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     

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     

Intracellular pH regulation in single cultured astrocytes from rat forebrain

We used the fluorescent pH-sensitive dye 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to monitor intracellular pH (pH_i) in single astrocytes cultured from the forebrain of neonatal rats. When exposed to a nominally CO₂/HCO₃^? -free medium buffered to pH 7.40 with HEPES at 37^°C, the cells had a mean pH_i of 6.89. Switching to a medium buffered to pH 7.40 with 5% CO₂ and 25 mM HCO₃^? caused the steady-state pH_i to increase by an average of 0.35, suggesting the presence of a HCO₃^? -dependent acid-extrusion mechanism. The sustained alkalinization was sometimes preceded by a small transient acidification. In experiments in which astrocytes were exposed to nominally HCO₃^?-free (HEPES-buffered) solutions, the application and withdrawal of 20 mM extracellular NH₄⁺ caused pH_i to fall to a value substantially below the initial one. pH_i spontaneously recovered from this acid load, stabilizing at a value ～ 0.1 higher than the one prevailing before the application of NH₄⁺. In other experiments conducted on cells bathed in HEPES-buffered solutions, removing extracellular Na⁺ caused pH_i to decrease rapidly by 0.5. Returning the Na⁺ caused pH_i to increase rapidly, indicating the presence of an Na⁺-dependent/HCO₃^?-independent acid-extrusion mechanism; the final pH_i after returning Na⁺ was ～ 0.08 higher than the initial value. This pH_i recovery elicited by returning Na⁺ was not substantially affected by 50 μM ethylisopropylamiloride (EIPA), but was speeded up by 50 μM 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS). Increasing [K⁺]_? from 5 to 25 mM caused pH_i to increase reversibly by ～ 0.2 in nominally CO₂/HCO₃^?-free solutions, and by ～ 0.1 in CO₂/HCO₃^?-containing solutions, although the initial pH_i was ～ 0.17 higher in the presence of CO₂/HCO₃^-. These results suggest the presence of a depolarization-induced alkalinization. Our results suggest the presence of both HCO₃^? dependent and -independent acid-base transport systems in cultured mammalian astrocytes, and indicate that astrocyte pH_i is sensitive to changes in either membrane voltage or [K⁺]₀ per se. © 1993 Wiley-Liss, Inc.     

Mechanisms of pHi regulation studied in individual neurons cultured from mouse cerebral cortex

Maintenance and regulation of intracellular pH (pH_i) was studied in single cultured mouse neocortical neurons using the fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Reversal of the Na⁺ gradient by reduction of the extracellular Na⁺ concentration ([Na⁺]_o) resulted in rapid intracellular acidification, inhibited by 5′-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na⁺/H⁺ exchange. In the presence of EIPA and/or 4′,4′-diisothiocyano-stilbene-2′,2′-sulfonic acid (DIDS), an inhibitor of Na⁺-coupled anion exchangers and Na⁺-HCO₃⁻ cotransport, a slow decline of pH_i was seen. Following intracellular acidification imposed by an NH₄Cl prepulse, pH_i recovered at a rapid rate, which was reduced by reduction of [Na⁺]_o and was virtually abolished by EIPA and DIDS in combination. Creating an outward Cl⁻ gradient by removal of extracellular Cl⁻ significantly increased the rate of pH_i recovery. In HCO₃⁻-free media, the pH_i recovery rate was reduced in control cells and was abolished at zero [Na⁺]_o and by EIPA. After intracellular alkalinization imposed by an acetate prepulse, pH_i recovery was unaffected by DIDS but was significantly reduced in the absence of extracellular Cl⁻, as well as in the presence of Zn²⁺, which is a blocker of proton channels. Together, this points toward a combined role of DIDS-insensitive Cl⁻/HCO₃⁻ and passive H⁺ influx in the recovery of pH_i after alkalinization. J. Neurosci. Res. 51:431–441, 1998. © 1998 Wiley-Liss, Inc.     

A depolarization-stimulated,bafilomycin-inhibitable H+ pump in hippocampal astrocytes

Relatively little is known about the mechanisms of pH_i regulation in mammalian glial cells. We analyzed pH_i regulation in rat hippocampal astrocytes in vitro using the pH-sensitive dye BCECF. All experiments were carried out in CO₂/HCO₃-free solutions. Recovery from NH⁺₄-induced acid loads was strongly dependent on the presence of extracellular Na⁺ and was inhibited by amiloride and its more specific analog EIPA, indicating the presence of Na⁺-H⁺ exchange in these cells. Removing bath Na⁺ or adding amiloride caused resting pH_i to shift in the acid direction. Even in the absence of bath Na⁺ or presence of Na⁺/H⁺ inhibitors, however, these astrocytes continued to show significant recovery from acid loads. The mechanism of this amiloride-insensitive and Na⁺-independent pH_i recovery process was sought and appeared to be a proton pump. In the absence of Na⁺, recovery from an acid load was completely blocked by the highly specific blocker of vacuolar-type (v-type) H⁺ ATPase, bafilomycin A₁ (BA₁). In normal Na⁺containing solutions, exposure to BA₁ caused a small acid shift in baseline pH_i and slowed recovery rate from NH⁺₄-induced acid loads by about 32%. The rate of Na⁺-independent pH_i recovery was increased by depolarization with 50 mM [K⁺] solution, and this effect was rapidly reversible and blocked by BA₁. These results indicate that, in CO₂/HCO^?₃-free solution, pH_i regulation in hippocampal astrocytes was mediated by Na⁺_?-H⁺ exchange and by a BA₁-inhibitable proton pump. Because the proton pump's activity was influenced by membrane potential, this acid exporting mechanism could contribute to the depolarization-induced alkalinization that is seen in astrocytes. Although v-type H⁺_?ATPase had been previously isolated from the brain, this is the first report indicating that it has a role in regulating pH_i in brain cells. © 1993 Wiley-Liss, Inc.     

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     

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.     

Metabotropic glutamate receptor agonists reduce glutamate release from cultured astrocytes

Astrocytes are thought to control extracellular glutamate concentrations ([Glu]_o) in the brain, thereby protecting neurons from excitotoxic injury. We investigated the effects of metabotropic glutamate receptor (mGluR) agonists on glutamate transport and [Glu]_o in primary hippocampal astrocytic cultures. Acute or chronic exposure of astrocytes to the mGluR agonist trans‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (trans‐ACPD) or its active isomer 1S,3R‐ACPD reduced [Glu]_o in a time‐ and dose‐dependent manner (44.5 ± 3.6% reductions of [Glu]_o in astrocytes from P0–P10 rats and 65.9 ± 4.1 % from rats P20 by 100 μM 1S,3R‐ACPD, EC₅₀ ∼ 5 μM). 1S,3R‐ACPD effects developed slowly (median effective at ∼60 min) and persisted for several hours after agonist removal. ACPD‐pretreated astrocytes established lower steady‐state [Glu]_o levels. ACPD effects persisted in the presence of the glutamate uptake inhibitors D ,L ‐threo‐β‐hydroxyaspartate (THA) and L ‐trans‐pyrrolidine‐2,4‐dicarboxylate (PDC) but were impaired by disruption of the transmembrane Na⁺, K⁺, or H⁺ gradients. In addition, 1S,3R‐ACPD had no effects on intracellular glutamate content and did not directly block glutamate transport. Furthermore, ACPD effects could be mimicked by glutamate per se and several other compounds presumed to be mGluR agonists, although (S)‐3,5‐dihydroxyphenylglycine (DHPG), (2S,2R,3R)‐2‐(2,3‐dicarboxycyclopropyl)glycine (DCG‐IV), and L ‐(+)‐2‐amino‐4‐phosphonobutyric acid (L ‐AP4) were without effect. These data suggest that glutamate and certain mGluR agonists may regulate [Glu]_o by modulating the transmembrane equilibrium of glutamate transport, especially by attenuating glutamate release. GLIA 25:270–281, 1999. © 1999 Wiley‐Liss, Inc.     

Astrocytes restrict discharge duration and neuronal sodium loads during recurrent network activity

Influx of sodium ions into active neurons is a highly energy‐expensive process which must be strictly limited. Astrocytes could play an important role herein because they take up glutamate and potassium from the extracellular space, thereby dampening neuronal excitation. Here, we performed sodium imaging in mouse hippocampal slices combined with field potential and whole‐cell patch‐clamp recordings and measurement of extracellular potassium ([K⁺]_o). Network activity was induced by Mg²⁺‐free, bicuculline‐containing saline, during which neurons showed recurring epileptiform bursting, accompanied by transient increases in [K⁺]_o and astrocyte depolarizations. During bursts, neurons displayed sodium increases by up to 22 mM. Astrocyte sodium concentration increased by up to 8.5 mM, which could be followed by an undershoot below baseline. Network sodium oscillations were dependent on action potentials and activation of ionotropic glutamate receptors. Inhibition of glutamate uptake caused acceleration, followed by cessation of electrical activity, irreversible sodium increases, and swelling of neurons. The gliotoxin NaFAc (sodium‐fluoroacetate) resulted in elevation of astrocyte sodium concentration and reduced glial uptake of glutamate and potassium uptake through Na⁺/K⁺‐ATPase. Moreover, NaFAc extended epileptiform bursts, caused elevation of neuronal sodium, and dramatically prolonged accompanying sodium signals, most likely because of the decreased clearance of glutamate and potassium by astrocytes. Our experiments establish that recurrent neuronal bursting evokes sodium transients in neurons and astrocytes and confirm the essential role of glutamate transporters for network activity. They suggest that astrocytes restrict discharge duration and show that an intact astrocyte metabolism is critical for the neurons' capacity to recover from sodium loads during synchronized activity. GLIA 2015;63:936–957     

Transporter‐mediated replacement of extracellular glutamate for GABA in the developing murine neocortex

During early development, cortical neurons migrate from their places of origin to their final destinations where they differentiate and establish synaptic connections. During corticogenesis, radially migrating cells move from deeper zone to the marginal zone, but they do not invade the latter. This “stop” function of the marginal zone is mediated by a number of factors, including glutamate and γ‐aminobutyric acid (GABA), two main neurotransmitters in the central nervous system. In the marginal zone, GABA has been shown to be released via GABA transporters (GAT)‐2/3, whereas glutamate transporters (EAATs) operate in the uptake mode. In this study, GABAergic postsynaptic currents (GPSCs) were recorded from Cajal‐Retzius cells in the marginal zone of murine neonatal neocortex using a whole‐cell patch‐clamp technique. Minimal electrical stimulation was applied to elicit evoked GPSCs using a paired‐pulse protocol. EAAT blockade with dl ‐threo‐b‐benzyloxyaspartic acid (dl ‐TBOA), a specific non‐transportable EAAT antagonist, abolishes constitutive GAT‐2/3‐mediated GABA release. In contrast to dl ‐TBOA, d ‐aspartate, an EAAT substrate, fails to block GAT‐2/3‐mediated GABA release. SNAP‐5114, a specific GAT‐2/3 antagonist, induced an elevation of intracellular sodium concentration ([Na⁺]_i) under resting conditions and in the presence of d ‐aspartate, indicating that GAT‐2/3 operates in reverse mode. In the presence of dl ‐TBOA, however, SNAP‐5114 elicited a [Na⁺]_i decrease, demonstrating that GAT‐2/3 operates in uptake mode. We conclude that EAATs via intracellular Na⁺ signaling and/or cell depolarization can govern the strength/direction of GAT‐mediated GABA transport.     

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.     

Octanol,a gap junction uncoupling agent,changes intracellular [H+] in rat astrocytes

Octanol rapidly closes gap junction channels but its mechanism of action is not known. Because intracellular [H⁺], pH_i, also affects the conductance of gap junctions, we studied octanol's effects on pH_i in cultured rat astrocytes, which are highly coupled cells. Octanol (1 mM) caused an acid shift in the pH_i of 90% of rat hippocampal astrocytes which averaged −0.19 ± 0.09 pH units in magnitude. In 58% of the cells tested, a biphasic change in pH_i was seen; octanol produced an initial acidification lasting ∼10 min that was followed by a persistent alkalinization. The related gap junction uncoupling agent, heptanol, had similar effects on pH_i. Octanol-induced changes in pH_i were similar in nominally HCO₃⁻-free and HCO₃⁻-containing solutions, although the rate of initial acidification was significantly greater in the presence of HCO₃⁻. The initial acidification was inhibited in the presence of the stilbene DIDS, an inhibitor of Na⁺/HCO₃⁻ cotransport, indicating that octanol caused acidification by blocking this powerful acid extruder. The alkalinization was inhibited by amiloride which blocks the Na⁺/H⁺ exchanger (NHE), an acid extruder, suggesting that the alkaline shift induced by octanol was caused by stimulation of NHE. As expected, octanol's effects on astrocytic pH_i were prevented by removal of external Na⁺, which blocks both Na⁺/HCO₃⁻ cotransport and NHE. Octanol had only small effects on intracellular Ca²⁺ (Ca²⁺_i) in astrocytes. Hepatocytes which, like astrocytes, are strongly coupled to one another, showed no change in pH_i with octanol application. Fluorescence recovery after photobleaching (FRAP) was used to study the effect of changes in astrocyte pH_i on degree of coupling in hippocampal astrocytes. Coupling was decreased by intracellular acid shifts ∼−0.2 pH units in size. Octanol's effects on astrocyte pH_i were complex but a prompt initial acidification was nearly always seen and could contribute to the uncoupling action of this drug in astrocytes. Because octanol uncouples hepatocytes without changing their pH_i, this compound clearly can influence gap junctional conductance independent of changes in pH_i. © 1996 Wiley-Liss, Inc.     

Intracellular acidification of the leech giant glial cell evoked by glutamate and aspartate

Glutamate is an excitatory receptor agonist in both neurones and glial cells, and, in addition, glutamate is also a substrate for glutamate transporter in glial cells. We have measured intracellular and extracellular pH changes induced by bath application of glutamate, its receptor agonist kainate, and its transporter agonist aspartate, in the giant neuropile glial cell in the central nervous system of the leech Hirudo medicinalis, using double-barrelled pH-sensitive microelectrodes. The giant glial cells responded to glutamate and aspartate (100–500 μM), and kainate (5–20 μM) with a membrane depolarization or an inward current, and with a distinct intracellular acidification. Glutamate and aspartate (both 500 μM) evoked a decrease in intracellular pH (pH_i) by 0.187 ± 0.081 (n = 88) and 0.198 ± 0.067 (n = 86) pH units, respectively. With a resting pH_i of 7.1 or 80 nM H⁺, these acidifications correspond to a mean increase of the intracellular H⁺ activity by 42 nM and 45 nM. Kainate caused a decrease of pH_i by 0.1 − 0.35 pH units (n = 15). The glutamate/aspartate-induced decrease in pH_i was not significantly affected by the glutamate receptor blockers kynurenic acid (1 mM) and 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX, 50–100 μM), which greatly reduced the kainate-induced change in pH_i. Extracellular alkalinizations produced by glutamate and aspartate were not affected by CNQX. Reduction of the external Na⁺ concentration gradually decreased the intracellular pH change induced by glutamate/aspartate, indicating half maximal activation of the acidifying process at 5–10 mM external Na⁺ concentration. When all external Na⁺ was replaced by NMDG⁺, the pH_iresponses were completely suppressed (glutamate) or reduced to 10% (aspartate). When Na⁺ was replaced by Li⁺, the glutamate- and aspartate-evoked pH_i responses were reduced to 18% and 14%, respectively. Removal of external Ca²⁺ reduced the glutamate- and aspartate-induced pH_i responses to 93 and 72%, respectively. The glutamate/aspartate-induced intracellular acidifications were not affected by the putative glutamate uptake inhibitor amino-adipidic acid (1 mM). DL-aspartate-β-hydroxamate (1 mM), and dihydrokainate (2 mM), which caused some pH_i decrease on its own, reduced the glutamate/aspartate-induced pH_i responses by 40 and 69%, respectively. The putative uptake inhibitor DL-threo-β-hydroxyaspartate (THA, 1 mM) induced a prominent intracellular acidification (0.36 ± 0.05 pH units, n = 9), and the pH_i change evoked by glutamate or aspartate in the presence of THA was reduced to less than 10%. The results indicate that glutamate, aspartate, and kainate produce substantial intracellular acidifications, which are mediated by at least two independent mechanisms: 1) via activation of non-NMDA glutamate receptors and 2) via uptake of the excitatory amino acids into the leech glial cell. © 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.     

Intracellular pH regulation in rat Schwann cells

We examined H⁺ and HCO₃^? transport mechanisms that are involved in the regulation of intracellular pH of Schwann cells. Primary cultures of Schwann cells were prepared from the sciatic nerves of 1–3-day-old rats. pH_i of single cells attached to cover slips was continuously monitored by measuring the absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein incorporated intracellularly. The average pH_i of neonatal Schwann cells bathed in HEPES mammalian solution was 7.17 ± 0.02 (n = 32). In the nominal absence of HCO₃^?, pH_i spontaneously recovered from an acute acid load induced by exposing the Schwann cells to 20 mM NH₄⁺ (NH₄⁺ prepulse). This pH_i recovery from the acute acid load was totally inhibited in the absence of external Na⁺ or in the presence of 1 mM amiloride. In both cases, the pH_i recovery was readily restored upon readdition of external Na⁺ or removal of amiloride. In the steady-state, addition of amiloride caused a small and slow decrease in pH_i which was readily reversed upon removal of amiloride. In the presence of HCO₃^?, removal of external Cl- caused pH_i to rapidly and reversibly increase by 0.23 = 0.03 (n = 15) and the initial rate of alkalinization was 20.6 ± 2.7 × 10-4 pH/sec. In the absence of external Na⁺, removal of bath Cl^? still caused pH_i to increase by 0.15 ± 0.02 and the initial rate of pH_i increase was not significantly altered. In the nominal absence of HCO₃^?, removal of bath Cl- caused pH_i to increase very slightly (0.05 ± 0.01) with an initial dpH_i/dt of only 4.4 ± 0.2 × 10^?4 pH/sec (n = 4). Addition of 100 μM DIDS did not inhibit the pH_i increase caused by removal of bath Cl^?. These data indicate that (1) Rat Schwann cells regulate their pH_i via an Na-H exchange mechanism which is moderately active at steady-state pH_i. (2) In the presence of HCO₃^?, there is a Na-independent Cl-HCO₃ (base) exchanger which also contributes to regulation of intracellular pH in Schwann cells. © 1994 Wiley-Liss, Inc.     

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.     

SHORT COMUNICATION Early sodium elevations induced by combined oxygen and glucose deprivation in pyramidal cortical neurons

We investigated the effects of oxygen (O₂)/glucose deprivation on intracellular sodium concentration ([Na⁺]_i) of cortical pyramidal cells in a slice preparation of rat frontal cortex. Intracellular recordings were combined with microfluorometric measurements of [Na⁺]_i using the Na⁺-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Deprivation of O₂/glucose caused an initial membrane hyperpolarization that was followed by a slowly developing large depolarization. Levels of [Na⁺]_i started to increase significantly during the phase of membrane hyperpolarization. Neither tetrodotoxin, a combination of ionotropic and metabotropic glutamate receptor antagonists (d -amino-phosphonovalerate, 6-cyano-7-nitroquinoxaline-2,3-dione plus S-methyl-4-carboxyphenylglycine) nor bepridil, an inhibitor of the Na⁺/Ca²⁺-exchanger, affected these responses to O₂/glucose. The present results demonstrate that, in cortical neurons, O₂/glucose deprivation induces an early rise in [Na⁺]_i which cannot be ascribed to the activity of voltage gated Na⁺-channels, glutamate receptors or of the Na⁺/Ca²⁺-exchanger.     

High effective cytosolic H+ buffering in mouse cortical astrocytes attributable to fast bicarbonate transport

Cytosolic H⁺ buffering plays a major role for shaping intracellular H⁺ shifts and hence for the availability of H⁺ for biochemical reactions and acid/base‐coupled transport processes. H⁺ buffering is one of the prime means to protect the cell from large acid/base shifts. We have used the H⁺ indicator dye BCECF and confocal microscopy to monitor the cytosolic H⁺ concentration, [H⁺]_i, in cultured cortical astrocytes of wild‐type mice and of mice deficient in sodium/bicarbonate cotransporter NBCe1 (NBCe1‐KO) or in carbonic anhydrase isoform II (CAII‐KO). The steady‐state buffer strength was calculated from the amplitude of [H⁺]_i transients as evoked by CO₂/HCO₃^? and by butyric acid in the presence and absence of CO₂/HCO₃^?. We tested the hypotheses if, in addition to instantaneous physicochemical H⁺ buffering, rapid acid/base transport across the cell membrane contributes to the total, “effective” cytosolic H⁺ buffering. In the presence of 5% CO₂/26 mM HCO₃^?, H⁺ buffer strength in astrocytes was increased 4–6 fold, as compared with that in non‐bicarbonate, HEPES‐buffered solution, which was largely attributable to fast HCO₃^? transport into the cells via NBCe1, supported by CAII activity. Our results show that within the time frame of determining physiological H⁺ buffering in cells, fast transport and equilibration of CO₂/H⁺/HCO₃^? can make a major contribution to the total “effective” H⁺ buffer strength. Thus, “effective” cellular H⁺ buffering is, to a large extent, attributable to membrane transport of base equivalents rather than a purely passive physicochemical process, and can be much larger than reported so far. Not only physicochemical H⁺ buffering, but also rapid import of HCO₃^? via the electrogenic sodium‐bicarbonate cotransporter NBCe1, supported by carbonic anhydrase II (CA II), was identified to enhance cytosolic H⁺ buffer strength substantially. GLIA 2015;63:1581–1594     

The influence of calcium transients on intracellular pH in cortical neurons in primary culture

The objective of this study was to assess the influence of Ca²⁺ influx on intracellular pH (pH_i) of neocortical neurons in primary culture. Neurons were exposed to glutamate (100–500 μM) or KCl (50 mM), and pH_i was recorded with microspectroflurometric techniques. Additional experiments were carried out in which calcium influx was triggered by ionomycin (2 μM) or the calcium ionophore 4-Br-A23187 (2 μM). Glutamate exposure either caused no, or only a small decrease in pH_i (ΔpH ≈ 0.06 units). When a decrease was observed, a rebound rise in pH_i above control was observed upon termination of glutamate exposure. In about 20% of the cells, the acidification was more pronounced (ΔpH ≈ 0.20 units), but all these cells had high control pH_i values, and showed gradual acidification. Exposure of cells to 50 mM KCl consistently increased pH_i. Since this increase was similar in the presence and nominal absence of HCO₃⁻, it probably did not reflect influx of HCO₃⁻ via a Na⁺-HCO₃⁻ symporter. Furthermore, since it occurred in the absence of external Ca²⁺ (or a measurable rise in Ca_i²⁺) it seemed independent of Ca²⁺ influx. It is tentatively concluded that the rise in pH_i was due to reduced passive influx of H⁺ along the electrochemical gradient, which is reduced by depolarization. In Ca²⁺-containing solutions, depolarization led to a rebound increase in pH_i above control. This, and the rebound found after glutamate transients, may reflect Ca²⁺-triggered phosphorylation and upregulation of the Na⁺/H⁺ antiporter which extrudes H⁺ from the cell. Ionomycin and 4-Br-A23187 gave rise to a large rise in Ca_i²⁺ and to alkalinization of the cell (ΔpH ≈ 0.5). Since amiloride or removal of Na⁺ from the external solution did not alter the rise in pH_i, it was probably not due to accelerated H⁺ extrusion. However, removal of Ca²⁺ from extracellular fluid prevented the rise, suggesting that it was secondary to Ca²⁺/2H⁺ exchange across plasma membranes.     

Voltage‐gated sodium channel Nav1.5 contributes to astrogliosis in an in vitro model of glial injury via reverse Na+/Ca2+ exchange

Astrogliosis is a prominent feature of many, if not all, pathologies of the brain and spinal cord, yet a detailed understanding of the underlying molecular pathways involved in the transformation from quiescent to reactive astrocyte remains elusive. We investigated the contribution of voltage‐gated sodium channels to astrogliosis in an in vitro model of mechanical injury to astrocytes. Previous studies have shown that a scratch injury to astrocytes invokes dual mechanisms of migration and proliferation in these cells. Our results demonstrate that wound closure after mechanical injury, involving both migration and proliferation, is attenuated by pharmacological treatment with tetrodotoxin (TTX) and KB‐R7943, at a dose that blocks reverse mode of the Na⁺/Ca²⁺ exchanger (NCX), and by knockdown of Na_v1.5 mRNA. We also show that astrocytes display a robust [Ca²⁺]_i transient after mechanical injury and demonstrate that this [Ca²⁺]_i response is also attenuated by TTX, KB‐R7943, and Na_v1.5 mRNA knockdown. Our results suggest that Na_v1.5 and NCX are potential targets for modulation of astrogliosis after injury via their effect on [Ca²⁺]_i. GLIA 2014;62:1162–1175