INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

POTASSIUM (K⁺) channels, a diverse family of membrane proteins, are known targets of transmitters, hormones, and growth factors leading to changes in membrane excitability, secretory function, and cell survival. There is also evidence that K⁺ channel activity is linked to cell cycle progression, possibly via its effect on membrane potential, cell volume regulation, intracellular [Ca²⁺], or levels of Cdk inhibitors (20, 38, 50). In cells of oligodendroglial (OLG) lineage, K⁺ channel expression is developmentally regulated. Proliferating OLG progenitor (OP) cells express predominantly delayed outward-rectifying K⁺ currents (I_K ), whereas postmitotic OLGs express predominantly inward-rectifying K⁺ currents (I_Kir) (43, 45, 46). Of the Shaker family (Kv1) subfamily, we found expression of Kv1.2, Kv1.4, Kv1.5, and Kv1.6 transcripts in OP cells, although antisense experiments reveal that OP I_K is encoded predominantly by Kv1.5 (2). Other subunits such as Kv1.3 and Kv3.1 have also been identified in OP cells by other investigators (9, 48).

There is a considerable but not total overlap in the repertoire of Kv channel subunits expressed by cells of OLG lineage and by other glial cells. Kv1.5 and Kv1.6 transcripts have also been identified in astrocytes (39, 40), whereas Schwann cells express Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv3.1 -subunits (10, 31, 42). Although there has not been a consensus regarding the contribution of specific Kv subunits to cell cycle progression, the correlation of I_K density with proliferation is uniformly observed in OP cells, astrocytes, and Schwann cells. Pharmacological blockers of I_K induce a decrease in the proliferation, whereas growth factors that act as mitogens enhance the expression of Kv channels (2, 11, 17, 27, 35, 49).

The goal of this study was to examine the regulation of Kv1.5 and Kv1.6 subunits by growth factors important in OLG development and to investigate the pathways involved. Platelet-derived growth factor (PDGF) plays a critical role in OLG development by stimulating the proliferation of OP cells. However, after a set number of divisions, these cells lose PDGF responsiveness and differentiate into OLGs (36). In contrast, basic fibroblast growth factor (bFGF) stimulates the proliferation indefinitely and upregulates PDGF- receptors in OP cells (30). Studies in fibroblasts and other cell types reveal that ligating the PDGF- receptor leads to autophosphorylation and recruitment of downstream molecules such as Src tyrosine kinases, phospholipase C- (PLC-), and phosphatidylinositol 3-kinase (PI 3-K) (for review, see Ref. 22). In OP cells, PDGF regulates the activation of ERK1/2, sphingosine kinase, and intracellular Ca²⁺ signaling (5, 13, 15, 51). We found that PDGF and bFGF increase I_K density in OP cells, which is accompanied by an upregulation of Kv1.5 and Kv1.6 transcripts. In addition, PDGF-induced upregulation of Kv1.5 involves Src family tyrosine kinases and sphingosine kinase but not ERK1/2 or PI 3-K pathways. There is a strong correlation between the percentage of proliferating cell nuclear antigen (PCNA)-positive cells and I_K density in OP cells treated with various inhibitors of PDGF-activated pathways.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Cell culture. Neonatal mixed glial cells were isolated according to the method described by McCarthy and deVellis (29). The collected cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). After 8-12 days, O-2A progenitors were detached by overnight shaking, collected, and preplated for 1 h to remove contaminating macrophages and astrocytes. Floating cells were collected and plated on poly-L-lysine-coated dishes. At this stage, cells were A2B5⁺. The culture medium was changed after 24 h to low-serum medium (0.5% FBS) supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml sodium selenite (ITS). Cells were treated with test agents on the same day 6 h later or the next day (2 days in vitro). Unless otherwise specified, electrophysiological experiments, RNA extraction, cell lysis, and immunofluorescence studies were done on OP cells that were 2 or 3 days in vitro.

RNase protection analysis. Total RNA was extracted from cultured OP cells and OLGs according to the method described by Chomczynski and Sacchi (12). Kv1.5 and Kv1.6 antisense cRNA probes were synthesized in vitro from the linearized cDNAs using T7 and T3 RNA polymerases (Stratagene, La Jolla, CA) , respectively, and [-³²P]UTP (4,000 Ci/mmol; Amersham, Amersham, UK). The template cDNA probes were derived by linearizing pBs/Kv1.5 and pBs/Kv1.6 plasmids with XbaI and XhoI, respectively. The labeled Kv1.5 and Kv1.6 cRNA probes (2.5 × 10⁵ cpm) of 430 and 563 base lengths, respectively, were hybridized separately with 20 µg of total RNA in 80% formamide, 40 mM PIPES (pH 6.4), 1 mM EDTA, and 0.4 M NaCl for 14 h at 45°C. To quantify the input RNA, a labeled cRNA probe encoding the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included in the hybridization mixture. The duplex RNA hybrids were then digested with RNase A and RNase T1. The RNase-resistant fragments were electrophoresed on 6% polyacrylamide, 7 M urea gels and autoradiographed. Data were quantified by scanning the labeled bands using a Umax Powerlook II densitometer (Taipei, Taiwan) and Adobe Photoshop software. The optical densities of Kv channel mRNA fragments were normalized to the GAPDH signal.

Reverse transcription-polymerase chain reaction. First-strand cDNA synthesis was carried out under the following conditions: 3-5 µg of total RNA were mixed in diethylpyrocarbonate-treated water with 0.5 µg of random hexamer primer (Pharmacia, Uppsala, Sweden). The mixture was incubated for 10 min at 70°C and chilled on ice. A first-strand reaction mix buffer containing 45 mM Tris (pH 8.3), 68 mM KCl, 15 mM dithiothreitol, 9 mM MgCl₂, 1.8 mM of each dNTP, 0.08 mg/ml BSA, 100 units of RNasin, and 50 units of cloned FPLC pure murine reverse transcriptase (Pharmacia) was then added and incubated for 1 h at 37°C. After the first-strand cDNA synthesis was completed, the reverse transcriptase was inactivated by heating the reaction to 95°C for 5 min. Five microliters of the first-strand cDNA synthesis reaction were used for PCR, which was carried out in a buffer containing 50 mM KCl, 10 mM Tris · HCl, pH 9.0 at 25°C, 0.1% Triton X-100, 0.2 µM each dNTP, 1 µM of each upstream and downstream primer, and 2.5 units of Taq DNA polymerase (Promega, WI).

Western blot analysis. Cultured OP cells were lysed in a lysis buffer containing 150 mM NaCl, 50 mM Tris · HCl (pH 8.0), 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM orthovanadate, 1 mM PMSF, and 2 µg/ml leupeptin. Samples (equal amount/lane) were resolved by 8% SDS-PAGE and electroblotted to polyvinylidene difluoride membranes. Blots were blocked with 5% nonfat milk in phosphate-buffered saline (PBS) or 5% BSA in 10 mM Tris (pH 7.5), 100 mM NaCl, 0.1% Tween 20 for 1 h at room temperature. The blots were incubated with polyclonal antibody against Kv1.5 (1:200) (Upstate Biotechnology, Lake Placid, NY) overnight at 4°C, washed three times, and then incubated for 1 h with goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:10,000-20,000 dilution) (Accurate, Westbury, NY). For experiments pertaining to ERK2 and Akt phosphorylation, polyclonal antibodies against these proteins obtained from New England Biolab (Beverly, MA) were used at 1:500-1,000 dilution. Immunoreactive proteins were visualized by enhanced chemiluminescence detection (ECL; Amersham) and then scanned and quantified using the NIH Image analysis program.

Electrophysiology. Current recordings were obtained by using the whole cell configuration of the patch-clamp technique as previously described (44). The pipette resistance ranged from 2-5 M. Cells were studied at room temperature. For recording of K⁺ currents, the bathing solution consisted of the following (in mM): 140 NaCl, 5.4 KCl, 2 CaCl₂, 1 MgCl₂, and 10 HEPES, pH 7.3 (normal bath solution). Pipette (intracellular) solutions contained (in mM) 140 KCl, 2 CaCl₂, 2 MgCl₂, 11 EGTA, and 10 HEPES, pH 7.3. For cell population studies, a dish of untreated OP cells was included for each set of experimental conditions to control for culture-to-culture variability in current densities. Current records were filtered at 2 kHz using an eight-pole Bessel filter and were sampled at 5 kHz.

Quantification of PCNA+ cells. Cells were fixed with acetone for 15 min, followed by methanol for 15 min, and were labeled with a monoclonal antibody against PCNA followed by fluorescein-conjugated goat anti-mouse IgG. This method has been shown to limit the PCNA immunoreactivity to the S phase (4). Scoring of PCNA⁺ nuclei was accomplished by examining 8-10 randomly selected, nonoverlapping microscopic fields (300-400 cells) on each coverslip.

Data analysis. Results were expressed as means ± SE with the number of experiments in parentheses. Statistical differences between control and test values for Kv transcript levels were analyzed by Student's t-test. For Kv protein levels, I_K density, and %PCNA⁺ cells, statistical significance was determined by using analysis of variance (ANOVA) followed by the Bonferroni method for multiple experiments. For data with unequal variance, Dunnett's T3 was used.

Materials. PDGF was obtained from R&D Systems (Minneapolis, MN). The other drugs were obtained from the following sources. PP2, PP3, wortmannin, U-73122, genistein, staurosporine, and LY-294002 were from Calbiochem (San Diego, CA). Mimosine and PD-98059 were from Sigma (St. Louis, MO). Dimethylsphingosine (DMS) and sphingosine-1-phosphate (SPP) were from Biomol (Plymouth, PA). BAPTA-AM and BCECF-AM were from Molecular Probes (Eugene, OR). Most of these agents, except for mimosine and genistein, were dissolved in either DMSO or ethanol and stored as aliquots. SPP was dissolved according to the manufacturer's instructions.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Macroscopic K⁺ currents were recorded from cultured neonatal rat OPs using the whole cell configuration of the patch-clamp technique. Pulses of 360-ms duration were stepped to various voltages from a holding potential of 80 mV and from 40 mV. Outward K⁺ currents activated at depolarized potentials from a holding potential of 40 mV were minimally inactivating and exhibited time-dependent activation resembling the delayed rectifier (I_K) as described previously (2, 45). An inactivating K⁺ current resembling the 4-aminopyridine (4-AP)-sensitive transient outward current (I_A) was observed when voltage pulses were stepped from a holding potential of 80 mV. Examples of I_K recorded from neonatal OPs and its inhibition by K⁺ channel blockers such as 4-AP and clofilium are shown in Fig. 1, A and B.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Regulation of IK by growth factors in oligodendroglial progenitor (OP) cells. A: examples of whole cell current recordings illustrating K+ currents (IK + IA) expressed by OP cells. Voltage current pulses of 360-ms duration were stepped from a holding potential (Vh) of 40 and 80 mV in 20-mV increments or decrements from the holding potential. B: inhibition of IK from OP cells by K+ channel blockers 4-aminopyridine (4-AP; 0.3 mM) and clofilium (Clof; 1 µM). Only 2 current traces recorded by voltage pulses to 60 mV are shown for clarity. C: increases in OP IK current density after 24-h treatment with PDGF (5-10 ng/ml) or basic fibroblast growth factor (bFGF; 5 ng/ml) but not with ciliary neurotrophic factor (CNTF; 10 ng/ml). *P < 0.00001 for overall ANOVA, Ctrl (control) vs. PDGF and Ctrl vs. bFGF; P > 0.05, Ctrl vs. CNTF. The effect of PDGF on IK was not mimicked by shorter exposure (1 h) to PDGF but was blocked by concomitant treatment for 24 h with mimosine (Mimo; 200 µM) or genistein (Genis; 20 µg/ml). *P < 0.0001, Ctrl vs. PDGF; P < 0.001, PDGF vs. PDGF + mimosine; P < 0.01, PDGF vs. PDGF + genistein. For IK density measurements, peak currents recorded by voltage pulses stepped from 40 mV to 0 mV were used.

We examined the effects of OP mitogens PDGF and bFGF on I_K. Incubation of neonatal OPs with PDGF (5-10 ng/ml) or bFGF (5 ng/ml) for 24 h in 0.5% FBS plus ITS resulted in an increase in I_K density compared with untreated cells (Fig. 1C). No effect was seen when cells were exposed to ciliary neurotrophic factor (CNTF; 10 ng/ml) for the same duration. I_K current density was 15.8 ± 1.2 pA/pF (n = 29) in control cells, 45.5 ± 6.3 pA/pF (n = 25) in PDGF-treated cells, 41.5 ± 4.3 pA/pF (n = 16) in bFGF-treated cells, and 11.9 ± 2.8 (n = 16) in CNTF-treated cells (P < 0.00001 for overall ANOVA, control vs. PDGF and control vs. bFGF; P > 0.05, control vs. CNTF). The increase in current density induced by PDGF or bFGF was accompanied by a decrease in total membrane capacitance, correlating with the effect of these growth factors on OLG morphology. Membrane capacitance was 32.7 ± 2.4 pF (n = 29) in control cells, 20.4 ± 1.5 pF (n = 25) in PDGF-treated cells, 19.9 ± 1.3 pF (n = 16) in bFGF-treated cells, and 43.4 ± 4.0 pF (n = 16) in CNTF-treated cells (P < 0.01, control vs. PDGF and control vs. bFGF; P > 0.05, control vs. CNTF). There was no increase in I_K density when OP cells were treated with PDGF for 1 h. Treatment with PDGF or FGF for 24 h had no effect on I_Kir density (data not shown).

Next, we examined whether the effect of PDGF on I_K current density could be prevented by concomitant treatment for 24 h with cell cycle inhibitors such as mimosine (200 µM) or by tyrosine kinase inhibitors such as genistein (20 µg/ml) (Fig. 1C). I_K density was 24.2 ± 3.5 pA/pF (n = 25) in control cells, 63.0 ± 6.0 pA/pF (n = 28) in PDGF-treated cells, 29.7 ± 4.4 pA/pF (n = 19) in cells treated with PDGF plus mimosine, and 35.4 ± 5.2 pA/pF (n = 17) in cells treated with PDGF plus genistein (P < 0.0001, control vs. PDGF; P < 0.001, PDGF vs. PDGF + mimosine; P < 0.01, PDGF vs. PDGF + genistein). Treatment with mimosine or genistein alone had no effect on I_K density [mimosine: 26.5 ± 3.8 pA/pF (n = 19); genistein: 27.2 ± 4.1 pA/pF (n = 15)]. These results support the concept that the enhancement of I_K by growth factors is linked to cell cycle control, a process that requires tyrosine kinases.

PDGF- and bFGF- induced increase in I_K was associated with an increase in Kv1.5 and Kv1.6 transcript levels. Figure 2 shows a significant increase in Kv1.5 and Kv1.6 mRNA transcripts following PDGF treatment of cultures as measured by both RNase protection analysis and RT-PCR (55 ± 17 and 120 ± 20% increase in Kv1.5 and Kv1.6 mRNAs, respectively, following PDGF treatment as measured by RT-PCR; n = 7, P < 0.01). Similar results were obtained upon bFGF treatment.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Upregulation of Kv1.5 and Kv1.6 channel transcripts by PDGF or bFGF. RNase protection (A) and semiquantitative RT-PCR analyses (B-D). OP cells were treated for 48 h with PDGF (10 ng/ml) or bFGF (10 ng/ml). In B and C, aliquots from control (C) or PDGF-treated samples (P) were removed at various PCR cycle numbers, resolved by 1.2% agarose gels, and processed for Southern blot analysis. In D, the density of the bands corresponding to Kv1.6 channel PCR fragments from control and PDGF-treated samples was normalized to the S16 signal for each given PCR cycle.

In subsequent studies, Western blot analysis was performed to investigate the role of PDGF-induced downstream signaling molecules in the regulation of Kv1.5 expression. Similar to Schwann cells (42), two Kv1.5-immunoreactive bands were observed in OP cells, a major 60-kDa and an additional 90-kDa species (Fig. 3A). Only the major band (60 kDa) was used for data analysis. At least three alternatively spliced Kv1.5 channel isoforms have been detected in other cell types (1). OP cells treated for 24 h with PDGF had an increase in Kv1.5 protein, which was attenuated by the addition of a Src family tyrosine kinase inhibitor, PP2 (5 µM). Expressed as percent control, Kv1.5 protein was 191.2 ± 23.3% in PDGF-treated cells, 91.2 ± 8.7% in PP2-treated cells, and 105.6 ± 7.3% in cells treated with both (n = 6 for each condition; P < 0.003, PDGF vs. PDGF + PP2). A brief exposure (15 min) to PDGF or PP2 had no effect on Kv1.5 protein level. Figure 3B shows the inhibitory action of PP2 on PDGF-induced phosphorylation of ERK1/2 and pAkt. However, PDGF-induced upregulation of Kv1.5 was not prevented by 24-h treatment with PI 3-K inhibitors such as LY-294002 (5 µM) or wortmannin (0.5 µM) or by treatment with PD-98059 (25-50 µM), an inhibitor of MEK/ERK pathway. Addition of the PLC- inhibitor U-73122 (2.5-5 µM) had no effect, whereas addition of the sphingosine kinase inhibitor DMS (1-5 µM) attenuated PDGF-induced upregulation of Kv1.5 protein (Fig. 4B). Sphingosine kinase inhibition results in the accumulation of sphingosine, a protein kinase C (PKC) inhibitor. Selective inhibition of PKC by low concentrations of staurosporine (10 nM) did not mimic the effect of DMS on Kv1.5, although both caused cell death at concentrations 100 nM and 10 µM, respectively. Figure 5 summarizes the corresponding effects of PP2, PP3, U-73122, and DMS on PDGF-induced increase in I_K density.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effect of the Src family inhibitor PP2 on Kv1.5 expression. A: Kv1.5 immunoreactive proteins (60 and 90 kDa) from OP cells treated with PDGF (10 ng/ml) for 24 h with or without PP2 (5 µM). Only the major band (60 kDa) was used for analysis in subsequent experiments. Brief exposure to PP2 or PDGF had no effect on Kv1.5. B: inhibitory effect of PP2 on PDGF-induced phosphorylation of ERK1/2 and Akt. Treatment duration was 24 h. Results are representative of 6 experiments for Kv1.5 protein and 3 experiments for pERK1/2 and pAkt.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effect of inhibitors of PDGF-mediated signaling pathways on Kv1.5 expression. A: inhibitors of phosphatidylinositol 3-kinase and MEK/ERK pathways did not block PDGF-induced upregulation of Kv1.5. LY, LY-294002 (5 µM); WT, wortmannin (0.5 µM); PD, PD-98059 (25-50 µM). Treatment duration was 24 h. In the bottom panel, the same blot was stripped and reprobed with anti-pAkt or anti-pERK antibodies to verify the effectiveness of WT and PD. B: PDGF-induced upregulation of Kv1.5 was mediated by the sphingosine kinase pathway but not by activation of PLC- or PKC. OP cells were treated for 24 h with PDGF with or without dimethylsphingosine (DMS; 5 µM), U-73122 (U7; 5 µM), or staurosporine (STS; 10 nM). Results from A and B are representative of at least 3 experiments for each condition, except for LY experiments (n = 2).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Effect of inhibitors of PDGF-mediated signaling pathways on OP IK density. A: PP2 (5 µM) vs. inactive analog PP3 (5 µM). P < 0.0001 for overall ANOVA; *P < 0.001, PDGF vs. Ctrl; **P < 0.004, PDGF vs. PDGF + PP2. B: U7 (5 µM) vs. DMS (1-5 µM). P < 0.0006 for overall ANOVA; *P < 0.001, PDGF vs. Ctrl; **P < 0.008, PDGF vs. PDGF + DMS.

Because sphingolipids are known to play an important role in Ca²⁺ signaling in cells of OLG lineage (16, 23), we examined the effect of the Ca²⁺ chelator BAPTA-AM in the regulation of Kv1.5. OP cells treated for 24 h with BAPTA-AM (5-10 µM) or DMS (1-5 µM) remained phase bright and did not exhibit morphological alterations such as loss of processes, as shown in Fig. 6A. Addition of BAPTA-AM (1-10 µM) prevented PDGF-induced increase in Kv1.5, whereas addition of a Ca²⁺-unrelated cell-permeable probe, BCECF-AM (1 µM), had no consistent effect (n = 3; data not shown). The effect of DMS and BAPTA-AM on Kv1.5 upregulation was not due to a nonspecific loss of proteins as a result of impending cell death, because there was no decrease in the phosphorylation of ERK1/2 or in the expression of total PCNA, a cell cycle protein (Fig. 6B). The effect of PDGF on Kv1.5 was not mimicked by exogenous SPP. OP cells treated for 24 h with SPP (10 µM) only had a slight increase in Kv1.5 protein (125.8 ± 5.5% of control, n = 5).

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Comparison of the effect of DMS and BAPTA-AM on OP morphology and Kv1.5 protein levels. A: examples of phase micrographs of cultured OP cells that were untreated (Ctrl) or treated for 24 h with PDGF (10 ng/ml) in the presence or absence of DMS (1 µM) or BAPTA-AM (5 µM). Bar represents 30 µm. B, top: Western blots illustrating the inhibitory action of DMS and BAPTA-AM on PDGF-induced Kv1.5 upregulation. DMS or BAPTA-AM did not attenuate the effect of PDGF on Kv1.5 on ERK1/2 phosphorylation or proliferating cell nuclear antigen (PCNA) protein levels. Bottom: bar graphs summarizing the results of PDGF on Kv1.5 in the presence or absence of DMS or BAPTA-AM. P < 0.00001 for overall ANOVA; *P < 0.00001, PDGF vs. PDGF + DMS; **P < 0.0005, PDGF vs. PDGF + BAPTA.

To investigate whether OP I_K density correlates with cellular proliferation, we quantified the percentage of OP cells with nuclear PCNA staining. Figure 7A summarizes the effect of various inhibitors on %PCNA⁺ cells. Agents that were effective inhibitors of Kv1.5 upregulation also attenuated PDGF-induced proliferation. For those agents with both electrophysiological and PCNA data, there was a good correlation between I_K density and %PCNA⁺ cells (r = 0.83), as depicted in Fig. 7B. The only discrepancy was observed with PDGF + PD-98059, where there was a modest difference from PDGF in terms of %PCNA⁺ cells (P < 0.03) but not in I_K density.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Effect of various inhibitors on PDGF-induced OP proliferation. A: bar graphs summarizing the inhibitory action of PP2 (5 µM), DMS (1 µM), and BAPTA-AM (5 µM) on PDGF-induced OP proliferation (%PCNA+ cells). Top: P < 0.00001 for overall ANOVA; *P < 0.00001, PDGF vs. Ctrl; **P < 0.001, PDGF vs. PDGF + PP2; ***P < 0.03, PDGF vs. PDGF + PD. Bottom: P < 0.00001 for overall ANOVA; *P < 0.00001, PDGF vs. Ctrl; **P < 0.001, PDGF vs. PDGF + DMS; *** P < 0.008, PDGF vs. PDGF + BAPTA. B: correlation between %PCNA+ cells and OP IK density in cultures treated with various inhibitors. For Ctrl and PDGF, the average from 2 sets of means was used for both IK density (see Fig. 5) and %PCNA+ cells (see A).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We have demonstrated that I_K density is upregulated in neonatal OP cells treated for 24 h with mitogens such as PDGF or bFGF, but not in cells treated with CNTF. PDGF-induced upregulation of I_K is associated with increased Kv1.5 and Kv1.6 transcript levels. In contrast, receptor activation induces a slow decline in the Kv1.5 current amplitude with a half-time of about 20 min when Kv1.5 and PDGF receptor or FGF receptor were coexpressed in Xenopus oocytes, an effect that is mediated by PLC (47). Thus peptide growth factors are capable of both rapidly altering cellular excitability and regulating ion channel expression. Furthermore, the functional consequence of rapid vs. long-term modulation may differ. The effect of growth factors is not necessarily restricted to a specific channel subtype. Acute application of bFGF or PDGF rapidly inhibits sodium currents in PC-12 cells through Ras and Src signaling pathways (24). An increase in the magnitude of both I_K and I_Na was observed in Schwann cells cultured with mitogens such as axon fragments or glial growth factors (49). We did not observe an increase in I_Kir density in OP cells treated with PDGF or bFGF .

We found that the effect of PDGF on I_K expression is blocked by mimosine and by genistein, suggesting that enhanced Kv gene expression is linked to cellular proliferation or cell cycle progression and involves tyrosine kinase signaling mechanisms. Interestingly, recent evidence indicates that K⁺ channel subunits are direct targets of Src family tyrosine kinases, but they can also function as SH3-dependent adaptors to induce tyrosine phosphorylation of adjacent subunits (25, 33). Tyrosine phosphorylation of Kv subunits leads to the suppression of K⁺ channel activity, except in Schwann cells where I_K is upregulated by Fyn-induced tyrosine phosphorylation of Kv2.1 (7, 26, 28, 41). Our data from PP2 and PP3 experiments reveal that Src family kinases are also involved in PDGF-induced increase in Kv1.5 protein. In addition, there is evidence that sphingosine kinase is involved, based on the finding that PDGF-induced upregulation of Kv1.5 protein and I_K density is inhibited by DMS. Inhibition of PLC-, PI 3-K, or ERK pathway did not abrogate the effect of PDGF, implying relative specificity of signaling pathways involved in Kv channel regulation.

Activation of sphingosine kinase converts sphingosine to SPP, which either acts as a second messenger or is released to act in a paracrine or autocrine manner at endothelial differentiation gene (EDG) receptors. Binding of SPP to EDG receptors has been shown to induce activation of a muscarinic K⁺ current in atrial myocytes and a Ca²⁺-activated K⁺ current in fibroblasts (8, 37). Our finding that exogenous SPP did not upregulate Kv1.5 to the same extent as PDGF could be interpreted in two ways: 1) a predominant second messenger role of SPP or 2) other pathways linked to tyrosine kinases are important in PDGF action in this regard. Analysis of sphingolipid production in transformed OLGs (CEINGE clone3) reveals that PDGF-induced synthesis of sphingosine and SPP is regulated by the cell cycle (15). Thus these sphingolipids are uniquely poised as potential candidates mediating Kv channel induction or regulation. Perhaps our finding that PDGF-induced enhancement of Kv1.5 expression involves both Src kinases and sphingosine kinase is not surprising given the bidirectional link between tyrosine kinases and SPP (6, 34).

Sphingolipids are known modulators of Ca²⁺ signaling in cells of OLG lineage (16, 23). Although PDGF-induced Kv1.5 upregulation was inhibited by chelation of intracellular Ca²⁺ with BAPTA-AM, it was not prevented by U-73122. The latter inhibits the formation of diacylglycerol and inositol 1,4,5-trisphosphate (IP₃), which activates PKC and intracellular Ca²⁺ release, respectively. One possible interpretation for these findings is that SPP induces Ca²⁺ release via a non-IP₃-mediated mechanism, as demonstrated by other investigators (21). The lack of effect of U-73122 and staurosporine would also imply that PKC is not involved in the upregulation of Kv1.5 by PDGF. Exactly how changes in intracellular Ca²⁺ influence Kv channel expression remains to be determined. Interestingly, the promoter region of Kv1.5 gene and Kv3.1 gene contains a cAMP response element (CRE) that can be activated by cAMP and Ca²⁺-dependent pathways (19, 32).

We found that PDGF-stimulated OP proliferation was inhibited by PP2, DMS, and BAPTA-AM but not by U-73122. Inhibition of MEK/ERK pathway by PD-98050 resulted in a modest but significant decrease in PDGF-induced OP proliferation but did not block PDGF-induced increase in I_K density. Another pathway that is not involved in PDGF-induced Kv1.5 upregulation is the PI 3-K pathway, which is thought to mediate PDGF-stimulated OP proliferation (3, 14). The finding that all effective inhibitors of PDGF-induced I_K or Kv1.5 upregulation attenuate OP proliferation but not vice versa suggests that 1) some pathways such as MEK/ERK and PI 3-K/Akt pathways may act via mechanisms that are independent of I_K upregulation, or 2) other Kv subunits such as Kv1.3 may be the target of PI 3-kinase pathway. Indeed, there is evidence that PDGF or FGF increases the expression of not only Kv1.5 but also Kv1.3 in in OP cells (9). Furthermore, a recent study showed that PI 3-K is involved in the upregulation of Kv1.1, Kv1.2, and Kv1.3 by another mitogen, insulin-like growth factor-1 (IGF-1), in HEK-293 cells (18). We conclude that 1) OP mitogens such as PDGF and bFGF enhance I_K expression, an effect that is prevented by cell cycle and tyrosine kinase inhibitors; 2) PDGF-induced upregulation of Kv1.5 is mediated via Src family tyrosine kinases and sphingosine kinase pathway, possibly converging at the level of intracellular Ca²⁺ signaling; and 3) there is a strong correlation between I_K density and cellular proliferation in OP cells treated with various inhibitors of PDGF-activated pathways, supporting the concept that I_K upregulation is crucial for cell cycle progression.