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Centre for Neuroscience and Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada
Submitted 27 August 2007; accepted in final form 19 January 2008
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ABSTRACT |
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INTRODUCTION |
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; Cummins and Waxman 1997; Jassar et al. 1993, 1994; Lhuillier and Dryer 2002; Martin-Caraballo and Dryer 2002). Various neurotrophic factors have therefore been studied as important determinants of ion channel expression in adult neurons and in cell lines (Chalazonitis et al. 1987; Fanger et al. 1997; Khorkova and Golowasch 2007; Levine et al. 1995; Pollock and Rane 1996; Pollock et al. 1990). Target-derived nerve growth factor (NGF) is responsible for the maintenance of tetrodotoxin (TTX)-sensitive and TTX-insensitive sodium currents (INa) and of N- and L-type calcium currents (ICa,N and ICa,L) in B-neurons of adult bullfrog sympathetic ganglia (BFSG) (Lei et al. 1997, 1998, 2001; Petrov et al. 2001). In serum-free, defined medium culture, NGF-induced increases in ICa are initiated via the mitogen-activated protein kinase (MAPK) pathway (Lei et al. 1998), whereas the transduction process underlying the NGF-induced increase in INa remains to be determined.
We have also demonstrated that luteinizing hormone releasing hormone (LHRH), a neurotransmitter released from preganglionic C-fibers in BFSG (Jan et al. 1979, 1980), is capable of regulating functional expression of Ca2+ channels (Ford et al. 2003a). Gonadotrophin receptors can couple via Gq/11, Gs, or Gi to various downstream effectors including ras-MAPK (Naor et al. 1998; Sim et al. 1995). The present experiments were therefore undertaken to determine 1) whether LHRH-induced increase of ICa in BFSG proceeds via the MAPK pathway in a similar fashion to NGF, 2) how G-protein–coupled LHRH receptors signal to the MAPK pathway, 3) the mechanism whereby NGF produces long-term increases in INa, and 4) whether LHRH has access to this transduction mechanisms so that it can produce long-term increases in INa in the same way as NGF. Parts of this work were published in preliminary reports (Ford and Smith 2000; Lu et al. 2002; Wong et al. 2006).
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METHODS |
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. Neurons were dissociated by incubation with trypsin (Sigma) and type 1A collagenase (Sigma) for 42–45 min, at 37°C. Final dissociation was accomplished by tituration with a Pasteur pipette. Cells were suspended in 3 ml of serum-free, modified L-15 medium (73% L-15, Gibco; pH 7.2), 10 mM glucose, 1 mM CaCl2, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10 µM cytosine arabinoside. The dissociated cells were then redistributed into 2.5 ml of medium in each of twenty 35-mm polystyrene tissue culture dishes. Dishes were placed in a light-proof, humidified chamber and maintained at room temperature (22°C) for 6–10 days as specified. Electrophysiology
Whole cell recordings were made from neurons cultured in the presence of chicken II LHRH (0.45 µM) for 6–9 days or NGF (200 ng/ml) for 9–10 days with or without various inhibitors of transduction pathways. Effects of LHRH on ICa and INa were compared with their effects in neurons cultured in the presence of inhibitors alone. Recordings were made from medium to large-sized cells with input capacitance >30 pF, which were almost certainly B-neurons (Kurenny et al. 1994). Current and voltage were recorded using an Axoclamp 2A amplifier in discontinuous, single-electrode voltage-clamp mode. INa was evoked by a series of depolarizing voltage commands from a holding potential of –85 mV. With low-resistance electrodes (2–5 M
), it was possible to use high switching frequencies (>30 kHz) with high clamp gains (>8 to <30 mV/nA). The fidelity of the clamp was confirmed by examining the voltage recording. Recordings from cells where the voltage trace was slow to rise or distorted were discarded. For recording INa, the solution in the bath (external) contained (in mM): NaCl, 97.5; tetraethylammonium (TEA)-Br, 20; MnCl2, 4; and Tris-Cl, 2.5 (pH 7.2) and the solution inside the pipette (internal) contained (in mM): CsCl, 103; NaCl, 9; TEA-Br, 5; Cs-HEPES, 2.5; Cs-EGTA, and 1 (pH 7.2). ICa was recorded using Ba2+ as a charge carrier (IBa). Currents were activated by incremental depolarizing voltage commands from a holding potential of –90 mV. To limit the amplitude, to lessen the rate of decay, and thereby to facilitate the analysis of tail currents, neurons were stepped to –40 mV at the conclusion of each depolarizing voltage command. The external solution contained (in mM): N-methyl-D-glucamine(NMG) chloride, 117.5; NMG-Hepes, 2.5; and BaCl2, 2.0 (pH 7.2). The internal solution consisted of (in mM): NMG-Cl, 76.5; Hepes, 2.5; Tris-BAPTA, 10; Tris-ATP, 5; and MgCl2, 4 (pH 7.2). Generally, external solutions were 250 mOsmol/kg and internal solutions were 240 mOsmol/kg.
During recording, the culture dishes were superfused with external solution at a flow rate of 2 ml/min. Neuronal input capacitance (Cin) was calculated by integrating the capacitative transient that accompanied a 10-mV depolarizing command from the holding potential. Current densities were expressed in terms of current per unit capacitance (i.e., pA/pF). All data are presented as means ± SE and Student's two-tailed t-test or ANOVA was used to assess statistical significance (P < 0.05).
Being highly hydrophobic, some inhibitors were dissolved in DMSO to make stock solutions. Stock solutions were dissolved in culture medium to make final desired concentrations. The final DMSO concentration used in all cases was 
0.1%. This concentration of DMSO did not affect IBa because currents from cells cultured in the presence of DMSO were not significantly different from controls (data not shown, n = 10). Inhibitors were used at five- to tenfold their published KD or IC50 values for their cognate enzymes. Cells treated with inhibitor plus chicken II LHRH (0.45 µM) or 200 ng/ml NGF were pretreated for 45 min with the inhibitor before addition of LHRH or NGF. NGF, LHRH, and/or enzyme inhibitors were added to medium at the time of dissociation and cells cultured in their presence for 6–10 days as specified. Medium containing wortmannin and PD-98059 was changed every 8 h, to maintain effective concentrations, since both of these chemicals are subject to hydrolysis in aqueous solutions. In an attempt to maintain activation yet avoid downregulation of protein kinase C (PKC), phorbol 12-myristate-13-acetate (PMA) and the negative control 4
-phorbol were used at 80 nM and applied to neurons for 1 h/day, followed by several washes to remove any residual drug.
Immunoblot analysis
This was done using modifications of methodology we previously developed to study mammalian sympathetic ganglia (Song and Posse de Chaves 2003). The VIIIth, IXth, and Xth paravertebral ganglia were removed from both sides of two adult bullfrogs and the neurons dissociated with trypsin and collagenase as described earlier. The cell suspension was plated into 10 wells of a 24-well dish at a density of 1.2 ganglia/well. The dissociated cells were cultured in L-15 medium for 5–6 days. At day 6, some treatment groups were given L-15 supplemented with 50 µM PD-98059 for 24 h to reduce basal phosphorylation of extracellularly regulated kinase (ERK). In these experiments, incubation with 100 nM LHRH began after two 5-min washes in L-15 to remove PD-98059. Cells were incubated for 10 min, 1 h, or 6 h in the presence of the peptide. For 6-day incubations, the LHRH-containing medium was replaced every other day. NGF experiments involved treatment of different groups of cells with 200 ng/ml NGF for 15 min, 1 h, and 6 h or 6 days with medium exchanges every other day. BFSG neurons cultured in 24-well dishes were washed with ice-cold L-15 medium prior to harvesting. Akt experiments included four treatment groups: 100 nM LHRH treatment for 10 min; 200 ng/ml NGF treatment for 15 min; 1 µM wortmannin treatment for 6 h; and 1 µM wortmannin pretreatment for 6 h followed by 200 ng/ml NGF incubation for 10 min. Protein kinase A (PKA) experiments involved incubation with Sp-cAMPS, or its inactive form Rp-cAMPS, for 1 h to activate PKA prior to examining ERK phosphorylation. PKC experiments involved incubation with PMA or its inactive form, 4-phorbol, for 1 h to activate PKC prior to examining ERK phosphorylation. The neurons were washed with ice-cold buffer with 1 mM Na2VO4 and 1 mM NaF to inhibit phosphatase activity. Cells from two wells of the same treatment were harvested with modified Laemmli sample buffer [40 nM Tris-HCl, pH 6.8, 0.002% bromophenol blue, 10% glycerol, 1% sodium dodecyl sulfate (SDS) and 4% 2-mercaptoethanol] and boiled for 2 min. Proteins were separated by SDS-PAGE on 10% polyacrylamide containing 0.1% SDS. After electrophoresis, proteins were transferred to "Immobilin" polyvinylidene difluoride (PVDF; Bio-RAD; Hercules, CA) membranes overnight at 4°C in 25 mM Tris (192 mM glycine, 16% methanol buffer, 0.1% SDS, and pH 8.3). Membranes were blocked for 1 h in 0.1% TTBS (Tris-buffered saline with 0.1% Tween-20) with 5% nonfat milk at room temperature and incubated in the primary antibody overnight at 4°C. The primary antibodies used were: rabbit polyclonal anti-phospho TrkA (Tyr490) (1:1,000), anti-phospho p42/44 MAPK (Thr202/Tyr204) (ERK1/2) (1:1,000), anti-phospho Akt (S473) (1:1,000), anti-phosphoPKC β11 Ser 660 (1:1,000), and anti-phosphoPKC 
Trp 514 (1:1,000) from Cell Signaling Technology (Beverly, MA); anti-pan-Trk polyclonal antibody Trk (C-14) (1:500), and polyclonal anti-ERK 1 (C-16) (1:1,000) from Santa Cruz Biotechnology (Santa Cruz, CA). Membranes underwent three washes in TBS, TTBS, and TBS followed by incubation with the secondary antibody (goat anti-rabbit IGg; Pierce, Brockville, Ontario, Canada) (1:2,000) in blocking buffer for 1 h at room temperature. Immunoreactivity was detected using enhanced chemoluminescence (ECL Plus Detection System; Amersham Biosciences, Piscataway, NJ). Equal protein loading was checked by probing for the nonphosphorylated forms of ERK 1 and TrkA or tubulin.
Data from immunoblots and activation assays (see following text) were scanned and their density assessed quantitatively using "Un-scan-it gel " software (Silk Scientific, Orem, UT). Changes in density were assessed relative to loading controls for three to four gels for each experiment. For presentation and comparison of data across multiple experiments, relative densities were normalized to those for the control situation in each series. Care was taken to avoid quantification of data from immunoblots that appeared to have saturated.
Ras activation assay
The Ras activation assay was performed according to the method of Herrmann et al. (1995). After treatments, cells were rinsed with ice-cold phosphate-buffered saline (PBS; with protease inhibitor cocktail, NaF, and Na2VO4), then immediately harvested and lysed in 300 µL of BOS buffer (50 nM Tris-HCl, pH 7.4, 200 mM NaCl, 1% Nonidet P-40, 10% glycerol, 10 mM NaF, 2.5 mM MgCl2, 1 mM EDTA). Protein assay was performed on the lysates and protein concentration was normalized. Equal amounts of GST-RBD (gift of Dr. Jim Stone, Department of Biochemistry, University of Alberta) construct precoupled to glutathione-agarose beads in BOS buffer were added to each concentration-normalized lysate group. Samples were incubated for 2 h at 4°C with gentle shaking to pull down all activated Ras. Samples were then rinsed three times followed by addition of 2 x sample buffer and boiling for 5 min to cleave glutathione-agarose beads from GST-RBD constructs with active Ras attached. Equal volumes of GST-RBD with bound active Ras in 2 x sample buffer for each experimental group were loaded for gel electrophoresis.
Anti-Ras antibody from Upstate Cell Signaling Solutions/Millipore (Billerica, MA) was used to detect active Ras across experimental groups and anti-GST antibody (Abcam, Cambridge, MA) was used to detect loading amounts.
Rap-1 activation assay
Procedures were followed according to EZ-Detect RAP1 activation kit (Pierce, Brockville, Ontario, Canada). Briefly, after treatments, cells were rinsed with ice-cold PBS (with protease inhibitor cocktail, NaF, and Na2VO4) then immediately harvested and lysed in 300 µL of lysis buffer. Protein assay was performed on the lysates and protein concentration was normalized. Equal amounts of GST-RalGDS-RBD construct were added to several SwellGel (immobilized glutathione disc) and each normalized lysate experimental groups was immediately added to each separate SwellGel-GST-RalGDS-RBD construct. Samples were incubated for 1 h at 4°C with gentle shaking to pull down activated Rap-1. Samples were then rinsed three times followed by addition of 2 x sample buffer and boiling for 5 min to cleave glutathione beads from GST-RalGDS-RBD construct with active Rap-1 attached. Equal volumes of GST-RalGDS-RBD with bound active Rap-1 in 2 x sample buffer for each experimental group were loaded for gel electrophoresis. Anti-Rap-1 antibody was used to detect active Rap-1 across experimental groups and anti-GST antibody was used to detect loading amounts.
Drugs and chemicals
The following drugs and reagents were from Biomol (Plymouth Meeting, PA): wortmannin, PP1, LY2940002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], PD-98059 (2'-amino-3'-methoxyflavone), H-89 (N-[2-(p-bromocinnamylamina)ethyl]-5-isoquinolinesulfonamide-2HCl), chelerythrine (chelerythrine chloride), U-73122 [1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione], U-73343 [1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-pyrrole-2,5-dione], Sp- and Rp-cAMPS (respectively, Sp- and Rp-adenosine-3',5'-cyclic monophosphorothioate triethylamine salt), PMA (phorbol 12-myristate-13-acetate), and 4-phorbol. Chicken II luteinizing hormone releasing hormone (LHRH) and nerve growth factor (NGF) were from Alomone Labs (Jerusalem, Israel). All other reagents were from Sigma–Aldrich (Oakville, Ontario, Canada) or Fisher Scientific (Edmonton, Alberta, Canada).
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RESULTS |
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Figure 1A illustrates a family of IBa currents recorded in a BFSG B-neuron after 6 days in defined medium culture. Figure 1B was recorded from another cell and illustrates the increase in IBa amplitude after 6-day culture with 0.45 µM LHRH. The bottom trace illustrates voltage recordings associated with this current trace. Figure 1C illustrates that the maximum current density, recorded at –10 mV from a holding potential of –90 mV, in 6-day cultured neurons was 113 ± 7 pA/pF (n = 49). This increased by 55% to 175 ± 16 pA/pF (n = 35; P < 0.02) in cells cultured for 6 days in the presence of 0.45 µM LHRH. We have shown previously that this LHRH-induced increase in IBa does not reflect a change in the voltage dependence of activation or inactivation. It does, however, reflect a selective increase in ICa,N in a transcription-dependent manner (Ford et al. 2003a). Exposure to LHRH may therefore induce synthesis or affect the trafficking of N-type Ca2 + channels.
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The involvement of mitogen-activated protein kinase/extracellular-regulated kinase kinase (MEK) in LHRH-mediated changes in ICa was examined using the inhibitor PD-98095 (10 µM) (Alessi et al. 1995). This substance, which prevents the NGF-induced increase in IBa in BFSG neurons (Lei et al. 1998), also blocked the LHRH-induced increase in current density (P < 0.05) (Fig. 1, D and E; Table 1). This finding is consistent with the hypothesis that the effect of LHRH on ICa involves the activation of MEK, and thus presumably the activation of MAPK.
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). It is therefore possible that LHRH may induce a transient activation of ERK that may signal the surface expression of new Ca2+ channels via a transcription-dependent process (Ford et al. 2003a). We therefore further characterized the time course of ERK phosphorylation and found that it was maximal after 10 min of LHRH treatment and returned to basal activation levels after 6 h of exposure to the peptide (Fig. 2C, lanes 2–5; summarized Fig. 2D). On the other hand, NGF induced sustained ERK activation throughout the duration of the experiment (Fig. 2F, lanes 2–4; data summarized in Fig. 2G). Elevated ERK phosphorylation was not the result of loading a greater amount of protein since equivalent amounts of total ERK and total TrkA were demonstrated by blotting with the corresponding antibodies. No phosphorylation of ERK was detected when LHRH was applied to the neurons in the continued presence of PD-98059 (data not shown).
Ligand binding to G-protein–coupled receptors (GPCRs) can lead to transactivation of Trk and other growth factor receptors (Rajagopal et al. 2004). Since phospho ERK1/2 is a well-defined downstream effector of TrkA (Kaplan and Stephens 1994; Sofroniew et al. 2001), it is possible that the observed effect of LHRH was mediated via transactivation of TrkA. This seemed unlikely because LHRH did not induce TrkA phosphorylation (Fig. 2C, lanes 3–5; summarized in Fig. 2E). By contrast, NGF (200 ng/ml) caused a clear and sustained activation of TrkA (Fig. 2F, lanes 2–4; summarized in Fig. 2H). Moreover, treatment with NGF, but not with LHRH, led to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is also downstream of TrkA (Fig. 2I, lane 3 vs. lane 2; summarized in Fig. 2J).
In all the immunoblot experiments performed, antibodies against phospho-ERK as well as antibodies against total ERK detected a single band with molecular weight corresponding to ERK 2 in BFSG, although the characteristic doublet was observed in rat neuronal samples processed in parallel (not shown). Since both antibodies (anti total ERK and anti phospho ERK) were unable to detect the band corresponding to ERK 1, it is possible that frogs do not express ERK 1. However, it is also possible that limited cross-reactivity of mammalian-origin antibodies with frog proteins plays a role or that amphibian ERK 1 has the same molecular weight as that of amphibian ERK 2.
Ras or Rap-1?
At least two different low molecular weight G-proteins, ras or rap-1, can participate in activation of MAPK by extracellular ligands (Sofroniew et al. 2001). Indeed, rap-1 has been implicated in regulation of Ca2+ channels by neurotropins in PC12 cells (Black et al. 2003). We examined ras and rap-1 activation using pulldown assays to evaluate their possible role in the action of LHRH. Figure 3, A (lanes 4 and 5) and B illustrates activation of ras by both LHRH and NGF. Neither substance activated rap-1 (Fig. 3, C, lanes 4 and 5 and D). Lane 1 in Fig. 3, A and C represents positive controls illustrating Ras and Rap-1 activation by GTP--S (see also Fig. 3, B and D).
How do LHRH receptors couple to MAPK?
In pituitary cell lines, GnRH (LHRH) receptor signaling to MAPK involves phospholipase C (PLC) and protein kinase C (PKC) (Naor et al. 2000; Reiss et al. 1997; Sim et al. 1995). Figure 4A (lane 2) illustrates activation of PKCβII by LHRH. Activation was demonstrated by using an antibody directed against the phosphorylated form of the enzyme. This yielded two bands at about 75 and 100 kDa, both of which were made more intense by LHRH. By contrast, LHRH failed to activate PKC. Quantification of the data for four replicate experiments is shown in Fig. 4B.
In electrophysiological experiments, we used the PKC inhibitor chelerythrin (Herbert et al. 1990) to examine the role of PKC in LHRH-induced potentiation of IBa. At 1 µM, chelerythrine prevented LHRH-mediated increases in current density because IBa recorded in the presence of chelerythrine plus LHRH was not significantly different from cells cultured for 6 days with chelerythrine alone (P > 0.05) (Table 1). These are the results that would be expected if LHRH signaling involves PKC.
The PLC inhibitor U-73122, together with its inactive structural analog U-73343, provides a specific way to investigate the role of PLC in signal transduction. Moreover, U-73122 has already been shown to inhibit agonist-induced PLC activation in BFSG neurons (Stemkowski et al. 2002). Inclusion of 20 µM U-73122 in the cultures prevented the increase in IBa produced by 0.45 µM LHRH (P > 0.05, Table 1). However, a substantial increase in Ca2+ current density still occurred when cells were maintained for 6 days in the presence of 0.45 µM LHRH and the inactive analogue, U-73343 (20 µM) compared with U-73343 alone (Table 1, P < 0.05). These findings implicate PLC in the action of LHRH.
In some pituitary cell lines, the actions of GnRH are mediated via PKA (Han and Conn 1999). Furthermore, signaling pathways involving PKA and/or cAMP are known to affect the ras-MAPK cascade (Impey et al. 1998; Naor et al. 2000). These findings prompted us to test the effect of the PKA inhibitor H-89 (Chijiwa et al. 1990). Cells treated for 6 days with H-89 plus LHRH did not show the increase in current density that was seen with LHRH alone (P < 0.05; Table 1). This finding raised the possibility that PKA is also involved in the LHRH response.
Gi-coupled GPCRs can activate MAPK via their β subunits and the transactivation of growth factor receptors. This effect proceeds via the nonreceptor tyrosine kinase, c-src, and the downstream activation of the phosphatidylinositol 3 kinase- (PI3K-) (Koch et al. 1991; Lopez-Ilasaca et al. 1997). The role of src kinase in LHRH-mediated Ca2+ channel regulation was investigated using PP1, an inhibitor of the src kinases p56lck and p59fyn (Hanke et al. 1996). PP1 (1.5 µM) failed to prevent LHRH from increasing IBa density because there was a significant increase (P < 0.05) in current density with PP1 plus LHRH compared with the current density seen in PP1 alone (Table 1). This result is consistent with the observation that LHRH failed to promote TrkA phosphorylation (Fig. 2, C, E, and I).
The role of PI3K in LHRH-mediated Ca2+ channel regulation was examined with the PI3K inhibitor, wortmannin (Yano et al. 1993). Like PP1, 100 nM wortmannin failed to prevent the increase in IBa density by LHRH (Table 1; P < 0.05). This excludes PI3K and c-src from the LHRH-mediated increases in Ca2+ channel expression.
These effects of inhibitors suggest that LHRH signals, through both PKC and PKA, to cause activation of MAPK that, in turn, leads to an increase in IBa density. To test this possibility further, we directly stimulated PKC or PKA in an attempt to mimic the action of LHRH. BFSG B-cells treated intermittently with the phorbol ester PMA (80 nM), for 1 h/day over a 6-day period to activate PKC (Ryves et al. 1991), exhibited IBa densities (71 ± 6 pA/pF; n = 17) that were significantly greater than those of neurons treated with 4-phorbol, the negative control for PMA (55 ± 4 pA/pF; n = 17; P < 0.05) (Fig. 1, F and G). This indicates that direct activation of PKC is sufficient to cause an increase in the level of Ca2+ channels expressed in BFSG.
A concern with the use of phorbol esters is that prolonged exposure may downregulate rather than activate PKC (Matthies et al. 1987). We hoped that the intermittent application protocol we developed for the electrophysiological experiments would promote sustained kinase activation for the whole 6-day experimental period. Figure 4, A (lanes 3–6) and B illustrates the effect of treatment for various time periods with PMA (80 nM). An exposure of 1 h promotes obvious phosphorylation of both the 75- and 100-kDa isoforms of PKCβII (Fig. 4, A, lanes 3 and 4 and B, top). By contrast, a continuous 24-h exposure or application according to the "intermittent" schedule used in our electrophysiological experiments (1 h/day for 6 days) failed to convincingly demonstrate activation of PKCβII (Fig. 4A, lanes 5 and 6). Our intermittent application protocol therefore did not achieve the sustained PKC activation we had anticipated. This may be of little consequence because the effect of LHRH on ERK phosphorylation is transient (Fig. 2, C and D). Thus the first 1-h exposure of neurons to PMA in the electrophysiological experiments and the transient activation of PKCβII likely initiated the genomic changes responsible for altered functional expression of ICa. Subsequent applications of PMA likely did not activate PKC but this is irrelevant if the first stimulus initiated the signal for altered channel expression.
PKA is "necessary but not sufficient" for LHRH activation of MAPK
Unlike PKC, the direct activation of PKA with the cAMP analogue Sp-cAMPS was not sufficient to increase IBa density. Current densities recorded from cells treated with Sp-cAMPS (57 ± 4 pA/pF; n = 41) were no different from the current density recorded from cells treated with the enantiomer Rp-cAMPS, a competitive inhibitor of the activation of PKA by cAMP (64 ± 3 pA/pF; n = 42; P > 0.05). This, taken with the results from the inhibition of PKA and PKC during stimulation with LHRH, suggests that both pathways are necessary in the regulation of Ca2+ channels by LHRH, yet only PKC on its own is sufficient to produce this effect.
To show that the lack of effect of extracellularly applied Sp-cAMPS on IBa was not due to its failure to penetrate neurons, we examined its ability to activate ERK by immunoblotting effects of Sp-cAMPS compared with Rp-cAMPS, PMA, and 4 phorbol (Fig. 4, C and D). The effect of Sp-cAMPS was weaker than that of PMA and Rp-cAMPS was ineffective. This confirms the effectiveness of extracellularly applied Sp-cAMPS and the fact that it is less effective than PMA may relate to the suggestion that PKA is necessary but not sufficient to alter the functional expression of Ca2+ channels.
The requirement for PKA activation for the action of LHRH is illustrated further by the experiments shown in Fig. 4, E and F. This shows that the competitive cAMP antagonist Rp-cAMPS attenuates LHRH activation of ERK.
Mechanism of NGF-induced increase in INa
We have shown that long-term exposure of BFSG B-neurons to 200 ng/ml nerve growth factor (NGF) results in a doubling of Na+ current density. This effect is transcription dependent and does not reflect changes in activation or inactivation kinetics (Lei et al. 2001).
Although the PI3K inhibitor wortmannin failed to prevent LHRH or NGF-induced increases in IBa (see Table 1 and Lei et al. 1998), it was highly effective in inhibiting NGF-induced increases in total INa. Relevant sample recordings are shown in Fig. 5, A, B, D, and E. Exposure to NGF for 10 days increased peak INa density at –5 mV from 299 ± 36 (n = 28) to 447 ± 53 pA/pF (n = 35; P < 0.03; Fig. 5C). By contrast, NGF failed to affect INa density in the presence of 1 µM wortmannin (peak INa density in wortmannin 344 ± 39; n = 32, in wortmannin + NGF 344 ± 49 pA/pF; n = 25; P > 0.85; Fig. 5F). The effect of NGF was also blocked by the more selective PI3K inhibitor LY294002 (10 µM). Peak INa in LY294002 was 329 ± 35 (n = 27) compared with 327 ± 34 pA/pF (n = 26; P < 0.95; Fig. 5G) in the presence of LY294002 + NGF. These results are consistent with the involvement of the PI3K pathway in the action of NGF on Na+ channel expression.
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Peak, total INa density was unaffected by LHRH. For 6-day cultures, maximum INa in the presence of 0.45 µM LHRH was 239 ± 35 pA/pF (n = 17) compared with 230 ± 16 pA/pF for controls (n = 20, P > 0.8). Similarly, for 9-day cultures, current density in LHRH was 276 ± 42 (n = 19) compared with 260 ± 35 pA/pF for controls (n = 19; P > 0.75). For 6-day cultures, there was no shift in the voltage dependence of gNa activation with LHRH treatment from –80 to +50 mV (n = 17 for LHRH and 20 for control). Also, for 6-day cultures, there was no shift in the h
curves with LHRH treatment (g/gmax) from –105 to +30 mV and stepping to +10 mV (n = 17 for LHRH and n = 20 for control; data not shown). Last, there was no change in the rate of onset of inactivation. For example, at 0 mV, 
for gNa inactivation after 6 days in LHRH was 1.8 ± 0.1 ms (n = 17) compared with 1.9 ± 0.1 ms in controls (n = 20, P > 0.2).
Since NGF produces effects through both the MAPK and PI3K pathways (Kaplan and Stephens 1994; Sofroniew et al. 2001), the present data suggest that the PI3K pathway is involved in regulation of INa and previous data suggest the MAPK pathway is involved in NGF regulation of ICa (Lei et al. 1998). Since LHRH regulates Ca2+ and not Na+ channels, it may be able to signal only through the MAPK pathway and not via the PI3K pathway. We tested this possibility by examining the effect of LHRH on phosphorylation of Akt, one of the downstream effectors of PI3K (Sofroniew et al. 2001). Figure 2, I (lane 2) and J shows that 10-min exposure to 100 nM LHRH failed to increase Akt phosphorylation, whereas a robust increase was seen in the presence of 200 ng/ml NGF (Fig. 2, I, lane 3 and J). The effect of NGF was attenuated by 6-h prior exposure to the PI3K inhibitor wortmannin (1 µM, data not shown).
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DISCUSSION |
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) it is suggested that downstream signaling from LHRH has selective access to MAPK and not to PI3K. These findings are summarized in Fig. 6.
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The suggestion that the effect of LHRH is independent of src and the PI3K pathway (Koch et al. 1991; Lopez-Ilasaca et al. 1997) but may instead involve PLC and the ras-MAPK pathway is supported by the lack of effect of the src kinase inhibitor PP1 and the PI3K inhibitor wortmannin as well as the positive results with U-73122 and PD-98059 (Table 1). Corroborative results emerge from the immunoblot analyses; LHRH promotes phosphorylation of ERK1/2 and not Akt, whereas NGF phosphorylates and activates both enzymes (Fig. 2). Another important result from the immunoblot experiments is that LHRH fails to activate TrkA. This provides additional evidence against a role for transactivation of growth factor receptors in the action of LHRH. There is good evidence, however, for the operation of this mechanism for other G-protein–coupled receptors in other systems (Rajagopal et al. 2004). Finally, the ability of LHRH and NGF to activate ras rather than rap-1 suggests that the former is involved in gonadotropin actions to increase Ca2+ current (Figs. 3, A and C and 6). Although surrogate growth factor receptors activate rap-1 in PC12 cells (Black et al. 2003) this does not appear to happen with native TrkA receptors in a sympathetic neuron.
Although U-73122 and PD-98059 prevent NGF-induced increases in IBa in BFSG neurons (Lei et al. 1998), the pharmacological approach always raises concerns about inhibitor concentration and specificity (Smith 1995). This issue may be especially relevant to studies on amphibian tissues using a series of agents that have been defined in terms of affinity and specificity in mammalian systems. Although "molecular" approaches such as RNA interference (Holen and Mobbs 2004) or introduction of dominant negative or constitutively active forms of ras (Fitzgerald 2000) might be considered, the lack of information on the Rana catesbeiana genome and the fact that suitable reagents are not readily available makes this approach difficult or infeasible. Since this study is a continuation of an extensive series of physiological studies (Ford et al. 2003a; Jassar et al. 1993; Lei et al. 1997, 1998, 2001; Petrov et al. 2001) on Rana catesbeina, it is not appropriate to change to a mammalian (Fitzgerald 2000; Fitzgerald and Dolphin 1997) or cell line system even though they are clearly more amenable to molecular biological approaches (Black et al. 2003).
Amphibian sympathetic ganglia contain both vasomotor C-neurons and B-neurons that project to mucous glands in the skin (Smith 1994). The present immunoblot experiments were done on cultures containing both neuron types, whereas the electophysiological experiments were confined to B cells. It is likely that NGF and LHRH modulate INa and ICa in a similar fashion in C-cells because these express LHRH receptors (Jones 1987) and, since they are vasomotor sympathetic neurons, they likely also express TrkA.
Role of G-proteins and protein kinases in the effect of LHRH
The present experiments were based on the premise that the neurotrophic effects of LHRH are mediated by a heterotrimeric G-protein. It is possible, however, that some other signaling process was involved. For example, internalization of peptide-bound receptors may trigger transduction processes (Miller and Lefkowitz 2001) or a G-protein–independent receptor, such as tyrosine kinase, may have been directly activated. These possibilities seem unlikely because all documented effects of LHRH in neurons, endocrine glands, and cancer cells seem to progress via receptors that exert their effects via heterotrimeric G-proteins (Cheng and Leung 2000; Grundker et al. 2001; Kakar et al. 2002; McArdle et al. 2002; Naor et al. 1998, 2000). Since it is generally accepted that LHRH signals via a GPCR to activate the ras-MAPK system in gonadotrophs (Han and Conn 1999; Reiss et al. 1997; Sim et al. 1995; Sundaresan et al. 1996), it is likely that a similar pathway exists in neurons.
LHRH (GnRH) receptors activate different heterotrimeric G-proteins/signal transduction pathways in different cell types (Conn et al. 1979; Kuphal et al. 1994). There are at least two mammalian GnRH receptor subtypes and coupling to Gq/11, Gs, and Gi has been reported (Hawes et al. 1993; Janovick and Conn 1994; Stanislaus et al. 1997; Ulloa-Aguirre et al. 1998). In bullfrogs, there are three distinct GnRH receptor subtypes, all of which appear capable of coupling to Gq (Wang et al. 2001). Within the sympathetic ganglia, LHRH activates PLC to increase inositol trisphosphate turnover and intracellular Ca2+ concentration (Pfaffinger et al. 1988) and to suppress M-type K+ current (Ford et al. 2003b, 2004), suggesting that the LHRH/GnRH receptor(s) in this tissue is/are also coupled to Gq/11. It is yet to be determined which of the three identified bullfrog GnRH receptors is/are involved in both this process and the neurotrophic effects described in the present work.
In some pituitary cell lines, the effect of GnRH on ras-MAPK is mediated via PKC (Reiss et al. 1997; Sundaresan et al. 1996). Direct stimulation of PKC with a phorbol ester is also known to activate MAPK (Reiss et al. 1997) and our electrophysiological results with U-73122 and chelerythrine suggest that the LHRH receptor signals via PLC and PKC in BFSG B-neurons (Table 1; Fig. 6). Furthermore, direct simulation of PKC by phorbol ester is sufficient to cause an increase in IBa (Fig. 1, F and G), suggesting that PKC is both "necessary and sufficient" to regulate BFSG Ca2+ channels. The use of isoform-specific antibodies implicate PKCβII and not PKC in the action of LHRH, although these findings do not exclude the participation of additional PKC isoforms. Because activation of PKC is known to affect inactivation of Na+ conductance (gNa) in a variety of neuronal systems (Franceschetti et al. 2000), one might predict that LHRH may have a similar effect in BFSG neurons, although this was not observed. It has been shown, however, that the membrane-bound 
PKC is involved in altering gNa inactivation (Chen et al. 2005) and it is possible that this particular isoform, like PKC, is not activated by the downstream effectors of LHRH actions.
Pathways also exist for cAMP to signal to ERK1/2 in neurons and PC12 cells. One is via cAMP/PKA/B-raf and the other via cAMP/Epac/rap1/B-raf (de Rooij et al. 1998; Grewal et al. 2000; Kawasaki et al. 1998; Vossler et al. 1997). The pituitary adenylate cyclase-activating polypeptide (PACAP) receptor Pac1 stimulates both a cAMP pathway and the PLC pathway to activate rap-1 and ras. These effectors act in a complex synergistic manner to activate ERK1/2. Cyclic AMP/PKA and ras are both permissive for rap-1 activation of ERK1/2. They are thus "necessary but not sufficient" for the action of Pac1 (Bouschet et al. 2003). If a similar mechanism exists for the LHRH receptor in BFSG, this would explain the finding that H-89 blocked the effects of LHRH, although the cAMP analogue Sp-cAMPS failed to mimic the effects of LHRH, even though it was able to promote a weak activation of ERK1/2 (Fig. 4, C and D). A permissive role for ras is supported by the fact that wild-type ras (p21ras) is also "necessary but not sufficient" to mediate neurotrophin induction of Ca2+ channels in PC12 cells (Pollock and Rane 1996).
There are some differences between the actions of NGF and LHRH on Ca2+ channels. Although LHRH exclusively increases ICa,N with no effect on activation or inactivation kinetics (Ford et al. 2003a), the effects of NGF are more complex and involve an increase in ICa,L, decreased inactivation of total ICa, and an increase in ICa,N (Lei et al. 1997). Mechanisms additional to ras-MAPK may be involved in NGF effects on ICa,L and inactivation processes. The differences also may relate to the lasting activation produced by NGF (Fig. 2, A, B, F, and H) compared with the transient ERK1/2 activation promoted by LHRH (Fig. 2, C and D). This transient activation is unlikely to reflect LHRH receptor internalization because an acute modulatory effect of LHRH on ICa (Elmslie et al. 1990) was preserved even after 6 days in the continued presence of the peptide (for details see Ford et al. 2003a).
Role of PI3K in NGF-induced increase in INa
Experiments with LY294002 and wortmannin support the involvement of the PI3K pathway in NGF-induced increases in Na+ current. Since downstream signaling from PI3K often proceeds through Akt (Sofroniew et al. 2001), the ability of NGF to phosphorylate this enzyme (Fig. 2, I and J) adds further support to the involvement of this pathway. A ras-independent increase in Na+ density in PC12 cells was previously reported (Fanger et al. 1993, 1997; Hilborn et al. 1998). However, in the latter report (Fanger et al. 1997) it was suggested that the ras-independent pathway may involve members of the src nonreceptor tyrosine kinase family rather than PI3K. One possible reason for this difference was that our experiments were done with native TrkA on intact adult neurons, whereas the experiments of Fanger et al. (1997) were done on PC12 cells expressing surrogate platelet-derived growth factor (PDGF) beta receptors with mutations that eliminate activation of specific signaling molecules. Alternatively, it could be argued that our data, which depend on the supposed selectivity of inhibitors, are less reliable than those obtained using molecular and cell biological techniques.
About 15% of the total INa in BFSG B-neurons is TTX resistant (Jassar et al. 1993) and is presumably a different gene product from the TTX-sensitive current (Goldin et al. 2000). We have shown previously that TTX-resistant INa is also upregulated by NGF in BFSG neurons (Lei et al. 2001). It would therefore be instructive to examine the associated transduction mechanism: Does the PI3K regulate both genes in parallel? Are the TTX-resistant and TTX-sensitive currents regulated by different transduction processes? Does the PI3K-mediated increase of total INa reflect a posttranslational modification or an action on trafficking of all Na+ channel types?
Lack of effect of LHRH on Na+ currents
The lack of effect of LHRH on INa is readily explained by the observation that the PI3K pathway involved in NGF-induced increases in INa is not activated by LHRH (Fig. 2, I and J). Thus the PI3K pathway appears to control functional expression of Na+ channels, whereas control of Ca2+ currents is exerted via the MAPK pathway. NGF, which has access to both pathways, increases both currents, whereas LHRH, which can access only the MAPK pathway via ras, regulates only Ca2+ currents.
Whereas functional upregulation of Ca2+ and Na+ channel function by NGF in BFSG persists as long as target-derived neurotrophin is available, the neurotrophic effect of LHRH is more labile because it depends on peptide release and thus neuronal activity in preganglionic nerve fibers. This may relate to the transient effect of LHRH compared with the sustained effect of NGF on ERK1/2 phosphorylation. Since neuropeptide release is favored by intense neuronal activity (Peng and Horn 1991), regulation of Ca2+ channels by LHRH may couple preganglionic activity to alterations in the electrical properties of postganglionic cells. The release of neuropeptides from preganglionic fibers also causes long-term increases in tyrosine hydroxylase activity and noradrenaline synthesis (McKeon and Zigmond 1993). Due to the dependence of neurotransmitter release on Ca2+ influx, increased Ca2+ channel availability at sympathetic postganglionic terminals may augment sympathetic outflow to target tissues. By contrast, control of Na+ channels is independent of ganglionic transmission and instead depends on the availability of target-derived NGF (Lei et al. 2001). This differential regulation of channel types is brought about by the ability of NGF to signal via MAPK and PI3K compared with the selective activation of ras-MAPK by LHRH.
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ACKNOWLEDGMENTS |
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Present address of C. P. Ford: Vollum Institute, Oregon Health Sciences University, Portland, OR 97239.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Smith, Department of Pharmacology, University of Alberta, 9.75 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7 (E-mail peter.a.smith{at}ualberta.ca)
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