Effect of amyloid beta-peptide on permeability transition pore: a comparative study

A potentially central factor in neurodegeneration is the permeability transition pore (PTP). Because of the tissue-specific differences in pore properties, we directly compared isolated brain and liver mitochondria responses to the neurotoxic A beta peptides. For this purpose, the following parameters were examined: mitochondrial membrane potential (Delta Psi m), respiration, swelling, ultrastructural morphology, and content of cytochrome c. Both peptides, A beta(25-35) (50 microM) and A beta(1-40) (2 microM), had a similar toxicity, exacerbating the effects of Ca(2+), although, per se, they did not induce (PTP). In liver mitochondria, A beta led to a drop in Delta Psi m and potentiated matrix swelling and disruption induced by Ca(2+). In contrast, brain mitochondria, exposed to the same conditions, demonstrated a higher capacity to accumulate Ca(2+) before the Delta Psi m drop and a slight increase of mitochondrial swelling compared with liver mitochondria. Furthermore, mitochondrial respiratory state 3 was depressed in the presence of A beta, whereas state 4 was unaltered, resulting in an uncoupling of respiration. In both types of mitochondria, A beta did not affect the content of cytochrome c. The Delta Psi m drop was reversed when Ca(2+) was removed by EGTA or when ADP plus oligomycin was present. Pretreatment with cyclosporin A or ADP plus oligomycin prevented the deleterious effects promoted by A beta and/or Ca(2+). It can be concluded that brain and liver mitochondria show a different susceptibility to the deleterious effect of A beta peptide, brain mitochondria being more resistant to the potentiation by A beta of Ca(2+)-induced PTP.     

Ca(2+)-ATPase and Na(+)-K(+)-ATPase content in skeletal muscle from malignant hyperthermia patients.

The purpose of this study was to determine the concentration of Ca(2+)-ATPase and Na(+)-K(+)-ATPase in biopsies from vastus lateralis muscle of 24 patients, who underwent a diagnostic contracture test for susceptibility to malignant hyperthermia (MH). Ca(2+)-ATPase was quantified as the Ca(2+)-dependent 32P incorporation in whole muscle homogenates. Na(+)-K(+)-ATPase was quantified as the [3H]ouabain-binding capacity in intact muscle samples. These methods avoid isolation of membranes, a procedure that may influence the results due to interindividual variation in recovery. The results show that both enzymes can be determined in (frozen) muscle biopsies weighing 50 mg. Neither the concentration of Ca(2+)-ATPase nor that of Na(+)-K(+)-ATPase differed in biopsies from subjects diagnosed as susceptible (MHS) or nonsusceptible (MHN) to MH. Our data support the view that changes in the concentration of Ca(2+)-ATPase and/or Na(+)-K(+)-ATPase do not play a primary role in the pathogenesis of MH.     

Differential effects of the mitochondrial uncoupling agent, 2,4-dinitrophenol, or the nitroxide antioxidant, Tempol, on synaptic or nonsynaptic mitochondria after spinal cord injury

We recently documented the progressive nature of mitochondrial dysfunction over 24 hr after contusion spinal cord injury (SCI), but the underlying mechanism has not been elucidated. We investigated the effects of targeting two distinct possible mechanisms of mitochondrial dysfunction by using the mitochondrial uncoupler 2,4-dinitrophenol (2,4-DNP) or the nitroxide antioxidant Tempol after contusion SCI in rats. A novel aspect of this study was that all assessments were made in both synaptosomal (neuronal)- and nonsynaptosomal (glial and neuronal soma)-derived mitochondria 24 hr after injury. Mitochondrial uncouplers target Ca(2+) cycling and subsequent reactive oxygen species production in mitochondria after injury. When 2,4-DNP was injected 15 and 30 min after injury, mitochondrial function was preserved in both populations compared with vehicle-treated rats, whereas 1 hr postinjury treatment was ineffective. Conversely, targeting peroxynitrite with Tempol failed to maintain normal bioenergetics in synaptic mitochondria, but was effective in nonsynaptic mitochondria when administered 15 min after injury. When administered at 15 and 30 min after injury, increased hydroxynonenal, 3-NT, and protein carbonyl levels were significantly reduced by 2,4-DNP, whereas Tempol only reduced 3-NT and protein carbonyls after SCI. Despite such antioxidant effects, only 2,4-DNP was effective in preventing mitochondrial dysfunction, indicating that mitochondrial Ca(2+) overload may be the key mechanism involved in acute mitochondrial damage after SCI. Collectively, our observations demonstrate the significant role that mitochondrial dysfunction plays in SCI neuropathology. Moreover, they indicate that combinatorial therapeutic approaches targeting different populations of mitochondria holds great potential in fostering neuroprotection after acute SCI.     

Suppression of sodium pump activity and an increase in the intracellular Ca concentration by dexamethasone in acidotic mouse brain

The effects of dexamethasone on adenosine 5'-triphosphatase (ATPase) activity and the intracellular Ca(2+) concentration ([Ca(2+)](i)) were investigated in acidotic mouse brain. Dexamethasone (3 mg/kg, i.p.) or vehicle was administered 3 h before decapitation ischemia, and the brain concentration of adenosine 5'-triphosphate (ATP) was determined 0.5-2 min after ischemia. The effects of dexamethasone (0.3-3 mg/kg, i.p.) on Na(+),K(+)-activated ATPase (Na(+),K(+)-ATPase) and Ca(2+)-ATPase activities were evaluated at pH 7.4 and 6.8. Changes in [Ca(2+)](i) in an acidic medium were determined in hippocampal slices by microfluorometry using rhod-2 acetoxymethyl ester as a Ca(2+) marker, and the effects of dexamethasone (240 microg/l) was evaluated. Decapitation ischemia for 0.5 and 1 min reduced the brain ATP contents to 32% and 16% of the basal level, respectively. Dexamethasone slightly suppressed the extent of the decrease in the ATP level. Although dexamethasone did not affect Na(+),K(+)-ATPase activity at pH 7.4, the activity was suppressed by dexamethasone (3 mg/kg) to 68% at pH 6.8. The activity of Ca(2+)-ATPase was not affected by dexamethasone at either pH 7.4 or pH 6.8. When the pH of the medium of the brain slices was changed from 7.4 to 6.8, almost no increase in [Ca(2+)](i) was observed in the control group. The dexamethasone treatment increased [Ca(2+)](i) in the CA1 field and dentate gyrus immediately after induction of the acidic medium, the effect being significant after 150 s. Because anaerobic glucose metabolism in the early stage of ischemia enhances intracellular lactic acidosis, the findings may suggest a mechanism for the aggravation of ischemic neuronal damage by glucocorticoids.     

Creatine increases hippocampal Na(+),K(+)-ATPase activity via NMDA-calcineurin pathway

Achievements made over the past few years have demonstrated the important role of the creatine and phosphocreatine system in the buffering and transport of high-energy phosphates into the brain; however, the non-energetic processes elicited by this guanidine compound in the hippocampus are still poorly understood. In the present study we disclosed that the incubation of rat hippocampal slices with creatine (10mM) for 30 min increased Na(+),K(+)-ATPase activity. In addition, intrahippocampal injection of creatine (5 nmol/site) also increased the above-mentioned activity. The incubation of hippocampal slices with N-methyl-d-aspartate (NMDA; MK-801, 10 μM) and NMDA Receptor 2B (NR2B; ifenprodil, 3 μM) antagonists but not with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA)/kainate antagonist (DNQX, 10 μM) and nitric oxide synthase inhibitor (NOS; l-NAME, 100 μM), blunted the effect of creatine on Na(+),K(+)-ATPase activity. Furthermore, the calcineurin inhibitor (cyclosporine A, 200 nM) as well as the Protein Kinase C (PMA, 100 nM) and Protein Kinase A (8-Br-cAMP, 30 μM) activators attenuated the creatine-induced increase of Na(+),K(+)-ATPase activity. In addition, the incubation of hippocampal slices with creatine (10mM) for 30 min increased calcineurin activity. The results presented here suggest that creatine increases Na(+),K(+)-ATPase activity via NMDA-calcineurin pathway, proposing an putative underlying non-energetic role of this guanidine compound. However, more studies are needed to assess the contribution of this putative alternative role in neurological diseases that present decreased Na(+),K(+)-ATPase activity.     

Arginine administration inhibits hippocampal Na(+),K(+)-ATPase activity and impairs retention of an inhibitory avoidance task in rats

In the present study we investigated the effect of acute administration of L-arginine (Arg) on hippocampal Na(+),K(+)-ATPase activity and on retrieval of step-down inhibitory avoidance in adult rats. The action of L-NAME on the effects produced by Arg was also tested. Sixty-day-old rats were treated with a single intraperitoneal injection of saline (group I, control), arginine (0.8 g/kg) (group II), L-NAME (2 mg/kg) (group III) or arginine (0.8 g/kg) plus L-NAME (2 mg/kg) (group IV). Na(+),K(+)-ATPase activity was significantly reduced in arginine-treated rats; this effect was prevented by L-NAME. Retrieval of the avoidance task was also significantly impaired by arginine, whereas the simultaneous injection of L-NAME prevented this effect. Present data strongly indicate that in vivo Arg administration reduces both Na(+),K(+)-ATPase activity and memory modulation in rats probably through NO formation.     

Mechanisms involved in morphine-induced activation of synaptosomal Na+,K+-ATPase

Morphine through mu-opioid receptors and G(i/o) proteins modulates several cellular effector systems; however, the mechanisms involved in the regulation of Na(+),K(+)-ATPase are not well known. We evaluated the effect of two mu-opioid receptor agonists on ouabain-sensitive Na(+),K(+)-ATPase activity in mice forebrain synaptosomes, and examined the modulation of this effect by antagonists of opioid receptors and a blocker of G(i/o) proteins. Incubation of synaptosomes with morphine (10(-9) to 10(-4) M) or buprenorphine (10(-10) to 10(-5) M) concentration-dependently stimulated Na(+),K(+)-ATPase activity, morphine being less potent but more efficacious than buprenorphine. Morphine did not displace [3H]ouabain from its binding site (Na(+),K(+)-ATPase) to forebrain membranes, whereas ouabain did so in a concentration-dependent manner. Naloxone, an opioid antagonist (10(-6) M), added to the synaptosomal medium, antagonized the enhancement of Na(+),K(+)-ATPase activity induced by morphine, producing a parallel shift to the right of the morphine concentration-response curve. Treatment with beta-funaltrexamine, a mu antagonist (2.5 and 10 microg/mouse, i.c.v.) and naloxonazine, a mu1 antagonist (35 mg/kg, s.c.), 24 h before the synaptosomes were obtained, produced a dose-dependent reduction in the E(max) of the morphine-induced increase in Na(+),K(+)-ATPase activity in vitro, but did not significantly modify its EC(50). Pertussis toxin (G(i/o) protein blocker) treatment at a dose of 0.5 microg/mouse, administered i.c.v. 5 days before the synaptosomes were obtained, completely abolished the enhancement of Na(+),K(+)-ATPase activity induced by morphine. A lower dose (0.25 microg/mouse) decreased the E(max) of morphine by 50% but did not significantly affect its EC(50). These results suggest that morphine indirectly enhances Na(+),K(+)-ATPase activity in the brain by activating mu-opioid receptors and G(i/o) proteins.     

Chronic treatment with glutaric acid induces partial tolerance to excitotoxicity in neuronal cultures from chick embryo telencephalons

Glutaryl-CoA dehydrogenase deficiency (GDD) is characterized biochemically by an accumulation of glutaric (GA) and 3-hydroxyglutaric (3-OH-GA) acids and clinically by the development of acute striatal degeneration. 3-OH-GA was recently shown to induce neuronal damage via N-methyl-D-aspartate (NMDA) receptors. The pathogenetic role of GA, however, remains unclear. We demonstrate that GA exerts a dual action in cultured chick embryo neurons. Short-term incubation with millimolar concentrations of GA induces a weak neuronal damage, adding to 3-OH-GA neurotoxicity. In contrast, chronic treatment with subtoxic, micromolar concentrations of GA results in partial tolerance to 3-OH-GA- and NMDA-induced cell damage. A downregulation of NMDA receptors, in particular of the NR2B subunit, is critically involved in this GA-induced effect, resulting in a reduced Ca(2+) increase and generation of reactive oxygen species after acute exposure to NMDA or 3-OH-GA. Furthermore, GA decreases Na(+)/K(+)-ATPase activity, which is prevented by glutathione, suggesting a modulation of NMDA receptor function via resting membrane potential and Na(+)-dependent glutamate transport. In contrast, GA does not inhibit mitochondrial respiratory chain and beta-oxidation of fatty acids, virtually excluding an activation of NMDA receptors secondary to ATP depletion. These results strongly suggest that GA modulates the NMDA receptor-mediated neurotoxicity of 3-OH-GA, providing an explanatory basis for the non-linear relationship between organic acid concentrations and disease progression in GDD patients. Furthermore, GA-induced downregulation of NMDA receptors might be involved in the delayed cerebral maturation of GDD patients, resulting in frontotemporal atrophy and a reduced opercularization, which are common neuroradiological findings in GDD patients.     

A Na+/Ca2+ exchanger isoform, NCX1, is involved in retinal cell death after N-methyl-D-aspartate injection and ischemia-reperfusion

We investigated the expression of Na(+)/Ca(2+) exchanger (NCX) and the functional role of NCX in retinal damage by using NCX1-heterozygous deficient mice (NCX1(+/-)) and SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy] phenoxy]-5-ethoxyaniline), a selective NCX inhibitor in vivo. We also examined the role of NCX in oxygen-glucose deprivation (OGD) stress with a retinal ganglion cell line (RGC-5) cell culture in vitro. The expression of NCX1 was confirmed and entirely localized in retina by immunoblotting and immunohistochemistry, respectively. NCX1(+/-) mice possessed significant protection against retinal damage induced by intravitreal injection of N-methyl-D-aspartate (NMDA). SEA0400 at 3 and 10 mg/kg significantly reduced NMDA- or high intraocular pressure-induced retinal cell damage in mice. Furthermore, SEA0400 reduced the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling)-positive cells and the expression of phosphorylated mitogen-activated protein kinases (ERK1/2, JNK, p38) induced by NMDA injection. In RGC-5, SEA0400 at 0.3 and 1 microM significantly inhibited OGD-induced cell damage. OGD-induced cell damage was aggravated by ouabain (a Na(+),K(+)-ATPase inhibitor) at 100 microM, and this increased damage was significantly reduced by SEA0400 at 1 microM. In conclusion, these results suggest that NCX1 may play a role in retinal cell death induced by NMDA and ischemia-reperfusion.     

Inhibition of Na, K-ATPase activity in rat striatum by guanidinoacetate

The aim of this work was to investigate the effect of guanidinoacetate (GAA), the principal metabolite accumulating in guanidinoacetate methyltransferase (GAMT)-deficiency, on Na(+), K(+)-ATPase, Mg(2+)-ATPase and acetylcholinesterase (AChE) activities in striatum of young rats. We also studied the kinetics of the inhibition of Na(+), K(+)-ATPase activity caused by guanidinoacetate. Guanidinoacetate did not alter acetylcholinesterase and Mg(2+)-ATPase activities, but significantly inhibited Na(+), K(+)-ATPase activity. The apparent K(m) and V(max) of Na(+), K(+)-ATPase for ATP as substrate were 0.20mM and 0.82nmol inorganic phosphate (Pi) released per min per mg of protein, respectively. K(i) value was 7.18mM, and the inhibition was of the uncompetitive type. The results also showed a competition between guanidinoacetate and argininic acid (AA), suggesting a common binding site for the guanidino compounds (GC) on the enzyme. It is proposed that Na(+), K(+)-ATPase inhibition by guanidinoacetate may be one of the mechanisms involved in the neuronal dysfunction observed in GAMT-deficiency and in other diseases which accumulate guanidinoacetate.     

Na+,K+-ATPase concentration and fiber type distribution after spinal cord injury

Complete spinal cord injury (SCI) is characterized, in part, by reduced fatigue-resistance of the paralyzed skeletal muscle during stimulated contractions, but the underlying mechanisms are not fully understood. The effects of complete SCI on skeletal muscle Na(+),K(+)-adenosine triphosphatase (ATPase) concentration, and fiber type distribution were therefore investigated. Six individuals (aged 32.0 +/- 5.3 years) with complete paraplegia (T4-T10; 1-19 years since injury) participated. There was a significantly lower Na(+),K(+)-ATPase concentration in the paralyzed vastus lateralis (VL) when compared to either the subjects' own unaffected deltoid or literature values (from our laboratory, utilizing the same methodology) of VL Na(+),K(+)-ATPase concentration for the healthy able-bodied (141.6 +/- 50.0, 213.4 +/- 23.9, 339 +/- 16 pmol/g wet wt., respectively; P < 0.05). There was also a significant negative correlation between the Na(+),K(+)-ATPase concentration in the paralyzed VL and years since injury (r = -0.75, P < 0.05). These findings are clinically relevant as they suggest that reductions in Na(+),K(+)-ATPase contribute to the fatigability of paralyzed muscle after SCI. Unexpectedly, the VL muscles of our subjects had a higher proportion of their area represented by type I fibers compared to literature values for the VL of the healthy able-bodied (52.6 +/- 25.3% vs. 36 +/- 11.3%, respectively; P < 0.05). As all our subjects had upper motor neuron injuries and, therefore, experienced muscle spasticity, our findings warrant further investigation into the relationship between muscle spasticity and fiber type expression after SCI.     

NMDA receptor antagonism does not inhibit induction of ischemic tolerance in gerbil brainin vivo

Effects of high and moderate affinity uncompetitive NMDA receptor antagonists (+)MK-801 and memantine on ischemic tolerance were compared in relation to telemetrically controlled brain temperature. The tolerance to an injurious 3 min test of global forebrain ischemia in Mongolian gerbils was induced 48 h earlier by 2 min preconditioning ischemia. Normothermic preconditioning was virtually harmless, and greatly reduced neurodegeneration evoked by test ischemia. In hyperthermic animals it was injurious and failed to induce tolerance. Memantine (5 mg/kg) and (+)MK-801 (3 mg/kg) injected i.p. 1 h before preconditioning did not inhibit ischemic tolerance in the normothermic gerbils, while in hyperthermic animals treated with (+)MK-801 ischemic tolerance was partially restored. Subchronic 3 day infusion of memantine (30 mg/kg/day) significantly decreased neurodegeneration, and preconditioning in the normothermic gerbils further reduced neuronal damage. Hyperthermia exacerbated preconditioning ischemia and in this way reduced expression of tolerance, while (+)MK-801 partially reversed this effect. Our results do not confirm previous reports on the role of NMDA receptors in the induction of ischemic tolerance in gerbils.     

Calpains are downstream effectors of bax-dependent excitotoxic apoptosis

Excitotoxicity resulting from excessive Ca(2+) influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca(2+) levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that (1) underwent excitotoxic necrosis, characterized by immediate deregulation of Ca(2+) homeostasis, a persistent depolarization of mitochondrial membrane potential (Δψ(m)), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca(2+) increases, sensitivity to bax gene deletion, and delayed Δψ(m) depolarization and Ca(2+) deregulation, or (3) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis but did not influence excitotoxic necrosis. Calpeptin failed to exert a protective effect in bax-deficient neurons but protected bid-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy using a calpain-sensitive F?rster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injury. Oxygen/glucose deprivation-induced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during bax-dependent apoptosis and in this setting function as downstream cell-death executioners.     

Mechanisms underlying the honokiol inhibition of evoked glutamate release from glutamatergic nerve terminals of the rat cerebral cortex

The effect of honokiol, an active component of Magnolia officinalis, on glutamate release from isolated nerve terminals (synaptosomes) was examined. Honokiol potently inhibited 4-aminopyridine (4-AP)-evoked glutamate release in a concentration-dependent manner, and this effect resulted from a reduction of vesicular exocytosis and not from an inhibition of Ca(2+)-independent efflux via glutamate transporter. The inhibitory action of honokiol was not due to decreasing synaptosomal excitability or directly interfering with the release process at some point subsequent to Ca(2+) influx, because honokiol did not alter the 4-AP-evoked depolarization of the synaptosomal plasma membrane potential or Ca(2+) ionophore ionomycin-induced glutamate release. Rather, examination of the effect of honokiol on cytosolic [Ca(2+)] revealed that the diminution of glutamate release could be attributed to a reduction in voltage-dependent Ca(2+) influx. Consistent with this, the honokiol-mediated inhibition of 4-AP-evoked glutamate release was completely prevented in synaptosomes pretreated with a wide-spectrum blocker of N-, P-, and Q-type Ca(2+) channels, omega-conotoxin MVIIC. In addition, honokiol modulation of 4-AP-evoked glutamate release appeared to involve a protein kinase C (PKC) signaling cascade, in so far as pretreatment of synaptosomes with the PKC inhibitors Ro318220 or GF109203X all effectively occluded the inhibitory effect of honokiol. Furthermore, honokiol attenuated 4-AP-induced phosphorylation of PKC. Together, these results suggest that honokiol effects a decrease in PKC activation, which subsequently attenuates the Ca(2+) entry through voltage-dependent N- and P/Q-type Ca(2+) channels to cause a decrease in evoked glutamate exocytosis. These actions of honokiol may contribute to its neuroprotective effect in excitotoxic injury.     

Differential responses to NMDA receptor activation in rat hippocampal interneurons and pyramidal cells may underlie enhanced pyramidal cell vulnerability

Hippocampal interneurons are generally more resistant than pyramidal cells to excitotoxic insults. Because NMDA receptors play a crucial role in neurodegeneration, we have compared the response to exogenous NMDA in CA1 pyramidal cells and interneurons of the stratum oriens using combined whole-cell patch-clamp recording and ratiometric Ca2+ imaging. In voltage-clamp, current-clamp or in nominally Mg2+-free medium, NMDA (10 microM; 3-5 min exposure in the presence of tetrodotoxin) induced a markedly larger inward current and Ca2+ rise in pyramidal cells than in interneurons. Pyramidal cells also showed a more pronounced voltage dependence in their response to NMDA. We hypothesized that this enhanced response to NMDA receptor activation in pyramidal cells could underlie their increased vulnerability to excitotoxicity. Using loss of dye as an indicator of degenerative membrane disruption, interneurons tolerated continuous exposure to a high concentration of NMDA (30 microM) for longer periods than pyramidal cells. This acute neurodegeneration in pyramidal cells was independent of intracellular Ca2+, because high intracellular BAPTA (20 mM) did not prolong survival time. Thus, a plausible explanation for the enhanced sensitivity of pyramidal neurons to excitotoxic insults associated with cerebral ischemia is their greater response to NMDA receptor activation, which may reflect differences in NMDA receptor expression and/or subunit composition.     

Suppression of sodium pump activity and an increase in the intracellular Ca2+ concentration by dexamethasone in acidotic mouse brain

The effects of dexamethasone on adenosine 5'-triphosphatase (ATPase) activity and the intracellular Ca(2+) concentration ([Ca(2+)](i)) were investigated in acidotic mouse brain. Dexamethasone (3 mg/kg, i.p.) or vehicle was administered 3 h before decapitation ischemia, and the brain concentration of adenosine 5'-triphosphate (ATP) was determined 0.5-2 min after ischemia. The effects of dexamethasone (0.3-3 mg/kg, i.p.) on Na(+),K(+)-activated ATPase (Na(+),K(+)-ATPase) and Ca(2+)-ATPase activities were evaluated at pH 7.4 and 6.8. Changes in [Ca(2+)](i) in an acidic medium were determined in hippocampal slices by microfluorometry using rhod-2 acetoxymethyl ester as a Ca(2+) marker, and the effects of dexamethasone (240 microg/l) was evaluated. Decapitation ischemia for 0.5 and 1 min reduced the brain ATP contents to 32% and 16% of the basal level, respectively. Dexamethasone slightly suppressed the extent of the decrease in the ATP level. Although dexamethasone did not affect Na(+),K(+)-ATPase activity at pH 7.4, the activity was suppressed by dexamethasone (3 mg/kg) to 68% at pH 6.8. The activity of Ca(2+)-ATPase was not affected by dexamethasone at either pH 7.4 or pH 6.8. When the pH of the medium of the brain slices was changed from 7.4 to 6.8, almost no increase in [Ca(2+)](i) was observed in the control group. The dexamethasone treatment increased [Ca(2+)](i) in the CA1 field and dentate gyrus immediately after induction of the acidic medium, the effect being significant after 150 s. Because anaerobic glucose metabolism in the early stage of ischemia enhances intracellular lactic acidosis, the findings may suggest a mechanism for the aggravation of ischemic neuronal damage by glucocorticoids.     

The dichotomy of memantine treatment for ischemic stroke: dose-dependent protective and detrimental effects

Excitotoxicity is a major contributor to cell death during the acute phase of ischemic stroke but aggressive pharmacological targeting of excitotoxicity has failed clinically. Here we investigated whether pretreatment with low doses of memantine, within the range currently used and well tolerated for the treatment of Alzheimer''s disease, produce a protective effect in stroke. A coculture preparation exposed to modeled ischemia showed cell death associated with rapid glutamate rises and cytotoxic Ca²⁺ influx. Cell death was significantly enhanced in the presence of high memantine concentrations. However, low memantine concentrations significantly protected neurons and glia via excitotoxic cascade interruption. Mice were systemically administered a range of memantine doses (0.02, 0.2, 2, 10, and 20 mg/kg/day) starting 24 hours before 60 minutes reversible focal cerebral ischemia and continuing for a 48-hour recovery period. Low dose (0.2 mg/kg/day) memantine treatment significantly reduced lesion volume (by 30% to 50%) and improved behavioral outcomes in stroke lesions that had been separated into either small/striatal or large/striatocortical infarcts. However, higher doses of memantine (20 mg/kg/day) significantly increased injury. These results show that clinically established low doses of memantine should be considered for patients ‘at risk'' of stroke, while higher doses are contraindicated.     

Chronic memantine does not block 3-nitropropionic acid-delayed ischaemic tolerance in rat hippocampal slices<Emphasis Type="Italic">ex vivo</Emphasis>

The moderate affinity uncompetitive NMDA receptor antagonist memantine, at concentrations found to be neuroprotective in animal models of chronic excitotoxicity, did not reduce ischaemic tolerance induced chemically with 3 nitropropionic acid (3-NP), but actually tended to enhance this effectex vivo. Injection of 3-NP (20 mg/kg i.p.) — 24 h prior to thein vitro experiment — significantly protected against hypoxia/hypoglycaemia-induced suppression of extracellular field excitatory postsynaptic potentials (fEPSPs) in rat hippocampal slices (62.2% vs. control of 16.8%), whereas 3 days pretreatment with memantine (20 mg/kg/day —Alzet minipumps) tended to enhance recovery further following 3-NP preconditioning (89.7%). This low dose of memantine had no effect on fEPSPs in the absence of preconditioning. As expected, 3 days pretreatment with a high dose of (+)MK-801 (dizocilpine; 2mg/kg/day-Alzet minipumps) tended to reduce ischaemic tolerance following 3-NP preconditioning (45.3%). We conclude that although NMDA receptors do seem to be involved in chemically-induced ischaemic tolerance, semi-chronic pre-treatment with therapeutically-relevant doses of memantine does not block ischameic tolerance.     

A link between hyperbilirubinemia, oxidative stress and injury to neocortical synaptosomes

Cytotoxicity by unconjugated bilirubin involves disturbances of membrane structure, excitotoxicity and cell death. These events were reported to trigger elevated free radicals production and impairment of calcium homeostasis, and to result in loss of cell membrane integrity. Therefore, this study was designed to investigate whether interaction of clinically relevant concentrations of free unconjugated bilirubin with synaptosomal membrane vesicles could be linked to oxidative stress, cytosolic calcium accumulation and perturbation of membrane function. Synaptosomal vesicles were prepared from gerbil cortical brain tissue and incubated with purified bilirubin (or=10%, P<0.05) and protein oxidation (>or=20%, P<0.01), ROS content (approximately 17%, P<0.01), and a decrease in GSH/GSSG ratio (>30%, P<0.01). In addition, synaptosomes exposed to bilirubin exhibited increased externalization of phosphatidylserine (approximately 10%, P<0.05), together with decreased flippase and NA(+),K(+)-ATPase (>or=15%, P<0.05) activities, events that were accompanied by enhanced intracellular calcium levels ( approximately 20%, P<0.01). The data obtained point out that interaction of unconjugated bilirubin with synaptosomal membrane vesicles leads to oxidative injury, loss of membrane asymmetry and functionality, and calcium intrusion, thus potentially contributing to the pathogenesis of encephalopathy by hyperbilirubinemia.