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.     

The role of glial cells in regulation of neurotransmitter amino acids in the external environment. I. Transmembrane electrical and ion gradients and energy parameters in cultured glial-derived cell lines

Studies of energy parameters and intracellular ion concentrations were carried out on two glial cell lines, one derived from an astrocytoma (C6) and the other from an oligodendroglioma (TR33B), to elucidate the mechanism of transport of amino acid neurotransmitters by glial cells. Respiratory rate was 2.7–2.9 nmol/min/mg dry wt.; cytochrome c at 0.035–0.041 nmol/mg dry wt., was 23–29% reduced with a calculated turnover number 4.7–5.1 e⁻/s at 23 °C. ATP levels were high, 5.0–6.5 mM and [CrP]/[Cr] was almost 2. Membrane potentials at[K⁺]_e = 5mM were approximately −90 mV for C6 cells and −72 mV for TR33B. [K⁺]_i was measured as approximately 100 mM for TR33B and 150 mM for C6 which indicated that the K⁺ diffusion potential was the major source of the membrane potential. [Na⁺]_i was 5.8 mM for C6 and 20 mM for TR33B cells while free calcium was about 100 nM. Near Nernstian relationships were found in both types of cell between [K⁺]_e and membrane potential over a range of 3.5–75 mM for TR33B and 5–110 mM for C6 cells. It is concluded that C6 and TR33B cell lines may be useful models for in vitro studies of some aspects of glial behavior.     

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

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

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.     

CNS control of active sodium transport in muscle during progressive hypokalemia in the rat

The degree of plasma hypokalemia was graded with the duration of a potassium deficient diet. The intracellular Na⁺ and K⁺ contents ([Na]_i and [K]_i) of the innervated soleus muscle of hypokalemic rats were determined and plotted as a function of plasma K⁺ concentration following a potassium deficient diet during 1–8 weeks. The progression of plasma hypokalemia was highly correlated with both [Na]_i accumulation and [K]_i loss in the muscle. Denervation of muscles in the hypokalemic rat resulted in a rapid activation of the Na-pump in the denervated soleus muscle regardless of the degree of plasma hypokalemia. This pump activation in the denervated muscle was chronically maintained throughout the hypokalemia. Restoration of a normal, K⁺ containing, diet for 4 days in hypokalemic rats resulted in a complete recovery of normal Na⁺ and K⁺ contents in both the innervated muscle and plasma, while the [Na]_i and [K]_i in the denervated muscle was unaffected. The relationship between the CNS-induced inhibition of the muscle Na-pump and the plasma K⁺ levels in hypokalemic rats is discussed.     

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.     

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.     

Secretion from rat neurohypophysial nerve terminals (neurosecretosomes) rapidly inactivates despite continued elevation of intracellular Ca

Cytoplasmic calcium concentration was measured in neurosecretory nerve terminals (neurosecretosomes) isolated from rat neurohypophyses by fura-2 fluorescence measurements and digital video microscopy. Hormone release and cytoplasmic calcium concentration were measured during depolarizations induced by elevated extracellular potassium concentration. During prolonged depolarizations with 55 mM [K⁺]₀, the cytoplasmic calcium concentration remained elevated as long as depolarization persisted, while secretion inactivated after the initial sharp rise. The amplitude and duration of the increase in [Ca²⁺]_i was dependent on the degree of depolarization such that upon low levels of depolarizations (12.5 mM or 25 mM [K⁺]₀), the calcium responses were smaller and relatively transient, and with higher levels of depolarization (55 mM [K⁺]₀) the responses were sustained and were higher in amplitude. Responses to low levels of depolarization were less sensitive to the dihydropyridine calcium channel blocker, nimodipine, while the increase in [Ca²⁺]_i induced by 55 mM [K⁺]₀ became transient, and was significantly smaller. These observations suggest that these peptidergic nerve terminals possess at least two different types of voltage-gated calcium channels. Removal of extracellular sodium resulted in a significant increase in [Ca²⁺]_i and secretion in the absence of depolarizing stimulus, suggesting that sodium-calcium exchange mechanism is operative in these nerve terminals. Although the [Ca²⁺]_i increase was of similar magnitude to the depolarization-induced changes, the resultant secretion was 10-fold lower, but the rate of inactivation of secretion, however, was comparable.     

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     

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.     

Inorganic phosphate enhances phosphonucleotide concentrations in cultured fetal rat cortical neurons

Our laboratory has recently characterized saturable Na⁺-dependent P_i import into cultured fetal rat cortical neurons and shown that a substantial fraction of the P_i so accumulated is incorporated into ATP. We now report that the ATP, NADPH and intracellular free P_i ([P_i]_i) concentrations of cultured fetal rat cortical neurons are dependent on the extracellular P_i concentration ([P_i]_e). [ATP], [NADPH] and [P_i]_i display a hyperbolic dependence upon [P_i]_e, being significantly increased after incubation with [P_i]_e of ≥10 μM, and maximal at ≥500 μM. Increases in both [ATP] and [NADPH] are abolished in the absence of glucose. In the absence of extracellular P_i, both [ATP] and [P_i]_i decline over time. Our data suggest that in cultured fetal rat cortical neurons [P_i]_e has a direct effect on glucose utilization, stimulating both ATP and NADPH synthesis via glycolysis and the pentose phosphate pathway.     

Microglia generate external proton and potassium ion gradients utilizing a member of the H/K ATPase family

An ion selective electrode in self-referencing mode was employed to detect ionic concentration gradients at the vicinity of microglia isolated from newborn rat brains. At 5 mM extracellular potassium concentration, a gradient of −9.43 ± 4.2 μM (n = 48) was recorded dissipating over a distance of 10 μm from the outer surface of the cell membrane. Pharmacological studies indicated that neither the Na⁺/K⁺-ATPase nor the inward rectifier potassium channel makes significant contributions to generation of this gradient. The recorded potassium gradient was found to be augmented by increase in extracellular potassium or proton concentrations and could be inhibited by Omeprazole (10 μM) and by the specific H⁺/K⁺-ATPase blocker SCH28080 (1 μM). These, along with the coexistence of a gradient of excess of protons, strongly suggest that a K/H ATPase is the major generator of both the potassium and the proton gradients. The Kd of the glial transporter for K ions is an order of magnitude higher (3.7 mM) than that of the epithelial H⁺/K⁺-ATPase. This is a first report of an H⁺/K⁺ transporter in microglia cells with a Kd in the physiological range of [K⁺]_out. Implications of the H⁺/K⁺-ATPase on potassium homeostasis in microglia under high extracellular potassium and low pH, as found at the site of brain injury, are discussed. GLIA 23:339–348, 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.     

Inhibition of [Ca2+]i Transients in Rat Adrenal Chromaffin Cells by Neuropeptide Y Role for a cGMP-dependent Protein Kinase-activated K+ Conductance

The effects of neuropeptide Y on the intracellular level of Ca²⁺ ([Ca²⁺]_i) were studied in cultured rat adrenal chromaffin cells loaded with fura-2. A proportion (16%) of cells exhibited spontaneous rhythmic [Ca²⁺]_i oscillations. In silent cells, oscillations could be induced by forskolin and 1,9–dideoxyforskolin. This action of forskolin was not modified by H-89, an inhibitor of protein kinase A. Spontaneous [Ca²⁺_i fluctuations and [Ca²⁺]_i fluctuations induced by forskolin- and 1,9-dideoxyforskolin were inhibited by neuropeptide Y. Increases in [Ca²⁺]_i induced by 10 and 20 mM KCI but not by 50 mM KCI were diminished by neuropeptide Y. However, neuropeptide Y had no effect on [Ca²⁺]_i increases evoked by (-)BAY K8644 and the inhibitory effect of neuropeptide Y on responses induced by 20 mM KCI was not modified by o-conotoxin GVIA, consistent with neither L- nor N-type voltage-sensitive Ca²⁺ channels being affected by neuropeptide Y. Rises in [Ca²⁺]_i provoked by 10 mM tetraethylammonium were not decreased by neuropeptide Y, suggesting that K⁺ channel blockade reduces the effect of neuropeptide Y. However, [Ca²⁺]_i transients induced by 1 mM tetraethylammonium and charybdotoxin were still inhibited by neuropeptide Y, as were those to 20 mM KCI in the presence of apamin. The actions of neuropeptide Y on [Ca²⁺]_i transients provoked by 20 and 50 mM KCI, 1 mM tetraethylammonium, (-)BAY K8644 and charybdotoxin were mimicked by 8–bromo-cGMP. In contrast, 8–bromo-CAMP did not modify responses to 20 mM KCI or 1 mM tetraethylammonium. The inhibitory effects of neuropeptide Y and 8–bromo-cGMP on increases in [Ca²⁺]_i induced by 1 mM tetraethylammonium were abolished by the Rp-8–pCPT-cGMPS, an inhibitor of protein kinase G, but not by H-89. A rapid, transient increase in cGMP level was found in rat adrenal medullary tissues stimulated with 1 μM neuropeptide Y. Rises in [Ca²⁺]_i produced by DMPP, a nicotinic agonist, but not by muscarine, were decreased by neuropeptide Y. Our data suggest that neuropeptide Y activates a K⁺ conductance via a protein kinase G-dependent pathway, thereby opposing the depolarizing action of K⁺ channel blocking agents and the associated rise in [Ca²⁺]_i.     

Carrier-mediated Cl− transport in cultured mouse oligodendrocytes

We studied the steady state and the regulation of intracellular Cl^? activity (aCl^?_i) and the mechanisms of KCl uptake in cultured oligodendrocytes from mouse spinal cord using Cl^?-selective microelectrodes. The majority of oligodendrocytes actively accumulated Cl^? above passive distribution (2–3 mM), few cells showed a passive Cl^? distribution. To identify the carriers mediating Cl^? uptake, oligodendrocytes were maintained in a solution with low extracellular Cl^? concentration ([Cl^?]₀) which resulted in a rapid decrease in aCl^?_i. The recovery of aCl^?_i above its passive distribution in normal [Cl^?]₀ was blocked in the absence of Na⁺ or in the presence of furosemide and of bumetanide, which has been reported to inhibit Na⁺/K⁺/Cl^? cotransport. We therefore conclude that Cl^? uptake is primarily due to the activity of a Na⁺/K⁺/Cl^? transport system. Cl^? uptake above passive distribution was not affected in HCO₃^?-free solution or in the presence of SITS and DIDS, indicating that Cl^?/HCO₃^? exchange is not involved in Cl^? uptake by oligodendrocytes. Elevation of [K⁺]₀ induced an increase in aCl^?_i and, as shown earlier, intracellular K⁺ activity. This K⁺-induced Cl^? uptake was not blocked by bumetanide, furosemide, SITS, or DIDS, suggesting that under conditions of raised [K⁺]₀ the combined uptake of K⁺ and Cl^? is not mediated by a carrier, but can be explained by the entry through channels driven by Donnan forces.     

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.     

Hyperpolarization induces a rise in intracellular sodium concentration in dopamine cells of the substantia nigra pars compacta

We investigated the effect of changes in membrane-voltage on intracellular sodium concentration ([Na⁺]_i) of dopamine-sensitive neurons of the substantia nigra pars compacta in a slice preparation of rat mesencephalon. Whole-cell patch-clamp techniques were combined with microfluorometric measurements of [Na⁺]_i using the Na⁺-sensitive probe, sodium-binding benzofuran isophthalate (SBFI). Hyperpolarization of spontaneously active dopamine neurons (recorded in current-clamp mode) caused the cessation of action potential firing accompanied by an elevation in [Na⁺]_i. In dopamine neurons voltage-clamped at a holding potential of ?60 mV elevations of [Na⁺]_i were induced by long-lasting (45–60 s) voltage jumps to more negative membrane potentials (–90 to ?120 mV) but not by corresponding voltage jumps to ?30 mV. These hyperpolarization-induced elevations of [Na⁺]_i were depressed during inhibition of I_h, a hyperpolarization-activated inward current, by Cs⁺. Hyperpolarization-induced elevations in [Na⁺]_i might occur also in other cell types which express a powerful I_h and might signal lack of postsynaptic activity.     

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.     

Effects of serotonin on extracellular potassium concentration in the rat hippocampal slice

The effects of serotonin (5-HT) on extracellular potassium concentration ([K⁺]₀) were measured with ion-selective microelectrodes in rat hippocampal slices. Electrical stimulation of an excitatory afferent system, the Schaffer collateral commissural pathway, caused a 2–4 mM rise in [K⁺]₀ in the stratum pyramidale of area CA1. 5-HT caused a 0.6–1.1 mM rise in [K⁺]₀. This rise was associated with hyperpolarization of neurons and cessation of their spontaneous spike discharge. Methysergide, a 5-HT antagonist, reduced the 5-HT effect. The change in [K⁺]₀ was highest in stratum moleculare and lowest in stratum pyramidale, the opposite gradient to that found with excitatory electrical stimulation. The 5-HT-induced [K⁺]₀ changes were maximal in CA1 stratum moleculare, intermediate in the dentate stratum granulare and almost non-existent in the CA3 stratum pyramidale.GABA, but not norepinephrine, produced a small (up to 0.5 mM) rise in [K⁺]₀ in stratum pyramidale. Extracellular calcium concentration measured with a Ca²⁺-sensitive microelectrode was reduced by electrical stimulation but unchanged by 5-HT or norepinephrine. It is suggested that 5-HT hyperpolarizes hippocampal cells by activation of sodium- and calcium-independent potassium channels, which cause a rise in [K⁺]₀.