Study on ATP concentration changes in cytosol of individual cultured neurons during glutamate-induced deregulation of calcium homeostasis.

For the first time, simultaneous monitoring of changes in the concentration of cytosolic ATP ([ATP]c), pH (pHc), and intracellular free Ca2+ concentration ([Ca2+]i) of the individual neurons challenged with toxic glutamate (Glu) concentrations was performed. To this end, the ATP-sensor AT1.03, which binds to ATP and therefore enhances the efficiency of resonance energy transfer between blue fluorescent protein (energy donor) and yellow-green fluorescent protein (energy acceptor), was expressed in cultured hippocampal neurons isolated from 1-2-day-old rat pups. Excitation of fluorescence in the acceptor protein allowed monitoring changes in pHc. Cells were loaded with fluorescent low-affinity Ca2+ indicators Fura-FF or X-rhod-FF to register [Ca2+]i. It was shown that Glu (20 µM, glycine 10 µM, Mg2+-free) produced a rapid acidification of the cytosol and decrease in [ATP]c. An approximately linear relationship (r(2) = 0.56) between the rate of [ATP]c decline and latency of glutamate-induced delayed calcium deregulation (DCD) was observed: higher rate of [ATP]c decrease corresponded to shorter DCD latency period. DCD began with a decrease in [ATP]c of as much as 15.9%. In the phase of high [Ca2+]i, the plateau of [ATP]c dropped to 10.4% compared to [ATP]c in resting neurons (100%). In the presence of the Na+/K+-ATPase inhibitor ouabain (0.5 mM), glutamate-induced reduction in [ATP]c in the phase of the high [Ca2+]i plateau was only 36.6%. Changes in [ATP]c, [Ca2+]i, mitochondrial potential, and pHc in calcium-free or sodium-free buffers, as well as in the presence of the inhibitor of Na+/K+-ATPase ouabain (0.5 mM), led us to suggest that in addition to increase in proton conductivity and decline in [ATP]c, one of the triggering factors of DCD might be a reversion of the neuronal plasma membrane Na+/Ca2+ exchange.

[Mathematical modelling of delayed calcium deregulation in brain neurons caused by hyperstimulation of glutamate receptors].

Mathematical model presented in this paper is based on the data obtained in experimental studies of the mechanism of glutamate-induced deregulation of Ca2+ homeostasis and mitochondrial depolarization in cultured nerve cells. According to our hypothesis the secondary Ca2+ increase during a prolonged glutamate challenge is mainly due to a profound mitochondrial depolarization which stops mitochondrial Ca2+ uptake in the face of continuous Ca2+ influx into the cell. It is supposed that a progressive decrease in mitochondrial NADH during glutamate exposure greatly enhances sensitivity of mitochondria to intracellular Ca-neurotoxicity. A system of equations developed in this work made it possible to provide a satisfactory simulation of most of experimental events observed in the present study.

Role of protein kinase C in Ca(2+) homeostasis disorders in cultured rat neurons during hyperstimulation of glutamate receptors.

The primary culture of rat cerebellar neurons was used to study protein kinase C activity, intracellular variations in calcium concentration ([Ca(2+)]i), changes in the mitochondrial potential, and neuronal death during hyperstimulation of glutamate receptors and after 24-h incubation with phorbol ester. Prolonged exposure of neurons to glutamate (100 microM, 45 min) was followed by the development of delayed calcium dysregulation. Protein kinase C activity depended on the time of cell incubation with glutamate. Protein kinase C activity increased in response to application of glutamate for 15 min. However, protein kinase C activity decreased after 45-min exposure to glutamate and development of delayed calcium dysregulation. Protein kinase C activity was nearly undetected after 24-h preincubation of neurons with phorbol ester. Under these conditions, delayed calcium dysregulation developed more slowly and was observed in a smaller number of neurons. Neuronal death decreased to 2+/-1%. Our results suggest that protein kinase C plays an important role in death of neurons, which exhibit delayed calcium dysregulation during glutamate treatment.

Changes in ATP content in cerebellar granule cells during hyperstimulation of glutamate receptors: possible role of NO and nitrite ions.

In primary 7-8-day culture of cerebellar granule cells, glutamate exposure (100 microM, 10-240 min) induced a 60-30% drop in ATP level; during the postglutamate period ATP level completely recovered after 24 h. Inhibition of NO-synthase with L-NAME during glutamate application resulted in less pronounced decrease in ATP level immediately after its application and had no effect on ATP recovery after 24 h. It was found that hyperstimulation of glutamate receptors elevates concentration of NO products (nitrites and nitrates), while NO2(-) ions can increase ATP content.

Arachidonic acid enhances intracellular [Ca2+]i increase and mitochondrial depolarization induced by glutamate in cerebellar granule cells.

Maturation of primary neuronal cultures is accompanied by an increase in the proportion of cells that exhibit biphasic increase in free cytoplasmic Ca2+ ([Ca2+]i) followed by synchronic decrease in electrical potential difference across the inner mitochondrial membrane (DeltaPsim) in response to stimulation of glutamate receptors. In the present study we have examined whether the appearance of the second phase of [Ca2+]i change can be attributed to arachidonic acid (AA) release in response to the effect of glutamate (Glu) on neurons. Using primary culture of rat cerebellar granule cells we have investigated the effect of AA (1-20 microM) on [Ca2+]i, DeltaPsim, and [ATP] and changes in these parameters induced by neurotoxic concentrations of Glu (100 microM, 10-40 min). At =10 microM, AA caused insignificant decrease in DeltaPsim without any influence on [Ca2+]i. The mitochondrial ATPase inhibitor oligomycin enhanced AA-induced decrease in DeltaPsim; this suggests that AA may inhibit mitochondrial respiration. Addition of AA during the treatment with Glu resulted in more pronounced augmentation of [Ca2+]i and the decrease in DeltaPsim than the changes in these parameters observed during independent action of AA; removal of Glu did not abolish these changes. An inhibitor of the cyclooxygenase and lipoxygenase pathways of AA metabolism, 5,8,11,14-eicosatetraynoic acid, increased the proportion of neurons characterized by Glu-induced biphasic increase in [Ca2+]i and the decrease in DeltaPsim. Palmitic acid (30 microM) did not increase the percentage of neurons exhibiting biphasic response to Glu. Co-administration of AA and Glu caused 2-3 times more pronounced decrease in ATP concentrations than that observed during the independent effect of AA and Glu. The data suggest that AA may influence the functional state of mitochondria, and these changes may promote biphasic [Ca2+]i and DeltaPsim responses of neurons to the neurotoxic effect of Glu.

The leading role of membrane Ca(2+)-ATPase in recovery of Ca(2+) homeostasis after glutamate shock.

Combined blockade of Na(+)/Ca(2+) exchange, Ca(2+) uptake by mitochondria and endoplasmic reticulum usually does not prevent recovery of the basal level of intracellular Ca(2+) after 1-min action of glutamate (100 microM) or K(+) (50 mM). However, replacement of Ca(2+) with Ba(2+), which cannot be transported by Ca(2+)-ATPase, considerably delayed the decrease in intracellular Ba(2+) after its rise caused by glutamate or potassium application in all examined cells, which attest to an important role of Ca(2+)-ATPase in Ca(2+) extrusion after the action of glutamate or K(+).

The leading role of mitochondrial depolarization in the mechanism of glutamate-induced disruptions in Ca2+ homeostasis.

Data obtained in studies of the nature of the correlation which we have previously observed [10,17] between mitochondrial depolarization and the level of disruption of Ca2+ homeostasis in cultivated brain neuronsare summarized. Experiments were performed on cultured cerebellar granule cells loaded with Fura-2-AM or rhodamine 123 to measure changes in cytoplasmic Ca2+ and mitochondrial potential during pathogenic treatments of the cells. Prolonged exposure to 100 microM glutamate induced a reversible increase in [Ca2+]i, which was accompanied by only a small degree of mitochondrial depolarization. A sharp increase in this mitochondrial depolarization, induced by addition of 3 mM NaCN or 300 microM dinitrophenol (DNP) to the glutamate-containing solution, resulted in further increase in [Ca2+]i, due to blockade of electrophoretic mitochondrial Ca2+ uptake. Prolonged exposure to CN- or DNP in the post-glutamate period maintained [Ca2+]i at a high level until the metabolic inhibitors were removed. In most cells, this plateau was characterized by low sensitivity to removal of external Ca2+, demonstrating that the mechanisms of Ca2+ release from neurons were disrupted. Addition of oligomycin, a blocker of mitochondrial ATP synthase/ATPase, to the solution containing glutamate and CN- or DNP eliminated the post-glutamate plateau. Parallel experiments with direct measurements of intracellular ATP levels ([ATP]) showed that profound mitochondrial depolarization induced by CN- or DNP sharply enhanced the drop in ATP due to glutamate, while oligomycin significantly weakened this effect of the metabolic inhibitors. Analysis of these data led to the conclusion that blockade of mitochondrial Ca2+ uptake and inhibition of ATP synthesis resulted from mitochondrial depolarization and plays a key role in the mechanism disrupting [Ca2+]i homeostasis after toxic exposure to glutamate.

Blocker studies of the functional architecture of the NMDA receptor channel.

Blockade of ion channels passing through the NMDA receptors of isolated rat hippocampus pyramidal neurons with tetraalkylammonium compounds, 9-aminoacridine, and Mg2+ was studied using patch-clamp methods in the whole-cell configuration. Currents through NMDA channels were evoked by application of 100 microM aspartate in magnesium-free medium containing glycine (3 microM) to neurons. Analysis of the kinetics, charge transfer, and relationships between the extent of suppression of stationary currents on the one hand and membrane potential, agonist concentration, and blocker concentration on the other showed that blockers had different effects on the closing, desensitization, and agonist dissociation of NMDA channels. The size of the blocker was found to be the decisive factor determining its action on the gating functions of NMDA channels: larger blockers prevented closure and/or desensitization of the channel; smaller blockers only had partial effects on these processes, while the smallest blockers had no effect at all. These experiments showed that the apparent affinity of the blocker for the channel (1/IC50) depended not only on the microscopic equilibrium dissociation constant (Kd), but also on the number of blocker binding sites, their mutual influences, and, of particular importance, the interaction of the blocker with the gating structures of the channel. These data led us to propose hypotheses relating to the geometry of the NMDA channel and the structure of its gating mechanism. The channel diameter at the level of activated gates was estimated to be 11 A.

[Effect of the potential antiparkinsonian agent adamantane derivative on ion channels of NMDA glutamate receptors]. / Vzaimodeistvie potentsial'nogo protivoparkinsonicheskogo sredstva proizvodnogo adamantana s ionnymi kanalami glutamatnykh retseptorov NMDA podtipa.

Interaction of a new potential antiparkinsonian drug, N-(2-adamantyl)hexamethyleneimine hydrochloride (A-7), with NMDA channels in acutely isolated rat hippocampal neuron culture was studied by the whole-cell patch-clamp technique. The currents through the NMDA channels were excited by aspartate (Asp) application in a magnesium-free glycine-containing (3 microM) medium. It was found that A-7 produced a concentration-dependent (IC50 = 11.8 +/- 0.6 microM) NMDA channel blocking. The blocking rate increased with the A-7 concentration, whereas the unblocking was concentration-independent. The degree of blocking was independent of the Asp concentration, but was markedly increased by hyperpolarization of the cell membrane. After Asp washout, A-7 remained trapped in the channel by the activation gate. The channel was unblocked upon the next Asp application. These data is evidence that the antiparkinsonian effect of A-7 is related to the NMDA-channel-blocking activity of the drug.

[The leading role of mitochondrial depolarization in the mechanism of glutamate-induced disorder in Ca(2+)-homeostasis]. / Vedushchaia rol' mitokhondrial'noi depoliarizatsii v mekhanizme glutamat-vyzvannogo narusheniia Ca(2+)-gomeostaza.

Digital fluorescence imaging techniques were employed to monitor changes in the cytoplasmic Ca2+ concentration and mitochondrial potential in fura-2 AM or rhodamine-123 loaded individual cerebellar granule cells during and following the Glu exposure. The data obtained suggests that the MD-induced blockade of the mitochondrial Ca2+ uptake and a reversal of the mitochondrial ATP-synthase play a critical role in the mechanism of the glutamate-induced disorder of neuronal Ca2+ homeostasis.

Exploration of the role of reactive oxygen species in glutamate neurotoxicity in rat hippocampal neurones in culture.

1. Exposure of hippocampal neurones to glutamate at toxic levels is associated with a profound collapse of mitochondrial potential and deregulation of calcium homeostasis. We have explored the contributions of reactive oxygen species (ROS) to these events, considered to represent the first steps in the progression to cell death. 2. Digital imaging techniques were used to monitor changes in cytosolic Ca2+ concentration ([Ca2+]c; fura-2FF) and mitochondrial potential (Deltapsim; rhodamine 123); rates of ROS generation were assessed using hydroethidium (HEt); and membrane currents were measured with the whole-cell configuration of the patch clamp technique. 3. Inhibitors of lipid peroxidation (trolox plus ascorbate) and scavengers of superoxide or hydrogen peroxide (manganese(III) tetrakis(4-benzoic acid) porphyrin (MnTBAP) and TEMPO plus catalase), had only minimal impact on the mitochondrial depolarisation and the sustained increase in [Ca2+]c during and following a 10 min exposure to glutamate. 4. The antioxidants completely suppressed ROS generated by xanthine with xanthine oxidase. No significant increase in ROS production was detected with HEt during a 10 min glutamate exposure. 5. A combination of antioxidants (TEMPO, catalase, trolox and ascorbate) delayed but did not prevent the glutamate-induced mitochondrial depolarisation and the secondary [Ca2+]c rise. However, this was attributable to a transient inhibition of the NMDA current by the antioxidants. 6. Despite their inability to attenuate the glutamate-induced collapse of Deltapsim and destabilisation of [Ca2+]c homeostasis, the antioxidants conferred significant protection in assays of cell viability at 24 h after a 10 min excitotoxic challenge. The data obtained suggest that antioxidants exert their protective effect against glutamate-induced neuronal death through steps downstream of a sustained increase in [Ca2+]c associated with the collapse of Deltapsi(m).

Mechanisms of destabilization of Ca2+-homeostasis of brain neurons caused by toxic glutamate challenge.

The long-term stimulation of mammalian central neurons with an excitatory neuromediator, glutamate, results in destabilization of Ca2+-homeostasis caused mainly by an impairment of the systems of excessive Ca2+ extrusion from the cytoplasm both into the environment (Na+/Ca2+-exchanger, Ca2+/H+ pump) and mitochondria. The data available suggest that inhibition of the mitochondrial Ca2+ uptake following the glutamate action is due to the strong depolarization of inner mitochondrial membrane caused by opening of the "large pore" in response to the Ca2+ overload and overproduction of free oxygen radicals and NO. The mechanism of deterioration of Ca2+ extrusion from the neuron into extracellular medium following the glutamate challenge has not been yet fully clarified. It is only known that some factors inhibiting or irreversibly altering the functions of Na+/Ca2+-exchanger and Ca2+/H+ pump are accumulated in the cell during the prolonged action of glutamate. They include lowering of ATP concentration and pHi, as well as overproduction of free oxygen radicals and products of lipid peroxidation. The exact contribution of these factors to the final destabilization of Ca2+ homeostasis is under study. A good correlation between the glutamate-induced mitochondrial depolarization and the failure of neurons to extrude excessive Ca2+ from the cytoplasm during the post-glutamate period indicates that at this period the mitochondrial dysfunction is critical for the destabilization of Ca2+ homeostasis.

[Study functional architecture of NMDA receptor channels with their blockers]. / Izuchenie funktsional'noi arkhitektury kanala NMDA-receptorov s pomoshch'iu blokatorov.

Blockade of ionic currents through NMDA receptor channels in acutely isolated rat hippocampal neurons by tetraalkylammonium compounds, 9-aminoacridine and Mg2+ was studied using whole-cell patch-clamp technique. The currents through NMDA channels were elicited by 100 microM aspartate application in a Mg(2+)-free 3 microM glycine-containing solution. An analysis of the kinetics, charge transfer and dependencies of the stationary current inhibition on the membrane potential and the agonist and the blocker concentrations showed that the blockers affect NMDA channel closure, desensitization and the agonist dissociation in different ways. The size of the blocker proved to be the determinant of the blocker action on the NMDA channel gating machinery: large blockers prevented the channel closure and/or desensitization, smaller ones only partly affected these processes, while the smallest did not affect at all. It was shown that the apparent blocker affinity to the channel, 1/IC50, depended not only on the microscopic dissociation constant, Kd, but also on the number of the blocker binding sites, their mutual dependence, and, which is much more important, on the blocker interaction with the channel gating machinery. Based upon the data obtained, there was advanced hypotheses on the NMDA channel geometry and the structure of its gating machinery. The diameter of the channel at the level of the activation gate was estimated as 11 A.

The mechanism of potentiation of the glutamate-induced neurotoxicity by serum albumin. A possible role of nitric oxide.

Potentiation of the delayed (Glu)-induced neurotoxicity by serum albumin (SA) was studied in experiments with cultured cerebellar granule cells. The delayed neuronal death (DND) was evaluated by counting neurons containing or excluding Trypan Blue 4 h after treatment with Glu. Cytoplasmic Ca2+ ([Ca2+]i) was measured in individual Fura-2-loaded neurons. It was shown that a 15-min application of bovine SA (4 mg/ml) together with Glu (100 microM, 10 microM glycine, Mg2+-free solution) enhanced DND in the culture 1.7 times (43.1+/-3.1%) with respect to the effect induced by Glu alone (24.6+/-0.6%). The bovine SA application did not change the dynamics of [Ca2+]i response during a short-term (1 min) and long-term (15 min) Glu-treatment. DND was prevented by simultaneous application of Glu and inhibitor of NO-synthase N omega-nitro-L-arginine methyl ester (L-NAME), 100 microM) (10.8+/-1.0%) as well as by the application of Glu with SA and L-NAME (9.8+/-1.2%). In order to evaluate the role of nitric oxide (NO) in the SA effect, the cells were incubated for 15 min with the NO-donors sodium nitroprusside (SNP, 10 and 100 microM) and sodium nitrite (NaNO2, 10 and 100 microM) together with SA and in its absence. SA also greatly enhanced the DND induced by SNP and NaNO2. Thus, the DND after simultaneous treatment with SA and SNP was 16.3+/-2.5% (10 microM) or 29.6+/-2.1% (100 microM), and 9.6+/-0.8% (10 microM) and 19.7+/-2.1% after treatment with SNP alone. Exposure to SA together with NaNO2 led to the DND increase up to 26.5+/-1.9% (10 microM) and 37.7+/-3.5% (100 microM) in comparison with 7.4+/-2.0% (10 microM) and 18.9+/-0.8% (100 microM) in experiments with NaNO2 alone. Taking into account the ability of NO and NO2 to oxidize unsaturated fatty acids and the ability of SA to bind them after their hydrolytic removal, we suggested that the SA-induced potentiation of Glu neurotoxicity resulted from exacerbation of the toxic effects of NO and other trace radicals on the neuronal membranes. This hypothesis was supported by the finding that SA also enhanced the neurotoxicity of the lipid prooxidant FeCl2. The simultaneous 15-min application of FeCl2 (10 microM) and SA caused a 51.5+/-4.0% increase in DND, which exceeded 2.4 times the effect produced by FeCl2 alone (21.3+/-2.3%).

Eosine-induced blockade of N-methyl-D-aspartate channels in acutely isolated rat hippocampal neurons.

Acutely isolated rat hippocampal neurons were voltage-clamped in the whole-cell configuration. The currents through N-methyl-D-aspartate (NMDA) channels were elicited by fast application of aspartate in a Mg(2+)-free 3 microM glycine-containing solution. Eosine, known as a potent reversible inhibitor of the plasma membrane Ca(2+) pump, proved to be able to induce a blockade of NMDA channels. The eosine-induced inhibition of NMDA-mediated currents enhanced with eosine concentration (IC(50) = 248 microM) but did not depend on the membrane potential, agonist (aspartate) or coagonist (glycine) concentrations, pH, or the presence of spermine, ethanol, and the disulfide-reducing agents dithiothreitol and glutathione. Zn(2+) inhibited NMDA channels with equal efficiency both in the presence and absence of eosine. These results suggest that eosine interacts with a new, previously unknown NMDA receptor regulatory site.

Probing of NMDA channels with fast blockers.

Using whole-cell patch-clamp techniques, we studied the interaction of open NMDA channels with tetraalkylammonium compounds: tetraethylammonium (TEA), tetrapropylammonium (TPA), tetrabutylammonium (TBA), and tetrapentylammonium (TPentA). Analysis of the blocking kinetics, concentration, and agonist dependencies using a set of kinetic models allowed us to create the criteria distinguishing the effects of these blockers on the channel closure, desensitization, and agonist dissociation. Thus, it was found that TPentA prohibited, TBA partly prevented, and TPA and TEA did not prevent either the channel closure or the agonist dissociation. TPentA and TBA prohibited, TPA slightly prevented, and TEA did not affect the channel desensitization. These data along with the voltage dependence of the stationary current inhibition led us to hypothesize that: (1) there are activation and desensitization gates in the NMDA channel; (2) these gates are distinct structures located in the external channel vestibule, the desensitization gate being located deeper than the activation gate. The size of the blocker plays a key role in its interaction with the NMDA channel gating machinery: small blockers (TEA and TPA) bind in the depth of the channel pore and permit the closure of both gates, whereas larger blockers (TBA) allow the closure of the activation gate but prohibit the closure of the desensitization gate; finally, the largest blockers (TPentA) prohibit the closure of both activation and desensitization gates. The mean diameter of the NMDA channel pore in the region of the activation gate localization was estimated to be approximately 11 A.

Blockade of NMDA channels in acutely isolated rat hippocampal neurons by the Na+/Ca2+ exchange inhibitor KB-R7943.

Neurons acutely isolated from the CA1 region of rat hippocampal slices using the 'vibrodissociation' method were voltage-clamped in the whole-cell configuration. The currents through NMDA channels were elicited by application of 100 microM aspartate (ASP) in a Mg2+-free solution in the presence of 3 microM glycine. The compound KB-R7943, (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) known as a Na+/Ca2+ exchange inhibitor was able to block effectively the NMDA channels. At a holding potential of -100 mV, the measurement of the concentration dependence of the stationary current blockade revealed the existence of two populations of NMDA channels differing by a high (IC50 = 0.8 microM) and low (IC50 = 11 microM) affinity for KB-R7943. The Hill coefficients indicated that one blocking molecule can bind to NMDA channels which have a high affinity for KB-R7943 and at least two blocking molecules can bind to the NMDA channels which have a low affinity for KB-R7943. When applied externally, KB-R7943 can bind to the low-affinity NMDA channels irrespective of whether or not these channels are activated by the agonist. The KB-R7943-induced blockade of the NMDA channel was partly voltage-dependent. Within the framework of the Woodhull model, the apparent value of delta calculated for the voltage-dependent binding of KB-R7943 was in the range of 0.26-0.41. The blocking action of KB-R7943 on NMDA channels did not depend either on ASP or glycine concentrations which indicated that the binding sites for KB-R7943 and those for the agonist and the coagonist did not overlap.

Glutamate-induced mitochondrial depolarisation and perturbation of calcium homeostasis in cultured rat hippocampal neurones.

1. The objective of this study was to clarify the relationships between loss of mitochondrial potential and the perturbation of neuronal Ca2+ homeostasis induced by a toxic glutamate challenge. Digital fluorescence imaging techniques were employed to monitor simultaneously changes in cytoplasmic Ca2+ concentration ([Ca2+]i) and mitochondrial potential (DeltaPsim) in individual hippocampal neurones in culture coloaded with fura-2 AM or fura-2FF AM and rhodamine 123 (Rh 123). 2. In most cells (96 %) at 6-7 days in vitro (DIV) and in a small proportion of cells (29 %) at 11-17 DIV the [Ca2+]i increase induced by exposure to 100 microM glutamate for 10 min was associated with a small mitochondrial depolarisation, followed by mitochondrial repolarisation, and a degree of recovery of [Ca2+]i following glutamate washout. In the majority of neurones at 11-17 DIV (71 %), exposure to glutamate for 10 min induced a profound mono- or biphasic mitochondrial depolarisation, which was clearly correlated with a sustained [Ca2+]i plateau despite the removal of glutamate. 3. Addition of glutamate receptor antagonists (15 microM MK-801 plus 75 microM 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX)) to the washout solution did not affect the post-glutamate [Ca2+]i plateau in neurones exhibiting a profound mitochondrial depolarisation but greatly improved [Ca2+]i recovery in those neurones undergoing only a small mitochondrial depolarisation, suggesting that the release of endogenous glutamate delays [Ca2+]i recovery in the postglutamate period. 4. Cyclosporin A (500 nM) or N-methyl Val-4-cyclosporin A (200 nM) delayed or even prevented the development of the second phase of mitochondrial depolarisation in cells at 11-17 DIV and increased the proportion of neurones exhibiting a small monophasic mitochondrial depolarisation and [Ca2+]i recovery upon glutamate removal. 5. We have thus described a striking correlation between mitochondrial depolarisation and the failure of cells to restore [Ca2+]i following a toxic glutamate challenge. These data suggest that mitochondrial dysfunction plays a major role in the deregulation of [Ca2+]i associated with glutamate toxicity.

Molecular size and hydrophobicity as factors which determine the efficacy of the blocking action of amino-adamantane derivatives on NMDA channels.

Neurons isolated from the CA-1 region of rat hippocampal slices by the "vibrodissociation" method were voltage-clamped in the whole cell configuration. The currents through NMDA channels were recorded in response to rapid application (solution exchange time <30 ms) of 100 microM aspartate (ASP) in a Mg2+-free solution in the presence of 3 microM glycine. When added to the ASP solution, amantadine as well as other amino-adamantane derivatives (AAD) produced an open-channel blockade of NMDA channels. Membrane hyperpolarization enhanced the AAD block. The affinity between NMDA channels and AAD was different for various AAD. The analysis of the experimental data led us to conclude that this affinity depended both on the molecular size of the blocker (calculated using HyperChem molecular modeling program) and on the blocker's hydrophobicity (calculated according to Hansch and Leo, 1979). The affinity between NMDA channels and AAD diminished with an increase in molecular size and raised with an increase in blocker's hydrophobicity. We propose an empirical equation which describes the dependence of affinity on the size and hydrophobicity of the blocker. The estimated critical diameter of the NMDA channel pore where the AAD blocking site is located proved to be about 17 A.

[Mechanisms of neuronal calcium homeostasis destabilization caused by hyperstimulation of glutamate receptors]. / Mekhanizmy destabilizatsii kal'tsievogo gomeostaza neirona pri giperstimuliatsii gliutamatnykh retseptorov.

The present paper summarizes the data obtained in studying the mechanisms of glutamate-induced deterioration of neuronal Ca2+ homeostasis. In the cultured mammalian central neurons, a short-term (< 1 min) glutamate (GLU, 100 mu) challenge is known to induce a readily reversible (transient) neuronal [Ca2+]i increase. In contrast, a long-term (15-30 min) GLU exposure leads to the appearance of high [Ca2+]i plateau or to the partial recovery of the increased [Ca2+]i. Experiments show that impaired [Ca2+]i recovery in the postglutamate period cannot be explained by the increased [Ca2+]i permeability of the neuronal membrane, as earlier considered. Moreover, a sustained elevation of [Ca2+]i during and after chronic GLU application is associated with a progressive decrease in Ca2+ permeability. The major cause of GLU-induced Ca2+ overload is the mitochondrial depolarization resulted from excessive Ca2+ influx into the mitochondria, the generation of free radicals and the opening of a "giant pore" in the inner mitochondrial membrane. This in turn suppresses both ATP synthesis and Ca2+ electrophoretic uptake into the mitochondrial matrix. In combination with [Ca2+]i-dependent acidification, this leads to the suppression of Ca2+ release from the cell via Na+/Ca2+ exchanger and Ca2+/H+ pump of the neuronal membrane. Therefore, [Ca2+]i recovery following a long-term GLU treatment becomes strongly or even irreversibly compromised.