Neurotensin modulates central muscarinic receptors, an effect which does not involve the high-affinity neurotensin receptor (NTS1).

Neurotensin (NT) is a tridecapeptide distributed in central and peripheral nervous systems, which can behave as a neurotransmitter or neuromodulator at central and peripheral levels. Herein we tested the potential effect of this peptide on quinuclidinyl benzilate ([3H]-QNB) binding to muscarinic receptor in rat CNS membranes. It was observed that NT decreased up to 50-70% ligand binding at 1x10(-7) M-1x10(-5) M concentration in cerebral cortex, cerebellum and striatum. In the hippocampus, NT exerted a biphasic effect, behaving as a stimulator in the presence of 1x10(-12) M-1x10(-10) M concentration but as an inhibitor at 1x10(-8) M-1x10(-5) M concentration. In order to test the involvement of high-affinity NT receptor (NTS1) in NT inhibitory effect, assays were carried out in the presence of 1x10(-6) M NT and/or SR 48692 (Sanofi-Aventis, U.S., Inc.), a specific antagonist for this receptor, dissolved in dimethylsulfoxide (DMSO) 10% v/v. As controls, membranes incubated with DMSO and/or NT 1x10(-6) M plus DMSO were processed. It was found that NT+DMSO decreased [3H]-QNB binding to cerebral cortex, cerebellum and hippocampal membranes by 49%, 32% and 53%, respectively. This inhibition was not observed with the DMSO control group. Membrane preincubation with 1x10(-6) M SR 48692 failed to alter NT effect on binding. SR 48692 at 1x10(-6) M concentration decreased the binding by 50% only in cerebral cortex membranes, suggesting a possible direct effect of the antagonist on muscarinic receptors in this area. It was therefore concluded that the high-affinity NT receptor may not be involved in ligand binding inhibition to muscarinic receptor by NT.

Modulation of aspartate release by ascorbic acid and endobain E, an endogenous Na+, K+ -ATPase inhibitor.

The isolation of a soluble brain fraction which behaves as an endogenous ouabain-like substance, termed endobain E, has been described. Endobain E contains two Na+, K+ -ATPase inhibitors, one of them identical to ascorbic acid. Neurotransmitter release in the presence of endobain E and ascorbic acid was studied in non-depolarizing (0 mM KCl) and depolarizing (40 mM KCl) conditions. Synaptosomes were isolated from cerebral cortex of male Wistar rats by differential centrifugation and Percoll gradient. Synaptosomes were preincubated in HEPES-saline buffer with 1 mM D-[3H]aspartate (15 min at 37 degrees C), centrifuged, washed, incubated in the presence of additions (60 s at 37 degrees C) and spun down; radioactivity in the supernatants was quantified. In the presence of 0.5-5.0 mM ascorbic acid, D-[3H]aspartate release was roughly 135-215% or 110-150%, with or without 40 mM KCI, respectively. The endogenous Na+, K+ -ATPase inhibitor endobain E dose-dependently increased neurotransmitter release, with values even higher in the presence of KCl, reaching 11-times control values. In the absence of KCl, addition of 0.5-10.0 mM commercial ouabain enhanced roughly 100% D-[3H]aspartate release; with 40 mM KCl a trend to increase was recorded with the lowest ouabain concentrations to achieve statistically significant difference vs. KCl above 4 mM ouabain. Experiments were performed in the presence of glutamate receptor antagonists. It was observed that MPEP (selective for mGluR5 subtype), failed to decrease endobain E response but reduced 50-60% ouabain effect; LY-367385 (selective for mGluR1 subtype) and dizocilpine (for ionotropic NMDA glutamate receptor) did not reduce endobain E or ouabain effects. These findings lead to suggest that endobain E effect on release is independent of metabotropic or ionotropic glutamate receptors, whereas that of ouabain involves mGluR5 but not mGluR1 receptor subtype. Assays performed at different temperatures indicated that in endobain E effect both exocytosis and transporter reversion are involved. It is concluded that endobain E and ascorbic acid, one of its components, due to their ability to inhibit Na+, K+ -ATPase, may well modulate neurotransmitter release at synapses.

Endobain E, a brain endogenous factor, is present and modulates NMDA receptor in ischemic conditions.

We have isolated from rat cerebral cortex an endogenous Na(+), K(+)-ATPase inhibitor, termed endobain E, which modulates glutamatergic N-methyl-d-aspartate (NMDA) receptor. This endogenous factor allosterically decreases [(3)H]dizocilpine binding to NMDA receptor, most likely acting as a weak channel blocker. In the present study we investigated whether endobain E is present in the cerebral cortex of rats subjected to ischemia and modulates NMDA receptor exposed to the same conditions. Ischemia-reperfusion was carried out by bilateral occlusion of common carotid arteries followed by a 15-min reperfusion period. Elution profile of brain soluble fraction showed that endobain E is present in cerebral cortex of ischemia-reperfusion rats. On assaying its effect on synaptosomal membrane Na(+), K(+)-ATPase activity and [(3)H]dizocilpine binding to cerebral cortex membranes prepared from animals without treatment, it was found that the endogenous modulator isolated from ischemia-reperfusion rats was able to inhibit both enzyme activity and ligand binding. On the other hand, endobain E prepared from rats without treatment also decreased binding to cerebral cortex or hippocampal membranes obtained from animals exposed to ischemia-reperfusion. Since ischemia decreases tissue pH and NMDA receptor activity varies according to proton concentration, pH influence on endobain E effect was tested. Endobain E ( approximately 80 mg original tissue) decreased [(3)H]dizocilpine binding 25% at pH 7.4 or 8.0 but 90% at pH 6.5. These results demonstrate that endobain E is present and also able to modulate NMDA receptor in the short-term period that follows cerebral ischemia and that its effect depends on proton concentration, suggesting greater NMDA receptor modulation by endobain E at low pH, typical of ischemic tissues.

The effect of endogenous modulator endobain E on NMDA receptor is interfered by Zn2+ but is independent of modulation by spermidine.

A brain endogenous factor, termed endobain E, allosterically decreases [3H]dizocilpine binding to NMDA receptor. Such effect depends on receptor activation by the coagonists glutamate and glycine and is interfered by channel blockers, suggesting its interaction with the inner surface of the associated channel. To further analyze endobain E effect on NMDA receptor, in the current study competitive [3H]dizocilpine binding assays to brain membranes were performed with Zn2+ to block the associated channel, as well as with spermidine (SPD), which exerts positive allosteric modulation of NMDA receptor. Partially or nonadditive effects on [3H]dizocilpine binding were recorded, respectively, in the presence of endobain E at a concentration that inhibits binding 25% plus IC25 Zn2+ or endobain E at a concentration that inhibits binding 50% plus IC50 Zn2+. With an endobain E concentration that decreases 25% ligand binding, SPD potentiated binding over a wide concentration range but failed to modify endobain E effect. Similarly, [3H]dizocilpine binding reduction over a wide endobain E concentration range remained unaltered by high SPD concentrations. Additive effects were observed with endobain E at a concentration that decreases binding 25% plus IC25 SPD site antagonists arcaine or ifenprodil. Zn2+ experiments indicated that endobain E effect is interfered by channel blockade produced by this ion. Although endobain E effect is dependent on NMDA receptor activation by glutamate and glycine, it proves independent of the positive modulation exerted by SPD. Thus the endogenous modulator seems not to interact at NMDA receptor polyamine site, favoring the hypothesis that endobain E binds inside the associated channel.

A comparative study between a brain Na+,K(+)-ATPase inhibitor (endobain E) and ascorbic acid.

In the search of Na+,K(+)-ATPase modulators, we have reported the isolation by gel filtration and HPLC of a brain fraction, termed endobain E, which highly inhibits Na+,K(+)-ATPase activity. In the present study we compared some properties of endobain E with those of ascorbic acid. Kinetic experiments assaying synaptosomal membrane K(+)-p-nitrophenylphosphatase (K(+)-p-NPPase) activity in the presence of endobain E or ascorbic acid showed that in neither case did enzyme inhibition prove competitive in nature versus K+ or p-NPP concentration. At pH 5.0, endobain E and ascorbic acid maximal UV absorbance was 266 and 258 nm, respectively; alkalinization to pH 14.0 led to absorption drop and shift for endobain E but to absorbance disappearance for ascorbic acid. After cysteine treatment, endobain E absorbance decreased, whereas that of ascorbic acid remained unaltered; iodine treatment led to absorbance drop and shift for endobain E but to absorbance disappearance for ascorbic acid. HPLC analysis of endobain E disclosed the presence of two components: one eluting with retention time and UV spectrum indistinguishable from those of ascorbic acid and a second, as yet unidentified, both exerting Na+,K(+)-ATPase inhibition.

Metabotropic glutamate receptor involvement in phosphoinositide hydrolysis stimulation by an endogenous Na(+), K(+)-ATPase inhibitor and ouabain in neonatal rat brain.

The mechanism of action of an endogenous Na(+), K(+)-ATPase inhibitor, termed endobain E, on phosphoinositide hydrolysis was studied in neonatal rat brain cortex and compared with that of ouabain. Lack of additivity for endobain E and glutamate paired stimulation on inositol phosphates accumulation suggested that they share at least a common step on inositol phosphate metabolism, as previously advanced for ouabain. In addition, Cd(2+) sensitivity of endobain E and ouabain effects strengthened the involvement of glutamate receptors. The participation of ionotropic glutamate receptors on endobain E- and ouabain-induced phosphoinositide hydrolysis seems untenable, since antagonists dizocilpine and CNQX proved unable to inhibit these effects. However, the endobain E effect was blocked by 2 x 10 (-4) M L-AP3 (an antagonist for group I mGluRs) when at least a 15-min preincubation protocol was employed. Maximal inhibition of endobain E effect (42%) occurred when L-AP3 preincubation was extended to 60 min, as already shown with glutamate, but only a trend to decrease was recorded with ouabain. At variance, the ouabain effect was reduced to 50% employing 5 x 10 (-4) M MCPG (a competitive antagonist for group I mGluRs), whereas no blockade was observed with endobain E or glutamate. In addition, MPEP (a selective mGluR5 antagonist) partially reduced ouabain, endobain E and glutamate responses and the selective mGluR1 antagonist LY367385 showed no activity at all. To sum up, the present findings support the involvement of mGluR5 in both endobain E and ouabain phosphoinositide hydrolysis stimulation in neonatal rat brain, in spite of dissimilar response to tested antagonists.

[3H]dizocilpine binding to N-methyl-D-aspartate (NMDA) receptor is modulated by an endogenous Na+, K+-ATPase inhibitor. Comparison with ouabain.

An endogenous Na+, K+-ATPase inhibitor termed endobain E has been isolated from rat brain which shares several biological properties with ouabain. This cardiac glycoside possesses neurotoxic properties attributable to Na+, K+-ATPase inhibition, which leads to NMDA receptor activation, thus supporting the concept that Na+/K+ gradient impairment has a critical impact on such receptor function. To evaluate potential direct effects of endobain E and ouabain on NMDA receptors, we assayed [3H]dizocilpine binding employing a system which excludes ionic gradient participation. Brain membranes thoroughly washed and stored as pellets ('non-resuspended' membranes) or after resuspension in sucrose ('resuspended' membranes) were employed. Membrane samples were incubated with 4 or 10 nM ligand with or without added endobain E or ouabain, in the presence of different glutamate plus glycine combinations, with or without spermidine. [3H]dizocilpine basal binding and Na+, K+- and Mg2+-ATPase activities proved very similar in 'non-resuspended' or 'resuspended' membranes. Endobain E decreased [3H]dizocilpine binding to 'resuspended' membranes in a concentration-dependent manner, attaining roughly 50% binding inhibition with the highest endobain E concentration assayed. Among tested conditions, only in 'resuspended' membranes, with 4 nM ligand and with 1x10(-8) M glutamate plus 1x10(-5) M glycine, was [3H]dizocilpine binding enhanced roughly +24% by ouabain (1 mM). After Triton X-100 membrane treatment, which drastically reduces Na+, K+-ATPase activity, the effect of ouabain on binding was lost whereas that of endobain E remained unaltered. Results indicate that not only membrane preparation but also treatment and storage are crucial to observe direct endobain E and ouabain effects on NMDA receptor, which are not attributable to changes in Na+, K+-ATPase activity or to Na+/K+ equilibrium alteration.

Seizure activity produces differential changes in adenosine A1 receptors within rat hippocampus.

Specific ligand binding to rat hippocampal adenosine A1 receptor after administration of the convulsant drug 3-mercaptopropionic acid (MP) was studied by means of a quantitative autoradiographic method. 2-Chloro-N6-[cyclopentyl-2,3,4,5-3H adenosine] ([3H]CCPA), a potent and selective A1 receptor ligand, was selected for binding studies. MP administration (150 mg/kg, i.p.), at seizure, caused significant increases in the following CA1 layers: pyramidal (45%), radiatum (18%) and lacunosum molecular (35%); in CA2 area, a significant decrease in stratum oriens (36%) and an increase in stratum radiatum (14%) and lacunosum molecular (33%) layers was observed. In CA3 area a rise in pyramidal (40%) and radiatum layers (26%), as well as in hillus (97%) was found. At postseizure, changes were restricted to CA1, CA2 and CA3 pyramidal layers and to CA1 lacunosum molecular layer, with increases ranging from 22 to 50%. These results show that [3H]CCPA binding is modified diversely in intrahippocampal layers and areas, thus indicating their dissimilar role in seizure activity.

K(+)-p-nitrophenylphosphatase inhibition by neurotensin involves high affinity neurotensin receptor: influence of potassium concentration and enzyme phosphorylation.

Neurotensin (NT), a 13-amino acid peptide, is widely distributed in the brain and peripheral tissues of several mammalian species including man. In adult rat brain NT can bind to two distinct sites, one of high and the other of low affinity, corresponding to NT(1) and NT(2) receptor, respectively; structurally unrelated to these two, a third NT receptor (NT(3)) has been described. We have previously shown that Na(+), K(+)-ATPase is inhibited by NT when using ATP as substrate. In order to determine whether K(+)-stimulated dephosphorylation of this enzyme is involved, we tested NT effect by using p-nitrophenylphosphate, a non-natural substrate. K(+)-p-nitrophenylphosphatase activity was inhibited 42% by NT at 8.6 x 10(-6) M using an incubation medium containing 2 mM KCl but was unaffected in the presence of 5 or 20 mM KCl; however, with such KCl concentrations, NT was enabled to inhibit enzyme activity ( congruent with 35%) provided a suitable ATP:NaCl mixture (0.6:45.0 mM) was added. Mg(2+)-p-nitrophenylphosphatase activity remained unaltered at all conditions tested. Since SR 48692, a selective non-peptide NT(1) antagonist, abolished NT effect, involvement of NT(1) receptor in enzyme inhibition is suggested.

In vitro dose dependent inverse effect of nantenine on synaptosomal membrane K+-p-NPPase activity.

The effect of nantenine, an aporphine alkaloid, on ATPase K+-dependent dephosphorylation was evaluated using p-nitrophenylphosphate (p-NPP) as substrate. Basal K+-p-NPPase activity was significantly increased with 3 x 10(-4) M, remained unchanged with 3 x 10(-6) M, 3 x 10(-5) M but was reduced with 7.5 x 10(-4) M and 1 x 10(-3) M nantenine, whereas Mg2+-p-NPPase activity was not modified. Kinetic studies showed that K+-p-NPPase inhibition by nantenine is competitive to KCl but non-competitive to substrate p-NPP, whereas K+-p-NPPase stimulation by nantenine is non-competitive to KCl but competitive to p-NPP. These data suggest that there may be two acceptor sites for nantenine in p-NPPase, one eliciting stimulation and the other inhibition of K+-dependent p-NPP hydrolysis. Considering the biphasic action of nantenine on seizures and the correlation between decreased ATPase activity and seizure development, alkaloid anticonvulsant effect observed at low nantenine doses is attributable to the stimulation of phosphatase activity whereas the convulsant effect at high alkaloid doses seems related to Na+, K+-ATPase inhibition.

An endogenous Na+, K+-ATPase inhibitor enhances phosphoinositide hydrolysis in neonatal but not in adult rat brain cortex.

The effect of an endogenous Na+, K+-ATPase inhibitor, termed endobain E, on phosphoinositide hydrolysis was studied in rat brain cortical prisms and compared with that of ouabain. As already shown for ouabain, a transient effect was obtained with endobain E; maximal accumulation of inositol phosphates induced by endobain E was 604 +/- 138% and 186 +/- 48% of basal values in neonatal and adult rats, respectively. The concentration-response plot for the interaction between endobain E and phosphoinositide turnover differed from that of ouabain, thus suggesting the involvement of distinct mechanisms. In the presence of endobain E plus ouabain at saturating concentrations, no additive effect was recorded, suggesting that both substances share at least a common step in their activation mechanism of inositol phosphates metabolism or that they enhance phosphatidylinositol 4,5-biphosphate breakdown from the same membrane precursor pool, until its exhaustion. Experiments with benzamil, a potent blocker of Na+/Ca2+ exchanger, showed that it partially and dose-dependently inhibited endobain E effect. These results indicate that the endogenous Na+, K+-ATPase inhibitor endobain E, like ouabain, is able to stimulate phosphoinositide turnover transiently during postnatal brain development.

How many endobains are there?

Oxidative metabolism is very active in brain, where large amounts of chemical energy as ATP molecules are consumed, mostly required to maintain cellular Na+/K+ gradients through the participation of the sodium pump (Na+,K+-ATPase), whose activity is selectively and potently inhibited by the alkaloid ouabain. Na+/K+ gradients are involved in nerve impulse propagation, in neurotransmitter release and cation homeostasis in the nervous system. Likewise, enzyme activity modulation is crucial for maintaining normal blood pressure and cardiovascular contractility as well as renal sodium excretion. The present article reviews the progress in disclosing putative ouabain-like substances, examines their denomination according to different research teams, tissue or biological fluid sources, extraction and purification, assays, biological properties and chemical and biophysical features. When data is available, comparison with ouabain itself is mentioned. Likewise, their potential action in normal physiology as well as in experimental and human pathology is summarized.

Nantenine and papaverine differentially modify synaptosomal membrane enzymes.

Papaverine (1-[(3,4-Dimethoxyphenyl) methyl]-6,7-dimethoxyisoquinoline) and nantenine (O-methyldomesticine) are chemically related isoquinoline alkaloids displaying similar dose-dependent sedative or convulsant effects, but seem to act differentially on synaptosomal membrane enzymes. Na+, K+-, Mg2+- and Ca2+-ATPase activities were inhibited by nantenine but not by papaverine, whereas acetylcholinesterase activity remained unchanged by nantenine but slightly enhanced by papaverine. Nantenine inhibited roughly both 20-50% Ca2+- and Mg2+-ATPase activities but 40-90% Na+, K+-ATPase activity. Kinetic analysis indicated that nantenine interacts with the substrate ATP for Ca2+-ATPase activity but that it competes with K+ for Na+, K+-ATPase activity. Given the roles of Na+, K+-ATPase and Ca2+-ATPase in cation transport and [Ca2+]i regulation, respectively, the inhibitory effect of nantenine upon these enzymes may explain its convulsant effect though not its sedative activity. The sedative action of both nantenine and papaverine is hardly attributable to an effect on the synaptosomal membrane enzymes assayed.

Neurotensin inhibits neuronal Na+,K+-ATPase activity through high affinity peptide receptor.

Neurotensin is a peptide present in mammalian CNS and peripheral tissues, which may play a major role in neurotransmission or neuromodulation, subserving diverse physiological functions. We studied the effect of added neurotensin on ATPase activities in synaptosomal membranes isolated from rat cerebral cortex. Neurotensin at 3 x 10(-8)-3 x 10(-6) M concentration decreased 20-44% Na+,K+-ATPase activity but failed to modify Mg2+-ATPase activity; lower neurotensin concentrations (3 x 10(-14)-3 x 10(-10) M) had no effect on enzyme activities. This inhibitory effect was abolished by neurotensin heating, by enzyme preincubation with neurotensin during periods exceeding 10 min, or by adding 1 x 10(-6) M SR 48692, a high affinity neurotensin receptor antagonist. Levocabastine, which blocks low affinity neurotensin receptor, failed to alter enzyme inhibition by the peptide. It is suggested that the sodium pump may be a target for neurotensin effects at neuronal level involving the participation of high affinity neurotensin receptor.

Calcitonin modifies ligand binding to muscarinic receptor in CNS membranes.

Calcitonin (CT) is a peptide produced by the thyroid gland, whose best described role is to prevent bone reabsorption, though it also participates in other biological functions through both central and peripheral mechanisms. CT is able to inhibit brain Na(+), K(+)-ATPase activity (Rodríguez de Lores Arnaiz, López Ordieres, Peptides 1997;18:613-5) and a relationship between such enzyme activity and cholinergic function has been suggested. Accordingly, we tested CT effect on [(3)H]-quinuclidinyl benzilate ([(3)H]-QNB) binding to rat CNS membranes to determine whether the peptide is able to modify the cholinergic muscarinic receptor as well. It was found that 1x10(-7)-1x10(-5) M CT decreased 20-70% ligand binding to hippocampal, cerebellar, cortical and striatal membranes. Scatchard analysis of saturation curves showed that 5x10(-6) M CT significantly modified binding kinetic constants, thus it increased roughly 220% K(d) values and decreased 20-36% B(max) values in cerebral cortical and cerebellar membranes. Since the peptide decreases affinity ligand binding and reduces the number of binding sites, CT may well be acting as a cholinergic modulator through a decrease in muscarinic receptor functionality.

Kinetics of Na+, K+-ATPase inhibition by an endogenous modulator (II-A).

We have previously reported the isolation by gel filtration and anionic exchange HPLC of two brain Na+, K+-ATPase inhibitors, II-A and II-E, and kinetics of enzyme interaction with the latter. In the present study we evaluated the kinetics of synaptosomal membrane Na+, K+-ATPase with II-A and found that inhibitory activity was independent of ATP (2-8 mM), Na+ (3.1-100 mM), or K+ (2.5-40 mM) concentration. Hanes-Woolf plots showed that II-A decreases Vmax in all cases; KM value decreased for ATP but remained unaltered for Na+ and K+, indicating respectively uncompetitive and noncompetitive interaction. However, II-A became a stimulator at 0.3 mM K+ concentration. It is postulated that brain endogenous factor II-A may behave as a sodium pump modulator at the synaptic region, an action which depends on K+ concentration.

Brain soluble fractions which modulate Na+, K+-ATPase activity likewise modify muscarinic receptor.

Two brain soluble fractions, named peaks I and II, which respectively stimulate and inhibit neuronal Na+, K+-ATPase activity, have been isolated by gel filtration in Sephadex G-50. Since cholinergic transmission seems related to such enzyme activity, in this study we evaluated the effect of brain peak I, peak II, a more purified fraction II-E and commercial ouabain, on specific binding of the muscarinic antagonist [3H]quinuclidinyl benzilate to membranes from rat cerebellum, hippocampus and cerebral cortex. We found that binding was increased by peak I and decreased by peak II, II-E and ouabain, all effects proving concentration-dependent. Since the changes exerted on the muscarinic receptor followed a pattern similar to the one already described for synaptosomal membrane Na+, K+-ATPase activity, both systems seem to interact at a functional level.

A brain Na+, K+-ATPase inhibitor (endobain E) enhances norepinephrine release in rat hypothalamus.

We have shown that synaptosomal membrane Na+, K+-ATPase activity is stimulated or inhibited by norepinephrine according to the presence or absence of a brain soluble fraction. Gel filtration of such soluble fraction has allowed the separation of two fractions, peaks I and II, able to stimulate and inhibit Na+, K+-ATPase activity, respectively. Peak II behaves much like ouabain, which has suggested the term endobain. From peak II, a subfraction termed II-E (endobain E), which highly inhibits Na+, K+-ATPase, has been separated by anionic exchange chromatography in a Synchropack AX-300 column. We determined the in vitro effect of endobain E obtained from rat cerebral cortex on neuronal norepinephrine release by incubating rat hypothalamic tissue in the presence of [3H]norepinephrine. Neuronal norepinephrine release was quantified as the factor above basal [3H]norepinephrine released to the medium at experimental and three post-experimental periods. Endobain E was found to increase norepinephrine release in a concentration-dependent fashion, reaching 200%, equivalent to the effect achieved with 400 microM ouabain. Ouabain effect persisted along three post-experimental periods whereas that of endobain E remained only during the first post-experimental period. These results led us to conclude that endobain increases norepinephrine release in hypothalamic neurons at the presynaptic nerve ending level, an effect resembling that of ouabain. It is postulated that endobain E may enhance catecholamine availability in the synaptic gap, leading to an increase in noradrenergic activity.

Regional vulnerability to oxidative stress in a model of experimental epilepsy.

We evaluated oxidative stress associated with a model of experimental epilepsy. Male Wistar rats were injected i.p. with 150 mg/kg convulsant 3-mercaptopropionic acid and decapitated in two stages: during seizures or in the post-seizure period. Spontaneous chemiluminescence, levels of thiobarbituric acid reactive substances, total antioxidant capacity and antioxidant enzyme activities were measured in cerebellum, hippocampus, cerebral cortex and striatum. In animals killed at seizure, increases of 42% and 90% were observed in spontaneous chemiluminescence of cerebellum and cerebral cortex homogenates, respectively, accompanied by a 25% increase in cerebral cortex levels of thiobarbituric acid reactive substances. In the post-seizure stage, emission completely returned to control levels in cerebral cortex and partly in cerebellum, thus showing oxidative stress reversibility in time. Hippocampus and striatum seemed less vulnerable areas to oxidative damage. A 30% decrease in glutathione peroxidase activity was only observed in cerebral cortex during seizures, while catalase and superoxide dismutase remained unchanged in all four areas during either stage. Likewise, total antioxidant capacity was unaffected in any of the studied areas. It is suggested that oxidative stress in this model of epilepsy arises from an increase in oxidant species rather than from depletion of antioxidant defences.

Effect of angiotensin-(1-7) on ATPase activities in several tissues.

The present investigation was undertaken to determine whether Ang-(1-7) is able to modify ATPase activities in membrane fractions prepared from several tissues. In the presence of 10(-6) M Ang-(1-7), total (Na , K+, Mg2+)-ATPase activity decreased 31% in rat atrium and 13% in sheep atrium but was unmodified in sheep liver, rat ventricle or crude brain membranes. In rat brain synaptosomal membranes, Ang-(1-7) at 10(-8) and 10(-7) M concentrations activated Na+, K+-ATPase 20 and 24%, respectively. Rat kidney Na+, K+-ATPase activity decreased roughly 40-70% with 10(-10)-10(-6) M Ang-(1-7)), but increased 22% with 10(-12) M peptide concentration, thus indicating a biphasic effect. Our findings showing that ATPase from several tissues responds differently to Ang-(1-7) are attributable to enzyme tissue specificity.