTP, but not 2'-Me-MPTP, causes a reduction (about 40%) in striatal dopamine content in rabbits of the "little silver-black" breed. The dopamine content of striata of "chinchilla" rabbits was not affected by either agent.

Alteration in rabbit brain endoplasmic reticulum Ca2+ transport by free oxygen radicals in vitro.

The involvement of free oxygen radicals in ischemia-reperfusion injury is generally accepted. We describe here the loss of efficiency of reticular membranes to sequester Ca2+ due to free oxygen radical damage in vitro. Based on experimental results we suggest that the primary effect of free oxygen radicals is alteration of the lipid component of the membrane manifested in the increase of passive Ca2+ leak. Prolonged treatment of microsomes with free oxygen radical generating systems also led to the decrease of Ca(2+)-ATPase activity which is caused as we suppose by modulation of the lipid-Ca2+ pump interactions. A protective effect of butylated hydroxytoluene on the depression in the Ca2+ uptake and Ca(2+)-ATPase activities supports our suggestions.

Effects of denervation on the contents of cholesterol and membrane systems involved in muscle contraction in rabbit fast-twitch sarcotubular system.

Denervated fast-twitch rabbit muscles were progressively losing their fresh weight and the yield of sarcotubular protein was increasing. The activity of Ca(2+)-ATPase was affected but very slightly, the basal Mg(2+)-ATPase and the Mg(2+)-ATPase/Ca(2+)-ATPase ratio however increased together with a simultaneous depression of the membrane-bound acetylcholinesterase activity. We did not observe any differences in density properties of sarcotubular fractions between control and denervated muscle. However, a relative enrichment in SM and H fraction could be seen after denervation with small changes in the content of the Ca(2+)-pump protein, increased levels of calsequestrin and cholesterol, mostly in the heavy and the SM fraction. After denervation the binding sites for 3H-PN-200-110 did not show any changes in receptor affinity, but the number of putative Ca(2+)-channels increased twice along with a depression of 3H-ouabain binding sites. We suggest that the denervation of fast-twitch muscle leads to the hypertrophy of the junctional sarcoplasmic reticulum and the T-system. Changes in the cholesterol content, in the number of putative Ca(2+)-channels and in Na+, K(+)-ATPase can affect the muscle contraction.

[Membrane-associated acetylcholinesterase in the sarcotubular system of skeletal muscles]. / Membránovo asociovaná acetylcholínesteráza zo sarkotubulárneho systému kostrového svalu.

Isolated membranes of the sarcotubular system of the skeletal muscle of the rabbit exhibit acetylcholinesterase activity. The activity of the enzyme depends on the intactness of the vesicles, it increases with time and after the action of detergents, suggesting that the enzyme may be bound to the membranes in a latent occluded form. The optimal conditions of the hydrolytic reaction were determined to be at the protein concentration of 35-40 micrograms/ml. The most suitable pH for acetylcholinesterase activity was found to be in the mildly alkaline region of pH = 8.0. The dependence of the activity of the enzyme on the concentration of the substrate displayed a hyperbolic course, the reciprocal dependence according to Lineweaver and Burk yielded the values KM = 0.69 mmol.l-1 and Vmax = 1.99 mumol/mg/h. The observed properties of acetylcholinesterase from the sarcotubular system show that the enzyme is associated with these membranes and after the action of detergents it has a well measurable activity.

Enzyme distribution and transport activities in electrophoretically isolated fractions of the muscle sarcotubular system.

A simple electrophoretic method is introduced allowing to isolate five fractions of skeletal muscle ST-system vesicles. In a previous study differences in lipid content, 3H-ouabain binding and in presence of triads in individual fractions (Lehotský et. al. 1986) were analysed. In the present study biochemical characterization was extended, and (in accordance with previous results) major differences were observed to exist between fraction 1 and fractions 3 and 4. SDS-PAGE showed that fractions 3 and 4 were enriched in a protein with m.w. 100 kD, these fractions showing the highest specific activities of (Mg2+ + Ca2+)-ATPase and oxalate-supported Ca2+-uptake; activities of Mg2+-ATPase and surface membrane marker enzymes were the lowest in these fractions. On the other hand, in fraction 1 the highest activities of Mg2+-ATPase and marker enzymes of the surface membrane were observed together with a decreased content of the 100 kD protein and activities of Ca2+ transport. It could be concluded that the method is suitable to differentiate between relatively pure SR (fractions 3 and 4) and fractions rich in sarcolemma or T-tubules components (fractions 1 and 5).