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.     

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.     

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.     

Intracellular pH regulation in cultured microglial cells from mouse brain

Intracellular pH (pH₁) and the mechanisms of pH₁ regulation have been investigated in cultured microglial cells from mouse brain using the pH-sensitive fluorescent dye 2′,7′-bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein (BCECF). Cells were acidified by a pulse of NH₄⁺ (4-5 min; 20 mM) and the subsequent pH₁ recovery from an acidification was studied. In HCO₃^--free saline, pH regulation was dependent on extracellular [Na⁺] and sensitive to amiloride, indicating the involvement of the Na⁺/H⁺ exchanger. In HCO₃^--containing solution 2 mM amiloride slowed but did not block pH₁ recovery; the recovery however was dependent on extracellular [Na⁺] and sensitive to 0.3 mM DIDS, suggesting the presence of Na⁺/HCO₃ cotransporter and/or Na⁺-dependent Cl^-/HCO₃^- exchanger. The involvement of a Na-dependent Cl^-/HCO₃^- exchanger was inferred from the observation that removal of Cl^- or application of 1 mM furosemide decreased but did not block the recovery rate. Increasing [K⁺]₀ resulted in an alkalinization by a process that was neither HCO₃^- nor Na⁺-dependent, nor DIDS- and amiloride-inhibitable. In conclusion, microglial cells express a distinct set of pH regulatory carriers which control for a defined level of pH₁. An increase in [K⁺]₀ can offset this level. © 1996 Wiley-Liss, Inc.     

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.     

Purine and pyrimidine nucleotides activate distinct signalling pathways in PC12 cells

The role of extracellular nucleotides in intracellular signalling and neurosecretion was assessed in PC12 cells. Activation of phospholipase C and increased [Ca²⁺]_i were mediated by purinoceptors with an agonist potency profile, ATP ～ UTP > 2-methylthioadenosine triphosphate (2-MeSATP), typical of P_2U. ATP also evoked a rapid acidification followed by a more gradual alkalinization (measured with 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF)), while UTP induced only a gradual alkalinization. The amiloride analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA) attenuated the alkalinization phase suggesting activation of the Na⁺/H⁺ exchanger by ATP and UTP. Using bisoxonol and [³H]tetraphenylphosphonium ([³H]TPP⁺) as potential-sensitive probes, we showed that while ATP rapidly depolarized PC 12 cells in an Na⁺ -dependent manner, UTP evoked a much reduced and delayed response. The potency profile (ATP ～ 2-MeSATP ～ adenosine 5′-0-(3thiotriphosphate) (ATPγS) ? UTP, α, beta;-methyleneATP) suggested involvement of a receptor subtype distinct from P_2U. Secretion of endogenous dopamine was also assessed. Those nucleotides that induced depolarization (ATP, 2-MeSATP, ATPγS) were also the most potent secretagogues. UTP was ineffective. Our results suggest that ATP stimulates distinct purinoceptor subtypes and induces neurosecretion through the activation of multiple signalling path ways. © 1995 Wily-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.     

Voltage-dependent Na+-HCO3− cotransporter and Na+/H+ exchanger are involved in intracellular pH regulation of cultured mature rat cerebellar oligodendrocytes

Intracellular pH (pH_i) was measured at 37°C in mature rat cerebellar oligodendrocytes dissociated in culture by using the pH-sensitive probe BCECF. Cells were identified by anti-galactocerebroside antibody. The mean steady-state pH_i was 7.02 in the absence of CO₂/bicarbonate (Hepes-buffered solution) at an external pH of 7.40 and 7.04 in 5% CO₂/25 mM bicarbonate-buffered solution at the same external pH; this value was modified neither by the removal of external chloride nor by the addition of the chloride-coupled transport blocker DIDS. In both external solutions steady-state pH_i values were strongly dependent on external pH. In Hepes-buffered solution pH_i recovery following an acid load required external Na⁺ and was completely inhibited by amiloride, indicating the presence of a Na⁺/H⁺ exchanger. In CO₂/bicarbonate-buffered solution amiloride partially reduced the pHi recovery rate, indicating the presence of a bicarbonate-dependent pH_i regulating mechanism. Membrane depolarization induced by increasing external K⁺ concentration elicited an alkalinization only in the presence of external Na⁺ and bicarbonate. Analysis of the calculated HCO₃ fluxes with respect to membrane potential indicated that these fluxes were mediated by a Na⁺-HCO₃ cotransport with a stoichiometry of 1:3. These results demonstrate that a Na⁺/H⁺ exchanger and a Na⁺ HCO₃ cotransporter are involved in pH_i regulation of mature oligodendrocytes. GLIA 19:74–84, 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.     

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.     

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.     

Characterization of Na+/H+ exchange activity in cultured rat hippocampal astrocytes

Astrocytes actively maintain their intracellular pH (pH_i) more alkaline than expected by passive distribution of H⁺. Acid extruding transporters such as the amiloride-sensitive Na⁺/H⁺ exchanger (NHE) are necessary for pH regulation. Currently, four mammalian NHEs (NHE1-NHE4) have been cloned, with a fifth (NHE5) partially cloned. We attempted to determine which isoform(s) of NHE was present in cultured hippocampal astrocytes using amiloride sensitivity and immunospecificity as criteria. In the absence of HCO₃⁻, amiloride blocked pH_i recovery after an acid load with an IC₅₀ of ∼3.18 μM, similar to values reported for the amiloride-sensitive isoforms NHE1 and NHE2. Immunoblotting with a highly specific antibody for NHE1 identified a 100 kDa protein, indicating the presence of NHE1 in whole brain, hippocampus, and cultured hippocampal astrocytes. Further probing for an additional amiloride-sensitive NHE failed to detect evidence of the presence of NHE4. Surprisingly, application of the potent analog of amiloride, ethylisopropylamiloride (EIPA), caused a reversible alkalinization of pH_i, suggesting the presence of an additional acid/base transport mechanism that is EIPA-sensitive. © 1996 Wiley-Liss, Inc.     

Single ion channel currents in neuropile glial cells of the leech central nervous system

The patch-clamp technique was used to investigate the activity of single ion channels in neuropile glial (NG) cells in the central nervous system (CNS) of the medicinal leech, Hirudo medicinalis. We found evidence for two distinct Cl^? channels that could be distinguished by their basic electrical properties and their responses to different inhibitors on single ion channels currents. In the Inside-out configuration in symmetrical Cl solutions, these channels showed current-voltage relationships with slight outward rectification, mean conductances of 70 and 80 pS, and reversal potentials near 0 mV. Significant permeability to Na⁺, K⁺, or SO₄^2? could not be detected. The open-state probability of the 70 pS Cl^? channel increased with membrane depolarization, whereas the open-state probability of the 80 pS Cl^? channel was voltage-independent. The application of the stilbene derivative DIDS (100 μM) to the cytoplasmic side of the glial cell membrane blocked both Cl^? channels. The activity of the 70 pS channel was blocked irreversibly by DIDS, whereas the activity of the 80 pS channel reappeared after wash-out of DIDS. Both channels were blocked reversibly by 1 mM Zn²⁺. K⁺ channels could only be observed occasionally in the soma membrane of the NG cells. We have characterized a 60 pS K⁺ channel with a high selectivity for K⁺ over Na⁺. The low density of K⁺ channels in the soma membrane may indicate a non-uniform distribution of this channel type in NG cells. © 1993 Wiley-Liss, Inc.     

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.     

P2U nucleotide receptor activation in rat glial cell line induces [Ca2+]i oscillations which depend on cytosolic pH

In single rat glioma cells, the signal transduction process activated by the UTP sensitive purinergic nucleotide receptor was studied by determining [Ca²⁺]_i by Fura-2 fluorescence and measuring pH by BCECF fluorescence to elucidate the control of [Ca²⁺]_i oscillations by intracellular pH. Addition of UTP for long time periods (some min) causes a [Ca²⁺]_i response composed of i) an initial large peak and a following sustained increase (160 s duration), and ii) subsequent regular [Ca²⁺]_i oscillations (amplitude 107 nM, frequency 1.5 oscillations per min). The maintenance of the [Ca²⁺]_i oscillations depends on the continued presence of agonist. The oscillations are abolished by reducing extracellular Ca²⁺ concentration. The interaction of UTP receptors and bradykinin receptors during the [Ca²⁺]_i oscillations was investigated because previous studies have already shown that the peptide causes comparable [Ca²⁺]_i oscillations. During [Ca²⁺]_i oscillations induced by UTP or bradykinin, long-term admission of both hormones (400–500 s) causes a large initial response superimposed on regular [Ca²⁺]_i oscillations. Short pulses (12 s) of the second agonist given in any phase of the oscillations induce large [Ca²⁺]_i peaks. In both cases, the following oscillations are not disturbed. The influence of cytosolic pH was studied by alkalinizing pH_i by application of NH₄Cl. [Ca²⁺]_i oscillations stop after addition of NH₄Cl. Recovery of NH₄Cl-induced alkalinization is reduced by furosemide. To the same degree, the interruption of [Ca²⁺]_i oscillations is significantly prolonged in the presence of furosemide. Thus cytosolic alkalinization suppresses hormone-induced [Ca²⁺]_i oscillations in rat glioma cells. The understanding of the molecular mechanism of this interference of pH should provide an important contribution for unravelling the function of cytosolic pH in cellular signal transduction. © 1996 Wiley-Liss, Inc.     

Intracellular acidosis of identified leech neurones produced by substitution of external sodium

The intracellular pH, pH_i, of identified neurones of the central nervous system of the leech Hirudo medicinalis L. was measured with double-barrelled neutral carrier pH-sensitive microelectrodes. The active regulation of pH_i of these neurons is due to amiloride-sensitive NaH exchange and hence requires extracellular Na, Na_o. We have measured a decrease of pH_i following the removal of Na_o. The rate of intracellular acidification in Na-free saline was similar to that in the presence of 2 mM amiloride suggesting that the acidification was due to inhibition of the NaH exchange. The rate of intracellular acidification depended on the Na substitute chosen; it was 0.02 ± 0.005 pH units/min (±S.D., n = 17) when Na was replaced by N-methyl-d-glucamine. A similar rate of acidification occurred with tris-hydroxymethyl-aminomethane (Tris) while the rate of acidification was higher with bis-2-hydroxymethyl-dimethyl-ammonium (BDA, 0.033 ± 0.016 pH units/min (±S.D., n = 7) and tetramethylammonium TMA, 0.046 ± 0.017 pH units/min (n = 3) as Na substitutes. A high, non-linear rate of intracellular acidification was observed, when Li, K or choline were used as Na substitute. The recovery of pH_i from acidification upon readdition of Na_o was fast, only when Li had replaced Na was the pH_i recovery considerably delayed. In conclusion, in all experiments using different Na substitutes the removal of Na_o caused a ssubstantial intracellular acidification presumably due to inhibition of NaH exchange. These changes in pH_i might be relevant for results obtained by experiments in which Na-free solutions are used.     

Both NKCC1 and anion exchangers contribute to Cl⁻ accumulation in postnatal forebrain neuronal progenitors

Neuronal progenitors are continuously generated in the postnatal rodent subventricular zone and migrate along the rostral migratory stream to supply interneurons in the olfactory bulb. Nonsynaptic GABAergic signaling affects the postnatal neurogenesis by depolarizing neuronal progenitors, which depends on an elevated intracellular Cl^? concentration. However, the molecular mechanism responsible for Cl^? accumulation in these cells still remains elusive. Using confocal Ca²⁺ imaging, we found that GABA depolarization‐induced Ca²⁺ increase was either abolished by bumetanide, a specific inhibitor of the Na⁺–K⁺–2Cl^? cotransporter, or reduced by partial replacement of extracellular Na⁺ with Li⁺, in the HEPES buffer but not in the CO₂/ buffer. GABA depolarization‐induced Ca²⁺ increase in CO₂/ buffer was abolished by a combination of bumetanide with the anion exchanger inhibitor DIDS or with the carbonic anhydrase inhibitor acetozalimide. Using gramicidin‐perforated patch‐clamp recording, we further confirmed that bumetanide, together with DIDS or acetozalimide, reduced the intracellular chloride concentration in the neuronal progenitors. In addition, with BrdU labeling, we demonstrated that blocking of the Na⁺–K⁺–2Cl^? cotransporter, but not anion exchangers, reduced the proliferation of neuronal progenitors. Our results indicate that both the Na⁺–K⁺–2Cl^? cotransporter and anion exchangers contribute to the elevated intracellular chloride responsible for the depolarizing action of GABA in the postnatal forebrain neuronal progenitors. However, the Na⁺–K⁺–2Cl^? cotransporter displays an additional effect on neuronal progenitor proliferation.     

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.