Principles of sodium homeostasis and sodium signalling in astroglia

Sodium homeostasis is at the center stage of astrocyte (and brain) physiology because the large inwardly directed Na⁺ gradient provides the energy for transport of ions, neurotransmitters, amino acids and many other molecules across the plasmalemma and endomembranes. Cell imaging with commercially available chemical indicators allows analysis of dynamic changes in intracellular Na⁺ concentration (Na⁺]_i), albeit further technological developments, such as genetically‐controlled or membrane targeted indicators or dyes usable for advanced microscopy (such as fluorescence‐lifetime imaging microscopy) are urgently needed. Thus, important questions related to the existence of Na⁺ gradients between different cellular compartments or occurrence of localised Na⁺ micro/nanodomains at the plasma membrane remain debatable. Extrusion of Na⁺ (and hence Na⁺ homeostasis) in astrocytes is mediated by the ubiquitously expressed Na⁺/K⁺‐ATPase (NKA), the major energy consumer of the brain. The activity of the NKA is counteracted by constant constitutive influx of Na⁺ through transporters such as the NKCC1 (Na⁺‐K⁺?2Cl^‐‐co‐transport) or the NBC (Na⁺?2 ‐co‐transport). In addition, Na⁺‐permeable ion channels at the plasma membrane as well as Na⁺‐dependent solute carrier transporters provide for Na⁺ influx into astrocytes. Activation of these pathways in response to neuronal activity results in an increase of [Na⁺]_i in astrocytes and there is manifold evidence for diverse signalling functions of these [Na⁺]_i transients. Thus, in addition to its established homeostatic functions, activity dependent fluctuations of astrocyte [Na⁺]_i regulate signalling cascades by feeding back on Na⁺‐dependent transporters. The Na⁺ signalling system may be ideally placed for fast coordinating signalling between neuronal activity and glial “homeostatic” Na⁺‐dependent transporters. GLIA 2016;64:1611–1627     

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.     

Astrocyte sodium signaling and the regulation of neurotransmission

The transmembrane Na⁺ concentration gradient is an important source of energy required not only to enable the generation of action potentials in excitable cells, but also for various transmembrane transporters both in excitable and non‐excitable cells, like astrocytes. One of the vital functions of astrocytes in the central nervous system (CNS) is to regulate neurotransmitter concentrations in the extracellular space. Most neurotransmitters in the CNS are removed from the extracellular space by Na⁺‐dependent neurotransmitter transporters (NeuTs) expressed both in neurons and astrocytes. Neuronal NeuTs control mainly phasic synaptic transmission, i.e., synaptically induced transient postsynaptic potentials, while astrocytic NeuTs contribute to the termination of phasic neurotransmission and modulate the tonic tone, i.e., the long‐lasting activation of extrasynaptic receptors by neurotransmitter that has diffused out of the synaptic cleft. Consequently, local intracellular Na⁺ ([Na⁺]_i) transients occurring in astrocytes, for example via the activation of ionotropic neurotransmitter receptors, can affect the driving force for neurotransmitter uptake, in turn modulating the spatio‐temporal profiles of neurotransmitter levels in the extracellular space. As some NeuTs are close to thermodynamic equilibrium under resting conditions, an increase in astrocytic [Na⁺]_i can stimulate the direct release of neurotransmitter via NeuT reversal. In this review we discuss the role of astrocytic [Na⁺]_i changes in the regulation of uptake/release of neurotransmitters. It is emphasized that an activation of one neurotransmitter system, including either its ionotropic receptor or Na⁺‐coupled co‐transporter, can strongly influence, or even reverse, other Na⁺‐dependent NeuTs, with potentially significant consequences for neuronal communication. GLIA 2016;64:1655–1666     

Astrocytic and neuronal oxidative metabolism are coupled to the rate of glutamate–glutamine cycle in the tree shrew visual cortex

Astrocytes play an important role in glutamatergic neurotransmission, namely by clearing synaptic glutamate and converting it into glutamine that is transferred back to neurons. The rate of this glutamate–glutamine cycle (V_NT) has been proposed to couple to that of glucose utilization and of neuronal tricarboxylic acid (TCA) cycle. In this study, we tested the hypothesis that glutamatergic neurotransmission is also coupled to the TCA cycle rate in astrocytes. For that we investigated energy metabolism by means of magnetic resonance spectroscopy (MRS) in the primary visual cortex of tree shrews (Tupaia belangeri) under light isoflurane anesthesia at rest and during continuous visual stimulation. After identifying the activated cortical volume by blood oxygenation level‐dependent functional magnetic resonance imaging, ¹H MRS was performed to measure stimulation‐induced variations in metabolite concentrations. Relative to baseline, stimulation of cortical activity for 20 min caused a reduction of glucose concentration by ?0.34 ± 0.09 µmol/g (p < 0.001), as well as a ?9% ± 1% decrease of the ratio of phosphocreatine‐to‐creatine (p < 0.05). Then ¹³C MRS during [1,6‐¹³C]glucose infusion was employed to measure fluxes of energy metabolism. Stimulation of glutamatergic activity, as indicated by a 20% increase of V_NT, resulted in increased TCA cycle rates in neurons by 12% ( , p < 0.001) and in astrocytes by 24% ( , p = 0.007). We further observed linear relationships between V_NT and both and . Altogether, these results suggest that in the tree shrew primary visual cortex glutamatergic neurotransmission is linked to overall glucose oxidation and to mitochondrial metabolism in both neurons and astrocytes.     

Glial Na+‐dependent ion transporters in pathophysiological conditions

Sodium dynamics are essential for regulating functional processes in glial cells. Indeed, glial Na⁺ signaling influences and regulates important glial activities, and plays a role in neuron‐glia interaction under physiological conditions or in response to injury of the central nervous system (CNS). Emerging studies indicate that Na⁺ pumps and Na⁺‐dependent ion transporters in astrocytes, microglia, and oligodendrocytes regulate Na⁺ homeostasis and play a fundamental role in modulating glial activities in neurological diseases. In this review, we first briefly introduced the emerging roles of each glial cell type in the pathophysiology of cerebral ischemia, Alzheimer's disease, epilepsy, Parkinson's disease, Amyotrophic Lateral Sclerosis, and myelin diseases. Then, we discussed the current knowledge on the main roles played by the different glial Na⁺‐dependent ion transporters, including Na⁺/K⁺ ATPase, Na⁺/Ca²⁺ exchangers, Na⁺/H⁺ exchangers, Na⁺‐K⁺‐Cl^? cotransporters, and Na⁺‐ cotransporter in the pathophysiology of the diverse CNS diseases. We highlighted their contributions in cell survival, synaptic pathology, gliotransmission, pH homeostasis, and their role in glial activation, migration, gliosis, inflammation, and tissue repair processes. Therefore, this review summarizes the foundation work for targeting Na⁺‐dependent ion transporters in glia as a novel strategy to control important glial activities associated with Na⁺ dynamics in different neurological disorders. GLIA 2016;64:1677–1697     

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.     

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.     

GFAP-positive hippocampal astrocytes in situ respond to glutamatergic neuroligands with increases in [Ca2+]i

It is becoming increasingly clear that astrocytes play very dynamic and interactive roles that are important for the normal functioning of the central nervous system. In culture, astrocytes express many receptors coupled to increases in intracellular calcium ([Ca²⁺]_i). In vivo, it is likely that these receptors are important for the modulation of astrocytic functions such as the uptake of neurotransmitters and ions. Currently, however, very little is known about the expression or stimulation of such astrocytic receptors in vivo. To address this issue, confocal microscopy and calcium sensitive fluorescent dyes were used to examine the dynamic changes in astrocytic [Ca²⁺]_i, within acutely isolated hippocampal slices. Astrocytes were subsequently identified by immunocytochemistry for glial fibrillary acidic protein. In this paper, we present data indicating that hippocampal astrocytes in situ respond to glutamate, kainate, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), 1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), N-methyl-D-aspartate (NMDA), and depolarization with increases in [Ca²⁺]_i. The increases in [Ca²⁺]_i occurred in both the astrocytic cell bodies and the processes. Temporally the changes in [Ca²⁺]_i were very dynamic, and various patterns ranging from sustained elevations to oscillations of [Ca²⁺]_i were observed. Individual astrocytes responded to neuroligands selective for both ionotropic and metabotropic glutamate receptors with increases in [Ca²⁺]_i. These findings indicate that astrocytes in vivo contain glutamatergic receptors coupled to increases in [C²⁺]_i and are able to respond to neuronally released neurotransmitters. (c) 1995 Wiley-Liss, Inc.     

Activation of alpha-2 adrenergic receptors stimulates GABA release by astrocytes

Norepinephrine is one of the key neurotransmitters in the hippocampus, but its role in the functioning of the neuroglial networks remains unclear. Here we show that norepinephrine suppresses NH₄Cl-induced oscillations of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in hippocampal neurons. We found that the inhibitory effect of norepinephrine against ammonium-induced [Ca²⁺]_i oscillations is mediated by activation of alpha-2 adrenergic receptors. Furthermore, UK 14,304, an agonist of alpha-2 adrenergic receptors, evokes a biphasic [Ca²⁺]_i elevation in a minor population of astrocytes. This elevation consists of an initial fast, peak-shaped [Ca²⁺]_i rise, mediated by G_iβγ subunit and subsequent PLC-induced mobilization of Ca²⁺ from internal stores, and a plateau phase, mediated by a Ca²⁺ influx from the extracellular medium through store-operated and TRPC3 channels. We show the correlation between the Ca²⁺ response in astrocytes and suppression of [Ca²⁺]_i oscillations in neurons. The inhibitory effect of UK 14,304 is abolished in the presence of gallein, an inhibitor of G_βγ-signaling. In turn, application of the agonist in the presence of the PLC inhibitor decreases the frequency and amplitude of [Ca²⁺]_i oscillations in neurons but does not suppress them. The same effect is observed in the presence of bicuculline, a GABA(A) receptor antagonist. We demonstrate that UK 14,304 application increases the frequency and amplitude of slow outward chloride currents in neurons, indicating the release of GABA by astrocytes. Thus, our findings indicate that the activation of astrocytic alpha-2 adrenergic receptors stimulates GABA release from astrocytes via G_iβγ subunit-associated signaling pathway, contributing to the suppression of neuronal activity.     

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.     

Coupling of AMPA receptors with the Na/Ca exchanger in cultured rat astrocytes

Astrocytes exhibit three transmembrane Ca²⁺ influx pathways: voltage-gated Ca²⁺ channels (VGCCs), the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) class of glutamate receptors, and Na⁺/Ca²⁺ exchangers. Each of these pathways is thought to be capable of mediating a significant increase in Ca²⁺ concentration ([Ca²⁺]_i); however, the relative importance of each and their interdependence in the regulation astrocyte [Ca²⁺]_i is not known. We demonstrate here that 100 μM AMPA in the presence of 100 μM cyclothiazide (CTZ) causes an increase in [Ca²⁺]_i in cultured cerebral astrocytes that requires transmembrane Ca²⁺ influx. This increase of [Ca²⁺]_i is blocked by 100 μM benzamil or 0.5 μM U-73122, which inhibit reverse-mode operation of the Na⁺/Ca²⁺ exchanger by independent mechanisms. This response does not require Ca²⁺ influx through VGCCs, nor does it depend upon a significant Ca²⁺ influx through AMPA receptors (AMPARs). Additionally, AMPA in the presence of CTZ causes a depletion of thapsigargin-sensitive intracellular Ca²⁺ stores, although depletion of these Ca²⁺ stores does not decrease the peak [Ca²⁺]_i response to AMPA. We propose that activation of AMPARs in astrocytes can cause [Ca²⁺]_i to increase through the reverse mode operation of the Na⁺/Ca²⁺ exchanger with an associated release of Ca²⁺ from intracellular stores. This proposed mechanism requires neither Ca²⁺-permeant AMPARs nor the activation of VGCCs to be effective.     

The cystine‐glutamate exchanger (xCT,Slc7a11) is expressed in significant concentrations in a subpopulation of astrocytes in the mouse brain

The cystine‐glutamate exchanger (xCT) promotes glutathione synthesis by catalyzing cystine uptake and glutamate release. The released glutamate may modulate normal neural signaling and contribute to excitotoxicity in pathological situations. Uncertainty, however, remains as neither the expression levels nor the distribution of xCT have been unambiguously determined. In fact, xCT has been reported in astrocytes, neurons, oligodendrocytes and microglia, but most of the information derives from cell cultures. Here, we show by immunohistochemistry and by Western blotting that xCT is widely expressed in the central nervous system of both sexes. The labeling specificity was validated using tissue from xCT knockout mice as controls. Astrocytes were selectively labeled, but showed greatly varying labeling intensities. This astroglial heterogeneity resulted in an astrocyte domain‐like labeling pattern. Strong xCT labeling was also found in the leptomeninges, along some blood vessels, in selected circumventricular organs and in a subpopulation of tanycytes residing the lateral walls of the ventral third ventricle. Neurons, oligodendrocytes and resting microglia, as well as reactive microglia induced by glutamine synthetase deficiency, were unlabeled. The concentration of xCT protein in hippocampus was compared with that of the EAAT3 glutamate transporter by immunoblotting using a chimeric xCT‐EAAT3 protein to normalize xCT and EAAT3 labeling intensities. The immunoblots suggested an xCT/EAAT3 ratio close to one (0.75 ± 0.07; average ± SEM; n = 4) in adult C57BL6 mice. Conclusions: xCT is present in select blood/brain/CSF interface areas and in an astrocyte subpopulation, in sufficient quantities to support the notion that system provides physiologically relevant transport activity.     

The system N transporter SN2 doubles as a transmitter precursor furnisher and a potential regulator of NMDA receptors

Activation of NMDA receptor requires two co‐agonists, glutamate and glycine. Despite its intrinsic role in brain functions molecular mechanisms involved in glutamate replenishment and identification of the origin of glycine have eluded characterization. We have performed direct measurements of glycine flux by SN2 (Slc38a5; also known as SNAT5), executed extensive electrophysiological characterization as well as implemented ratiometric analyses to show that SN2 transport resembles SN1 in mechanism but differ in functional implications. We report that rat SN2 mediates electroneutral and bidirectional transport of glutamine and glycine at perisynaptic astroglial membranes. Sophisticated coupled and uncoupled movements of H⁺ differentially associate with glutamine and glycine transport by SN2 and regulate pH_i and the release mode of the transporter. Consequently, SN2 doubles as a transmitter precursor furnisher and a potential regulator of NMDA receptors. © 2012 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.     

Intracellular pH regulation in primary rat astrocytes and C6 glioma cells

We used the pH-sensitive fluorescent dye BCECF to study intracellular pH (pH_i) regulation in primary cultures of rat astrocytes and C6 glioma cells. Both cell types contain three pH-regulating transporters: (1) alkalinizing Na⁺/H⁺ exchange; (2) alkalinizing Na⁺ + HCO₃ ?/Cl_? exchange; and (3) acidifying Cl^?/HCO₃ ^? exchange. Na⁺/H⁺ exchange was most evident in the absence of CO₂; recovery from acidification was Na⁺ dependent and amiloride sensitive. Exposure to CO₂ caused a cell alkalinization that was inhibited by DIDS, dependent on external Na⁺, and inhibited 75% in the absence of Cl^? (thus mediated by Na⁺ + HCO₃ ^?/Cl^? exchange). When pH_i was increased above the normal steady-state pH_i, a DIDS-inhibitable and Na⁺ -independent acidifying recovery was evident, indicating the presence of Cl^? /HCO₃ ^? exchange. Astrocytes, but not C6 cells, contain a fourth pH-regulating transporter, Na⁺ ?HCO₃ ^? cotransport; in the presence of CO₂, depolarization caused an alkalinization of 0.12 ⁺_? 0.01 (n = 8) and increased the rate of CO₂-induced alkalinization from 0.23 ± 0.02 to 0.42 ± 0.03 pH unit/min. Since C6 cells lack the Na⁺ -HCO₃ ⁺ cotransporter, they are an inferior model of pH_i regulation in glia. Our results differ from previous observations in glia in that: (1) Na⁺ /H⁺ exchange was entirely inhibited by amiloride; (2) Na⁺ + HCO₃ ^?/Cl^? exchange was present and largely responsible for CO₂?induced alkalinization; (3) Cl^? /HCO₃ ^? exchange was only active at pH_i values above steady state; and (4) depolarization-induced alkalinization of astrocytes was seen only in the presence of CO₂.     

Effects of Dibutyryl Cyclic AMP on Na+, K+-ATPase Activity and Intracellular Na+ and K+ in Primary Cultures of Astrocytes from DBA and C57 Mice

Summary: Effects of chronic treatment of dibutyryl cyclic AMP (db-cyclic AMP) on Na⁺, K⁺-ATPase activity in cell homogenates and intracellular N a f and K+ contents [(Na⁺)_i and (K⁺)_i] were studied in primary cultures of astrocytes derived from cerebral cortex of neonatal audiogenic seizure-susceptible DBA and audiogenic seizure-resistant C57 mice. Na⁺, K⁺-ATPase activity in cell homogenates was greater and (Na⁺)_i was less in DBA astrocytes than in C57 astrocytes. There was no difference in (K⁺)_i between astrocytes from DBA and C57 mice. Addition of db-cyclic AMP to the medium from day 14 to day 21 in culture (final concentration 0.25 mM) increased Na⁺, K⁺-ATPase activity in cell homogenates and decreased (Na⁺)_i, but had no significant effect on (K⁺)_i in astrocytes from either DBA or C57 mice. Chronic treatment with db-cyclic AMP altered cell growth. Protein and DNA content of cultured astrocytes from both DBA and C57 mice was decreased. DNA was more affected than protein. Modifying K⁺ and Na⁺ concentration in medium altered Na⁺, K⁺-ATPase activity in cell homogenates as well as (Na⁺)_i and (K⁺)_i in cultured astrocytes of both DBA and C57 mice. Changes in (Na⁺)_i and (K⁺)_i at different K⁺ concentrations in medium paralleled those in Na⁺, K⁺-ATPase activity in cell homogenates. Results indicate that the ability to transport Na⁺ across the cell membrane and the response of Na⁺, K⁺-ATPase to db-cyclic AMP and to the changes in K + in medium of cultured astrocytes from audiogenic seizure-susceptible DBA mice are sufficient.     

Involvement of Na+–Ca2+ Exchanger in Reperfusion-induced Delayed Cell Death of Cultured Rat Astrocytes

In some cells, Ca²⁺ depletion induces an increase in intracellular Ca²⁺ ([Ca²⁺]_i) after reperfusion with Ca²⁺-containing solution, but the mechanism for the reperfusion injury is not fully elucidated. Using an antisense strategy we studied the role of the Na⁺-Ca²⁺ exchanger in reperfusion injury in cultured rat astrocytes. When astrocytes were perfused in Ca²⁺-free medium for 15–60 min, a persistent increase in [Ca²⁺]_i was observed immediately after reperfusion with Ca²⁺-containing medium, and the number of surviving cells decreased 3–5 days latter. The increase in [Ca²⁺]_i was enhanced by low extracellular Na⁺ ([Na⁺]_o) during reperfusion and blocked by the inhibitors of the Na⁺-Ca²⁺ exchanger amiloride and 3,4-dichlorobenzamil, but not by the Ca²⁺ channel antagonists nifedipine, Cd²⁺ and Ni²⁺. Treatment of astrocytes with antisense, but not sense, oligodeoxynucleotide to the Na⁺-Ca²⁺ exchanger decreased Na⁺–Ca²⁺ exchanger protein level and exchange activity. The antisense oligomer attenuated reperfusion-induced increase in [Ca²⁺]ⁱ and cell toxicity. The Na⁺-Ca²⁺ exchange inhibitors 3,4-dichlorobenzamil and ascorbic acid protected astrocytes from reperfusion injury partially, while the stimulators sodium nitroprusside and 8-bromo-cyclic GMP and low [Na⁺]_o exacerbated the injury. Pretreatment of astrocytes with ouabain and monensin caused similar delayed glial cell death. These findings suggest that Ca²⁺ entry via the Na⁺–Ca²⁺ exchanger plays an important role in reperfusion-induced delayed glial cell death.     

Importance of astrocytes for potassium ion (K+) homeostasis in brain and glial effects of K+ and its transporters on learning

Initial clearance of extracellular K⁺ ([K⁺]_o) following neuronal excitation occurs by astrocytic uptake, because elevated [K⁺]_o activates astrocytic but not neuronal Na⁺,K⁺-ATPases. Subsequently, astrocytic K⁺ is re-released via Kir4.1 channels after distribution in the astrocytic functional syncytium via gap junctions. The dispersal ensures widespread release, preventing renewed [K⁺]_o increase and allowing neuronal Na⁺,K⁺-ATPase-mediated re-uptake. Na⁺,K⁺-ATPase operation creates extracellular hypertonicity and cell shrinkage which is reversed by the astrocytic cotransporter NKCC1. Inhibition of Kir channels by activation of specific PKC isotypes may decrease syncytial distribution and enable physiologically occurring [K⁺]_o increases to open L-channels for Ca²⁺, activating [K⁺]_o-stimulated gliotransmitter release and regulating gap junctions. Learning is impaired when [K⁺]_o is decreased to levels mainly affecting astrocytic membrane potential or Na⁺,K⁺-ATPase or by abnormalities in its α2 subunit. It is enhanced by NKCC1-mediated ion and water uptake during the undershoot, reversing neuronal inactivity, but impaired in migraine with aura in which [K⁺]_o is highly increased. Vasopressin augments NKCC1 effects and facilitates learning. Enhanced myelination, facilitated by astrocytic-oligodendrocytic gap junctions also promotes learning.     

Relationship between L-glutamate-regulated intracellular Na+ dynamics and ATP hydrolysis in astrocytes

Summary. Glutamate uptake into astrocytes and the resulting increase in intracellular Na⁺ (Na⁺_i) have been identified as a key signal coupling excitatory neuronal activity to increased glucose utilization. Arguments based mostly on mathematical modeling led to the conclusion that physiological concentrations of glutamate more than double astrocytic Na⁺/K⁺-ATPase activity, which should proportionally increase its ATP hydrolysis rate. This hypothesis was tested in the present study by fluorescence monitoring of free Mg²⁺ (Mg²⁺_i), a parameter that inversely correlates with ATP levels. Glutamate application measurably increased Mg²⁺_i (i.e. decreased ATP), which was reversible after glutamate washout. Na⁺_i and ATP changes were then directly compared by simultaneous Na⁺_i and Mg²⁺ imaging. Glutamate increased both parameters with different rates and blocking the Na⁺/K⁺-ATPase during the glutamate-evoked Na⁺_i response, resulted in a drop of Mg²⁺_i levels (i.e. increased ATP). Taken together, this study demonstrates the tight correlation between glutamate transport, Na⁺ homeostasis and ATP levels in astrocytes.