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     

Gap junctions equalize intracellular Na+ concentration in astrocytes

Gap junctions between glial cells allow intercellular exchange of ions and small molecules. We have investigated the influence of gap junction coupling on regulation of intracellular Na⁺ concentration ([Na⁺]_i) in cultured rat hippocampal astrocytes, using fluorescence ratio imaging with the Na⁺ indicator dye SBFI (sodium-binding benzofuran isophthalate). The [Na⁺]_i in neighboring astrocytes was very similar (12.0 ± 3.3 mM) and did not fluctuate under resting conditions. During uncoupling of gap junctions with octanol (0.5 mM), baseline [Na⁺]_i was unaltered in 24%, increased in 54%, and decreased in 22% of cells. Qualitatively similar results were obtained with two other uncoupling agents, heptanol and α-glycyrrhetinic acid (AGA). Octanol did not alter the recovery from intracellular Na⁺ load induced by removal of extracellular K⁺, indicating that octanol's effects on baseline [Na⁺]_i were not due to inhibition of Na⁺, K⁺-ATPase activity. Under control conditions, increasing [K⁺]_o from 3 to 8 mM caused similar decreases in [Na⁺]_i in groups of astrocytes, presumably by stimulating Na⁺, K⁺-ATPase. During octanol application, [K⁺]_o-induced [Na⁺]_i decreases were amplified in cells with increased baseline [Na⁺]_i, and reduced in cells with decreased baseline [Na⁺]_i. This suggests that baseline [Na⁺]_i in astrocytes “sets” the responsiveness of Na⁺, K⁺-ATPase to increases in [K⁺]_o. Our results indicate that individual hippocampal astrocytes in culture rapidly develop different levels of baseline [Na⁺]_i when they are isolated from one another by uncoupling agents. In astrocytes, therefore, an apparent function of coupling is the intercellular exchange of Na⁺ ions to equalize baseline [Na⁺]_i, which serves to coordinate physiological responses that depend on the intracellular concentration of this ion. GLIA 20:299–307, 1997. © 1997 Wiley-Liss, Inc.     

Molecular mechanisms of K+ clearance and extracellular space shrinkage—Glia cells as the stars

Neuronal signaling in the central nervous system (CNS) associates with release of K⁺ into the extracellular space resulting in transient increases in [K⁺]_o. This elevated K⁺ is swiftly removed, in part, via uptake by neighboring glia cells. This process occurs in parallel to the [K⁺]_o elevation and glia cells thus act as K⁺ sinks during the neuronal activity, while releasing it at the termination of the pulse. The molecular transport mechanisms governing this glial K⁺ absorption remain a point of debate. Passive distribution of K⁺ via Kir4.1-mediated spatial buffering of K⁺ has become a favorite within the glial field, although evidence for a quantitatively significant contribution from this ion channel to K⁺ clearance from the extracellular space is sparse. The Na⁺/K⁺-ATPase, but not the Na⁺/K⁺/Cl⁻ cotransporter, NKCC1, shapes the activity-evoked K⁺ transient. The different isoform combinations of the Na⁺/K⁺-ATPase expressed in glia cells and neurons display different kinetic characteristics and are thereby distinctly geared toward their temporal and quantitative contribution to K⁺ clearance. The glia cell swelling occurring with the K⁺ transient was long assumed to be directly associated with K⁺ uptake and/or AQP4, although accumulating evidence suggests that they are not. Rather, activation of bicarbonate- and lactate transporters appear to lead to glial cell swelling via the activity-evoked alkaline transient, K⁺-mediated glial depolarization, and metabolic demand. This review covers evidence, or lack thereof, accumulated over the last half century on the molecular mechanisms supporting activity-evoked K⁺ and extracellular space dynamics.     

Astrocyte ERK phosphorylation precedes K+‐induced swelling but follows hypotonicity‐induced swelling

Hypotonicity following water intoxication and/or salt loss leads to mainly astrocytic brain swelling. Astrocytic swelling also occurs following brain trauma or ischemia, together with an increase in extracellular K⁺ ([K⁺]_o), stimulating a bumetanide/furosemide/ethacrynic acid‐inhibitable cotransporter, NKCC1, that accumulates Na⁺ and K⁺ together with 2 Cl^‐ and osmotically obliged water. Either type of swelling may become fatal and is associated with phosphorylation of extracellular regulated kinases 1 and 2 (ERK_1/2). Only the swelling associated with elevated [K⁺]_o, leads to an increase in astrocytic proliferation and in expression of the astrocytic marker, glial fibrillary acidic protein. These differences prompted us to investigate key aspects of the molecular pathways between hypotonicity‐induced and high‐K⁺‐mediated swelling in primary cultures of mouse astrocytes. In the latter Ca²⁺‐mediated, AG1478‐inhibitable transactivation of the epidermal growth factor (EGF) receptor leads, via bumetanide‐inhibitable activation of the mitogen activated protein (MAP) kinase pathway to ERK phosphorylation and to NKCC1‐mediated swelling. In the former, inhibition of the MAP kinase pathway, but not of EGF receptor activation, abolishes ERK phosphorylation, but has no effect on swelling, indicating that activation of ERK is a result, not a cause, of the swelling.     

Characteristics of activity-dependent potassium accumulation in mammalian peripheral nerve in vitro

Ion-sensitive microelectrodes were used to study the behavior of extracellular ions in rat sciatic nerve during and following activity. Nerve stimulation produced increases in [K⁺]_o that were dependent upon the frequency and duration of stimulation; no change in extracellular pH occurred with stimulation. Increases in [K⁺]_o dependent on axonal discharge since they were blocked by inhibiting sodium channels with tetrodotoxin. At 22°C, stimulation could induce increases in [K⁺]_o of several mM; at 36°C, stimulation rarely produced increases in [K⁺]_o greater than 1mM. Stimulated increases in [K⁺]_o dissipated very slowly (i.e. t_1/2 = 50–100s) and the rate of dissipation was not significantly affected by anoxia, changes in temperature, changes in extracellular pH, or the application of a blocker of Na⁺, K⁺-ATPase (ouabain) or a K⁺ channel blocker (Ba²⁺). In comparison to the central nervous system, neural activity in rat sciatic nerve produced smaller increases in [K⁺]_o and these increases dissipated much more slowly. The primary mechanism of K⁺ dissipation appeared to be diffusion, probably facilitated by the larger extracellular space in peripheral nerve compared to the central nervous system, but impeded by diffusion barriers imposed by the blood-nerve barrier.     

Activity‐dependent astrocyte swelling is mediated by pH‐regulating mechanisms

During neuronal activity in the mammalian brain, the K⁺ released into the synaptic space is initially buffered by the astrocytic compartment. In parallel, the extracellular space (ECS) shrinks, presumably due to astrocytic cell swelling. With the Na⁺/K⁺/2Cl^? cotransporter and the Kir4.1/AQP4 complex not required for the astrocytic cell swelling in the hippocampus, the molecular mechanisms underlying the activity‐dependent ECS shrinkage have remained unresolved. To identify these molecular mechanisms, we employed ion‐sensitive microelectrodes to measure changes in ECS, [K⁺]_o and [H⁺]_o/pH_o during electrical stimulation of rat hippocampal slices. Transporters and receptors responding directly to the K⁺ and glutamate released into the extracellular space (the K⁺/Cl^? cotransporter, KCC, glutamate transporters and G protein‐coupled receptors) did not modulate the extracellular space dynamics. The ‐transporting mechanism, which in astrocytes mainly constitutes the electrogenic Na⁺/ cotransporter 1 (NBCe1), is activated by the K⁺‐mediated depolarization of the astrocytic membrane. Inhibition of this transporter reduced the ECS shrinkage by ～25% without affecting the K⁺ transients, pointing to NBCe1 as a key contributor to the stimulus‐induced astrocytic cell swelling. Inhibition of the monocarboxylate cotransporters (MCT), like‐wise, reduced the ECS shrinkage by ～25% without compromising the K⁺ transients. Isosmotic reduction of extracellular Cl^? revealed a requirement for this ion in parts of the ECS shrinkage. Taken together, the stimulus‐evoked astrocytic cell swelling does not appear to occur as a direct effect of the K⁺ clearance, as earlier proposed, but partly via the pH‐regulating transport mechanisms activated by the K⁺‐induced astrocytic depolarization and the activity‐dependent metabolism.     

Regulatory volume increase in astrocytes exposed to hypertonic medium requires β1‐adrenergic Na+/K+‐ATPase stimulation and glycogenolysis

The cotransporter of Na⁺, K⁺, 2Cl^–, and water, NKKC1, is activated under two conditions in the brain, exposure to highly elevated extracellular K⁺ concentrations, causing astrocytic swelling, and regulatory volume increase in cells shrunk in response to exposure to hypertonic medium. NKCC1‐mediated transport occurs as secondary active transport driven by Na⁺/K⁺‐ATPase activity, which establishes a favorable ratio for NKCC1 operation between extracellular and intracellular products of the concentrations of Na⁺, K⁺, and Cl^– × Cl^–. In the adult brain, astrocytes are the main target for NKCC1 stimulation, and their Na⁺/K⁺‐ATPase activity is stimulated by elevated K⁺ or the β‐adrenergic agonist isoproterenol. Extracellular K⁺ concentration is normal during regulatory volume increase, so this study investigated whether the volume increase occurred faster in the presence of isoproterenol. Measurement of cell volume via live cell microscopic imaging fluorescence to record fluorescence intensity of calcein showed that this was the case at isoproterenol concentrations of ≥1 µM in well‐differentiated mouse astrocyte cultures incubated in isotonic medium with 100 mM sucrose added. This stimulation was abolished by the β₁‐adrenergic antagonist betaxolol, but not by ICI118551, a β₂‐adrenergic antagonist. A large part of the β₁‐adrenergic signaling pathway in astrocytes is known. Inhibitors of this pathway as well as the glycogenolysis inhibitor 1,4‐dideoxy‐1,4‐imino‐D‐arabinitol hydrochloride and the NKCC1 inhibitors bumetanide and furosemide abolished stimulation by isoproterenol, and it was weakened by the Na⁺/K⁺‐ATPase inhibitor ouabain. These observations are of physiological relevance because extracellular hypertonicity occurs during intense neuronal activity. This might trigger a regulatory volume increase, associated with the post‐excitatory undershoot. © 2014 Wiley Periodicals, Inc.     

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.     

Serotonin,norepinephrine, and acetylcholine differentially affect astrocytic potassium clearance to modulate somatosensory signaling in male mice

Changes in extracellular potassium ([K⁺]_e) modulate neuronal networks via changes in membrane potential, voltage-gated channel activity, and alteration to transmission at the synapse. Given the limited extracellular space in the central nervous system, potassium clearance is crucial. As activity-induced potassium transients are rapidly managed by astrocytic Kir4.1 and astrocyte-specific Na⁺/K⁺-ATPase, any neurotransmitter/neuromodulator that can regulate their function may have indirect influence on network activity. Neuromodulators differentially affect cortical/thalamic networks to align sensory processing with differing behavioral states. Given serotonin (5HT), norepinephrine (NE), and acetylcholine (ACh) differentially affect spike frequency adaptation and signal fidelity (“signal-to-noise”) in somatosensory cortex, we hypothesize that [K⁺]_e may be differentially regulated by the different neuromodulators to exert their individual effects on network function. This study aimed to compare effects of individually applied 5HT, NE, and ACh on regulating [K⁺]_e in connection to effects on cortical-evoked response amplitude and adaptation in male mice. Using extracellular field and K⁺ ion-selective recordings of somatosensory stimulation, we found that differential effects of 5HT, NE, and ACh on [K⁺]_e regulation mirrored differential effects on amplitude and adaptation. 5HT effects on transient K⁺ recovery, adaptation, and field post-synaptic potential amplitude were disrupted by barium (200 µM), whereas NE and ACh effects were disrupted by ouabain (1 µM) or iodoacetate (100 µM). Considering the impact [K⁺]_e can have on many network functions; it seems highly efficient that neuromodulators regulate [K⁺]_e to exert their many effects. This study provides functional significance for astrocyte-mediated buffering of [K⁺]_e in neuromodulator-mediated shaping of cortical network activity.     

Glial contribution to seizure: K activation of (Na, K)-ATPase in bulk isolated glial cells and synaptosomes of epileptogenic cortex

Glial and neuronal (Na⁺, K⁺)-ATPase have dissimilar apparent affinities for K⁺ ions. Glial (Na⁺, K⁺)-ATPase is maximally activated by 20 mM K⁺ while neuronal (Na⁺, K⁺)-ATPase is maximally stimulated by 3–5 mM K⁺. Because this glial property may play an important role in the clearance of [K⁺]₀ during seizures, we investigated the K⁺ activation of (Na⁺, K⁺)-ATPase within bulk isolated glial cells and synaptosomes isolated from epileptogenic brains. The primary focus (F), the homolateral brain area around the focus (PF) and the mirror (M) or secondary focus induced by freezing lesions were studied.Results show that both glial and synaptosomal enzyme activities in the epileptogenic state change in comparison with controls, i.e. sham-operated cats. In F and M., glial enzyme decreased reaction velocities between 3 and 18 mM K⁺. In PF, maximum velocities increased in glial (Na⁺, K⁺)-ATPase. These results indicate that in actively firing epileptogenic tissue (F, M), glial (Na⁺, K⁺)-ATPase decreased rate reactions while the catalytic activity was increased in cortical tissues surrounding the focus. These phenomena appeared early, i.e. 1–3 days after production of the freezing lesion, and was associated with a sharp decrease in absolute levels of enzyme activity.Synaptosomal (Na⁺, K⁺)-ATPase from controls always exhibited a saturation curve at 3–6 mM K⁺. Synaptosomal enzyme activities in the primary (F) lesion increased slightly 24 h after lesion production, then progressively decreased 3 days after lesion production. No significant changes were seen in M and PF.     

Exacerbation of Epilepsy by Astrocyte Alkalization and Gap Junction Uncoupling

Seizures invite seizures. At the initial stage of epilepsy, seizures intensify with each episode; however, the mechanisms underlying this exacerbation remain to be solved. Astrocytes have a strong control over neuronal excitability and the mode of information processing. This control is accomplished by adjusting the levels of various ions in the extracellular space. The network of astrocytes connected via gap junctions allows a wider or more confined distribution of these ions depending on the open probability of the gap junctions. K⁺ clearance relies on the K⁺ uptake by astrocytes and the subsequent diffusion of K⁺ through the astrocyte network. When astrocytes become uncoupled, K⁺ clearance becomes hindered. Accumulation of extracellular K⁺ leads to hyperexcitability of neurons. Here, using acute hippocampal slices from mice, we uncovered that brief periods of epileptiform activity result in gap junction uncoupling. In slices that experienced short-term epileptiform activity, extracellular K⁺ transients in response to glutamate became prolonged. Na⁺ imaging with a fluorescent indicator indicated that intercellular diffusion of small cations in the astrocytic syncytium via gap junctions became rapidly restricted after epileptiform activity. Using a transgenic mouse with astrocyte-specific expression of a pH sensor (Lck-E²GFP), we confirmed that astrocytes react to epileptiform activity with intracellular alkalization. Application of Na⁺/HCO₃^– cotransporter blocker led to the suppression of intracellular alkalization of astrocytes and to the prevention of astrocyte uncoupling and hyperactivity intensification both in vitro and in vivo. Therefore, the inhibition of astrocyte alkalization could become a promising therapeutic strategy for countering epilepsy development.SIGNIFICANCE STATEMENT We aimed to understand the mechanisms underlying the plastic change of forebrain circuits associated with the intensification of epilepsy. Here, we demonstrate that first-time exposure to only brief periods of epileptiform activity results in acute disturbance of the intercellular astrocyte network formed by gap junctions in hippocampal tissue slices from mice. Moreover, rapid clearance of K⁺ from the extracellular space was impaired. Epileptiform activity activated inward Na⁺/HCO₃^– cotransport in astrocytes by cell depolarization, resulting in their alkalization. Our data suggest that alkaline pH shifts in astrocytes lead to gap junction uncoupling, hampering K⁺ clearance, and thereby to exacerbation of epilepsy. Pharmacological intervention could become a promising new strategy to dampen neuronal hyperexcitability and epileptogenesis.     

Involvement of the glutamate transporter and the sodium–calcium exchanger in the hypoxia-induced increase in intracellular Ca in rat hippocampal slices

Hippocampal slices prepared from adult rats were loaded with fura-2 and the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the CA1 pyramidal cell layer was measured. Hypoxia (oxygen–glucose deprivation) elicited a gradual increase in [Ca²⁺]_i in normal Krebs solution. At high extracellular sodium concentrations ([Na⁺]_o), the hypoxia-induced response was attenuated. In contrast, hypoxia in low [Na⁺]_o elicited a significantly enhanced response. This exaggerated response to hypoxia at a low [Na⁺]_o was reversed by pre-incubation of the slice at a low [Na⁺]_o prior to the hypoxic insult. The attenuation of the response to hypoxia by high [Na⁺]_o was no longer observed in the presence of antagonist to glutamate transporter. However, antagonist to Na⁺–Ca²⁺ exchanger only slightly influenced the effects of high [Na⁺]_o. These observations suggest that disturbance of the transmembrane gradient of Na⁺ concentrations is an important factor in hypoxia-induced neuronal damage and corroborates the participation of the glutamate transporter in hypoxia-induced neuronal injury. In addition, the excess release of glutamate during hypoxia is due to a reversal of Na⁺-dependent glutamate transporter rather than an exocytotic process.     

Mechanistic hypotheses for nonsynaptic epileptiform activity induction and its transition from the interictal to ictal state--computational simulation

Purpose: The aim of this work is to study, by means of computational simulations, the induction and sustaining of nonsynaptic epileptiform activity. Methods: The computational model consists of a network of cellular bodies of neurons and glial cells connected to a three‐dimensional (3D) network of juxtaposed extracellular compartments. The extracellular electrodiffusion calculation was used to simulate the extracellular potential. Each cellular body was represented in terms of the transmembrane ionic transports (Na⁺/K⁺ pumps, ionic channels, and cotransport mechanisms), the intercellular electrodiffusion through gap‐junctions, and the neuronal interaction by electric field and the variation of cellular volume. Results: The computational model allows simulating the nonsynaptic epileptiform activity and the extracellular potential captured the main feature of the experimental measurements. The simulations of the concomitant ionic fluxes and concentrations can be used to propose the basic mechanisms involved in the induction and sustaining of the activities. Discussion: The simulations suggest: The bursting induction is mediated by the Cl^? Nernst potential overcoming the transmembrane potential in response to the extracellular [K⁺] increase. The burst onset is characterized by a critical point defined by the instant when the Na⁺ influx through its permeable ionic channels overcomes the Na⁺/K⁺ pump electrogenic current. The burst finalization is defined by another critical point, when the electrogenic current of the Na⁺/K⁺ pump overcomes its influx through the channels.     

Ammonium‐evoked alterations in intracellular sodium and pH reduce glial glutamate transport activity

The clearance of extracellular glutamate is mainly mediated by pH‐ and sodium‐dependent transport into astrocytes. During hepatic encephalopathy (HE), however, elevated extracellular glutamate concentrations are observed. The primary candidate responsible for the toxic effects observed during HE is ammonium (NH₄⁺/NH₃). Here, we examined the effects of NH₄⁺/NH₃ on steady‐state intracellular pH (pH_i) and sodium concentration ([Na⁺]_i) in cultured astrocytes in two different age groups. Moreover, we assessed the influence of NH₄⁺/NH₃ on glutamate transporter activity by measuring D ‐aspartate‐induced pH_i and [Na⁺]_i transients. In 20–34 days in vitro (DIV) astrocytes, NH₄⁺/NH₃ decreased steady‐state pH_i by 0.19 pH units and increased [Na⁺]_i by 21 mM. D ‐Aspartate‐induced pH_i and [Na⁺]_i transients were reduced by 80–90% in the presence of NH₄⁺/NH₃, indicating a dramatic reduction of glutamate uptake activity. In 9–16 DIV astrocytes, in contrast, pH_i and [Na⁺]_i were minimally affected by NH₄⁺/NH₃, and D ‐aspartate‐induced pH_i and [Na⁺]_i transients were reduced by only 30–40%. Next we determined the contribution of Na⁺, K⁺, Cl^?‐cotransport (NKCC). Immunocytochemical stainings indicated an increased expression of NKCC1 in 20–34 DIV astrocytes. Moreover, inhibition of NKCC with bumetanide prevented NH₄⁺/NH₃‐evoked changes in steady‐state pH_i and [Na⁺]_i and attenuated the reduction of D ‐aspartate‐induced pH_i and [Na⁺]_i transients by NH₄⁺/NH₃ to 30% in 20–34 DIV astrocytes. Our results suggest that NH₄⁺/NH₃ decreases steady‐state pH_i and increases steady‐state [Na⁺]_i in astrocytes by an age‐dependent activation of NKCC. These NH₄⁺/NH₃‐evoked changes in the transmembrane pH and sodium gradients directly reduce glutamate transport activity, and may, thus, contribute to elevated extracellular glutamate levels observed during HE. © 2008 Wiley‐Liss, Inc.     

Synaptic impairment induced by paroxysmal ionic conditions in neocortex

Purpose: Seizures are associated with a reduction in extracellular Ca²⁺ concentration ([Ca²⁺]_o) and an increase in extracellular K⁺ concentration ([K⁺]_o). The long‐range synchrony observed between distant electrodes during seizures is weak. We hypothesized that changes in extracellular ionic conditions during seizures are sufficient to alter synaptic neuronal responses and synchrony in the neocortex. Methods: We obtained in vivo and in vitro electrophysiologic recordings combined with microstimulation from cat/rat neocortical neurons during seizures and seizure‐like ionic conditions. In vitro the [K⁺]_o was 2.8, 6.25, 8.0, and 12 mm and the [Ca²⁺]_o was 1.2 and 0.6 mm . Key Findings: During seizures recorded in vivo, we observed abolition of evoked synaptic responses. In vitro, the membrane potential of both regular‐spiking and fast‐spiking neurons was depolarized in high [K⁺]_o conditions and hyperpolarized in high [Ca²⁺]_o conditions. During high [K⁺]_o conditions, changes in [Ca²⁺]_o did not affect membrane potential. The synaptic responsiveness of both regular‐spiking and fast‐spiking neurons was reduced during seizure‐like ionic conditions. A reduction in [Ca²⁺]_o to 0.6 mm increased failure rates but did not abolish responses. However, an increase in [K⁺]_o to 12 mm abolished postsynaptic responses, which depended on a blockade in axonal spike propagation. Significance: We conclude that concomitant changes in [K⁺]_o and [Ca²⁺]_o observed during seizures contribute largely to the alterations of synaptic neuronal responses and to the decrease in long‐range synchrony during neocortical seizures.     

Effect of K ions on kinetic properties of the (Na, K)-ATPase (EC 3.6.1.3) of bulk isolated glial cells, perikarya and synaptosomes from rabbit brain cortex

Progress curves of the enzymatic reactions show that ATPases of bulk isolated glial cells, perikarya and synaptosomes exhibit hysteretic change. Initial velocities of enzyme activities were therefore obtained according to the equation valid for the hysteretic model.The (Na⁺, K⁺)-ATPase activities of the same brain fractions were measured before or after NaI treatment. Only glial and synaptosomal enzyme could be adequately extracted by using this procedure. Attempts to purify the (Na⁺, K⁺)-ATPase from brain perikarya by NaI extraction were unsuccessful.In order to determine the effect of the K⁺ ions on enzymic physiological efficiency (phys. eff.; i.e., the ratio V_max/K_mapp) the variation of (Na⁺, K⁺)-ATPase activities from each brain fraction was measured as a function of Mg·ATP²⁻ concentration in the presence of 5 and 20 mM K⁺ ions. High K⁺ ion concentrations (20mM) increased the physiological efficiency of glial enzyme and decreased the same kinetic parameter in neuronal (perikaryal as well as synaptosomal) enzyme preparations.Results are discussed in relation to a possible distribution of distinct enzyme in different brain cell populations as well as a possible role of glial cells in an active regulation of K⁺ ion extracellular fluid in the CNS.     

Control of the Na+, K+-ATPase under normal and pathological conditions

The Na⁺, K⁺-ATPase is an important enzyme in determining the ionic milieu of the cerebromicrovasculature and neurons. The effect of hypertension or aging on this enzyme, as well as its susceptibility to regulation by fatty acids or aluminum, is the focus of this study. A significant increase (34%) in the apparent affinity constant (K _D) but no change in the maximum binding capacity (B _max) for [³H]ouabain binding to the cerebromicrovascular Na⁺, K⁺-ATPase occurs after induction of acute hypertension. In addition, long chain unsaturated fatty acids stimulate the binding of [³H]ouabain to the enzyme in microvessels from normotensive and hypertensive rats. The synaptosomal Na⁺, K⁺-ATPase is sensitive to aluminum. AlCl₃ (1–100 μM) inhibits the K⁺-dependent-p-nitrophenylphosphatase (K⁺-NPPase) activity of the Na⁺, K⁺-ATPase in a dose-dependent manner. AlCl₃ (100 μM) decreases theV _max by 14% but does not alter theK _M, suggestive of noncompetitive inhibition. The enzyme from aged brain displays a greaterV _max, but shows the same susceptibility to AlCl₃ as the enzyme from younger brain. In summary, disruption of the Na⁺, K⁺-ATPase may underlie, at least in part, abnormalities of nerve and vascular cell function in disorders where elevated concentrations of fatty acids or metal ions are involved.     

High-frequency fatigue in rat skeletal muscle: Role of extracellular ion concentrations

High-frequency fatigue (HFF), the decline of force during continuous tetanic stimulation (lasting 4–40 s), was studied in isolated bundles of rat skeletal muscle fibers. HFF was slower in slow-twitch soleus fibers than in fast-twitch red or white sternomastoid fibers; denervation accelerated fatigue in soleus. Maximal 200-mmol/L potassium contractures of normal amplitude were induced in fatigued fibers, suggesting that crossbridge cycling and the voltage activation of excitation–contraction coupling could still occur maximally, but that activation by action potentials was impaired. An increase in [Na⁺]_o slowed HFF, while a small increase in [K⁺]_o or reduction in [Cl^?]_o accelerated HFF. Increasing the tetanic stimulation frequency exacerbated fatigue. Recovery from HFF proceeded rapidly since force increased markedly within a few seconds when stimulation ceased. These results support the hypothesis that a redistribution of Na⁺, K⁺, and Cl^? across the transverse tubular membranes during repeated action potential activity induces fatigue by reducing the amplitude and conduction of action potentials. © 1995 John Wiley & Sons, Inc.     

Effects of mammalian brain extracts and chlormadinone acetate on neuronal Na, K-ATPase and electrogenic Na, K-pump activity in vitro

Acid-acetone extracts of brain (from beef and guinea pig) and chlormadinone acetate (CMA) were compared with ouabain for their ability to inhibit the electrogenic Na⁺,K⁺-pump and the Na⁺,K⁺-ATPase of neuronal tissues. The membrane potential of neurones in the paravertebral sympathetic ganglion of the bullfrog was recorded in K⁺-free Ringer's solution by means of the sucrose gap technique. The potassium activated hyperpolarization (K_H⁺), induced by the re-introduction of potassium, was used as an index of electrogenic Na⁺,K⁺-pumping. The K_H⁺ was blocked by 1 μM ouabain. Na⁺,K⁺-ATPase activity was measured in microsomal membrane preparations of frog and beef brain using a continuous spectrophotometric assay. Although ouabain consistently inhibited beef brain Na⁺,K⁺-ATPase (IC₅₀ = 2.2 μM), acid-acetone extracts prepared from guinea pig and beef brain produced only partial inhibition. Neither of the extracts significantly reduced the K_H⁺ of the frog ganglion. CMA inhibited Na⁺,K⁺-ATPase prepared from bullfrog brain and spinal cord with slightly greater potency (IC₅₀ = 4.5 μM) than did ouabain (IC₅₀ = 10 μM). In contrast, electrogenic Na⁺,K⁺-pumping (i.e. the K_H⁺) in the frog ganglion was not affected by this steroid. It is concluded that although both the extracts and CMA inhibited Na⁺,K⁺-ATPase, neither can be considered ouabain-like due to their failure to affect the electrogenic Na⁺,K⁺-pump in situ.     

The ouabain-induced [Ca]i increase in leech Retzius neurones is mediated by voltage-dependent Ca channels

In leech Retzius neurones the inhibition of the Na⁺–K⁺ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca²⁺]_i). To elucidate the mechanism of this increase we investigated the changes in [Ca²⁺]_i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na⁺–K⁺ pump in bathing solutions of different ionic composition. The results show that Na⁺–K⁺ pump inhibition induced a [Ca²⁺]_i increase only if the cells depolarized sufficiently in the presence of extracellular Ca²⁺. Specifically, the relationship between [Ca²⁺]_i and the membrane potential upon Na⁺–K⁺ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca²⁺ channels by raising the extracellular K⁺ concentration. It is concluded that the [Ca²⁺]_i increase caused by inhibiting the Na⁺–K⁺ pump in leech Retzius neurones is exclusively due to Ca²⁺ influx through voltage-dependent Ca²⁺ channels.