Brain Research 957 (2002) 1–11 www.elsevier.com / locate / brainres
Research report
Impaired activity of volume-sensitive anion channel during lactacidosis-induced swelling in neuronally differentiated NG108-15 cells Shin-ichiro Mori a,b,c , Shigeru Morishima a,b , *, Mayumi Takasaki c , Yasunobu Okada a,b a
Department of Cell Physiology, National Institute for Physiological Sciences, Myodaiji-cho, Okazaki 444 -8585, Japan b CREST of Japan Science and Technology Corporation, Okazaki 444 -8585, Japan c Department of Anesthesiology, Miyazaki Medical College, Miyazaki 889 -1692, Japan Accepted 7 August 2002
Abstract Acidosis coupled to lactate accumulation, called lactacidosis, occurs in cerebral ischemia or trauma and is known to cause persistent swelling in neuronal and glial cells. It is therefore possible that mechanisms of cell volume regulation are impaired during lactacidosis. Here we tested this possibility using neuronally differentiated NG108-15 cells. These cells responded to a hypotonic challenge with osmotic swelling followed by a regulatory volume decrease (RVD) under physiological pH conditions in the absence of lactate. Under normotonic conditions, sustained cell swelling without subsequent RVD was induced by exposure to lactate-containing solution with acidic pH (6.4 or 6.2), but not with physiological pH (7.4). Under whole-cell patch-clamp, osmotic swelling was found to activate outwardly rectifying Cl 2 currents in cells exposed to control hypotonic solution. A Cl 2 channel blocker, NPPB, inhibited both RVD and the swelling-activated Cl 2 current. RVD and the volume-sensitive Cl 2 current were also markedly inhibited by lactacidosis (pH 6.4 or 6.2), but neither by application of lactate with physiological pH (7.4) nor by acidification without lactate (pH 6.2). RT-PCR analysis showed mRNA expression of two isoforms of proton-coupled monocarboxylate transporters, MCT1 and MCT8, in differentiated NG108-15 cells. Thus, we conclude that persistence of neuronal cell swelling under lactacidosis is coupled to an impairment of the activity of the volume-sensitive Cl 2 channel and to dysfunction of RVD. It is also suggested that the volume-sensitive Cl 2 channel is inhibited by intracellular acidification induced by MCT-mediated proton influx under lactacidosis. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: Lactacidosis; Cell swelling; Regulatory volume decrease; Volume-sensitive Cl 2 channel; Neuron
1. Introduction Cerebral ischemia or trauma results in not only intracellular and extracellular acidosis mainly due to proton liberation from hydrolysis of high-energy phosphates greater than their synthesis but also lactate accumulation through enhanced anaerobic glycolysis [34,61]. In the ischemic brain, extracellular pH is known to fall to 6.8–5.8 in proportion to the amount of lactate accumulated [23,48]. *Corresponding author. Tel.: 181-564-55-7731; fax: 181-564-557735. E-mail address:
[email protected] (S. Morishima).
A significant role of acidosis coupled to lactate accumulation (called lactacidosis) in the formation of cytotoxic brain edema, giving rise to irreversible damage of nerve and glial cells, is widely accepted [26,61]. Lactacidosis was actually demonstrated to lead to swelling in cultured neuronal cells [64], undifferentiated neuroblastoma3 glioma hybrid cells [1], astrocytes [21,22,24,31,50,52,62] and spinal microglia [36]. The mechanism of normotonic swelling induced by lactacidosis has been suggested to involve osmolyte uptake via several pathways, such as lactate influx via the H 1 monocarboxylate symporter [31], Na 1 influx via the Na 1 / H 1 antiporter [1,21,22,50], Cl 2 influx via the Cl 2 / HCO 32
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antiporter [24,50,53], and Na 1 , K 1 and Cl 2 uptake via the Na 1 –K 1 –2Cl 2 symporter [53]. However, most cell types are known to be capable of regulatory volume decrease (RVD) after cell swelling [17,27,43]. This suggests the possible involvement of inactivation of the volume regulatory mechanism in lactacidosis-induced cell swelling [60]. However, no direct evidence has been provided for this inference. In the present study, therefore, we aimed to answer the question of whether dysfunction of the RVD is associated with lactacidosis-induced swelling in neuronally differentiated cells. Swelling-activated Cl 2 channels are known to serve as a pathway for volume-regulatory anion efflux in a large number of cell types [40,42,65]. Also, there is some evidence that volume-sensitive anion channels in glial [8,20,55–57] and neuroblastoma CHP-100 cells [5] mediate swelling-induced release of organic osmolytes, which contributes significantly to volume regulation in brain cells [25,46]. Thus, we also investigated whether the activity of volume-sensitive Cl 2 channel is impaired in neuronally differentiated NG108-15 cells under lactacidosis conditions. A preliminary account of part of these data has appeared in abstract form [35].
2. Materials and methods
2.1. Cell culture In all the experiments, a mouse neuroblastoma3rat glioma hybrid cell line, NG108-15, was used after induction of neuronal differentiation. Cells were grown in highglucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 mM hypoxanthine, 1 mM aminopterin, 16 mM thymidine (HAT), and 10% of fetal bovine serum (FBS: ICN Biomedicals, Aurora, OH, USA) at 37 8C in 5% CO 2 and 95% air. To obtain differentiated cells, they were seeded onto glass coverslips coated with 0.01% polyethylenimine and then incubated in DMEM with 1% FBS, HAT, and 1 mM dibutyl adenosine 39, 59-cyclic monophosphate (db-cAMP) for 4–5 days before use. Neuronal differentiation could be morphologically assessed by extension of neurite-like processes. Functional differentiation was testified to by the generation of action potential and development of voltage-gated Na 1 channel current under whole-cell current-clamp and voltage-clamp (data not shown).
2.2. Chemicals and solutions Anion channel blockers, 4, 49-diisothiocyanatostilbene2, 29-disulfonic acid (DIDS) and 5-nitro-2-(3phenylpropylamino)benzoic acid (NPPB), were purchased from Sigma (St. Louis, MO) and Tocris (Ballwin, MO, USA), respectively. A blocker of monocarboxylate-proton cotransporter (MCT), a-cyano-4-hydroxycinnamate
(CHC), and that of Na 1 / H 1 exchanger, amiloride, were obtained from Sigma. Stock solutions of 1 and 0.1 M of CHC and the other drugs, respectively, were prepared with DMSO. The concentration of DMSO never exceeded 0.1% and had no effect on the cell size or Cl 2 currents. Whole-cell recordings were taken using a K 1 -, Na 1 and Ca 21 -free bathing solution and a K 1 -free, Cs 1 -rich, TEA1 -containing pipette solution to eliminate K 1 , Na 1 and Ca 21 currents. The isotonic control bathing solution contained (in mM) 80 N-methyl-D-glucamine (NMDG), 70 HCl, 10 NaCl, 5 MgCl 2 , and 10 piperazine-1,4-bis(2ethanesulfonic acid) (PIPES), and the pH and osmolality were adjusted to 7.4 and 320 mOsm / kg?H 2 O by adding NMDG and mannitol, respectively. Hypotonic control bathing solution was prepared by reducing the mannitol concentration to adjust the osmolality to 280 mOsm / kg? H 2 O. Low-Cl 2 hypotonic solution was prepared by replacing Cl 2 with gluconate 2 , Br 2 or I 2 . Hypotonic acidic solution was prepared by adjusting the pH of hypotonic control solution to 6.2. Lactate-containing acidic solutions of pH 6.2 and 6.4 were made by adding 7.5 and 6.5 mM lactic acid, respectively, to isotonic or hypotonic control solution. Acetate-containing acidic solution of pH 6.2 was made by adding 7.5 mM acetic acid to isotonic or hypotonic control solution. In some experiments, hypotonic solution containing 7.5 mM lactate, titrated to pH 7.4 with NMDG was employed. The composition of pipette solution was (in mM): 70 CsCl, 20 tetraethylammonium chloride (TEA-Cl), 5 MgCl 2 , 60 D(2)-mannitol, 5 EGTA, 1 HEPES, 5 Na 2 -ATP, and 5 creatinine phosphate sodium salt, and pH was adjusted to 7.2 with CsOH. Volume measurements were taken in isotonic or hypotonic solution in the absence or presence of lactate. The bathing buffer composition (in mM) was 110 NaCl, 5 KCl, 2 CaCl 2 , 0.5 MgCl 2 , 1 NaH 2 PO 4 , 25 NaHCO 3 , 5 glucose, and 0 or 25 lactate. In some experiments, the lactate-free solution containing 24 mM acetate was used. The pH of solution was adjusted to 7.460.05 or 6.260.08, respectively, by bubbling with 73% N 2 , 21% O 2 and 5% CO 2 . The osmolality was adjusted to 350 or 280 mOsm / kg?H 2 O by adding 5 or 70 mM mannitol. The osmolality was measured with a freezing-point depression osmometer (Vogel GmbH, Germany).
2.3. Electrophysiology Differentiated NG108-15 cells adhered to a coverslip were transferred to a recording chamber which was mounted on an inverted differential interference contrast microscope (Eclipse TE300, Nikon, Tokyo, Japan). Currents were measured under the whole-cell configuration at room temperature (24–26 8C) using a patch-clamp amplifier, Axopatch 200B (Axon Instruments, Foster City, CA, USA). Pipettes filled with pipette solution had a resistance of 1.5–2.5 MV. After attaining the whole-cell configuration, the series resistance was less than 10 MV
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and was compensated for by 80–70% to minimize voltage errors. Pulse generation and data acquisition were done on-line using an A / D and D/A interface, Digidata1200 and pCLAMP7 software (Axon Instruments). Current signals were filtered at 2 kHz and sampled at 5 kHz. The holding potential was set at 0 mV to inactivate voltage-gated Na 1 and Ca 21 channels. The time course of current change was monitored by applying repetitive alternating step pulses of 400 ms in duration from 0 to 640 mV every 15 s. To obtain the current–voltage (I–V) relationship, cells were first pre-hyperpolarized from the holding potential to 2100 mV for 200 ms, and successive step pulses of 800 ms in duration were applied from 2100 to 1120 mV in 20-mV increments. In some experiments, the I–V curves were measured by ramp voltage-clamp. Ramp command pulses were applied for 2 s from 280 to 180 mV. An agarose bridge containing 150 mM NMDGCl was used for a reference electrode to minimize junction potential changes when the bath Cl 2 concentration was changed. Remaining junction potentials were calculated using the CLAMPEX7 program (Axon Instruments), and compensated for the study on anion permeability.
2.4. RT-PCR Poly(A)1 RNA was extracted from differentiated NG108-15 cells grown in a culture bottle, the bottom of which was coated with 0.01% polyethylenimine, using a Magnetic Porous Glass (MPG) Direct mRNA Purification Kit (CPG, Lincoln Park, NJ, USA). In brief, the cells were lysed in extraction-hybridization buffer (100 mM Tris– HCl, 500 mM LiCl, 10 mM EDTA, 5 mM DTT, pH 8.0), and the poly(A)1 RNA was hybridized with MPG streptavidin plus biotinylated oligo(dT) 25 . The poly(A)1 RNA was washed three times with 500 ml of washing buffer (10 mM Tris–HCl, 150 mM LiCl, 1 mM EDTA, pH 8.0). Then the poly(A)1 RNA was collected magnetically and eluted with 20 ml of 2 mM EDTA solution. The mRNA sequences of rat MCT1, MCT2, MCT3, MCT4 and mouse MCT8 were obtained from the GeneBank database. The primer sets for MCT1, MCT2, MCT3, MCT4, and MCT8 were designed to amplify the bases 416–1189, 392–985, 123–757, 577–1100, and 1008–1517 in their coding sequences, respectively. The oligonucleotide sequences of forward and reverse primers and the expected RT-PCR product size are as follows: for MCT1, 59-TCTACAAGAAGCGACCATTGG-39, 59GGAGGACAGGACAACATTCC-39, and 774 bp; for MCT2, 59-CATTCAACCTGCAACCAGC-39, 59-CAGTGAACATGATTGCCACG-39, and 594 bp; for MCT3, 59-CCTCCTCCGAGAGCTCAAGC-39, 59-AGTTCACCAGCAGGATAGCC-39, and 635 bp; for MCT4, 59TGTGCTGCACTCATGAGACC-39, 59-TGTGCTGCACTCATGAGACC-39, and 524 bp; and for MCT8, 59CTTCAACATGCGTGTATTCCG-39, 59-AAGGCCACATGGTAATCACC-39, and 510 bp. As a positive control, a
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fragment of mRNA of glyceraldehyde 3-phosphate dehydrogenase (G-3-PDH) was amplified. Sequences of the primers (Clontech, Palo Alto, CA, USA) are 59-ACCACAGTCCATGCCATCAC-39 and 59-TCCACCACCCTGTTGATGTA-39, and the expected RT-PCR product size is 452 bp. The Superscript Preamplification System for First Strand cDNA Synthesis (Gibco BRL, Rockville, MD, USA) was used in the reverse transcription. PCR was performed with Ampli Taq Gold (Perkin Elmer, Norwalk, CT, USA) for 30 PCR cycles at 94 8C (1 min) / 45 8C (2 min) / 72 8C (3 min) using a programmable cycler (GeneAmp PCR System 9600, Perkin-Elmer). As a negative control, RT-PCR without the addition of reverse transcriptase was performed simultaneously. Sequencing of the PCR products was performed using ABI PRISM姠 Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer). PCR was repeated twice to acquire enough PCR products for sequencing, and the products after the second PCR were gel extracted and purified with GeneClean II (BIO101, La Jolla, CA, USA).
2.5. Cell size measurements Cell size was measured at room temperature under an inverted microscope (Nikon Diaphot 300) by a high-speed automatic image analyzing technique, as described previously [37]. In brief, cell images were digitally enhanced and digitized in bitmap files. Surroundings of cells were traced, and the planar area was measured as an indication of the cell size.
2.6. Statistical analysis Data are given as mean6S.E.M. Comparison of two experimental groups was done using Student’s t-test, whereas multiple comparisons were done using an analysis of variance (ANOVA) followed by Dunnett’s test. Statistical significance was considered to be at P , 0.05.
3. Results
3.1. Effects of lactacidosis on cell size and RVD Exposure of neuronally differentiated NG108-15 cells to lactate-containing isotonic solution with pH 6.2 induced a gradual increase in the cell size which reached a plateau level of swelling within 30–50 min, as shown in Fig. 1A (filled circles). In contrast, the cells failed to respond significantly with swelling to lactate at pH 7.4 (open circles). During the first 30 min after lactate application at pH 6.2, swelling proceeded more rapidly in the presence of Cl 2 channel blocker, NPPB (0.1 mM, filled diamonds), than that in the absence of NPPB (filled circles). However, the plateau level of peak swelling with NPPB was not significantly different from that without NPPB. These
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mechanism, because CHC is known to block MCT at a low concentration (IC 50 0.17–0.43 mM) [16] and inhibit Cl 2 / HCO 2 3 exchanger only at a high concentration (IC 50 2.4 mM) [14]. Persistent swelling was also observed in pH 6.2 isotonic solution containing 24 mM acetate, which is a membrane permeable buffer (data not shown, n 5 10). This fact suggests that intracellular acidification and osmolyte accumulation are both involved in persistent normotonic swelling. Differentiated NG108-15 cells responded to exposure to a hypotonic control solution with rapid swelling followed by slow volume recovery (Fig. 1B, open circles). RVD was markedly inhibited by a Cl 2 channel blocker, NPPB (0.1 mM: open squares). Under lactacidosis conditions, in contrast, the pre-swollen cells responded to a hypotonic challenge with additional swelling but never thereafter exhibited RVD (filled circles).
3.2. Effects of lactacidosis on volume-sensitive Cl 2 current
Fig. 1. Effects of lactacidosis and hypotonic stress on cell size in neuronally differentiated NG108-15 cells. Relative cell size is the ratio of planar area of a single cell at a given time to that at time zero. Each symbol represents the mean value for 10–19 cells (vertical bars: S.E.M.). *, P , 0.05 vs. data obtained for lactate-containing acidic (pH 6.2) solution (filled circles). (A) Lactate-containing isotonic acidic solution at pH 6.2 was applied during the time period indicated by the horizontal bar, in the absence (filled circles) or presence of 1 mM CHC (filled squares), 0.1 mM amiloride (filled triangles) or 0.1 mM NPPB (filled diamonds). For control experiments (open circles), lactate-containing isotonic solution of pH 7.4 was applied. (B) Hypotonic solution of pH 7.4 or 6.2 was applied during the time period indicated by the horizontal bar, in the absence (control, pH 7.4: open circles) or presence of lactate (pH 6.2: filled circles) or 0.1 mM NPPB (pH 7.4: open squares). A hypotonic challenge was applied after 30 min preincubation with lactate at pH 6.2 or with NPPB at pH 7.4. The mean values of relative cell size at time zero were 160.06, 1.0060.06 and 1.0560.03 for control (pH 7.4), NPPB (pH 7.4) and lactacidosis (pH 6.2), respectively. Note that RVD is attained in the control experiments where lactate-free hypotonic solution of pH 7.4 was applied in the absence of NPPB.
results may suggest that full inhibition of NPPB-sensitive volume-regulatory Cl 2 channels was attained in the late, but not beginning, phase of lactacidosis-induced swelling. Amiloride (0.1 mM) largely abolished lactacidosis-induced swelling (filled triangles), suggesting an involvement of the Na 1 / H 1 exchanger (NHE) in the mechanism of swelling. Lactacidosis-induced swelling of differentiated NG108-15 cells was partially suppressed by CHC (1 mM: filled squares). This result suggests an involvement of MCT-mediated lactate uptake, in part, in the swelling
An inhibitory effect of NPPB on the RVD suggests that the cells functionally express volume-sensitive Cl 2 channels in differentiated NG108-15 cells. In fact, osmotic swelling was found to activate whole-cell ionic currents under conditions where K 1 , Na 1 and Ca 21 currents were eliminated in differentiated NG108-15 cells, as shown in Fig. 2A. Under these conditions, hypotonic stimulation induced sustained swelling, although RVD did not occur because of the whole-cell configuration. The current amplitude increased with the increase in cell size. Swelling-activated currents exhibited slight time-dependent inactivation at potentials more positive than 160– 180 mV (Fig. 2B: b). The I–V relation recorded by step-pulse voltage-clamp exhibited outward rectification even under symmetrical Cl 2 conditions, as depicted in Fig. 2C. Upon reduction of the extracellular Cl 2 concentration by substitution with gluconate 2 , the reversal potential (Erev ) was found to shift to positive potentials. The Erev shift per 10-fold decrease in the extracellular Cl 2 concentration was 246.1 mV (Inset in Fig. 2C), indicating that the current was largely selective to Cl 2 , and the permeability ratio of gluconate 2 to Cl 2 was estimated to be 0.24, assuming lack of cation permeability. The ramp voltage-clamp also showed an outwardly rectifying I–V relationship under symmetrical Cl 2 conditions (Fig. 2D). Upon replacement of 70% of Cl 2 with I 2 and Br 2 in hypotonic bath solution, the Erev value shifted from 20.3560.09 mV to 21.5860.49 and 25.1860.44 mV (n54), respectively (Fig. 2D). Thus, the permeability coefficients normalized to that for Cl 2 were estimated to be 1.09 for Br 2 and 1.33 for I 2 , corresponding to Eisenman’s sequence I [67]. The volume-sensitive outwardly rectifying (VSOR) Cl 2 current was reversibly blocked by NPPB in a concentration-dependent manner (Fig. 3A). The current responses
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Fig. 2. Swelling-activated whole-cell currents and their anion selectivity in neuronally differentiated NG108-15 cells exposed to control or low Cl 2 hypotonic solution at 280 mOsm / kg?H 2 O. Arrowheads represent zero-current level. (A) Representative time courses of current activation and cell size change before, during and after exposure to control hypotonic solution (pH 7.4). The current was monitored by applying alternating step pulses to 640 mV every 15 s from a holding potential of 0 mV. Step pulses from 2100 to 1120 mV in 20-mV increments were applied at a and b. Gain of chart recorder was changed to one-fourth at b. Micrographs show representative cell images under a differential interference-contrast microscope, at times indicated by arrows. The relative cell size was calculated as the ratio of planar area of a single cell at a given time to that before attaining the whole-cell configuration. The data represent 16 similar observations. (B) Representative traces of current responses to step pulses (protocol given in c) recorded at a and b in A. (C) Effect of changes in extracellular Cl 2 concentration on I–V relationships measured by step pulse application. Inset: Relation between the reversal potential (Erev ) and logarithm of the external Cl 2 concentration ([Cl 2 ] o ). The slope of the fitted line is 246.1 mV/ decade. Each symbol represents the mean6S.E.M. (vertical bars) of five observations. (D) Effects of anion substitutions on I–V curves measured by ramp pulse application. 70% of external Cl 2 was substituted with Br 2 or I 2 . Each curve represents four similar observations.
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Fig. 3. Effects of NPPB (A) and DIDS (B) on volume-sensitive Cl 2 currents in neuronally differentiated NG108-15 cells. Upper panels: representative records of currents during application of alternating pulses (640 mV) or of step pulses (from 2100 to 1120 mV in 20-mV increments), before exposure to control hypotonic solution, during exposure in the absence or presence of a Cl 2 channel blocker, and after exposure. Lower panels: current responses to step pulses (the protocol given in Fig. 2Bc) recorded at a and b in the upper panels as well as the I–V relationships (c) where open and filled circles represent the mean6S.E.M. (vertical bar) of six observations in the absence (control) and presence, respectively, of a Cl 2 channel blocker. Arrowheads represent zero-current level. *, P , 0.05 vs. corresponding control data (open circles).
to step pulses (a, b) and the I–V curve (c) show that NPPB-induced inhibition was independent of voltage. In contrast, DIDS reversibly inhibited VSOR Cl 2 currents in a manner dependent not only on concentration but also on voltage (Fig. 3B). Outward currents were preferentially blocked, and the inactivation kinetics at positive potentials became more prominent and accelerated by DIDS.
Effects of lactacidosis on the VSOR Cl 2 current were examined in differentiated NG108-15 cells by applying lactate-containing acidic hypotonic solution after attaining steady-state activation of Cl 2 currents under whole-cell patch-clamp. At pH 6.4, lactacidosis induced inhibition of the VSOR Cl 2 current, in a completely reversible manner, after transiently augmenting the current without affecting
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the extent of swelling, as shown in Fig. 4A. Full inhibition was produced within 60 min after application of lactic acid at pH 6.4, and the time required for a half-maximal inhibition was 4606127 s (n54). The current responses to step pulses (a, b) and the I–V curves (c) show that the inhibiting effect was independent of voltage. In contrast, the VSOR Cl 2 current was virtually unaffected by addition of lactate with maintenance of the pH at 7.4 (Fig. 4B). At pH 6.2, lactacidosis inhibited the VSOR Cl 2 current more rapidly with a half-maximal inhibition time of 190628 s (n53), as shown in Fig. 4C, after transiently augmenting the current. In the absence of lactate, acidic hypotonic solution at pH 6.2 also produced a transiently enhancing effect but brought about a far less inhibitory effect on the VSOR Cl 2 current (Fig. 4D). The current was never completely inhibited even after over 60 min of exposure to acidic hypotonic solution devoid of lactate, where the extrapolated value for half-maximal inhibition time was 12866356 s (n53). Such mutual dependence between protons and lactate suggests that the VSOR Cl 2 channel was inhibited by intracellular acidification induced by lactate-dependent proton uptake. This inference was supported by the fact that similar inhibition of whole-cell VSOR Cl 2 currents was induced by application of pH 6.2 solution buffered with a membrane-permeable buffer acetate (7.5 mM: data not shown, n55).
3.3. Expression of MCT mRNA It is well known that lactate-dependent proton uptake by MCT decreases the cytosolic pH in neurons and astrocytes [38] as well as other mammalian cells [10,19]. Among a family of eight MCTs hitherto cloned, MCT1, MCT2, MCT3, MCT4 and MCT8 are known to be expressed in mouse and rat [16]. Thus, we examined molecular expression of these five MCTs in differentiated NG108-15 cells, which were derived from mouse neuroblastoma and rat glioma cells, by RT-PCR. As presented in Fig. 5, single bands corresponding only to RT-PCR products (expected lengths 774 and 510 bp) from MCT1 and MCT8 reversetranscribed RNA (RT(1)) were detected. However, PCR products of MCT2, MCT3 and MCT4 were not detected, whereas the PCR product of house-keeping enzyme, G-3PDH, was consistently detected. Also, no PCR product was amplified when reverse transcriptase was omitted from the reaction (RT(2)). The nucleotide sequence of the PCR products was found to be completely identical to the corresponding regions in MCT1 and MCT8 (data not shown).
4. Discussion Here we report the following new findings in neuronally differentiated NG108-15 cells. First, the cells express both
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volume-sensitive outwardly rectifying (VSOR) Cl 2 channels, which are involved in a regulatory volume decrease (RVD), and genes of proton-monocarboxylate symporter isoforms, MCT1 and MCT8, which mediate cotransport of H 1 and lactate 2 . Second, lactacidosis-induced cytosolic acidification inhibits VSOR Cl 2 channel activity, thereby abrogating RVD. Third, lactacidosis-induced persistent cell swelling, which may be the result of Na 1 uptake via Na 1 / H 1 exchange and by lactate 2 uptake via MCT, is coupled to impairments of RVD capacity and VSOR Cl 2 channel activity.
4.1. Neuronal cell RVD and its impairment under lactacidosis One of the most essential cell functions is regulation of cell volume, which must be adjusted not only in the face of fluctuations in intra- and extracellular osmolarity, but also in association with cell proliferation, migration and death [28,45]. Most cell types are known to exhibit RVD after osmotic swelling upon a hypotonic challenge [17,27,43]. The RVD capability has been observed in a number of neuronal cells, including sympathetic neurons [18,29,51], cerebellar granule neurons [47,49], CA1 stratum radiatum of hippocampal slices [12], cerebral cortex neurons [13], and sensory trigeminal neurons [66] as well as neuroblastoma cells [4,6,9,30,32]. Undifferentiated neuroblastoma3glioma NG108-15 cells have also been shown to possess the RVD ability [54]. In the present study, the volume regulation was demonstrated to be preserved after neuronal differentiation by direct measurements of cell size, being in agreement with previous indirect observations with fluorescence probes [2]. Furthermore, the present study provides evidence for the first time that RVD is impaired in differentiated NG108-15 cells under lactacidosis conditions.
4.2. Neuronal volume-sensitive Cl 2 channel and its impairment under lactacidosis In a variety of cell types, RVD is known to be a result of parallel activation of K 1 and Cl 2 channels [17,44,59]. Among a number of types of volume-regulatory anion channel, the VSOR Cl 2 channel with an intermediate single-channel conductance is known to be most ubiquitously expressed and to serve as a volume-regulatory anion efflux pathway in a large number of cell types [40,42,65]. The VSOR Cl 2 channel has been shown to be functionally expressed in neuronal cell types, including sympathetic neurons [29], carotid body types I cells [11] and cerebellar granule neurons [49] as well as some neuroblastoma cells [3,7,9]. The VSOR Cl 2 current was also observed in undifferentiated neuroblastoma3glioma NG108-15 cells [15,54], though the properties have not been studied in detail. The present study, for the first time, showed the preservation of the VSOR Cl 2 channel activity after
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well known that cytosolic pH decreases are readily produced by lactate-dependent proton uptake [21,31,39] via MCT in mammalian cells [10,19,38]. In fact, the present RT-PCR study provided evidence for molecular expression of MCT1 and MCT8 in neuronally differentiated NG10815 cells. Also, our preliminary studies using BCECF demonstrated that lactacidosis readily induced reduction of intracellular pH in differentiated NG108-15 cells (S. Mori, S. Morishima and Y. Okada, unpublished observations). That cytosolic acidification is responsible for inhibition of the VSOR Cl 2 channel was also proved by the fact that whole-cell VSOR Cl 2 currents in differentiated NG108-15 cells were rapidly suppressed by applying acidic (pH 6.2) hypotonic solution containing a membrane-permeable pH buffer, acetate.
Fig. 5. RT-PCR analysis of MCT mRNA expression in neuronally differentiated NG108-15 cells. The primer pairs for MCT1, MCT2, MCT3, MCT4 and MCT8 were applied in the presence (1) or absence (2) of reverse transcriptase (RT). The first lane contains size markers. The last lane represents G-3-PDH-specific PCR product (predicted size to be 452 bp) as a positive control of the RT-PCR, respectively.
4.3. Lactacidosis-induced neuronal cell swelling and its pathophysiological significance
neuronal differentiation in NG108-15 cells and characterized the channel properties. The Cl 2 channel current was found to exhibit phenotypical properties of VSOR Cl 2 current [42], such as outward rectification, weak-field anion selectivity corresponding to Eisenman’s type I sequence, voltage-dependent blocking by DIDS, and voltage-independent sensitivity to NPPB as well as inactivation kinetics at large positive potentials. It is suggested that the VSOR Cl 2 channel is involved in the RVD mechanism in differentiated NG108-15 cells, because RVD was blocked by NPPB. Exposure to lactate-containing acidic hypotonic solution was found to give rise to biphasic effects on the VSOR Cl 2 current in differentiated NG108-15 cells: rapid enhancement and subsequent slow inhibition. Both effects would be independent of ATP depletion, because intracellular (pipette) solution contained 5 mM ATP as well as 5 mM creatinine phosphate, and could not be explained by lactate alone, because lactate-containing solution at pH 7.4 never induced these effects. The early-phase upregulating effect appears to be due to protonation of extracellular sites by which the single-channel amplitude of VSOR Cl 2 channel current was reported to increase in endothelial cells [58]. The late-phase downregulation appears to be caused by intracellular acidification, which was previously shown to inhibit VSOR Cl 2 channel activity in undifferentiated NG108-15 cells [15] and in endothelial cells [58], as it is
Previous in vitro studies showed that extracellular lactacidosis causes normotonic swelling in glial cells [21,22,24,31,36,50,52,63] and in neuroblastoma cells [64]. Sustained swelling was also observed in undifferentiated NG108-15 cells exposed to lactate-containing acidic solution [1]. Here we demonstrated lactacidosis-induced normotonic swelling in neuronally differentiated NG108-15 cells. In light of the suppressive effects of an MCT blocker (CHC) and an NHE blocker (amiloride) observed in the present study, it is likely that neuronal cell swelling under lactacidosis was caused by MCT-mediated lactate 2 and H 1 uptake together with NHE-mediated Na 1 uptake stimulated by H 1 accumulated in the cytosol, as was the case for lactacidosis-induced swelling in glial cells [62]. Lactacidosis-induced swelling in neuronally differentiated NG108-15 cells persisted without exhibiting RVD. This fact may not be simply explained only by persistent osmolyte uptake, which overrides volume-regulatory osmolyte extrusion, because the plateau swelling level was not significantly affected by NPPB, which inhibits volumesensitive Cl 2 channels and thereby RVD. Actually, we demonstrated that both swelling-induced activation of the VSOR Cl 2 channel and the RVD function were impaired under lactacidosis. Swelling of neuronal and glial cells, which is responsible for brain edema, is a common finding in cerebral ischemia, seizures and severe head injury [61]. Lac-
Fig. 4. Effects of lactacidosis, lactate at neutral pH, and acidosis without lactate on volume-sensitive Cl 2 currents in neuronally differentiated NG108-15 cells. Pulse protocols and arrowheads are the same as in Fig. 2. The current traces represent four similar observations. (A) Upper panel: a representative record before, during and after exposure to hypotonic solution, with exposure to acidic lactate-containing solution (pH 6.4) for a period of time during the hypotonic exposure. Gain of chart recorder was changed to one-fifth at a. Lower panel: current responses to step pulses recorded at a and b in the upper panel, and I–V relationships (c) recorded at a and b. Open and filled circles represent the mean6S.E.M. (vertical bars) of four observations under control and lactacidosis conditions, respectively. *, P , 0.05 vs. corresponding control. (B–D) A representative record before and during exposure to hypotonic solution, with exposure to lactate-containing neutral solution of pH 7.4 (B), lactate-containing acidic solution of pH 6.2 (C), and lactate-free acidic solution of pH 6.2 (D) for a period of time during the hypotonic exposure. Gain of chart recorder was changed to one-fourth upon application of step pulses from 2100 to 1120 mV.
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tacidosis-induced swelling may be an essential element in the formation of cytotoxic brain edema [26,61], because cerebral ischemia, seizure and trauma are associated with the enhancement of anaerobic metabolism. ATP depletion associated with ischemia and anoxia is also known to be a causal factor in cell swelling. The resulting reduction in Na 1 pump activity should impair the pump-leak balance mechanism, which is responsible for steady-state volume regulation and counterbalances intracellular oncotic pressure [17,33]. Under ATP-deficient conditions, cell swelling persists because VSOR Cl 2 channel activity is inhibited by both reduction of free ATP and elevation of free Mg 21 within the cell [41,42]. VSOR Cl 2 channel activity and hence, RVD, were in fact found to be impaired in cerebellar granule neurons under ischemic or hypoxic conditions [49]. We conclude that lactacidosis and ATP depletion causes, in a cumulative manner, cytotoxic and persistent cell swelling (a necrotic volume increase [45]) in neuronal and glial cells under ischemia not only by introducing osmolytes into the cells but also by downregulating volume-regulatory anion channels.
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Acknowledgements This work was supported by a grant from the Salt Science Foundation. We thank K. Dezaki for helpful discussion, M. Ohara and E.L. Lee for technical assistance, and T. Okayasu for secretarial assistance.
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