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Previous Article | Next Article
Volume 17, Number 8, Issue of April 15, 1997 pp. 2669-2682
Copyright ©1997 Society for NeuroscienceK+ Channel Expression and Cell Proliferation Are Regulated by Intracellular Sodium and Membrane Depolarization in Oligodendrocyte Progenitor Cells
Peter Knutson, Cristina A. Ghiani, Jia-Min Zhou, Vittorio Gallo, and Chris J. McBain Laboratory of Cellular and Molecular Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4495
ABSTRACT
The effects of a variety of antiproliferative agents on voltage-dependent K+ channel function in cortical oligodendrocyte progenitor (O-2A) cells were studied. Previously, we had shown that glutamate receptor activation reversibly inhibited O-2A cell proliferation stimulated by mitogenic factors and prevented lineage progression by attenuating outward K+ currents in O-2A cells. We now show that the antiproliferative actions of glutamate receptor activation are Ca2+-independent and arise from an increase in intracellular Na+ and subsequent block of outward K+ currents. In support of this mechanism, agents that acted to depolarize O-2A cells or increase intracellular sodium similarly had an antiproliferative effect, attributable at least in part to a reduction in voltage-gated K+ currents. Also, these effects were reversible and Ca2+-independent. Chronic treatment with glutamate agonists was without any long-term effect on K+ current function. Cells cultured in elevated K+, however, demonstrated an upregulation of inward rectifier K+ currents, concomitant with an hyperpolarization of the resting membrane potential. This culture condition therefore promoted a current phenotype typical of pro-oligodendroblasts. Finally, cells chronically treated with the mitotic inhibitor retinoic acid displayed a selective downregulation of outward K+ currents. In conclusion, signals that affect O-2A cell proliferation do so by regulating K+ channel function. These data indicate that the regulation of K+ currents in cells of the oligodendrocyte lineage plays an important role in determining their proliferative potential and demonstrate that O-2A cell K+ current phenotype can be modified by long-term depolarization of the cell membrane.
Key words: potassium channels; O-2A progenitors; cell proliferation; glial development; depolarization; lineage progression
INTRODUCTION
In the mammalian CNS, both types of macroglial cells, astrocytes and oligodendrocytes, express virtually all the membrane channels that are found in neurons (for review, see Newman and Reichenbach, 1996
; Ransom and Orkand, 1996
O-2A cell proliferation is modulated by a variety of factors (McMorris and Dubois-Dalcq, 1988
Because of the emerging role that K+ channels play in glial development, we sought to determine the following: (1) the mechanism of blockade of these channels on GluR activation; (2) which functional subtypes of K+ channels are expressed in oligodendrocyte lineage cells during their proliferative and postmitotic stages; (3) how these distinct subtypes of K+ channels are affected by acute treatment with antimitotic agents acting through different mechanisms; and (4) whether chronic treatment with antiproliferative agents induce changes in the functional expression of K+ channels in O-2A cells. Our data indicate that membrane depolarization by itself and the subsequent reduction of delayed rectifier and transient K+ currents are sufficient to inhibit O-2A proliferation and lineage progression. Finally, we show that culturing O-2A cells in depolarizing concentrations of K+ not only blocks their proliferation but also promotes expression of a K+ channel phenotype that is typical of preoligodendroblasts, i.e., causes a strong upregulation of inward rectifier K+ channels.
MATERIALS AND METHODS
Materials. All cell culture media were obtained from Life Technologies (Gaithersburg, MD). Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT). Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) were obtained from Upstate Biotechnology (Lake Placid, NY). Kainic acid, glutamate, TTX, veratridine, and all-trans RA were obtained from Sigma (St. Louis, MO), and AMPA and DNQX were obtained from Tocris Cookson (Bristol, UK). A23187 was obtained from Calbiochem (San Diego, CA). [Methyl-3H]thymidine was obtained from Amersham Life Science (Arlington Heights, IL). All secondary fluorochrome-conjugated antibodies used for immunocytochemistry were obtained from Cappel-Organon Teknika (Durham, NC).
Cell cultures. Purified cortical O-2A progenitor cultures were prepared by modifications of previously described methods (Armstrong et al., 1990
Cultures enriched with different cell types were characterized immunocytochemically by using cell type-specific antibodies (see below). Cell cultures used for immunostaining were grown on glass coverslips precoated with poly-D-ornithine. In cortical cultures enriched in O-2A progenitors, >95% of the cells were labeled by the monoclonal LB1, anti-GD3 antibody (Levi et al., 1986
Cell proliferation assays. Cell proliferation was assayed as described previously (Gallo et al., 1996
Immunocytochemistry and counting of cell cultures. The primary antibodies used were LB1 (Levi et al., 1986
Electrophysiology. For electrophysiological experiments, cells were cultured either with 10 ng/ml PDGF (proliferating O-2A progenitor cells) or with PDGF for 2 d and then in N1 + O.5% FBS for 3 d (pro-oligodendroblasts). Cells were perfused with media of the following composition (in mM): NaCl 160, KCl 2.5, CaCl2 1.5, MgSO4 1.5, and glucose 10; HEPES 10; TTX 0.5-1 µM. In experiments in which Ca2+ was omitted from the recording solution, 1 mM Co2+ or 200 µM Cd2+ was substituted. In the experiments using 45 mM [K+]o, K+ was substituted by an equimolar amount for [Na+]o. In experiments involving NMDG substitution, 160 mM NMDG was substituted for Na+. The solution was buffered to pH 7.3 using HCl to give a final [Cl ]o of 160 mM.
Tight-seal (>5 G ) whole-cell recordings (Hamill et al., 1981
-aminoethylether, EGTA 0.6; buffered to pH 7.4 and ~275 mOsm. Glutathione (5 mM) was included and Mg2+ excluded from the intracellular solution to prevent a loss of N-type inactivation resulting from cysteine oxidation (Ruppersberg et al., 1991
70 and
where g/gmax is the conductance normalized to its maximum value, V is the membrane potential, V1/2 is the membrane potential at which the current amplitude is half maximum, and k is a constant. For the construction of activation curves of the sustained current component (e.g., see Fig. 1) the sum of two independent Boltzmann equations was used. All drug solutions were added directly to the bath via the perfusion system in known concentrations. All data are expressed as the mean ± SEM.
![g/g<SUB><UP>max</UP></SUB>=[1+<UP>exp</UP> {V<SUB>1/2</SUB>−V/k}]<SUP>−1</SUP>,](/content/vol17/issue8/fulltext/2669/img001.gif)
Fig. 1. Both transient and sustained voltage-dependent outward K+ current phenotypes are observed in O-2A progenitor cells. A, Outward currents were activated by test potentials up to +70 mV (10 mV increments, 0.1 Hz, Vhold =
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RESULTS
Voltage-dependent potassium currents in O-2A progenitor cells
Sustained current component Previous studies have characterized the variety of voltage-dependent currents present in A2B5+ O-2A progenitor cells (Sontheimer et al., 1989Inward rectifying K+ currents (Kir) GD3+ O-2A cells displayed Kir with properties similar to those described previously by Barres et al. (1990)
At a holding potential of
Table 1. A comparison of 0-2A and pro-oligodendroblast K+ current properties
O-2A (n = 47) O4+ (n = 11) RMP (mV) Membrane capacitance (pF) 10.6 ± 0.6 35.0 ± 2.6 Sustained currenta (pA/pF) 150.7 ± 12.6 8.9 ± 2.9 Transient currenta (pA/pF) 148.2 ± 18.3c ND Kir (pA/pF)b a Currents amplitude measured at +70 mV. bCurrent amplitude measured at 
Fig. 3. The block of outward K+ currents by GluR activation in O-2A cells is [Na+]i-dependent. A, Addition of kainate (KA, 200 µM) reversibly attenuated the sustained current by 48.5 ± 5.3% (n = 11). Currents were activated by a test pulse to +70 mV (with or without a prepulse to
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Fig. 4. An elevation of extracellular K+ reduces outward and augments inward K+ currents in O-2A cells. Elevation of [K+]o from 2.5 to 45 mM reduced outward currents (A) and augmented the inward rectifying current (B). The magnitudes of both the sustained current reduction and the Kir augmentation were as predicted from a simple change in the K+ driving force. Aii, The current amplitude was reduced by 52 ± 5% (n = 6), a value close to the calculated value (56%). Bii, Likewise, the observed augmentation of the Cs+-sensitive Kir was 440 ± 60% (n = 5), a value close to the predicted value of 469%.
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Transient current properties When depolarizing test pulses were preceded by a prepulse to
The voltage-dependence of activation of O-2A cell outward currents has not been analyzed previously. Figure 1B illustrates the mean conductance-voltage relationship of the sustained current obtained from 25 representative O-2A cells. The sum of two Boltzmann distributions was required to adequately describe the voltage-dependence of activation (see Materials and Methods); the mean half-activation potentials of the two currents were 
Fig. 2. Kir currents in O-2A progenitor cells are revealed at negative test potentials. Kir were activated by either one of two protocols. A, A ramp protocol (inset) delivered from
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K+ current properties in pro-oligodendroblasts
We reported previously that on attainment of the O4+ pro-oligodendroblast phenotype, a downregulation of all outward K+ currents was observed (Gallo et al., 1996
The observations that K+ currents in the O-2A lineage are developmentally downregulated and that the antiproliferative actions of glutamate occur through a reversible blockade of voltage-dependent K+ channels (Gallo et al., 1996 [Na+]i-dependent block of K+ currents in O-2A cells
In our previous study, we demonstrated that AMPA receptor activation attenuated O-2A cell K+ currents and suggested that the antiproliferative action of GluR activation may result from a mechanism involving a blockade or a downregulation of K+ channels. A study by Kettenmann and co-workers (Borges and Kettenmann, 1995
In the present experiments, we first confirmed our initial finding that GluR activation blocks K+ currents in O-2A cells and then extended this observation to analyze the role of depolarization and [Na+]i in the current attenuation. Figure 3 demonstrates that at a test potential of +70 mV and in the presence of extracellular Na+ ions, 200 µM kainate attenuated the sustained current by 48.5 ± 5.3% (n = 11). In addition, we now show that the isolated transient current was also blocked by GluR activation (mean current block, 41.2 ± 9%, n = 6) (Fig. 3E). The block of the K+ currents by kainate was weakly voltage-dependent; the block of the sustained current at +70 mV (48%) was similar to that observed at 0 mV (mean current block, 46.0 ± 6.0%, n = 6), but was greater than that seen at
Voltage-dependent Na+ currents have been described previously in cultured O-2A cells (Sontheimer et al., 1989
We next investigated the impact of directly depolarizing O-2A cells with high [K+]o (45 mM) on K+ currents in O-2A cells. As expected, elevation of [K+]o from 2.5 to 45 mM decreased outward currents and augmented the inward rectifying current (Fig. 4). Both the magnitude of the reduction of the sustained current and the augmentation of the Kir were as predicted from a simple change in the K+ driving force. The calculated reversal potential (EKcalc) for K+ currents in 45 mM [K+]o was The inhibition of O-2A cell proliferation by depolarization is Ca2+-independent
The data illustrated above indicate that activation of GluR receptors, an elevation in [Na+]i, and membrane depolarization with elevated [K+]o all reduce K+ currents in proliferating O-2A cells. Activation of GluRs in the same cells also inhibits their proliferation (Gallo et al., 1996
Fig. 5. Membrane depolarization and increased [Na+]i inhibit O-2A cell proliferation[3H]thymidine incorporation assays. A, High extracellular K+ inhibits O-2A cell proliferation. B, Veratridine inhibits O-2A cell proliferation. Cells were plated in 24-well plates at a density of 30,000 cells/well and cultured in DME-N1 + 0.5% FBS with PDGF and/or bFGF (both 10 ng/ml). Veratridine was added at a concentration of 10 or 50 µM, whereas the high K+ media contained 25 or 45 mM KCl, respectively. [3H]thymidine (0.5 µCi/ml) was added to the cultures 2 hr after plating the cells. After 22 hr, [3H]thymidine incorporation was measured by trichloroacetic acid precipitation and scintillation counting. Averages of three experiments in triplicate ± SEM are shown. A, *p < 0.001, **p < 0.05 compared with their respective controls (Student's t test). B-D, Proliferation of cells treated with veratridine (50 and 10 µM) was significantly different from controls (p < 0.05 and p < 0.001, respectively). The antiproliferative effects of high K+ and veratridine are reversible. Time course after removal of high K+- (C) or veratridine-containing (D) medium. Progenitor cells were cultured in PDGF (10 ng/ml) in the absence (control condition) or presence of 45 mM K+- or veratridine-containing (20 µM) medium. After 22 hr, all cells were shifted to fresh culture medium without high K+ or veratridine, but containing PDGF (10 ng/ml) and [3H]thymidine (0.5 µCi/ml). At 22 hr, before the shift to low -K+ or veratridine-free medium, high K+ and veratridine inhibited [3H]thymidine incorporation by 70 and 47%, respectively. Cells were harvested after 6, 12, and 24 hr after shift to low-K+ or veratridine-free medium, and [3H]thymidine incorporation was measured by trichloroacetic acid precipitation and scintillation counting. Averages ± SEM (n = 3) are shown.
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O-2A cells cultured with PDGF and 45 mM K+ for 24 hr, and then [3H]thymidine-pulsed in a low-K+ medium containing PDGF, reentered S-phase with a temporal pattern similar to cells that were never exposed to depolarizing concentrations of K+ (Fig. 5C). A similar pattern was also observed for cells cultured with PDGF and veratridine for 24 hr, and then [3H]thymidine-pulsed in a veratridine-free medium containing PDGF (Fig. 5D). It can be concluded, therefore, that the inhibitory effects of depolarization on O-2A cell proliferation were reversible.
The Na+/K+ exchange inhibitor ouabain, which causes a raise in the intracellular Na+ concentration, dose-dependently inhibited proliferation of O-2A cells cultured with PDGF or bFGF. Table 2 shows that ouabain (10-1000 µM) caused a 20-80% dose-dependent inhibition of both PDGF- and bFGF-stimulated O-2A cell proliferation.
Table 2. Ouabain inhibits O-2A cell proliferation
[3H]thymidine incorporation (% over N1, no growth factors) PDGF bFGF Control 324 ± 30 310 ± 15 Ouabain 10 260 ± 26** 283 ± 8 Ouabain 100 59 ± 8* 75 ± 4* Ouabain 1000 38 ± 6* 50 ± 4* O-2A progenitor cells were purified and cultured as described previously at a density of 3 × 104 cells/cm2 in DME-N1 medium, containing 0.5% FBS (Gallo and Armstrong, 1995
The antiproliferative effects of GluR agonists, agents that increase [Na+]i and elevated [K+]o, were independent of extracellular Ca2+. Figure 6A shows that in a nominally Ca2+-free culture medium, O-2A cell proliferation stimulated by mitogenic factors was still strongly inhibited by kainate, veratridine, and high [K+]o. In the nominally Ca2+-free medium, basal and growth factor-stimulated O-2A cell proliferation was lower than in the presence of Ca2+. [3H]thymidine incorporation in O-2A cells cultured in Ca2+-free N1 was 35.7 ± 1.7% of N1 + Ca2+ (n = 11). PDGF- and bFGF-stimulated O-2A cell proliferation in the absence of Ca2+ was 40.7 ± 4.3 and 41 ± 2.1% (n = 12 each), respectively, of that measured in a Ca2+-containing medium. However, growth factor-stimulated O-2A cell proliferation over control (N1) was unaltered by the absence of extracellular Ca2+ (Fig. 6A). These findings indicate that extracellular Ca2+ plays an important role in O-2A cell proliferation, but is not involved in the inhibitory effects of GluR agonists, agents that increase [Na+]i or elevate [K+]o. Consistent with this conclusion, the ionophore A23187, which strongly stimulates transmembrane Ca2+ influx in O-2A cells (Raff et al., 1988 
Fig. 6. The inhibitory effects of GluR agonists, agents that increase [Na+]i and membrane depolarization on O-2A cell proliferation, are independent on extracellular Ca2+. A, Absence of extracellular Ca2+ does not prevent kainate-, veratridine-, and high K+-induced inhibition of O-2A cell proliferation. Cells were plated in 24-well plates at a density of 30,000 cells/well and cultured in DME-N1 + 0.5% FBS with PDGF and/or bFGF (both 10 ng/ml), in the presence or absence of extracellular Ca2+ (see Materials and Methods for media composition). Veratridine was added at a concentration of 25 µM, kainate was 100 µM, whereas the high K+ media contained 45 mM KCl. [3H]thymidine (0.5 µCi/ml) was added to the cultures 2 hr after plating the cells. After 22 hr, [3H]thymidine incorporation was measured by trichloroacetic acid precipitation and scintillation counting. Averages of two experiments in triplicate ± SEM are shown. All treatments (kainate, veratridine, and high K+) were significantly different from their respective controls, in both the presence and absence of Ca2+ (p < 0.001, Student's t test). B, Treatment with the Ca2+ ionophore A23187 does not modify O-2A cell proliferation. Cells were plated in a DME-N1 medium containing Ca2+ with PDGF and/or bFGF (both 10 ng/ml), in the presence or absence of A23187 (1-30 nM) or AMPA (100 µM). [3H]thymidine (0.5 µCi/ml) was added to the cultures 2 hr after plating the cells. After 22 hr, [3H]thymidine incorporation was measured by trichloroacetic acid precipitation and scintillation counting. Averages of three experiments in triplicate ± SEM are shown.
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Membrane depolarization prevents O-2A lineage progression
Our previous studies demonstrated that activation of GluRs in O-2A cells inhibited their lineage progression to the pro-oligodendroblast stage, identified by the monoclonal antibody O4 (Gallo et al., 1996
Fig. 7. Depolarization with high extracellular K+ prevents O-2A lineage progression, as detected by staining with O4 antibody. O-2A progenitor cells were purified and cultured on coverslips in DME-N1 medium with 0.5% FBS with PDGF (10 ng/ml), or bFGF (10 ng/ml), or PDGF + bFGF. K45 indicates that cells were cultured in the same medium with the addition of 45 mM K+ (see Materials and Methods for media composition). After 48 hr, cells were immunostained with O4 antibody and counted. Averages ± SEM obtained from three experiments (n = 30) are shown. The total number of cells counted for each culture condition ranged from 1020 to 2046; *p < 0.001; **p < 0.005 (Student's t test).
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Chronic GluR activation does not alter K+ current properties
Previous studies have indicated that cells of the oligodendrocyte lineage display changes in their K+ current phenotype that are related to their proliferative potential and their developmental stage (Sontheimer et al., 1989
Treatment of cultures with either 100 µM kainate or 100 µM AMPA for 24-48 hr had little effect on O-2A cell resting membrane parameters (Table 3). The mean current densities of both the sustained and Kir currents in O-2A cells were unaltered (Table 3). Likewise, the voltage-dependence of activation of the sustained current components were unaltered (data not shown). In kainate-treated cultures, the mean half-activation potentials of the sustained current components were
Table 3. Effects of chronic treatment with antiproliferative agents on K+ current expression
Control cells (n = 23) Kainate-treated cells (n = 10) 45 mM [K+]o-treated cells (n = 21) RA-treated cells (n = 13) RMP (mV) Membrane capacitance (pF) 10.0 ± 0.7 9.1 ± 0.9 11.7 ± 0.7 9.2 ± 1.2 Sustained currenta (pA/pF) 168.0 ± 14.7 168.2 ± 48.5 227.5 ± 22.9 118.9 ± 17.7 Transient currenta (pA/pF) 131.6 ± 19.1 32.9 ± 8.5 169.0 ± 24.2 51.3 ± 16.2 Kir (pA/pF)b 41.3 ± 16.5d a Currents amplitude measured at +70 mV. bCurrent amplitude measured at Culturing O-2A cells in 45 mM [K+]o modifies the K+ current phenotype
Cells cultured in 45 mM [K+]o displayed physiological properties distinct from those of control O-2A cells. When cells cultured in 45 mM [K+]o were recorded in standard extracellular recording conditions (i.e., 2.5 mM [K+]o, see Materials and Methods), the resting membrane potential was significantly more negative than that seen in control control cultured cells, whereas no significant change in cell capacitance was observed (Table 3). This shift in the membrane potential likely reflected the upregulation of the Kir current density observed (Fig. 8B; Table 3), because Kir play a dominant role in setting resting membrane potential (Duffy et al., 1995
Fig. 8. Chronic exposure of O-2A cells to 45 mM [K+]o upregulates the Kir current. Cells cultured in 45 mM [K+]o for 48 hr possessed properties distinct from those observed in control cultured O-2A cells. After removal of the elevated K+ culturing medium, cells were perfused with a control solution (i.e., 2.5 mM K+) for electrophysiological recordings. The resting membrane potential of cells cultured in 45 mM [K+]o was significantly more negative than that seen in control (Table 3). A, An increase in the sustained current density was observed after culture in 45 mM [K+]o for 48 hr (see also Table 1). Ai, Representative current traces obtained from two different cells cultured under control and 45 mM [K+]o conditions demonstrate the upregulation of the sustained current component after chronic exposure to 45 mM [K+]o. Aii, Summary histograms of data obtained at a test pulse of +70 mV reveals an upregulation of both the current amplitude and the current density when normalized for any changes in membrane capacitance. Bi, Similarly, an upregulation of the Kir current density was also observed after chronic exposure to 45 mM [K+]o culturing conditions (Bi, 1, 3, 4) (see also Table 3). Bi, 2, Normalization of the Kir currents obtained in control (n = 8) versus the 45 mM [K+]o (n = 6) revealed no change in the current-voltage relationship of the Kir after chronic 45 mM [K+]o culture. Aii, Bii, Summary histograms of sustained and Kir current data obtained from both control (2.5 mM [K+]o) and 45 mM [K+]o culturing conditions reveal an upregulation of both the total current and the current density in 45 mM [K+]o culturing conditions. Data shown in the histogram was obtained from a current step to
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RA reversibly inhibits O-2A cell proliferation and downregulates K+ currents
Finally, we analyzed the effects of a different anti-proliferative agent, RA, on K+ channel function. RA is known to regulate oligodendrocyte precursor development (Barres et al., 1993
Fig. 9. RA reversibly inhibits O-2A cell proliferation and prevents lineage progression. A, RA inhibits O-2A cell proliferation. Cells were plated in 24-well plates. After 2 hr, PDGF and/or bFGF (both 10 ng/ml), as well as RA (0.1-3 µM), were added to the cultures, together with [3H]thymidine (0.5 µCi/ml). After 22 hr, [3H]thymidine incorporation was measured by trichloroacetic acid precipitation and scintillation counting. Averages ± SEM obtained from three to six experiments run in triplicate are shown. All concentrations of RA significantly (p < 0.05) inhibited O-2A cell proliferation, except for 0.01 µM in bFGF and P + F (Student's t test). B, The antiproliferative effects of RA are reversible. Progenitor cells were cultured in PDGF (10 ng/ml) in the absence or presence of RA (1 µM). After 22 hr, all cells were shifted to fresh culture medium, without RA, containing PDGF (10 ng/ml) and [3H]thymidine (0.5 µCi/ml). Cells were harvested after 6, 12, and 24 hr, and [3H]thymidine incorporation was determined by trichloroacetic acid precipitation and scintillation counting. Averages ± SEM (n = 3) are shown. At 22 hr, before the shift to RA-free medium, RA inhibited [3H]thymidine incorporation by 47%. C, Treatment with RA prevents O-2A lineage progression, as detected by staining with O4 antibody. O-2A progenitor cells were purified and cultured on coverslips in DME-N1 medium with 0.5% FBS with PDGF (10 ng/ml), or bFGF (10 ng/ml), or PDGF + bFGF. RA (1 µM) was added to the culture medium 2 hr after plating. After 48 hr, cells were immunostained with O4 antibody and counted. Averages ± SEM obtained from three experiments (n = 20) are shown. The total number of cells counted for each culture condition ranged from 683 to 2176; *p < 0.001 (Student's t test).
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Consistent with previous reports (Barres et al., 1994
Like the other antiproliferative agents tested, RA also altered O-2A K+ currents. In cells cultured for periods of 24-48 hr in 1 µM RA-containing media, both a downregulation of the transient current component and the sustained current density were observed in O-2A cells recorded in the absence of RA (Fig. 10). The mean current density reductions were 61 and 30%, respectively, (Table 3) and were not associated with any changes in the resting membrane potential or cell capacitance (Table 3). Similar to the other antiproliferative agents, RA had no effect on the voltage-dependent properties of activation of either the sustained or the transient current components. In cells treated with RA, two sustained current components still could be resolved, possessing half-activations of 
Fig. 10. Long-term treatment with RA downregulates both the transient and sustained outward K+ currents in O-2A cells. A, B, Cells cultured in RA-containing (1 µM) media for 48 hr displayed a downregulation of both the isolated sustained current (Ai) and transient current density (Aii) (see also Table 3). Representative traces from two different cells in both control and RA culture conditions are depicted in Ai and Bi to illustrate the downregulation of both the transient and sustained currents in RA culturing conditions. Aii, Bii, Summary histograms of the effects of chronic exposure to RA on both the isolated sustained and transient current components. Both the transient and the sustained current densities were significantly reduced after chronic exposure to RA. Aiii, Biii, RA had no effect on the voltage-dependent properties of activation of either the sustained or the transient current components. Aiii, Two sustained current components could be resolved, possessing half-activations of
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DISCUSSION
The present report extends our previous findings in O-2A cells that GluR activation causes an indirect blockage of voltage-dependent K+ channels and through this mechanism, a reduction in cell proliferation (Gallo et al., 1996
Similar to GluR activation, agents that directly increase [Na+]i or depolarize the cell membrane caused marked antiproliferative effects on cultured O-2A progenitor cells. Our experiments indicate that all of these agents act through a similar mechanism to inhibit O-2A cell proliferation, i.e., through the reduction of voltage-dependent outward K+ currents. First, elevation of [Na+]i with the alkaloid veratridine or depolarization with high concentrations of [K+]o ions caused a reduction of K+ currents in O-2A cells, similar to that observed on GluR activation. Second, both veratridine and high [K+]o markedly and reversibly inhibited O-2A cell proliferation in the same concentration range that inhibited K+ currents in O-2A cells. Third, the antiproliferative effects of kainate, veratridine, and high [K+]o were independent of extracellular Ca2+ ions. Finally, another agent, ouabain, that acts to increase [Na+]i suppressed both the sustained and transient K+ currents and inhibited O-2A cell proliferation. These findings not only confirm that modulation of voltage-gated K+ channels can modify the proliferative state of O-2A cells, but indicate further that glial development can be controlled by signals that cause changes in the cell membrane potential and/or increase [Na+]i.
RA has been shown to regulate oligodendrocyte development in different areas of the nervous system. Our analysis in cortical O-2A cells confirms previous findings that both cell proliferation and lineage progression are partially inhibited by RA (Barres et al., 1994
The present results provide some insight into the role of voltage-gated outward currents in an otherwise electrically inexcitable (i.e., absence of action potentials) cell type. The observation that the activation threshold for outward K+ currents (
Previous studies have demonstrated that membrane depolarization and an increase in [Na+]i can stimulate a mitogenic response in cultured central neurons (Cone and Cone, 1976
An important finding of the present study is that chronic exposure of O-2A cells to elevated [K+]o causes a long-term change in K+ channel current expression, such that the resulting current phenotype is similar to that observed previously in pro-oligodendroblast (Barres et al., 1989
Similarly, RA partially reproduced a shift to a pro-oligodendroblast phenotype by selectively downregulating outward K+ currents, but did not promote lineage progression to the O4+ stage. These results not only show that plastic changes in voltage-dependent K+ currents can be triggered by distinct extracellular signals in O-2A cells, but indicate additionally that the voltage-dependent channel phenotype of oligodendrocyte lineage cells does not correlate with their differentiation stage.
Depolarizing [K+]o not only affects glial cell proliferation (Canady et al., 1990
FOOTNOTES
Received Dec. 23, 1996; revised Jan. 28, 1997; accepted Jan. 30, 1997.
This work was supported by a National Institute of Child Health and Human Development (NICHD) Pre-IRTA Fellowship (P.K.) and partially supported by a fellowship from the National Research Council of Italy (C.A.G.). We thank Sotirios Keros for providing the Origin 4.0 macros used for the data analysis, Alex Eisen for providing the data concerning the effects of ouabain on outward K+ currents, Dr. Mark Mayer for critically reading this manuscript, and Drs. James Russell and Peter Simpson for communicating their data concerning the effects of A23187 on O-2A intracellular calcium levels.
Correspondence should be addressed to Dr. Chris J. McBain, Laboratory of Cellular and Molecular Neurophysiology, Room 5A72, Building 49, NICHD, National Institutes of Health, 49 Convent Drive, Bethesda, MD 20892-4495.
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