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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Hypoxia induced by Na2S2O4 increases [Na]i in mouse glomus cells, an effect depressed by cobalt. Experiments with Na-selective microelectrodes and voltage-clamping

The intracellular sodium concentration ([Na⁺]_i) and resting potential (E_m) of cultured mouse glomus cells (clustered and isolated) were simultaneously measured with intracellular Na⁺-sensitive and conventional, KCl-filled, microelectrodes. Results obtained in clustered and isolated cells were similar. During normoxia (PO₂ 122 Torr), [Na⁺]_i was 12–13 mM corresponding to a Na⁺ equilibrium potential (E_Na) of about 58 mV. Em was about −42 mV. Hypoxia, induced by Na₂S₂O₄ 1 mM (PO₂ 10 Torr), depolarized the cells by about 20 mV, [Na⁺]_i increased by 21 mM and E_Na dropped to about 35 mV. One millimolar of CoCl₂ depressed, or blocked, the effects of Na₂S₂O₄ on [Na⁺]_i but did not affect hypoxic depolarization. Voltage-clamping at −70 mV, while delivering pulses of different amplitudes, produced only small (about 10 pA) and slow TTX-insensitive inward currents. Fast and large (TTX-sensitive) inward currents were not detected. The cell conductance (measured with voltage ramps) was less than 1 nS. It was not affected by hypoxia but was depressed by cobalt. Voltage ramps elicited small inward currents in control and hypoxic solutions that were much smaller than those induced by barium (presumably enhancing calcium currents). Also, normoxic and hypoxic currents had lower thresholds and their troughs were at more negative voltages than in the presence of Ba²⁺. All currents were blocked by 1 mM CoCl₂ suggesting that, at this concentration, cobalt exerted a nonspecific effect on glomus membrane channels. Hypoxia induced a large [Na⁺]_i increase (presumably through inflow), but very small voltage-gated inward currents. Thus, Na⁺ increases (inflow) probably occurred by disturbing a Na⁺/K⁺ exchange mechanism and not by activation of voltage-gated channels.     

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.     

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     

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.     

Voltage-dependent Ca influx into identified leech neurones

We determined the relationships between the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and the membrane potential (E_m) of six different neurones in the leech central nervous system: Retzius, 50 (Leydig), AP, AE, P, and N neurones. The [Ca²⁺]_i was monitored by using iontophoretically injected fura-2. The membrane depolarization evoked by raising the extracellular K⁺ concentration ([K⁺]_o) up to 89 mM caused a persistent increase in [Ca²⁺]_i, which was abolished in Ca²⁺-free solution indicating that it was due to Ca²⁺ influx. The threshold membrane potential that must be reached in the different types of neurones to induce a [Ca²⁺]_i increase ranged between −40 and −25 mV. The different threshold potentials as well as differences in the relationships between [Ca²⁺]_i and E_m were partly due to the cell-specific generation of action potentials. In Na⁺-free solution, the action potentials were suppressed and the [Ca²⁺]_i/E_m relationships were similar. The K⁺-induced [Ca²⁺]_i increase was inhibited by the polyvalent cations Co²⁺, Ni²⁺, Mn²⁺, Cd²⁺, and La³⁺, as well as by the cyclic alcohol menthol. Neither the polyvalent cations nor menthol had a significant effect on the K⁺-induced membrane depolarization. Our results suggest that different leech neurones possess voltage-dependent Ca²⁺ channels with similar properties.     

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.     

Properties of Single Calcium-activated Potassium Channels of Large Conductance in Rat Hippocampal Neurons in Culture

Patch-clamp recordings were made on rat hippocampal neurons maintained in culture. In cell-attached and excised inside-out and outside-out patches a large single-channel current was observed. This channel had a conductance of 220 and 100 pS in 140 mM [K⁺]_i/140 mM [K⁺]_o and 140 mM [K⁺]_i/3 mM [K⁺]_o respectively. From the reversal potential the channel was highly selective for K⁺, the P_K+/P_na+ ratio being 50/1. Channel activity was voltage-dependent, the open probability at 100 mM [Ca²⁺]_i increasing by e-fold for a 22 mV depolarization. It was also dependent on [Ca²⁺]_i at both resting and depolarized membrane potentials. Channel open states were best described by the sum of two exponentials with time constants that increased as the membrane potential became more positive. Channel activity was sensitive to both external (500 μM) and internal (5 mM) tetraethylammonium chloride. These data are consistent with the properties of maxi-K⁺ channels described in other preparations, and further suggest a role for maxi-channel activity in regulating neuronal excitability at the resting membrane potential. Channel activity was not altered by 8-chlorophenyl thio cAMP, concanavalin A, pH reduction or neuraminidase. In two of five patches lemakalim (BRL 38227) increased channel activity. Internal ruthenium red (10 μM) blocked the channel by shortening the duration of both open states. This change in channel gating was distinct from the ‘mode switching’ seen in two patches, where a channel switched spontaneously from normal activity typified by two open states to a mode where only short openings were represented.     

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.     

Blockade of K+ channels induced by AMPA/kainate receptor activation in mouse oligodendrocyte precursor cells is mediated by NA+ entry

AMPA/kainate receptor activation in cultured oligodendrocyte precursor cells from embryonic mouse cortex leads to a blockade of delayed rectifying K⁺ currents. In the present study, we provide evidence using the patch-clamp technique in the whole-cell configuration that the mechanism linking kainate receptor activation and K⁺ conductance blockade is due to the receptor-mediated Na⁺ entry: (1) The blockade was not observed in Na⁺ -free bathing solution nor when intracellular [Na⁺] was elevated by dialzying the cell with a pipette solution containing high [Na⁺]. (2) Elevation of intracellular [Na⁺] alone led to a blockade of outward currents in contrast to cells dialyzed by sucrose. High [Li⁺]_i also reduced the outward currents, and in Li⁺-containing bathing solution the kainate-induced blockade of K⁺ channels was more pronounced. Probably, Li⁺ accumulates intracellularly after permeation through the receptor pore due to slower extrusion mechanisms. Experiments with GTPγS or GDPβS and pertussis toxin indicated that GTP-binding protein-mediated mechanisms were not of importance for the kainate-induced K⁺ conductance blockade. Our data suggest that in glial precursor cells AMPA/kainate receptor activation leads to an intracellular [Na⁺] increase which blocks delayed rectifying K⁺ channels. © 1995 Wiley-Liss, Inc.     

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     

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca and glycolysis

To elucidate the mechanism of pH_i changes induced by membrane depolarization, the variations in pH_i and [Ca²⁺]_i induced by a number of depolarizing agents, including high K⁺, veratridine, N-methyl-

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-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pH_i and an elevation of intracellular calcium ([Ca²⁺]_i) in the CA1 pyramidal cell layer. The increases in [Ca²⁺]_i caused by the depolarizing agents almost completely disappeared in the absence of Ca²⁺ (0 mM Ca²⁺ with 1 mM EGTA). In Ca²⁺ free media, pH_i acid shifts produced by high K⁺, veratridine or NMDA were attenuated by 10–25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pH_i acid shifts induced by both high K⁺ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K⁺, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na⁺–K⁺ ATPase activity. A Ca²⁺-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H⁺ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.     

Differential inhibition of catecholamine secretion by amitriptyline through blockage of nicotinic receptors,sodium channels,and calcium channels in bovine adrenal chromaffin cells

We investigated the effects of amitriptyline, a tricyclic antidepressant, on [³H]norepinephrine ([³H]NE) secretion and ion flux in bovine adrenal chromaffin cells. Amitriptyline inhibited [³H]NE secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) and 70 mM K⁺. The half maximal inhibitory concentration (IC₅₀) was 2 μM and 9 μM, respectively. Amitriptyline also inhibited the elevation of cytosolic calcium ([Ca²⁺]_i) induced by DMPP and 70 mM K⁺ with IC₅₀ values of 1.1 μM and 35 μM, respectively. The rises in cytosolic sodium ([Na⁺]_i) and [Ca²⁺]_i induced by the Na⁺ channel activator veratridine were also inhibited by amitriptyline with IC₅₀ values of 7 μM and 30 μM, respectively. These results suggest that amitriptyline at micromolar concentrations inhibits both voltage-sensitive calcium (VSCCs) and sodium channels (VSSCs). Furthermore, submicromolar concentrations of amitriptyline significantly inhibited DMPP-induced [³H]NE secretion and [Ca²⁺]_i rise, but not veratridine- or 70 mM K⁺-induced responses, suggesting that nicotinic acetylcholine receptors (nAChR) as well as VSCCs and VSSCs can be targeted by amitriptyline. DMPP-induced [Na⁺]_i rise was much more sensitive to amitriptyline than the veratridine-induced rise, suggesting that the influx of Na⁺ and Ca²⁺ through the nAChR itself is blocked by amitriptyline. Receptor binding competition analysis showed that binding of [³H]nicotine to chromaffin cells was significantly affected by amitriptyline at submicromolar concentrations. The data suggest that amitriptyline inhibits catecholamine secretion by blocking nAChR, VSSC, and VSCC. Synapse 29:248–256, 1998. © 1998 Wiley-Liss, Inc.