Pharmacological Characterization of Na+ Influx via Voltage-Gated Na+ Channels in Spinal Cord Astrocytes
Christine R. Rose1, 2,
Bruce R. Ransom3, and
Stephen G. Waxman1, 2
1 Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06520; 2 Neuroscience Research Center, Veterans Affairs Hospital, West Haven, Connecticut 06516; and 3 Department of Neurology, University of Washington School of Medicine, Seattle, Washington 98195-6465
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ABSTRACT |
Rose, Christine R., Bruce R. Ransom, and Stephan G. Waxman. Pharmacological characterization of Na+ influx via voltage-gated Na+ channels in spinal cord astrocytes. J. Neurophysiol. 78: 3249-3258, 1997. Spinal cord astrocytes display a high density of voltage-gated Na+ channels. To study the contribution of Na+ influx via these channels to Na+ homeostasis in cultured spinal cord astrocytes, we measured intracellular Na+ concentration ([Na+]i) with the fluorescent dye sodium-binding benzofuran isophthalate. Stellate and nonstellate astrocytes, which display Na+ currents with different properties, were differentiated. Baseline [Na+]i was 8.5 mM in these cells and was not altered by 100 µM tetrodotoxin (TTX). Inhibition of Na+ channel inactivation by veratridine (100 µM) evoked a [Na+]i increase of 47.1 mM in 44% of stellate and 9.7 mM in 64% of nonstellate astrocytes. About 30% of cells reacted to veratridine with a [Na+]i decrease of ~2 mM. Qualitatively similar [Na+]i changes were caused by aconitine. The effects of veratridine were blocked by TTX, amplified by (
-)scorpion toxin and usually were readily reversible. Veratridine-induced [Na+]i increases were reduced upon membrane depolarization with elevated extracellular [K+]. Recovery to baseline [Na+]i was unaltered during blocking of K+ channels with 4-aminopyridine. [Na+]i increases evoked by the ionotropic non-N-methyl-D-aspartate receptor agonist kainate were not altered by TTX. Our results indicate that influx of Na+ via voltage- gated Na+ channels is not a prerequisite for glial Na+,K+-ATPase activity in spinal cord astrocytes at rest nor does it seem to be involved in [Na+]i increases evoked by kainate. During pharmacological inhibition of Na+ channel inactivation, however, Na+ channels can serve as prominent pathways of Na+ influx and mediate large perturbations in [Na+]i, suggesting that Na+ channel inactivation plays an important functional role in these cells.
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INTRODUCTION |
Glial cells maintain a steep inwardly directed electrochemical gradient for Na+ due to the activity of Na+,K+-ATPase (Rose and Ransom 1996a
). This Na+ gradient is essential for intraglial ion homeostasis and also energizes the reuptake of transmitters like glutamate (Nicholls and Attwell 1990
). Changes in intraglial Na+ concentration ([Na+]i), especially [Na+]i increases that result in a reduction of the glial transmembrane Na+ gradient, are, therefore, likely to influence synaptic transmission significantly by altering transmitter reuptake and/or promoting reverse operation of the transmitter carriers in the brain (Attwell et al. 1993
; Mennerick and Zorumski 1994
; Nicholls and Attwell 1990
; Rothstein et al. 1996
). Despite this critical role of glial [Na+]i homeostasis for nervous system function, the regulation of [Na+]i and factors that lead to changes in glial [Na+]i are understood poorly. Because most of the previous studies had to rely on indirect methods such as radioisotope uptake and did not determine [Na+]i directly (e.g., Kimelberg et al. 1989
), conclusions about functional consequences of the glial Na+ changes have been limited.
One possible route of Na+ entry into glial cells is provided by voltage-gated Na+ channels (Berwald-Netter et al. 1983
; Bevan et al. 1985
). The presence of these channels in glial cells in vitro and in vivo is accepted widely, but their function remains obscure (Barres et al. 1989
, 1990
; Black et al. 1994b
; Oh et al. 1994
; Ransom and Sontheimer 1992
; Ritchie 1992
; Sontheimer et al. 1991
, 1996
; Steinhäuser et al. 1994
). Na+ channels have been proposed to play a critical role in intracellular Na+ homeostasis in astrocytes by serving as a pathway for Na+ influx that maintains cellular Na+,K+-ATPase activity (Sontheimer et al. 1994
). Moreover, in several types of glial cells, the glutamatergic agonist kainate evokes a membrane depolarization via its action on ionotropic receptors and by reducing K+ channel conductance (Gallo et al. 1996
; Jabs et al. 1994
; Müller et al. 1992
; Robert and Magistretti 1997
), suggesting that it might produce a depolarization strong enough to activate glial voltage-gated Na+ channels. However, the effects of Na+ channel activity on [Na+]i in glial cells have not been investigated in detail.
In the present study, we examined the contribution of Na+ influx via Na+ channels to [Na+]i homeostasis in spinal cord astrocytes in culture, by analyzing changes in [Na+]i during pharmacological manipulation of Na+ channels, using fluorescence ratio imaging with the Na+ indicator dye sodium-binding benzofuran isophthalate (SBFI). This optical imaging method allows the on-line measurement of absolute Na+ concentrations in individual cells with high accuracy. [Na+]i changes of ~1 mM can be resolved (Rose and Ransom 1996a
,b
, 1997a
,b
). Cultured spinal cord astrocytes were chosen for this study because they are characterized by an unusually high density of Na+ channels (Sontheimer et al. 1992
). Electrophysiological studies have revealed that two morphological subtypes of spinal cord astrocytes in vitro, stellate and nonstellate cells, express two distinct Na+ currents differing in tetrodotoxin (TTX)-sensitivity, steady state inactivation and current/voltage relationships (Sontheimer and Waxman 1992
), which could result in different characteristics of channel-mediated [Na+]i changes.
Our results confirm that a large percentage of spinal cord astrocytes in vitro possess voltage-gated Na+ channels. Na+ influx via these channels is, however, apparently not required to maintain Na+,K+-ATPase activity during resting conditions nor is it detectable during kainate-induced depolarization. Pharmacological inhibition of Na+ channel inactivation leads to substantial [Na+]i increases, which are much larger in stellate compared with nonstellate astrocytes probably because of differences in cell morphology and/or Na+ channel expression. These results indicate that Na+ channels do not play a major role in [Na+]i homeostasis under resting conditions but can serve as prominent pathways of Na+ influx into spinal cord astrocytes when channel inactivation is removed.
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METHODS |
Cell cultures
Astrocyte cell cultures were prepared as described previously (Sontheimer et al. 1992
). In summary, rat pups (Sprague-Dawley rats, postnatal day 0) were anesthetized by CO2 narcosis and decapitated. Spinal cords were removed and exposed to papain (30 U/ml for 30 min; Worthington). After trituration, the dissociated cells were plated onto poly-L-ornithine/laminin-coated (Sigma) coverslips in complete medium (Earle's minimum essential medium; JRH Biosciences) containing 10% fetal bovine serum (Hyclone), 6 mM glucose, and penicillin/streptomycin (120 U/ml and 120 µg/ml, respectively; Sigma). Cells were maintained at 37°C in a 5% CO2-95% humid air atmosphere and half the medium was exchanged every 3-4 days.
Measurements were made using cells between 6 and 10 days in culture, when Na+ channel expression is at its maximum in cultured spinal cord astrocytes (Sontheimer et al. 1992
). At this time in culture, cells had not reached confluence. Two astrocyte cell types, which express two different types of Na+ currents (Sontheimer and Waxman 1992
; Sontheimer et al. 1992
), were differentiated morphologically and identified according to criteria published earlier (Sontheimer et al. 1992
). Stellate astrocytes were characterized by round, small somata and numerous processes and represented ~5-10% of cells on a given coverslip. Nonstellate astrocytes were composed of round, nonprocess-bearing cells ("pancake" cells, ~5%) and polygonal cells ("flat" astrocytes, ~80-90%) (cf. Fig. 1 in Sontheimer et al. 1992
). We did not observe differences in baseline [Na+]i nor differences in [Na+]i changes in reaction to drugs between pancake (n = 22) and flat astrocytes (n = 281).

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| FIG. 1.
Influence of tetrodotoxin (TTX) and veratridine on [Na+]i. A: baseline [Na+]i was very stable in spinal cord astrocytes if no experimental manipulation was performed. Perfusion with 100 µM TTX (15 min) did not alter baseline [Na+]i. B-D: application of 100 µM veratridine (Ver, indicated by bars) caused a reversible increase in [Na+]i in 44% of stellate (B) and 64% of nonstellate (C and D) astrocytes. Veratridine-induced [Na+]i increase was larger in stellate compared with nonstellate cells and was rapidly reversible upon removal of the drug (B and D); in some cells, recovery started while the drug was still present (C). D: a 2nd veratridine application during this experiment elicited similar changes in [Na+]i as the 1st one. In this, and all other illustrations, traces reflect [Na+]i measurements from single cells.
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Solutions and chemicals
The standard saline used for the experiments contained (in mM) 133.6 NaCl, 5 KCl, 1.2 MgSO4, 1 CaCl2, 2 NaH2PO4, 25 N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES), and 10 glucose, titrated to a pH of 7.4 with NaOH. CO2/HCO
3-buffered saline contained only 120.6 mM NaCl but 23 mM NaHCO3 and no HEPES. It was bubbled continuously with 5% CO2-95% O2, resulting in a pH of 7.36 as measured by a pH meter. In salines with increased [K+], [Na+] was reduced reciprocally to maintain constant osmolarity.
The solutions for calibration of intracellular SBFI's sensitivity to Na+ contained (in mM) 150 (K+ + Na+), 30 Cl
, 120 gluconic acid, and 10 HEPES and were titrated to pH 6.9 (for measurements in HEPES-buffered saline) or pH 7.15 (CO2/HCO
3-buffered saline) with KOH (see below). Calibration solutions contained 3 µM gramicidin and 10 µM monensin for equilibration of extra- and intracellular [Na+]. Calibration solutions used for testing the pH sensitivity were titrated to pH 6.2, 6.6, 7.0, and 7.4 with KOH.
Drugs and chemicals were purchased from Sigma and added to the saline shortly before use. Gramicidin, monensin, veratridine, and aconitine were prepared as 1 M stock solutions in dimethyl sulfoxide (Sigma) and stored in the freezer. Acetoxymethylester of sodium-binding benzofuran isophthalate (SBFI-AM) was obtained from Teflabs.
[Na+]i measurements
[Na+]i measurements were performed as described earlier (Rose and Ransom 1996a
). Cells were incubated with SBFI-AM (20 µM; 90 min) in HEPES-buffered saline containing 0.1% Pluronic (BASF) and then transferred to an experimental chamber (volume ~500 µl), where they were perfused with saline at a flow rate of ~1 ml/min. For purposes of comparison with earlier studies (Sontheimer and Waxman 1992
; Sontheimer et al. 1992
, 1994
), experiments were carried out at room temperature (20-22°C). The experimental chamber was mounted on the stage of a Nikon-Diaphot-TMD inverted microscope equipped with an oil-immersion objective (Nikon Fluor 40/1.30). Cells were excited every 10 s at 345 and 390 nm and emission fluorescence >510 nm was collected, averaging 10 video frames. Emission of single cells (cell soma and prominent processes) was quantified with a computer program from Georgia Instruments (Roswell). Autofluorescence and dye bleaching were negligible during the experiments.
To calibrate SBFI's sensitivity to changes in [Na+]i, cells were perfused with calibration solutions containing different Na+ concentrations and the ionophores gramicidin (3 µM) and monensin (10 µM) to equilibrate extra- and intracellular [Na+]. During perfusion of these calibration solutions, the 345/390 nm fluorescence ratio of intracellular SBFI changed monotonically with changes in [Na+]i (not shown), with a slope of 0.16 per 10 mM Na+ from 0-30 mM [Na+]i (n = 47) and a slope of 0.07 per 10 mM Na+ >30 mM [Na+]i (n = 40). Calibration properties were the same in stellate and nonstellate cells. Comparison of the ratio values obtained in Na+-free saline with those obtained during perfusion with Na+-free calibration solution confirmed that the calibration solutions did not induce artifacts due to their restricted ionic composition (see earlier text).
As observed earlier (Rose and Ransom 1996a
, 1997a
), intracellular SBFI was sensitive to changes in intracellular pH. In astrocytes that were clamped to a [Na+]i of 15 mM at a pH of 7.1, acidification to pH 6.7 resulted in a decrease of the ratio signal, mimicking an apparent [Na+]i decrease by 3.2 ± 1.0 mM. Alkalinization to pH 7.5 mimicked an apparent [Na+]i increase by 3.8 ± 1.6 mM (n = 50; not shown).
Two- or three-point calibrations of [Na+]i were performed within the astrocytes after each experiment by perfusing the cells with calibration solutions containing 0 and 30 mM [Na+], or 0, 30, and 50 mM [Na+], respectively. Results are presented as Na+ concentrations, obtained by direct comparison with the calibration solutions containing known Na+ concentrations (cf. Rose and Ransom 1996a
). Experiments were repeated on at least four different coverslips, each allowing analysis of
15 individual cells. Due to the low density of stellate astrocytes (see above), only one to two stellate cells usually could be imaged during one experiment. Data are presented as means ± SD and were analyzed statistically using Student's t-test where appropriate (significance level: P < 0.005, unless stated otherwise).
 |
RESULTS |
Baseline [Na+]i and influence of TTX
Baseline [Na+]i of cultured spinal cord astrocytes was 8.5 ± 4.3 mM (n = 382) in standard, HEPES-buffered saline and did not differ significantly between stellate (9.0 ± 5.8, n = 79) and nonstellate cells (8.3 ± 3.7, n = 303; see METHODS). [Na+]i was significantly higher in CO2/HCO
3-buffered saline (11.4 ± 4.2 mM; n = 61), indicating a contribution of Na+-HCO
3-cotransport and/or Na+-dependent Cl
/HCO
3-exchange to baseline [Na+]i in these cells (cf. Rose and Ransom 1996a
).
Bath application of TTX at concentrations much higher than the IC50 for TTX-induced block of glial voltage-gated Na+-channels (10 or 100 µM, 15-30 min) (Sontheimer and Waxman 1992
) did not alter [Na+]i (n = 18 stellate, 103 nonstellate cells; Fig. 1A; cf. Figs. 2 and 7, A and C). This suggested that influx of Na+ via voltage-gated Na+ channels was not necessary to maintain baseline [Na+]i in these astrocytes.

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| FIG. 2.
Influence of TTX on veratridine-induced [Na+]i changes. A nonstellate (A) and a stellate (B) cell taken from an experiment in which veratridine was applied at 50 µM are shown. Note the repetitive spike-like [Na+]i transients in A. TTX (10 µM) reversibly blocked both veratridine-induced increases and decreases in [Na+]i, indicating that veratridine's effects were dependent on its action on voltage-gated Na+ channels.
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| FIG. 7.
Influence of TTX on kainate-induced [Na+]i changes. A: non-N-methyl-D-aspartate receptor agonist kainate (KA) evoked a reversible [Na+]i increase in the majority of astrocytes as shown here for a nonstellate cell. Kainate-induced [Na+]i increase was not altered by TTX (10 µM), indicating that it was not dependent on Na+ influx via Na+ channels. B: in several nonstellate astrocytes, kainate induced a decrease that could precede a [Na+]i increase. C: kainate-induced [Na+]i decrease was not altered by TTX. Kainate was applied at 1 mM for either 1 min (A) or 2 min (B and C).
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Influence of veratridine on [Na+]i
To verify that voltage-gated Na+ channels were present in our cultured spinal cord astrocytes and to test whether influx of Na+ via voltage-gated Na+ channels could significantly alter their [Na+]i, we applied the lipophilic neurotoxin veratridine, which binds to the Na+ channel receptor site 2 and causes a persistent activation of the channels by blocking their inactivation (Catterall 1992
). In squid Schwann cells (Villegas et al. 1976
), rat glioma cells (Reiser and Hamprecht 1983
), and rat astrocytes (Bowman et al. 1984
), veratridine causes reversible membrane depolarizations, providing some of the first evidence for the existence of Na+ channels in these glial cells.
Application of veratridine at 50 µM for 4 min caused [Na+]i to change in only 17% of astrocytes (n = 97; Fig. 2), whereas 78% of cells responded when veratridine was applied at a concentration of 100 µM for
5 min (n = 138). Therefore, the latter concentration and application duration were chosen for all subsequent experiments (unless stated otherwise). During veratridine application, we did not observe changes in the fluorescence emission after excitation with the Na+-insensitive wavelength 345 nm. This indicated that veratridine did not cause detectable cell swelling in spinal cord astrocytes in contrast to its effects reported for cortical neurons (Churchwell et al. 1996
).
Veratridine (100 µM,
5 min) induced an increase in [Na+]i in 44% of stellate (n = 41; Fig. 1B) and 64% of nonstellate cells (n = 97; Fig. 1, C and D). The average amplitudes of these veratridine-induced increases in [Na+]i differed significantly between the two cell types: [Na+]i increased by 47.1 ± 38.6 mM in stellate cells that displayed a rise in [Na+]i in response to veratridine but increased by only 9.7 ± 15.6 mM in nonstellate astrocytes that displayed a [Na+]i rise (Fig. 3).

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| FIG. 3.
Histogram summarizing [Na+]i increases caused by veratridine and ( -)scorpion toxin. Shown are mean values ± SD of [Na+]i increases ( Na+i) and percentage of cells that responded with an increase in [Na+]i to the drugs applied. [Na+]i increases were always significantly larger in stellate compared with nonstellate astrocytes (as indicated by the asterisk). Ver: veratridine, ScTx: scorpion toxin.
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Except for three stellate cells that experienced a very large increase in [Na+]i (>70 mM), all cells were able to recover rapidly and completely from the veratridine-induced [Na+]i load (Fig. 1). The recovery often started while the drug was still present in the bath. In eight nonstellate cells, veratridine evoked transient [Na+]i elevations that peaked in 20-30 s and then immediately started to recover; up to two of these spike-like increases in [Na+]i were observed in response to veratridine (Fig. 2A, cf. Fig. 1C). The effects of veratridine were reproducible with repetitive applications during a single experiment in both stellate and nonstellate cells, and the response did not desensitize with maximally three applications performed (interval between applications
15 min; Fig. 1D).
Surprisingly, veratridine induced a transient decrease in [Na+]i by ~2 mM in 39% of stellate and 27% of nonstellate astrocytes investigated (Fig. 4A). This [Na+]i decrease could precede a veratridine-induced [Na+]i increase; a biphasic signal was observed in 7% of stellate and 17% of nonstellate cells (Fig. 4B).

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| FIG. 4.
[Na+]i decreases induced by veratridine. A: veratridine (100 µM) induced a small, reversible decrease in [Na+]i in ~1/3 of cells investigated. B: veratridine-induced [Na+]i decrease could precede a veratridine-induced increase in [Na+]i, resulting in a biphasic [Na+]i change. Measurements from 2 nonstellate cells are shown.
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To determine whether the effect of veratridine was mediated by an effect on Na+ channels, we studied astrocytes that had been exposed to both veratridine and TTX. Both the veratridine-induced increase and the decrease in [Na+]i were blocked totally and reversibly by 10 µM TTX (n = 2 stellate, 10 nonstellate cells; Fig. 2, A and B). This indicated that the effects of veratridine were solely dependent on its action on voltage-gated Na+ channels and not related to a possible nonspecific alteration of K+ or Ca2+ channel conductances as reported earlier (Romey and Lazdunski 1982
; Verheugen et al. 1994
).
Influence of aconitine on [Na+]i
In addition to veratridine, the effects of aconitine, another alkaloid modulator of Na+ channel inactivation that binds at receptor site 2 (Catterall 1992
), were tested. Aconitine, applied at 200 µM for 15 min, had effects that were qualitatively similar to those of veratridine, causing either an increase or a decrease in [Na+]i (n = 9 stellate; n = 97 nonstellate cells; not shown).
The overall percentage of cells reacting to the drug (72% of stellate and 37% of nonstellate astrocytes) and the absolute amplitude of the aconitine-induced [Na+]i increases (~6 mM) were, however, much smaller compared with those induced by veratridine. These effects of aconitine were reversible in the majority of cells. As observed with veratridine, recovery was usually fast and could occur while aconitine was still present.
Effects of (
-)scorpion toxin
(
)-Scorpion toxins are polypeptide toxins from scorpion venoms that slow or block the inactivation of Na+ channels by binding to receptor site 3 (Catterall 1992
). In addition, their binding enhances the effects of lipophilic neurotoxins like veratridine or aconitine acting at receptor site 2 (Catterall 1977
, 1980
). We tested whether this cooperative action between the two receptor sites also was present on spinal cord astrocyte Na+ channels by using crude scorpion toxin from the venom of Leirus quinquestriatus, whose main active component is an (
)-scorpion toxin (Catterall 1976
).
Scorpion toxin (50 µg/ml, 5 min) alone induced [Na+]i changes that were similar to those caused by veratridine, although smaller in amplitude. [Na+]i reversibly increased by ~10 mM in 78% of stellate cells (n = 9; Figs. 3 and 5B), whereas it rose by only ~2 mM in 10% of nonstellate astrocytes (n = 21; Fig. 3). Twenty percent of both stellate and nonstellate cells responded with a reversible decrease in [Na+]i by <2 mM (Fig. 5D), and a biphasic decrease-increase in [Na+]i was elicited in one cell of each type (not shown).

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| FIG. 5.
Effects of ( -)scorpion toxin. Venom of Leirus quinquestriatus (ScTx; 50 µg/ml), the main active compound of which is an ( )-scorpion toxin, had only small effects when applied alone (B and D). Actions of scorpion toxin and veratridine (100 µM), however, were amplified when both drugs were applied together. A and B: combined application of veratridine and scorpion toxin caused large [Na+]i increases in stellate astrocytes, which were irreversible in ~1/2 the cells. C and D: nonstellate astrocytes experienced a much smaller increase in [Na+]i with combined application of veratridine and scorpion toxin and could usually recover from this [Na+]i load.
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Combined application of scorpion toxin and veratridine amplified the effects that each drug had alone (Fig. 3). In addition to increasing the amplitude of [Na+]i changes, the percentage of responding cells was increased. All stellate cells tested (n = 18) and 95% of nonstellate cells (n = 37) reacted with changes in [Na+]i when the two drugs were applied together. Again, the toxins altered [Na+]i more profoundly in stellate compared to nonstellate cells. Seventy-two percent of stellate cells reacted with a fast increase in [Na+]i by as much as 71.5 ± 36.5 mM (Figs. 3 and 5, A and B); this increase was only reversible in about half the cells. In contrast, [Na+]i increased by 23.9 ± 34.3 mM in 89% of nonstellate astrocytes. This [Na+]i increase was reversible in 92% of cells (Figs. 3 and 5, C and D). In addition to these effects, a decrease in [Na+]i by ~2 mM was observed in 33 and 19% of stellate and nonstellate cells, respectively (Fig. 5D).
Effects of membrane depolarization
Veratridine has been reported to bind only to voltage-gated Na+ channels in the open state (Catterall 1977
; Sutro 1986
). We tested the effect of a membrane depolarization, which significantly increases Na+ channel inactivation in spinal cord astrocytes (Sontheimer and Waxman 1992
), on veratridine-induced [Na+]i changes. Cells were depolarized by elevating the external [K+] concentration ([K+]e) from its standard value of 5 mM to 40 mM (with reciprocal reduction in saline [Na+]). This caused a transient increase in [Na+]i by ~5 mM in 5 of 18 cells investigated, whereas [Na+]i transiently decreased by 4 mM in one cell (not shown).
The amplitude of the veratridine-induced decrease in [Na+]i observed in one cell was unaltered during elevation of [K+]e to 40 mM. In contrast, elevation of [K+]e reduced the amplitude of the veratridine-induced [Na+]i increase by ~70% in both stellate (n = 9) and nonstellate (n = 9) cells (Fig. 6A). Switching back to normal saline (5 mM [K+]e) immediately after removal of veratridine caused a second rise in [Na+]i (Fig. 6B), presumably because the drug was still present and the membrane repolarized, reducing the percentage of inactivated Na+ channels and increasing the driving force for inward movement of Na+. These results confirm that the effects of veratridine strongly depended on the astrocytic membrane potential and the availability of open Na+ channels.

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| FIG. 6.
Influence of membrane depolarization on veratridine-induced [Na+]i changes. A: membrane depolarization by saline containing 40 mM [K+] caused a strong reduction in the veratridine-induced [Na+]i increase as shown here for a stellate astrocyte. B: this recording illustrates that switching back to standard saline containing 5 mM [K+] immediately after the veratridine-pulse caused a 2nd increase in [Na+]i in a stellate cell, presumably because the membrane repolarized (see text). Veratridine was applied at 100 µM.
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Effects of 4-aminopyridine
The effects of lipophilic alkaloids like veratridine are only slowly reversible in nerve and muscle cells if the drugs are applied for >1 min, presumably because they tend to partition in the lipid bilayer (Strichartz et al. 1987
; Ulbricht 1969
). In agreement with this view, we recently showed that cultured hippocampal neurons were unable to recover from [Na+]i increases caused by 3 min application of veratridine (Rose and Ransom 1997a
). In contrast, spinal cord astrocytes rapidly and immediately recovered from even massive [Na+]i loads caused by veratridine application of
30 min, and recovery sometimes took place while the drug still was present (see earlier text).
To investigate if the rapid recovery from [Na+]i increases caused by veratridine in spinal cord astrocytes resulted from a membrane hyperpolarization due to opening of K+ channels, allowing the closing of veratridine-modified Na+ channels (Sutro 1986
), we applied 4-aminopyridine (4-AP), which blocks voltage-activated outward K+ currents in spinal cord astrocytes (Sontheimer et al. 1992
). Upon perfusion with 5 mM 4-AP, [Na+]i transiently increased by ~3 mM in 5 of 30 cells tested (not shown).
In stellate astrocytes (n = 12) the veratridine-induced [Na+]i increase was augmented by ~40% during 4-AP perfusion, but this change was not statistically significant (P < 0.25). The [Na+]i increase was unaltered by 4-AP in nonstellate cells (n = 18, not shown). Likewise, 4-AP did not change the amplitude of the veratridine-induced [Na+]i decrease in the astrocytes (n = 10, not shown). The slope of recovery from the veratridine-induced [Na+]i increase was not reduced by 4-AP (not shown). These results indicated that the reversibility of veratridine-induced [Na+]i changes was not primarily related to 4-AP-sensitive K+ channels.
Contribution of Na+ channels to kainate-induced increases in [Na+]i
The results presented above showed that influx of Na+ via voltage-gated Na+ channels can lead to significant increases in [Na+]i in spinal cord astrocytes. We investigated if these channels might mediate a detectable Na+ influx into the astrocytes during application of the glutamatergic non-N-methyl-D-aspartate receptor agonist kainate, which has been shown to strongly increase [Na+]i in hippocampal astrocytes after activation of receptor-activated ion channels (Rose and Ransom 1996b
). Kainate was applied at a concentration of 1 mM.
Kainate (1 min) induced an increase in [Na+]i by 83.8 ± 32.1 mM in six of seven stellate astrocytes that we tested; four cells were able to recover from this kainate-induced [Na+]i increase within <20 min. In nonstellate astrocytes (16 of 29 cells investigated), kainate (1 min) caused a significantly smaller [Na+]i increase (30.7 ± 38.4 mM) from which all cells recovered within 20 min (Fig. 7A). Surprisingly, a decrease in [Na+]i by 4.5 ± 4.8 mM was observed in four nonstellate cells, and it preceded the kainate-induced [Na+]i increase in two cases (Fig. 7, B and C).
Perfusion with 10 µM TTX did not influence the amplitude nor the time course of kainate-induced [Na+]i changes (n = 16; Fig. 7, A and C). This indicated that influx of Na+ via voltage-gated Na+ channels did not significantly contribute to the observed [Na+]i increases.
 |
DISCUSSION |
Contribution of Na+ flux via Na+ channels to baseline [Na+]i
Our experiments confirm earlier results (Sontheimer et al. 1992
) in indicating that a high percentage of cultured spinal cord astrocytes express Na+ channels, which is consistent with the demonstration of Na+ channel mRNA and protein in these cells (Black et al. 1994b
, 1995
; Oh et al. 1994
). Although differences between astrocytes in vitro and in vivo might exist, it is important to note that spinal cord astrocytes in situ express Na+ channel protein at levels that are detectable by immunocytochemistry (Black et al. 1994a
). The observation that some cultured spinal cord astrocytes hyperpolarize upon removal of extracellular Na+ (Ransom et al. 1996
) or application of TTX (Sontheimer et al. 1994
) suggested that these cells display a resting Na+ conductance. The presence of open Na+ channels at resting potential also was indicated in the present study, because veratridine, which binds preferably to open Na+ channels (Catterall 1977
; Sutro 1986
), induced [Na+]i increases in ~50% of cells.
Because of the steep inwardly directed gradient for Na+, the presence of "open" Na+ channels will result in channel-mediated influx of Na+ into the astrocytes, which could influence [Na+]i. The channel-mediated Na+ influx can be estimated as follows: as a result of overlapping ms
and h
curves, Na+ channel open probability is 0.001 in nonstellate cells at
60 mV (m3h model), and 0.0001 in stellate cells at
45 mV (m4h), enabling "window currents" at these potentials (Sontheimer and Waxman 1992
). When depolarized, the astrocytes display a peak Na+ current density of 100 pA/pF, with an estimated open probability of 0.25 (Sontheimer et al. 1992
), representing a current of 10 mA/cm2, and flux of 6*1014 Na+ ions/s*cm2. Na+ influx via window currents, therefore, would amount maximally to 4 pmol/s*cm2 and 0.4 pmol/s*cm2, respectively. Assuming that these cells experience a similar total Na+ influx as squid axons (30 pmol/s*cm2) (Hodgkin and Keynes 1955
), Na+ flux via window currents would account for 15% (nonstellate) and 1.5% (stellate) of the total Na+ influx. This calculation is, however, only valid if the membrane potential matches the potentials at which window currents occur. Cultured spinal cord astrocytes display membrane potentials of about
30 mV (nonstellate) and
60 mV (stellate), at which window currents are smaller (Sontheimer and Waxman 1992
). Moreover, window currents may be largest at this stage of development in vitro (6-10 days) because Na+ channel density is downregulated with time in culture (Sontheimer et al. 1992
) and depends on cell-cell interactions (Thio et al. 1993
). Furthermore, astrocytes in situ usually display membrane potentials close to the K+ equilibrium potential (approximately
80 mV) (Ballanyi et al. 1987
), at which channel inactivation is nearly complete (Sontheimer et al. 1992
).
It recently was suggested that channel-mediated Na+ influx is required for Na+,K+-ATPase activity in spinal cord astrocytes, because prolonged TTX exposure caused cell death and preliminary [Na+]i measurements indicated an immediate [Na+]i decrease induced by TTX (Sontheimer et al. 1994
). In the present study, in contrast, TTX had no effect on [Na+]i. Given the low estimated contribution of maximal channel-mediated Na+ flux to total Na+ influx (15%; see earlier text), any effect on [Na+]i while blocking this influx by TTX might have been obscured by Na+ influx via other pathways such as Na+-dependent transporters (e.g., Na+/K+/2Cl
-cotransport). Alternatively, the lack of a demonstrable TTX effect might be due to the absence of significant channel-mediated Na+ influx. Irrespective of this, because TTX did not alter [Na+]i, we conclude that Na+ channel activity is not a prerequisite for Na+,K+-ATPase activity in our cultures. Because cultured spinal cord astrocytes display the highest Na+ channel density described for astrocytes thus far (Sontheimer et al. 1992
) and because channel open probability is likely to be smaller in vivo, our results call the role of Na+ channel activity in regulating astrocytic Na+,K+-ATPase into question.
Na+ influx via Na+ channels after inhibition of inactivation and membrane depolarization
Modulation of Na+ channel inactivation by veratridine caused [Na+]i increases in the majority of spinal cord astrocytes, indicating that Na+ channels mediate significant Na+ influx when inactivation is removed. During application of drugs that inhibited channel inactivation, the increase in SBFI ratio indicated [Na+]i increases to >100 mM in some cells (cf. Fig. 5). The SBFI ratio responds to [Na+]i changes
140 mM [Na+]i and is largely insensitive to changes in intracellular [Ca2+]i (Rose and Ransom 1996a
). Moreover, we did not detect volume changes accompanying channel-mediated [Na+]i influx (as revealed by the fluorescence emission after excitation with 345 nm). Finally, pHi is most likely to increase due to depolarization-induced alkalinization during [Na+]i influx (Ransom and Sontheimer 1992
), which would cause a Na+-independent decrease in the SBFI ratio (and an apparent [Na+]i decrease; see METHODS). It seems, therefore, reasonable to conclude that the absolute [Na+]i did, indeed, rise to these very high values.
Veratridine-induced [Na+]i changes were blocked by TTX, verifying their dependence on veratridine's action on Na+ channels. This also was confirmed by the qualitatively similar effects of aconitine and by the amplification of veratridine-induced [Na+]i increases by (
-)scorpion toxin. Veratridine's effects on [Na+]i also indicated that Na+ channels can exist in the open state at resting potential (see earlier text). Veratridine binding to these channels and concomitant [Na+]i influx probably depolarized the astrocytes and caused opening of additional Na+ channels, promoting further veratridine binding and accounting for the large increases in [Na+]i.
Veratridine-induced [Na+]i increases were reversible even when [Na+]i rose to 50-60 mM, demonstrating that astrocytes possess robust mechanisms for [Na+]i homeostasis. In contrast, veratridine's effects are virtually irreversible in hippocampal neurons (Rose and Ransom 1997a
). The reversibility of veratridine-induced [Na+]i changes in spinal cord astrocytes may be attributable to membrane repolarization because veratridine-modified Na+ channels close with hyperpolarization below resting potential (Strichartz et al. 1987
; Sutro 1986
). However, because [Na+]i recovery was not significantly delayed by 4-AP, recovery was probably not due to repolarization that resulted from the opening of K+ channels.
Because the Na+ channel density of spinal cord astrocytes at 6-10 days in vitro and hippocampal neurons is similar (Sontheimer et al. 1992
), the difference in veratridine's reversibility indicates that astrocytic Na+ channels have a different affinity for this drug. This could be attributable to a different channel open probability at resting potential (see earlier text) or to structural differences in receptor site 2. Although astrocytic and neuronal Na+ currents differ in terms of their electrophysiological properties (Sontheimer and Waxman 1992
), it is unclear whether structural differences exist; neurons and glia express some Na+ channel subtypes in common (Black et al. 1994b
; Gautron et al. 1992
; Oh and Waxman 1994
; Oh et al. 1994
; Schaller et al. 1995
; Waxman and Black 1996
).
TTX did not alter the amplitude of [Na+]i changes evoked by the glutamatergic agonist kainate, indicating that Na+ influx via voltage-gated Na+ channels is not a major contributor to kainate-induced [Na+]i increases, despite the strong depolarization that kainate probably caused (hippocampal astrocytes depolarize by ~25 mV with 1 mM kainate) (Backus et al. 1989
). Our results, however, do not exclude a local contribution of Na+ influx via voltage-gated Na+ channels to [Na+]i increases in fine cellular processes, because only somata and larger processes were investigated.
Differences between stellate and nonstellate astrocytes
Stellate astrocytes experienced much larger [Na+]i changes than nonstellate cells during inhibition of Na+ channel inactivation. This might be caused by a higher Na+ channel density and/or surface/volume ratio in stellate compared with nonstellate astrocytes. Moreover, the two cell types could differ in Na+,K+-ATPase activity, which, however, would probably not completely account for the difference in amplitude but rather influence recovery from [Na+]i increases. Different [Na+]i changes also could be influenced by differences in gap junctional coupling between the astrocytes (Sontheimer et al. 1990
) because Na+ easily passes gap junctions (Rose and Ransom 1997
b). Although most experiments were done in subconfluent cultures, coupling via fine processes can not be excluded and could have dampened [Na+]i changes.
The difference in neurotoxin-induced [Na+]i changes in the two types of astrocytes also could be explained by different drug affinity. As discussed above, different veratridine-binding can arise because of differences in open channel probability and/or structural differences at receptor site 2. If resting potentials do not fall within the domain where overlapping of m
and h
curves occurs, Na+ channel open probability will be low (Sontheimer and Waxman 1992
), resulting in weak binding of veratridine. The more positive membrane potential of nonstellate astrocytes compared with stellate cells is likely to result in a lower open probability of Na+ channels and could partly account for the weaker effects of veratridine on nonstellate cells. The dependence of veratridine's effects on membrane potential is illustrated by the observation that sustained, K+-induced depolarization (and the resultant reduction of channel open probability) diminished veratridine-induced [Na+]i increases in both astrocyte types. Additionally, the more negative membrane potential of stellate astrocytes (see earlier text) provides a greater driving force for Na+ entry.
A similar pattern of [Na+]i changes was observed with kainate, which evoked significantly higher [Na+]i increases in stellate compared with nonstellate cells. This could be related to different expression levels of
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors but also could be caused by a larger surface/volume ratio of stellate compared with nonstellate cells. Our results do not allow us to differentiate between these possibilities, and further studies are needed to elucidate the mechanisms that underlie the differences in [Na+]i changes between the two cell types.
Mechanisms underlying [Na+]i decreases
Unexpectedly, pharmacological Na+ channel inactivation, as well as kainate application, caused a small [Na+]i decrease in ~30% of astrocytes. A [Na+]i decrease can be achieved by reduction of Na+ influx, which was, however, probably not the case with these manipulations. Another mechanism that will decrease [Na+]i is activation of Na+,K+-ATPase. Na+,K+-ATPase is primarily stimulated by increases in [Na+]i, but cells also can respond to changes in Na+ influx by increasing Na+,K+-ATPase activity without visible [Na+]i increase (Haber et al. 1987
). The occurrence of biphasic [Na+]i changes in some cells indicated that this might have been the mechanism underlying the observed [Na+]i decreases.
Conclusions
The main objective of our study was to analyze the contribution of Na+ influx via Na+ channels to intracellular Na+ homeostasis in spinal cord astrocytes. This was based on the hypothesis that influx of Na+ via Na+ channels was necessary to maintain a constant baseline [Na+]i as proposed in an earlier investigation (Sontheimer et al. 1994
). Our study verifies that the majority of cultured spinal cord astrocytes possess voltage-gated Na+ channels and indicates the presence of open Na+ channels at resting potential. Influx of Na+ via voltage-gated Na+ channels, however, does not contribute to baseline [Na+]i and is not a major contributor for Na+,K+-ATPase activity at rest nor does it seem to contribute to [Na+]i changes caused by kainate. Pharmacological inhibition of Na+ channel inactivation, in contrast, leads to [Na+]i increases, which are much larger in stellate compared with nonstellate astrocytes, probably because of differences in cell morphology and/or Na+ channel expression. Na+ channels therefore can serve as prominent pathways of Na+ influx into spinal cord astrocytes when channel inactivation is removed. Because increases in [Na+]i can alter many astrocyte functions, our results suggest that Na+ channel inactivation plays an important functional role in these cells.
 |
ACKNOWLEDGEMENTS |
We thank Dr. J. A. Black for help in beginning the astrocyte cultures.
This work was supported in part by National Institute of Neurological Disorders and Stroke Grants NS-15589 to B. R. Ransom and National Multiple Sclerosis Society Grant RG 1912 and the Medical Research Service, Veterans Administration to S. G. Waxman. C. R. Rose was supported by fellowships from the Deutsche Forschungsgemeinschaft (Ro 1130/1-2) and the Eastern Paralyzed Veterans Association.
 |
FOOTNOTES |
Present address and address for reprint request: C. R. Rose, I. Physiologisches Institut, Universität des Saarlandes, D-66421 Homburg/Saar, Germany.
Received 11 April 1997; accepted in final form 8 August 1997.
 |
REFERENCES |
-
ATTWELL, D.,
BARBOUR, B.,
SZATKOWSI, M.
Nonvesicular release of neurotransmitter.
Neuron
11: 401-407, 1993.[Medline]
-
BACKUS, K. H.,
KETTENMANN, H.,
SCHACHNER, M.
Pharmacological characterization of the glutamate receptor in cultured astrocytes.
J. Neurosci. Res.
22: 274-282, 1989.[Medline]
-
BALLANYI, K.,
GRAFE, P.,
TEN BRUGGENCATE, G.
Ion activities and potassium uptake mechanisms of glial cells in guinea-pig olfactory cortex slices.
J. Physiol. (Lond.)
382: 159-174, 1987.[Abstract]
-
BARRES, B. A.,
CHUN, L.L.Y.,
COREY, D. P.
Glial and neuronal forms of the voltage-dependent sodium channel: characteristics and cell-type distribution.
Neuron
2: 1375-1388, 1989.[Medline]
-
BARRES, B. A.,
CHUN, L.L.Y.,
COREY, D. P.
Ion channels in vertebrate glia.
Annu. Rev. Neurosci.
13: 441-474, 1990.[Medline]
-
BERWALD-NETTER, Y.,
BEAUDOIN, D.,
COURAND, F.
Contribution to the characterization of astrocyte membrane properties (Abstract).
J. Neurochem.
41: S3, 1983.
-
BEVAN, S.,
CHIU, S. Y.,
GRAY, P. T.,
RITCHIE, J. M.
The presence of voltage-gated sodium, potassium and chloride channels in rat cultured astrocytes.
Proc. R. Soc. Lond. B Biol. Sci.
225: 299-313, 1985.[Medline]
-
BLACK, J. A.,
WESTENBROEK, R.,
RANSOM, B. R.,
CATTERALL, W. A.,
WAXMAN, S. G.
Type II sodium channels in spinal cord astrocytes in situ: immunocytochemical observations.
Glia
12: 219-227, 1994a.[Medline]
-
BLACK, J. A.,
WESTENBROEK, R.,
MINTURN, J. E.,
RANSOM, B. R.,
CATTERALL, W. A.,
WAXMAN, S. G.
Isoform-specific expression of sodium channels in astrocytes in vitro: immunocytochemical observations.
Glia
14: 133-144, 1995.[Medline]
-
BLACK, J. A.,
YOKOYAMA, S.,
WAXMAN, S. G.,
OH, Y.,
ZUR, K. B.,
SONTHEIMER, H.,
HIGASHIDA, H.,
RANSOM, B. R.
Sodium channel mRNAs in cultured spinal cord astrocytes: in situ hybridization in identified cell types.
Mol. Brain. Res.
23: 235-245, 1994b.[Medline]
-
BOWMAN, C. L.,
KIMELBERG, H. K.,
FRANGAKIS, M. V.,
BERWALD-NETTER, Y.,
EDWARDS, C.
Astrocytes in primary culture have chemically activated sodium channels.
J. Neurosci.
4: 1527-1534, 1984.[Abstract]
-
CATTERALL, W. A.
Purification of a toxic protein from scorpion venom which activates the action potential Na+ ionophore.
J. Biol. Chem.
251: 5528-5536, 1976.[Abstract]
-
CATTERALL, W. A.
Activation of the action potential Na+ ionophore by neurotoxins. An allosteric model.
J. Biol. Chem.
252: 8669-8676, 1977.[Medline]
-
CATTERALL, W. A.
Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes.
Annu. Rev. Pharmacol. Toxicol.
20: 15-43, 1980.[Medline]
-
CATTERALL, W. A.
Cellular and molecular biology of voltage-gated sodium channels.
Physiol. Rev.
72: S15-S48, 1992.[Medline]
-
CHURCHWELL, K. B.,
WRIGHT, S. H.,
EMMA, F.,
ROSENBERG, P. A.,
STRANGE, K.
NMDA receptor activation inhibits neuronal volume regulation after swelling induced by veratridine-stimulated Na+-influx in rat cortical cultures.
J. Neurosci.
16: 7447-7457, 1996.[Abstract/Free Full Text]
-
GALLO, V.,
ZHOU, J. M.,
MCBAIN, C. J.,
WRIGHT, P.,
KNUTSON, P. L.,
ARMSTRONG, R. C.
Oligodendrocyte progenitor cell proliferation and lineage progression are regulated by glutamate receptor-mediated K+ channel block.
J. Neurosci.
16: 2659-2670, 1996.[Abstract]
-
GAUTRON, S.,
DOS SANTOS, G.,
PINTO-HENRIQUE, D.,
KOULAKOFF, A.,
GROS, F.,
BERWALD-NETTER, Y.
The glial voltage-gated sodium channel: cell- and tissue-specific mRNA expression.
Proc. Natl. Acad. Sci. USA
89: 7272-7276, 1992.[Abstract]
-
HABER, R. S.,
PRESSLEY, T. A.,
LOEB, J. N.,
ISMAIL, B. F.
Ionic dependence of active Na-K transport: "clamping" of cellular Na+ with monensin.
Am. J. Physiol.
253: F26-F33, 1987.[Abstract/Free Full Text]
-
HODGKIN, A. L.,
KEYNES, R. D.
Active transport of cations in giant axons from sepia and loligo.
J. Physiol. (Lond.)
128: 28-60, 1955.[Medline]
-
JABS, R.,
KIRCHHOFF, F.,
KETTENMANN, H.,
STEINHÄUSER, C.
Kainate activates Ca2+-permeable glutamate receptors and blocks voltage-gated K+ currents in glial cells of mouse hippocampal slices.
Pflügers Arch.
426: 310-319, 1994.[Medline]
-
KIMELBERG, H. K.,
PANG, S.,
TREBLE, D. H.
Excitatory amino acid-stimulated uptake of 22Na+ in primary astrocyte cultures.
J. Neurosci.
9: 1141-1149, 1989.[Abstract]
-
MENNERICK, S.,
ZORUMSKI, C. F.
Glial contribution to excitatory neurotransmission in cultured hippocampal cells.
Nature
368: 59-62, 1994.[Medline]
-
MÜLLER, T.,
MÖLLER, T.,
BERGER, T.,
SCHNITZER, J.,
KETTENMANN, H.
Calcium entry through kainate receptors and resulting potassium-channel blockade in Bergmann glial cells.
Science
256: 1563-1566, 1992.[Medline]
-
NICHOLLS, D.,
ATTWELL, D.
The release and uptake of excitatory amino acids.
Trends Pharmacol. Sci.
11: 462-468, 1990.[Medline]
-
OH, Y.,
BLACK, J. A.,
WAXMAN, S. G.
The expression of rat brain voltage-sensitive Na+ channel mRNA's in astrocytes.
Mol. Brain. Res.
23: 57-65, 1994.[Medline]
-
OH, Y.,
WAXMAN, S. G.
The
1 subunit mRNA of the rat brain Na+ channel is expressed in glial cells.
Proc. Natl. Acad. Sci. USA
91: 9985-9989, 1994.[Abstract/Free Full Text] -
RANSOM, B. R.,
SONTHEIMER, H.
The neurophysiology of glial cells.
J. Clin. Neurophys.
9: 224-251, 1992.[Medline]
-
RANSOM, C. B.,
SONTHEIMER, H.,
JANIGRO, D.
Astrocytic inwardly rectifying potassium currents are dependent on external sodium ions.
J. Neurophysiol.
76: 626-630, 1996.[Abstract/Free Full Text]
-
REISER, G.,
HAMPRECHT, B.
Sodium-channels in non-excitable glioma cells, shown by the influence of veratridine, scorpiontoxin, and tetrodotoxin on membrane potential and on ion transport.
Pflügers Arch.
397: 260-274, 1983.[Medline]
-
RITCHIE, J. M.
Voltage-gated ion channels in Schwann cells and glia.
Trends Neurosci.
15: 345-351, 1992.[Medline]
-
ROBERT, A.,
MAGISTRETTI, P. J.
AMPA/kainate receptor activation blocks K+ currents via internal Na+ increase in mouse cultured stellate astrocytes.
Glia
20: 38-50, 1997.[Medline]
-
ROMEY, G.,
LAZDUNSKI, M.
Lipid-soluble toxins thought to be specific for Na+ channels block Ca2+ channels in neuronal cells.
Nature
297: 79-80, 1982.[Medline]
-
ROSE, C. R.,
RANSOM, B. R.
Intracellular Na+ homeostasis in cultured rat hippocampal astrocytes.
J. Physiol. (Lond.)
491: 291-305, 1996a.[Abstract]
-
ROSE, C. R.,
RANSOM, B. R.
Mechanisms of H+ and Na+ changes induced by glutamate, kainate, and D-aspartate in rat hippocampal astrocytes.
J. Neurosci.
16: 5393-5404, 1996b.[Abstract/Free Full Text]
-
ROSE, C. R.,
RANSOM, B. R.
Regulation of intracellular sodium in cultured rat hippocampal neurones.
J. Physiol. (Lond.)
499: 573-587, 1997a.[Abstract]
-
ROSE, C. R.,
RANSOM, B. R.
Gap junctions equalize intracellular Na+ concentrations in astrocytes.
Glia
20: 299-307, 1997.[Medline]
-
ROTHSTEIN, J. D.,
DYKES-HOBERG, M.,
PARDO, C. A.,
BRISTOL, L. A.,
JIN, L.,
KUNCL, R. W.,
KANAI, Y.,
HEDIGER, M. A.,
WANG, Y.,
SCHIELKE, J. P.,
WELTY, D. F.
Knockout of glutamate transports reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.
Neuron
16: 675-686, 1996.[Medline]
-
SCHALLER, K. L.,
KRZEMIEN, D. M.,
YAROWSKY, P. J.,
KRUEGER, B. K.,
CALDWELL, J. H. A
novel, abundant sodium channel expressed in neurons and glia.
J. Neurosci.
15: 3231-3242, 1995.[Abstract]
-
SONTHEIMER, H.,
BLACK, J. A.,
RANSOM, B. R.,
WAXMAN, S. G.
Ion channels in spinal cord astrocytes in vitro. I. Transient expression of high levels of Na+ and K+ channels.
J. Neurophysiol.
68: 985-1000, 1992.[Abstract/Free Full Text]
-
SONTHEIMER, H.,
BLACK, J. A.,
WAXMAN, S. G.
Voltage-gated Na+ channels in glia: properties and possible functions.
Trends Neurosci.
19: 325-331, 1996.[Medline]
-
SONTHEIMER, H.,
FERNANDEZ-MARQUES, E.,
ULLRICH, N.,
PAPPAS, C. A.,
WAXMAN, S. G.
Astrocyte Na+ channels are required for maintenance of Na+/K+-ATPase activity.
J. Neurosci.
14: 2464-2475, 1994.[Abstract]
-
SONTHEIMER, H.,
MINTURN, J. E.,
BLACK, J. A.,
WAXMAN, S. G.,
RANSOM, B. R.
Specificity of cell-cell coupling in rat optic nerve astrocytes in vitro.
Proc. Natl. Acad. Sci. USA
87: 9833-9837, 1990.[Abstract]
-
SONTHEIMER, H.,
RANSOM, B. R.,
CORNELL-BELL, A. H.,
BLACK, J. A.,
WAXMAN, S. G.
Na+ current expression in rat hippocampal astrocytes in vitro: alterations during development.
J. Neurophysiol.
65: 3-19, 1991.[Abstract/Free Full Text]
-
SONTHEIMER, H.,
WAXMAN, S. G.
Ion channels in spinal cord astrocytes in vitro. II. Biophysical and pharmacological analysis of two Na+ current types.
J. Neurophysiol.
68: 1001-1011, 1992.[Abstract/Free Full Text]
-
STEINHÄUSER, C.,
KRESSIN, K.,
KUPRIJANOVA, E.,
WEBER, M.,
SEIFERT, G.
Properties of voltage-activated sodium and potassium currents in mouse hippocampal glial cells in situ and after acute isolation from tissue slices.
Pflügers Arch.
428: 610-620, 1994.[Medline]
-
STRICHARTZ, G.,
RANDO, T.,
WANG, G. K.
An integrated view of the molecular toxicology of sodium channel gating in excitable cells.
Annu. Rev. Neurosci.
10: 237-267, 1987.[Medline]
-
SUTRO, J.
Kinetics of veratridine action on Na channels of skeletal muscle.
J. Gen. Physiol.
87: 1-24, 1986.[Abstract]
-
THIO, C. L.,
WAXMAN, S. G.,
SONTHEIMER, H.
Ion channels in spinal cord astrocytes in vitro. III. Modulation of channel expression by coculture with neurons and neuron-conditioned medium.
J. Neurophysiol.
69: 819-831, 1993.[Abstract/Free Full Text]
-
ULBRICHT, W.
The effect of veratridine on excitable membranes of nerve and muscle.
Ergeb. Physiol. Biol. Chem. Exp. Pharmakol.
61: 18-71, 1969.[Medline]
-
VERHEUGEN, J.A.H.,
OORTGIESEN, M.,
VIJVERBERG, H. P.
Veratridine blocks voltage-gated potassium current in human T lymphocytes and in mouse neuroblastoma cells.
J. Membr. Biol.
137: 205-215, 1994.[Medline]
-
VILLEGAS, J.,
SEVCIK, C.,
BARNOLA, F. V.,
VILLEGAS, R.
Gryanotoxin, veratridine, and tetrodotoxin-sensitive sodium pathways in the Schwann cell membrane of squid nerve fibres.
J. Gen. Physiol.
67: 369-380, 1976.[Abstract]
-
WAXMAN, S. G.,
BLACK, J. A.
Expression of mRNA for a sodium channel in subfamily 2 in spinal sensory neurons.
Neurochem. Res.
21: 395-401, 1996.[Medline]