Vulnerability of Medium Spiny Striatal Neurons to Glutamate: Role of Na+/K+ ATPase

In Huntington's disease neuronal degeneration mainly involves medium-sized spiny neurons. It has been postulated that both excitotoxic mechanisms and energy metabolism failure are implicated in the neuronal degeneration observed in Huntington's disease. In central neurons, >40% of the energy released by respiration is used by Na⁺/K⁺ ATPase to maintain ionic gradients. Considering that impairment of Na⁺/K⁺ ATPase activity might alter postsynaptic responsivity to excitatory amino acids (EAAs), we investigated the effects of the Na⁺/K⁺ ATPase inhibitors, ouabain and strophanthidin, on the responses to different agonists of EAA receptors in identified medium-sized spiny neurons electrophysiologically recorded in the current- and voltage-clamp modes. In most of the cells both ouabain and strophanthidin (1–3 μM) did not cause significant change in the membrane properties of the recorded neurons. Higher doses of either ouabain (30 μM) or strophanthidin (30 μM) induced, per se, an irreversible inward current coupled to an increase in conductance, leading to cell deterioration. Moreover, both ouabain (1–10 μM) and strophanthidin (1–10 μM) dramatically increased the membrane depolarization and the inward current produced by subcritical concentrations of glutamate, AMPA and NMDA. These concentrations of Na⁺/K⁺ ATPase inhibitors also increased the membrane responses induced by repetitive cortical activation. In addition, since it had previously been proposed that dopamine mimics the effects of Na⁺/K⁺ ATPase inhibitors and that dopamine agonists differentially regulate the postsynaptic responses to EAAs, we tested the possible modulation of EAA-induced membrane depolarization and inward current by dopamine agonists. Neither dopamine nor selective dopamine agonists or antagonists affected the postsynaptic responses to EAAs. Our experiments show that impairment of the activity of Na⁺/K⁺ ATPase may render striatal neurons more sensitive to the action of glutamate, lowering the threshold for the excitotoxic events. Our data support neither the role of dopamine as an ouabain-like agent nor the differential modulatory action of dopamine receptors on the EAA-induced responses in the striatum.     

Ciliary neurotrophic factor (CNTF) activation of astrocytes decreases spreading depolarization susceptibility and increases potassium clearance

Waves of spreading depolarization (SD) have been implicated in the progressive expansion of acute brain injuries. SD can persist over several days, coincident with the time course of astrocyte activation, but little is known about how astrocyte activation may influence SD susceptibility. We examined whether activation of astrocytes modified SD threshold in hippocampal slices. Injection of a lentiviral vector encoding Ciliary neurotrophic factor (CNTF) into the hippocampus in vivo, led to sustained astrocyte activation, verified by up‐regulation of glial fibrillary acidic protein (GFAP) at the mRNA and protein levels, as compared to controls injected with vector encoding LacZ. In acute brain slices from LacZ controls, localized 1M KCl microinjections invariably generated SD in CA1 hippocampus, but SD was never induced with this stimulus in CNTF tissues. No significant change in intrinsic excitability was observed in CA1 neurons, but excitatory synaptic transmission was significantly reduced in CNTF samples. mRNA levels of the predominantly astrocytic Na⁺/K⁺‐ATPase pump α2 subunit were higher in CNTF samples, and the kinetics of extracellular K⁺ transients during matched synaptic activation were consistent with increased K⁺ uptake in CNTF tissues. Supporting a role for the Na⁺/K⁺‐ATPase pump in increased SD threshold, ouabain, an inhibitor of the pump, was able to generate SD in CNTF tissues. These data support the hypothesis that activated astrocytes can limit SD onset via increased K⁺ clearance and suggest that therapeutic strategies targeting these glial cells could improve the outcome following acute brain injuries associated with SD. GLIA 2015;63:91–103     

Selective vascular permeability to digoxin and the inhibition of Na,K adenosine triphosphatase in the brain stem

The purpose of the present investigation was to provide evidence that the area postrema served as a portal of entry into the caudal medulla for cardiac glycosides. In [studies utilizing] young adult male Sprague-Dawley rats, indicator-dilution experiments suggested that digoxin, at cardiotoxic doses of 3 mg/kg, did not cross the blood-brain barrier. Assays of [³H]digoxin uptake, however, demonstrated significantly higher concentrations of digoxin in the brain stem compared with the cerebral hemisphere. In addition, microchemical assays of sodium-potassium activated adenosine triphosphatase (Na⁺,K⁺ATPase) activity, after intramuscular injection of digoxin (3 mg/kg), showed significant inhibition only in the brain stem. Finally, microchemical assays of Na⁺,K⁺ATPase activity were carried out in serial frozen sections of medulla in digoxin-treated rats. Only sections of medulla at the level of the area postrema showed significant inhibition of Na⁺,K⁺ATPase activity. Inhibition of Na⁺,K⁺ATPase activity has been used as a marker of cardiac glycoside binding to plasma membrane-bound proteins, and neurons possess a relatively high density of high-affinity receptors for cardiac glycosides with Na⁺,K⁺ATPase activity. Therefore, these studies suggest that the modified blood-brain barrier in area postrema serves as a portal of entry for cardiac glycosides into the caudal medulla, allowing them to bind to Na⁺,K⁺ATPase on the plasma membrane of neurons which, at least in part, play an important role in chronotropic regulation of cardiac function.     

Different mechanisms of Ca2+ regulation that influence synaptic transmission: Comparison between crayfish and Drosophila neuromuscular junctions

A brief historical background on synaptic transmission in relation to Ca²⁺ dynamics and short‐term facilitation is described. This study focuses on the mechanisms responsible for the regulation of intracellular calcium concentration ([Ca²⁺]_i) in high output terminals of larval Drosophila compared to a low‐output terminal of the crayfish neuromuscular junction (NMJ). Three processes; plasmalemmal Na⁺/Ca²⁺ exchanger [NCX], Ca²⁺‐ATPase (PMCA), and sarcoplasmic/endoplasmic Ca²⁺‐ATPase (SERCA) are important in regulating the [Ca²⁺]_i are examined. When the NCX is compromised by reduced [Na⁺]_o, no consistent effect occurred; but a NCX blocker KB‐R7943 decreased the excitatory postsynaptic potential (EPSP) amplitudes. Compromising the PMCA with pH 8.8 resulted in an increase in EPSP amplitude but treatment with a PMCA specific inhibitor carboxyeosin produced opposite results. Thapsigargin exposure to block the SERCA generally decreases EPSP amplitude. Compromising the activity of the above Ca²⁺ regulating proteins had no substantial effects on short‐term depression. The Kum^170TS strain (with dysfunctional SERCA), showed a decrease in EPSP amplitudes including the first EPSP within the train. Synaptic transmission is altered by reducing the function of the above three [Ca²⁺]_i regulators; but they are not consistent among different species as expected. Results in crayfish NMJ were more consistent with expected results as compared to the Drosophila NMJ. It is predicated that different mechanisms are used for regulating the [Ca²⁺]_i in high and low output synaptic terminals. Synapse 63:1100–1121, 2009. © 2009 Wiley‐Liss, Inc.     

N-methyl-d-aspartate and hypoxia induced Ca-changes in the CA1 region of the hippocampal slice

Hypoxic neuronal depolarization was accompanied by a large decrease in extracellular [Ca²⁺]. After reoxygenation, the time at which [Ca²⁺] normalized was correlated with the extent of recovery of N-methyl-d-aspartate (NMDA) and synaptic responses. There was no evidence that the NMDA receptor system was more disrupted following hypoxia than the receptors involved in synaptic transmission. The Na⁺/K⁺ pump appeared to be better able to recover from hypoxia than the NMDA responses or synaptic transmission.     

Energetic demands of the Na+/K+ ATPase in mammalian astrocytes

Cultured astrocytes and cell lines derived therefrom maintain a high energy level ([ATP]/[ADP]) through operation of oxidative phosphorylation and glycolysis. The contribution from the latter to total ATP production is 25–32%. A powerful Na⁺/K⁺ pump maintains potassium, sodium, and calcium gradients out of equilibrium. [Na⁺]_i is about 20 mM, [K⁺]_i is 130 mM and [Ca²⁺]_i is less than 100 nM. Under non-stimulated conditions, the Na⁺/K⁺ ATPase consumes 20% of astrocytic ATP production. Inhibition of the pump by ouabain decreases energy expenditure, raises [creatine phosphate]/[creatine], and leads to a leakage of sodium, potassium, and calcium ions. Decrease in the pump function via a fall in [ATP] also collapses ion gradients; the rate and extent of the fall correlates positively with cellular energy state. Under “normal” conditions (i.e., when ATP production pathways are not inhibited), there appears to be no preferential utilization of energy produced by either glycolysis or oxidative phosphorylation for the support of pump function. GLIA 21:35–45, 1997. © 1997 Wiley-Liss, Inc.     

Contributions of the Na+/K+‐ATPase,NKCC1, and Kir4.1 to hippocampal K+ clearance and volume responses

Network activity in the brain is associated with a transient increase in extracellular K⁺ concentration. The excess K⁺ is removed from the extracellular space by mechanisms proposed to involve Kir4.1‐mediated spatial buffering, the Na⁺/K⁺/2Cl^? cotransporter 1 (NKCC1), and/or Na⁺/K⁺‐ATPase activity. Their individual contribution to [K⁺]_o management has been of extended controversy. This study aimed, by several complementary approaches, to delineate the transport characteristics of Kir4.1, NKCC1, and Na⁺/K⁺‐ATPase and to resolve their involvement in clearance of extracellular K⁺ transients. Primary cultures of rat astrocytes displayed robust NKCC1 activity with [K⁺]_o increases above basal levels. Increased [K⁺]_o produced NKCC1‐mediated swelling of cultured astrocytes and NKCC1 could thereby potentially act as a mechanism of K⁺ clearance while concomitantly mediate the associated shrinkage of the extracellular space. In rat hippocampal slices, inhibition of NKCC1 failed to affect the rate of K⁺ removal from the extracellular space while Kir4.1 enacted its spatial buffering only during a local [K⁺]_o increase. In contrast, inhibition of the different isoforms of Na⁺/K⁺‐ATPase reduced post‐stimulus clearance of K⁺ transients. The astrocyte‐characteristic α2β2 subunit composition of Na⁺/K⁺‐ATPase, when expressed in Xenopus oocytes, displayed a K⁺ affinity and voltage‐sensitivity that would render this subunit composition specifically geared for controlling [K⁺]_o during neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na⁺/K⁺‐ATPase accounted for the stimulus‐induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity‐induced extracellular K⁺ recovery in native hippocampal tissue while Kir4.1 and Na⁺/K⁺‐ATPase serve temporally distinct roles. GLIA 2014;62:608–622     

ATPase and carbonic anhydrase activities of bulk-isolated neuron, glia and synaptosome fractions from rat brain

The (Na⁺ + K⁺) ATPase, carbonic anhydrase and HCO₃⁻-stimulated ATPase activities of bulk-isolated neuronal perikarya, glial and synaptosome fractions from 20–26-day-old rats were studied. The effects of varying K⁺, Na⁺ and ATP concentrations were investigated to determine limits on how these enzymes might respond to changes in the levels of these substances in vivo.The (Na⁺ + K⁺) ATPase activity of all three fractions had a similar high affinity for K⁺, withK_m values in the range of0.7–1.7mM. TheK_m for Na⁺ was around 10-fold higher, in the range of10–15mM.K_m values for ATP were also not markedly different between the different fractions, being1.2mM for the neurons and1.8mM for the glia and synaptosome fractions, respectively. The V_max at infinite [ATP] in the presence of10mM K⁺ was 2.9-fold higher for the glia as compared to the neuron fraction and 1.5-fold higher than the synaptosome fraction. At infinite K⁺ in the presence of3.3mM ATP the corresponding figures were 3.0 and 2.1. Arrhenius plots of (Na⁺ + K⁺) ATPase activity were different for the neuron and glia fractions as compared to the synaptosome fraction,, suggesting subtle differences in themembrane environment and/or the enzyme molecule itself.The HCO₃⁻-stimulated ATPase activity was only 14% higher in the glial fraction compared to the neurons and was variably stimulated by added K⁺ at concentrations< 10mM. The glial-enriched cell fraction had a 2-fold higher specific carbonic anhydrase activity than the neuron fraction, but the combined activity of these fractions only represented< 2% of the total brain activity. Total brain carbonic anhydrase activity was not stimulated by added K⁺ in the range of5–50mM K⁺.These data indicate that none of the enzymes studied are likely to directly respond to extracellular K⁺ levels in excess of10mM, and therefore would only be secondarily involved in cerebrocortical swelling caused by increasing K⁺ concentrations20mM. The relation of these findings to previous work on increased (Na⁺ + K⁺) ATPase and carbonic anhydrase activity in glial cells, the role of glial cells in passive or mediated transport processes related to increased levels of extracellular K⁺ and the suitability of bulk-isolated glia and neuron-enriched fractions as experimental models are discussed.     

Effect of hyperphenylalaninemia on Na + K-ATPase in rat brain

We examined the effect of experimental hyperphenylalaninemia in rats on Na⁺ + K⁺-ATPase activity in various subcellular fractions from brain. The hyperphenylalaninemia was induced by treatment with phenylalanine plus p-chlorophenylalanine. The data indicated that the activity of Na⁺ + K⁺-ATPase in mitochondria and synaptosomes from brains of hyperphenylalaninemic rats was significantly reduced. We suggest that the reduction in ATPase activity is related to the reduction in unsaturated fatty acid in brain phospholipids known to occur in experimental hyperphenylalaninemia. In view of the central role of ATPase activity in neuronal function, it is possible that the reduction in the ATPase activity may be implicated in the functional abnormalities observed in experimental hyperphenylalaninemia and perhaps in the genetic disease phenylketonuria in humans.     

Na+/K+‐ATPase coupled to endothelin receptor type B stimulates peripheral nerve regeneration via lactate signalling

We have recently demonstrated that endothelin (ET) is functionally coupled to Na_x, a Na⁺ concentration‐sensitive Na⁺ channel for lactate release via ET receptor type B (ET_BR) and is involved in peripheral nerve regeneration in a sciatic nerve transection–regeneration mouse model. Na_x is known to interact directly with Na⁺/K⁺‐ATPase, leading to lactate production in the brain. To investigate the role of Na⁺/K⁺‐ATPase in peripheral nerve regeneration, in this study, we applied ouabain, a Na⁺/K⁺‐ATPase inhibitor, to the cut site for 4 weeks with an osmotic pump. While functional recovery and nerve reinnervation to the toe started at 5 weeks after axotomy and were completed by 7 weeks, ouabain delayed them by 2 weeks. The delay by ouabain was improved by lactate, and its effect was blocked by α‐cyano‐4‐hydroxy‐cinnamic acid (CIN), a broad monocarboxylate transporter (MCT) inhibitor. In primary cultures of dorsal root ganglia, neurite outgrowth of neurons and lactate release into the culture medium was inhibited by ouabain. Conversely, lactate enhanced the neurite outgrowth, which was blocked by CIN, but not by AR‐C155858, a MCT1/2‐selective inhibitor. ET‐1 and ET‐3 increased neurite outgrowth of neurons, which was attenuated by an ET_BR antagonist, ouabain and 2 protein kinase C inhibitors. Taken together with the finding that ET_BR was expressed in Schwann cells, these results demonstrate that ET enhanced neurite outgrowth of neurons mediated by Na⁺/K⁺‐ATPase via ET_BR in Schwann cells. This study suggests that Na⁺/K⁺‐ATPase coupled to the ET‐ET_BR system plays a critical role in peripheral nerve regeneration via lactate signalling.     

Astrocyte‐mediated infantile‐onset leukoencephalopathy mouse model

Astrocytes have recently been shown to provide physiological support for various brain functions, although little is known about their involvement in white matter integrity. Several inherited infantile‐onset leukoencephalopathies, such as Alexander disease and megalencephalic leukoencephalopathy with subcortical cysts (MLC), implicate astrocytic involvement in the formation of white matter. Several mouse models of MLC had been generated by knocking out the Mlc1 gene; however, none of those models was reported to show myelin abnormalities prior to formation of the myelin sheath. Here we generated a new Mlc1 knockout mouse and a Mlc1 overexpressing mouse, and demonstrate that astrocyte‐specific Mlc1 overexpression causes infantile‐onset abnormalities of the white matter in which astrocytic swelling followed by myelin membrane splitting are present, whereas knocking out Mlc1 does not, and only shows myelin abnormalities after 12 months of age. Biochemical analyses demonstrated that MLC1 interacts with the Na⁺/K⁺ ATPase and that overexpression of Mlc1 results in decreased activity of the astrocytic Na⁺/K⁺ pump. In contrast, no changes in Na⁺/K⁺ pump activity were observed in Mlc1 KO mice, suggesting that the reduction in Na⁺/K⁺ pump activity resulting from Mlc1 overexpression causes astrocytic swelling. Our infantile‐onset leukoencephalopathy model based on Mlc1 overexpression may provide an opportunity to further explore the roles of astrocytes in white matter development and structural integrity. We established a novel mouse model for infantile‐onset leukoencephalopathy by the overexpression of Mlc1. Mlc1 overexpression reduced activity of the astrocytic sodium pump, which may underlie white matter edema followed by myelin membrane splitting. GLIA 2016 GLIA 2017;65:150–168     

Na+, K+-ATPase and ouabain binding activities of chick embryo dorsal root ganglia supported by and deprived of nerve growth factor

Recently we have shown that nerve growth factor (NGF) influences the coupled movements of Na⁺ and K⁺ across the membrane of chick embryo dorsal root ganglion (DRG) and other target cells. These ionic effects of NGF are consistent with a model in which NGF acts through the classical Na⁺,K⁺-ATPase pump. Direct evidence for NGF-induced alteration of this pump has been sought in the present study through two approaches. In one approach, DRG cell dissociates were incubated for 6 hours without NGF (to allow development of the ionic defect), and [³H]ouabain binding to the cells was measured before and during a delayed administration of NGF. No differences were detected in either total binding or binding time. In the second approach, intact DRG or DRG dissociates were incubated for 6 hours with or without NGF, or received NGF after 6 hours of deprivation, and Na⁺,K⁺-ATPase (ouabain-sensitive) activity was measured in the corresponding microsomal preparations. Activity levels were found to be the same in all cases, and were unchanged by addition of NGF directly to the enzyme preparations. Different concentrations of Na⁺, K⁺, or ATP affected in identical manners the enzyme preparations from NGF-treated and NGF-deprived ganglia, speaking against NGF-imposed changes in the affinities of the corresponding enzyme sites. Also unsuccessful were attempts to reveal NGF-related differences by testing ouabain-sensitive ATPase activity (1) in the presence of varying concentrations of cyclic AMP or of Ca²⁺, (2) after treatment with Triton X--100 or in the presence of vanadate, or (3) on addition of a 100,000g DRG extract. These negative findings are discussed in terms of the Na⁺,K⁺-ATPase hypothesis for NGF action.     

Characterization of neuromuscular synapse function abnormalities in multiple Duchenne muscular dystrophy mouse models

Duchenne muscular dystrophy (DMD) is an X‐linked myopathy caused by dystrophin deficiency. Dystrophin is present intracellularly at the sarcolemma, connecting actin to the dystrophin‐associated glycoprotein complex. Interestingly, it is enriched postsynaptically at the neuromuscular junction (NMJ), but its synaptic function is largely unknown. Utrophin, a dystrophin homologue, is also concentrated at the NMJ, and upregulated in DMD. It is possible that the absence of dystrophin at NMJs in DMD causes neuromuscular transmission defects that aggravate muscle weakness. We studied NMJ function in mdx mice (lacking dystrophin) and wild type mice. In addition, mdx/utrn^+/? and mdx/utrn^?/? mice (lacking utrophin) were used to investigate influences of utrophin levels. The three Duchenne mouse models showed muscle weakness when comparatively tested in vivo, with mdx/utrn^?/? mice being weakest. Ex vivo muscle contraction and electrophysiological studies showed a reduced safety factor of neuromuscular transmission in all models. NMJs had ~ 40% smaller miniature endplate potential amplitudes compared with wild type, indicating postsynaptic sensitivity loss for the neurotransmitter acetylcholine. However, nerve stimulation‐evoked endplate potential amplitudes were unchanged. Consequently, quantal content (i.e. the number of acetylcholine quanta released per nerve impulse) was considerably increased. Such a homeostatic compensatory increase in neurotransmitter release is also found at NMJs in myasthenia gravis, where autoantibodies reduce acetylcholine receptors. However, high‐rate nerve stimulation induced exaggerated endplate potential rundown. Study of NMJ morphology showed that fragmentation of acetylcholine receptor clusters occurred in all models, being most severe in mdx/utrn^?/? mice. Overall, we showed mild ‘myasthenia‐like’ neuromuscular synaptic dysfunction in several Duchenne mouse models, which possibly affects muscle weakness and degeneration.     

Activation of α-adrenoceptors indirectly facilitates sodium pumping in frog motoneurons

The effects of clonidine on Na⁺ pumping in motoneurons of the isolated frog spinal cord was investigated using sucrose gap recordings from ventral roots. Na⁺ pump activity, induced in motoneurons either by tetanizing the dorsal root or by rapidly exposing the cord to normal medium following 30 min in K⁺-free Ringer's solution (K⁺-activated hyperpolarizations), was increased by application of clonidine (100 μM). These actions of clonidine were blocked by the preferential α₂-adrenergic antagonist yohimbine, but not by α₁-adrenergic antagonist prazosin or the β-blocker propranolol. Clonidine's effects on Na⁺ pumping appeared to be indirect (presumably via interneurons) because its effects on K⁺-activated hyperpolarizations were reduced by tetrodotoxin (TTX) or high concentrations of Mg²⁺. This indirect mechanism involved activation of non-NMDA excitatory amino acid receptors. Thus, in the presence of clonidine, CNQX, but not APH, limited the ability of clonidine to enhance K⁺-activated hyperpolarizations. The AMPA receptor may play a role in the process. K⁺-activated hyperpolarizations were augmented by the presence of AMPA; NMDA had no effect. The present results are consistent with the idea that activation of α₂-adrenoceptors produces the following: the release of excitatory amino acids from interneurons; the activation of non-NMDA receptors on motoneurons; increased Na⁺ influx and loading and increased Na⁺ pump activity.     

Changes of the Na/K ATPase activity in the cerebral cortical microvessels of rat after single intraperitoneal administration of mercuric chloride: histochemical demonstration with light and electron microscopy

Summary Since inorganic mercury salts only poorly penetrate the cerebral microvascular endothelial cells comprising the blood-brain barrier (BBB), their neurotoxicity may be predicted to result from interference with BBB transport enzymes. In the present study, we tested the effect of mercuric chloride (HgCl₂) on Na⁺/K⁺ ATPase activity, a key enzyme involved in the ion transport in and out of the brain. Routine histochemical staining in conjunction with light and electron microscopy was used to evaluate the changes in the Na⁺/K⁺ ATPase activity in cerebral cortical microvesels of rats who received a single intraperitoneal injection of 6 mg/kg HgCl₂. At 1 h after HgCl₂ administration, light microscopy revealed uniform reduction of the Na⁺/K⁺ ATPase reaction in all cortical layers. Electron microscopy confirmed the enzyme reaction to be very weak to completely absent in both the luminal and abluminal endothelial cell membranes, and the luminal plasmalemma showed invaginations and pinocytic vesicles indicative of changes in its transport functions. The enzyme inhibition coincided with, and was likely to contribute to, profound perivascular swelling, involving mainly the astrocytic endfeet. The enzyme activity showed a partial recovery 18 h after HgCl₂ treatment, mainly in cortical layers II and III. After 5 days, the recovery of the enzyme activity appeared complete as observed by light and electron microscopy. The recovery of the microvascular Na⁺/K⁺ ATPase coincided with the appearance of a strongly positive Na⁺/K⁺ ATPase reaction in the adjacent astrocytic processes and with the diminution of perivascular swelling.Supported by the Medical Research Centre Project no. 7     

Taurine binding to membranes from rat brain regions

[³H]-taurine binding to membranes from different regions from rat brain was studied. Binding to membranes from cerebral cortex and its subcellular fractions, hypothalamus, olfactory bulb and cerebellum was measured. Binding to membranes from dorsal root ganglion was also determined. Na⁺-dependent taurine binding was consistently observed in all the membranes except those from dorsal root ganglion. A K_D = 4.06 μM was obtained for binding to membranes from cerebral cortex. Na⁺-dependent taurine binding was displaced by 20 μM strychnine or bicuculline. Na⁺-independent taurine binding with properties corresponding to a postsynaptic interaction could not be detected in any of the regions studied. The possibility of Na⁺-dependent taurine binding, representing binding to uptake sites or to postsynaptic receptors for GABA and glycine, is discussed.     

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, lowaffinity site parameters were affected by both ischemia and ischemia followed by reperfusion. Maximal velocity (V _max) decreased by 43 and 42% after ischemia and after ischemia/reperfusion, respectively. The apparentK _m 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⁺ (K _a K⁺) increased by 60% after ischemia and by 146% after ischemia/reperfusion. On the other hand, we found thatK _aNa⁺ decreased significantly after ischemia/reperfusion (16%). We concluded that it is the dephosphorylation step of the ATPase reation 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.     

AMPA/kainate receptor activation blocks K+ currents via internal Na+ increase in mouse cultured stellate astrocytes

Cultured mouse cortical astrocytes of the stellate type were studied by using the patch-clamp technique in whole-cell configuration. The astrocytes express at least two types of outwardly rectifying K⁺ channels which mediate a transient and a sustained current. Activation of AMPA receptors by kainate leads to a substantial blockade of both types of K⁺ currents. The blockade is absent when Na⁺ is withdrawn from the external medium, suggesting that it is caused by constant Na⁺ influx through AMPA receptors. The presence of high Na⁺ solutions in the pipette induces a blockade of both K⁺ currents which is very similar to the blockade induced by kainate, supporting thus the view that the mechanism of the blockade of K⁺ channels by kainate involves Na⁺ increases in the submembrane area. The blockade occurs between 20 and 40 mM [Na⁺]_i, which is within the physiological range of [Na⁺]_i in astrocytes. The data may suggest that the blockade of K⁺ channels by high [Na⁺]_i conditions could provide a mechanism to prevent K⁺ leakage from the astrocytes into the extracellular space during periods of intense neuronal activity. GLIA 20:38-50, 1997. © 1997 Wiley-Liss, Inc.