 |
INTRODUCTION |
The differentiation, survival, and long-term maintenance of sympathetic neurons and small, nociceptive, sensory neurons depends on an adequate supply of nerve growth factor (NGF) (Davies 1996
). The neurotrophic activity of NGF is exerted via the high-affinity (TrkA) receptor (Kaplan et al. 1991
; Klein et al. 1991
) and/or the low affinity (p75) receptor (Chao et al. 1986
; Radeke et al. 1987
). Ligand-mediated dimerization of TrkA is followed by autophosphorylation of specific tyrosine residues (Jing et al. 1992
; Kaplan et al. 1991
; Schlessinger and Ullrich 1992
) that then bind to Src homology 2 (SH2) domains on cytoplasmic adaptor proteins. This leads to activation of phosphatidylinositol-3-kinase (PI3K), phospholipase C-
, (PLC-
), Ras adaptor proteins (Shc, Grb2, and SOS) (Koch et al. 1991
; Schlessinger and Ullrich 1992
), or Suc-associated nucleotide neurotrophic factor-induced tyrosine phosphorylated target (SNT) (Kaplan and Stephens 1994
). These enzymes initiate transduction processes that operate independently or in concert to generate various aspects of the neurotrophic response.
Stimulation of PI3K and the production of D-3-phosphorylated phosphoinositides (Whitman et al. 1987
, 1988
) is involved in growth-factor-mediated mitogenesis (Jhun et al. 1994
; Karnitz et al. 1995
), prevention of apoptosis (Scheid et al. 1995
; Yao and Cooper 1995
), and promotion of neurite outgrowth in pheochromocytoma (PC12) cells (Kimura et al. 1994
).
PLC-
catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol trisphosphate (InsP3). DAG activates protein kinase C and InsP3 promotes transient release of Ca2+ from the endoplasmic reticulum (Berridge and Irvine 1989
). Combined with Ras, the PLC-
pathway is proposed to be responsible for NGF-induced cell differentiation (Obermeier et al. 1994
) and neurite outgrowth (Stephens et al. 1994
).
Ras (p21Ras) is a low molecular weight monomeric G-protein that initiates a cascade of events that lead to the activation of mitogen-activated protein kinase (MAPK). Substrates for MAPK include nuclear transcription factors (Gille et al. 1992
; Pulverer et al. 1991
), phospholipase A2 (Nemenoff et al. 1993
), and kinases, such as ribosomal S6 kinase II (Sturgill et al. 1988
). Like the PI3K and PLC-
pathways, Ras, MAPK, and their downstream products are important in growth factor-mediated neuronal survival (Borasio et al. 1993
; Nobes and Tolkovsky 1995
; Nobes et al. 1996
), in the differentiation of PC12 cells (Hagag et al. 1986
; Kremer et al. 1991
) and in neurite outgrowth (Fukuda et al. 1995
).
NGF binding to the low-affinity p75 receptor activates certain protein kinases (Canossa et al. 1996
; Volonte et al. 1993) and generates intracellular signals, such as ceramide (Dobrowsky et al. 1994
) and adenosine 3',5'-cyclic monophosphate (Knipper et al. 1993
). Interaction of NGF with the p75 receptor is also thought to facilitate actions mediated via TrkA (Barker and Shooter 1994
; Benedetti et al. 1993
; Hantzopoulos et al. 1994
; Hempstead et al. 1991
; Verdi et al. 1994
).
The overall response of PC12 cells to NGF reflects the differentiation of an undifferentiated neuroendocrine cell into a "sympatheticlike" neuron. In the absence of NGF, PC12 cells continue to proliferate and continue to exhibit a chromaffin cell phenotype. NGF-induced differentiation therefore involves termination of cell division, initiation of neurite outgrowth, increased expression of Na+ and Ca2+ channel currents (Streit and Lux 1987
; Usowicz et al. 1990
), and the assumption of an electrically excitable, neuronal phenotype. NGF is also required for the maintenance of normal Ca2+ channel properties in adult sympathetic neurons (Lei et al. 1997a
). When B-cells of adult bullfrog sympathetic ganglia (BFSG) are plated at low density in defined medium, serum-free, neuron-enriched culture, Ca2+ channel activity (IBa current density) decreases by ~50% within 6-9 days. Inclusion of NGF in these cultures maintains IBa density at control levels for
16 days. Also, subcutaneous injection of NGF into the areas of the skin that receive sympathetic B-fiber innervation increases Ca2+ channel activity in the B-cell bodies. By contrast, injection of NGF antibodies decreases cell body Ca2+ current (Lei et al. 1997a
). Although p21Ras-mediated signaling and the induction of the immediate early genes c-fos and c-jun (Cavelié et al. 1995; Pollock and Rane 1995) were implicated in the NGF-induced increase in ICa that is associated with differentiation in PC12 cells, nothing is known about the mechanism(s) through which NGF continues to control Ca2+ channel function in adult sympathetic neurons. We therefore used Trk antibodies and p75 antibodies to examine the roles of the high- and low-affinity NGF receptors in Ca2+ channel regulation. Specific enzyme inhibitors were then used to identify which pathway(s) is/are necessary for the regulation of Ca2+ channel currents by NGF in adult BFSG neurons. Some of the results appeared in abstract form (Lei et al. 1996
, 1997b
,c
).
 |
METHODS |
Tissue culture
Neurons dissociated from adult bullfrog paravertebral sympathetic ganglia were maintained for
2 wk in serum-free, low-density, defined-medium culture as previously described by Lei et al. (1997a)
. The neuron-enriched cultures that were used for some of our previous experiments were used for all experiments in this study. The total yield of ganglion cells from each frog were therefore preplated into two or three 35-mm culture dishes. After 1-2 h, most of the nonneuronal cells adhered to the bottom of the dishes, and the nonadherent cells, which were primarily neurons, were harvested, redistributed to 30 culture dishes (35 mm), and cultured in fresh medium (3 ml/dish). The culture medium consisted of diluted L-15 medium (73%) supplemented with 10 mM glucose, 1 mM CaCl2, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 µM cytosine arabinoside.
Electrophysiology
Whole cell Ca2+ channel currents were recorded by means of discontinuous single electrode voltage-clamp (Lei et al. 1997
) with the use of Ba2+ as the charge carrier (IBa). For recording, tissue culture medium was replaced with external solution containing (in mM) 117.5 N-methyl-D-glucamine (NMG) chloride, 2.5 NMG-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2.0 BaCl2, 2.0, pH 7.2. The "internal" solution in the patch pippette consisted of (in mM) 76.5 NMG-Cl, 2.5 HEPES, 10 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, 5 Tris-ATP, 4 MgCl2, pH 7.2. Input capacitance (Cin) was used as an index of cell size. This was determined by integrating the capacitive transient current elicited by 10 mV depolarizing voltage command. Recordings were made from B-cells that were identified on the basis of their size (Cin > 40 pF) (Lei et al. 1997a
). IBa was evoked by a series of depolarizing voltage steps (10-mV increments) from a holding potential of
80 mV. Peak current generally occurred at 0 mV for cultured cells with or without NGF. All measurements are expressed in terms of current density (peak current at 0 mV divided by Cin). Measurements were made from
20 cells for each experimental condition. Culture dishes containing cells exposed to either dimethylsulfoxide (0.1% DMSO; vehicle) alone or DMSO plus NGF were prepared, and their currents were measured to provide an assurance of batch quality. The data from all these experiments were pooled and used as blank controls (n = 85) and positive controls (n = 83) for each experimental group.
Chemicals
NGF (2.5 s) and anti-p75 antibody were from Alomone Labs., Jerusalem, Israel. The anti-p75 was a mouse IgG1 monoclonal antibody against rat p75. Anti-Trk Ig-G antibody and negative control Ig-G antibody (raised against Aspergillus niger glucose oxidase) were from Zymed Laboratories (purchased from Inter-Medico, Markham, ON, Canada). Leibovitz's L-15 medium and penicillin-streptomycin antibiotics were from GIBCO BRL. The following chemicals were from Biomol (Plymouth Meeting, PA): genistein, daidzein, lavendustin A, wortmannin, LY29002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), U-73122 (1-(6((17
- 3-methoxyestra - 1 , 3 , 5 (10) - trien - 17 - yl)amino)hexyl)1H-pyrrole-2,5-dione), U-73343 (1-(6-((17
-3-methoxyestra1,3,5(10) - trien - 17 - yl)amino)hexyl) - 2,5 - pyrrolidine - dione),
hydroxyfarnesylphosphonic acid (
-HFA), perillic acid (PA), and actinomycin D. All other chemicals were from Sigma (St. Louis, MO). Because many of the inhibitors used to probe NGF signal transduction are highly hydrophobic, they were dissolved in DMSO to make stock solutions. The stock solutions were diluted in culture medium to make the desired final concentrations. The final DMSO concentration in the culture medium that was used in the blank (NGF-free) or positive control (NGF-treated) groups was 0.1%. This concentration of DMSO did not affect Ca2+ currents. Inhibitors were used at 5-10 times their published KD or IC50. Cells cultured with enzyme inhibitor plus NGF (200 ng/ml) were pretreated with a medium containing the same concentration of the inhibitor for 1 h before replacement with the medium containing both inhibitor and NGF. The culture medium was changed daily to maintain the effective concentrations of the inhibitors. Because wortmannin and PD98059 are susceptible to rapid hydrolysis in aqueous solution, medium containing either of these substances was exchanged every 5-8 h. Continuous inhibition of PI3K and mitogen-activated protein kinase kinase (MAPKK) activities can be achieved in this way (Kimura et al. 1994
; Virdee and Tolkovsky 1996
). Neurons were cultured at room temperature in a light-proof environment to avoid photolysis of potentially light-sensitive reagents. Electrophysiological analysis was carried out after 6 days in culture because the effect of NGF was significant at that time (Lei et al. 1997a
). Data are presented as means ± SE and were considered significant when P < 0.05 using one-way analysis of variance or Student's unpaired, two-tailed, t-test as appropriate.
 |
RESULTS |
Transcription-dependence of NGF response
An RNA-synthesis inhibitor (cordycepin, 20 µM) or a DNA-transcription inhibitor (actinomycin D, 0.01 µg/ml) was used to examine whether NGF-induced enhancement of IBa involved alteration in gene expression. Although IBa density was almost doubled after 6-day exposure to NGF (200 ng/ml), current densities recorded in the presence of NGF plus cordycepin or NGF plus actinomycin D were statistically indistinguishable from control (Table 1).
Role of tyrosine-kinase in NGF response
Tyrosine kinase-mediated phosphorylation is of potential importance in the signal transduction mechanism of NGF because of the inherent tyrosine kinase activity of TrkA and the possibility that downstream nonreceptor tyrosine kinases such as c-Src, c-Yes, and Fyn may be activated (Kremer et al. 1991
; Vaillancourt et al. 1995
). The tyrosine kinase inhibitors genistein and lavendustin A were used to test this possibility. IBa density recorded after 6 days in the presence of 20 µM genistein was no different (P > 0.05) from that seen in cells cultured with 20 µM daidzein, an inactive analog. However, at this concentration, genistein completely blocked the increase of the current density induced by 6-day culture with NGF (Table 1). Typical data records of IBa from cells studied in the presence of genistein or daidzein with and without NGF are shown in Fig. 1. Lavendustin A had similar effects. At 1 µM, it blocked NGF-induced increase in current density but had no effects (P > 0.05) on the control current density.

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| FIG. 1.
Blockade of nerve growth factor (NGF)-induced enhancement of IBa density by the tyrosine kinase inhibitor genistein but not by the inactive isomer daidzen. Currents evoked by a series of steps in 10-mV increments from a holding potential of 80 to +70 mV. Top record: typical recordings of IBa after 6 dAYS in the presence of 20 µM diadzen or genistein. Bottom record: IBa recorded after 6 days in the presence of 20 µM diadzen or genistein + NGF (200 ng/ml).
|
|
The actions of higher concentrations of inhibitors were more complex. Genistein (100 µM) reduced the current in control cells by 79% (Table 1) and unexpectedly failed to prevent the NGF-induced increase in current. IBa density increased from ~9 nA/pF in 100 µM genistein to ~ 22 nA/pF in the presence of 100 µM genistein plus NGF (P < 0.05, Table 1). Similarly, a high concentration of lavendustin A (10 µM) reduced the current density of control cells by ~58%. NGF slightly increased in current in the presence of 10 µM lavendutin A, but the amplitude of this effect failed to attain statistical significance (0.05 >P > 0.1; Table 1).
NGF response is related to the activation of Trk
Because intracellular, nonreceptor tyrosine kinases may be involved in the regulation of Ca2+ channels by NGF, the above results do not necessarily implicate TrkA. An anti-Trk antibody that was a rabbit Ig-G raised against mouse Trk extracellular epitopes was therefore used to see whether it mimicked the effect of NGF. This antibody mimics the action of NGF on neuronal survival (Clary et al. 1994
; Lefcort et al. 1996
). This may reflect cross-linking of two TrkA monomers by the double-armed-structure of Ig-G to promote receptor dimerization and activation. To test whether the antibody was effective in our system, explant cultures of BFSG were prepared according to the methods described by Traynor et al. (1992)
. Although addition of NGF to single dissociated BFSG neurons in defined medium fails to enhance neurite ougrowth (Lei et al. 1997a
), explants of BFSG (in serum-rich medium) exhibit a robust growth response (Kelly et al. 1989
). Figure 2A shows photomicrographs of control explants treated with 20 µg/ml control Ig-G antibody and explants treated with 20 µg/ml Trk antibody. The antibody enhanced the outgrowth of neurites and therefore mimicked the action of NGF. Anti-Trk Ig-G (20 µg/ml) also enhanced IBa density in single dissociated cells in defined medium by 72% (P < 0.01, t-test), which was comparable with the effect of NGF. Sample data records are shown in Fig. 2B, and the numerical data are summarized in Fig. 2C.

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| FIG. 2.
Anti-Trk Ig-G increased IBa density and promoted neurite outgrowth in explants. Currents evoked by a series of steps in 10-mV increments from a holding potential of 80 to + 70 mV. A, top: BFSG explant cultured in the presence of negative control Ig-G antibody (20 µg/ml) for 15 days. A, bottom: another bullfrog sympathetic ganglia (BFSG) explant cultured in the presence of anti-Trk Ig-G antibody (20 µg/ml) for 15 days. Note profuse outgrowth of neurites from the explant. B: examples of IBa recorded from a cell treated with negative control Ig-G antibody and a cell treated with anti-Trk Ig-G antibody. C: histogram to summarize effects on IBa density. **a P < 0.01 compared with the current density of control cells; **b P < 0.01 compared with the current density of the cells treated with negative control Ig-G antibody.
|
|
Anti-p75 antibody did not block the NGF response
The observation that activation of Trk leads to the induction of Ca2+ channels provides only collaborative evidence for its involvement in the action of NGF. Additional evidence for the involvement of Trk requires exclusion of the alternate possibility that NGF exerts its actions through the low-affinity p75 receptor. This possibility was tested with the use of a monoclonal antibody directed against the extracellular domain of the p75 neurotrophin receptor that blocks NGF binding (Cortazzo et al. 1996
; Huber and Chao 1995
). Neurons were pretreated for 30 min with medium containing 20 µg/ml anti-p75 antibody. The medium was then replaced with medium containing the same concentration of anti-p75 antibody plus NGF and cultured for 6-9 days. The effect of NGF on current density was unchanged (Table 1) in the presence of anti-p75 antibody, suggesting that the effect of NGF on Ca2+ channel current may be independent of the p75 receptor.
Inhibitors PI3K and PLC-
do not block NGF response
The role of PI3K in NGF-mediated Ca2+ channel regulation was investigated with the use of wortmannin and LY294002. Wortmannin, a highly potent PI3K inhibitor, acts by binding to the p110 subunit of the enzyme (Yano et al. 1993
). LY294002, on the other hand, behaves as a competitive inhibitor for the ATP binding site of PI3K and abolishes PI3K activity both in vivo and in vitro at low micromolar concentrations but has no inhibitory effect against phosphatidyl-inositol-4-kinase (PI4K) nor a number of intracellular serine/threonine or tyrosine kinases (Vlahos et al. 1994
). Both wortmannin (100 nM) and LY294002 (100 µM) were unable to block the effects of NGF on IBa density (Table 1), although these concentrations promote complete inhibition of PI3K activity in other systems (Cheatham et al. 1994
; Vlahos et al. 1994
), including PC12 cells (Kimura et al. 1994
).
Two PLC-
inhibitors, U-73122 and neomycin, were used to test whether activity of PLC-
was involved in the action of NGF. U-73122 together with its control inactive enantiomer, U-73343, is a useful tool to investigate the involvement of PLC in signal transduction (Bleasdale et al. 1990
; Thompson et al. 1991
; Yule and Williams 1992
). At a concentration of 100 µM (the highest concentration tested), U-73122 did not attenuate the effects of NGF (Table 1). Neomycin, a polycationic aminoglycoside antibiotic, binds strongly to phosphatidyl-inositol-4,5-bisphosphate (PIP2) (Lang et al. 1977
) and prevents its further metabolism (Downes and Michell 1981
; Schacht 1976
). PLC-mediated inositol phospholipid turnover is therefore selectively inhibited by neomycin (Carney et al. 1985
; Griffin et al. 1980
). Inclusion of 100 µM neomycin in the culture medium failed to affect the NGF-mediated enhancement of Ca2+ current density (Table 1). Thus activity of neither PI3K nor PLC-
seems necessary for the induction of Ca2+ channel currents by NGF.
Blockade of NGF response by inhibitors of posttranslational modification of Ras
Ras proteins are synthesized as cytosolic precursors and must undergo posttranslational modification at a region of their C-terminal before they become biologically functional. These modifications include farnesylation at the cysteine residue mediated by farnesyl-protein transferase (FPTase), followed by proteolytic cleavage and methyl esterification of the new C-terminal cysteine. These modifications are essential for anchoring Ras proteins to the plasma membrane (Hancock et al. 1990
) and for a number of their biological activities (Hancock et al. 1990
; Qui et al. 1991
; Schater et al. 1989
).
Farnesylation of Ras can be impaired with the use of specific FPTase inhibitors (Gibbs et al. 1993
; Kohl et al. 1993
) such as
-hydroxyfarnesylphosphonic acid (
-HFA), which acts by competition with the farnesyl diphosphate substrate both in vitro (Pompliano et al. 1992
) and in vivo (Gibbs et al. 1993
). Alternatively, PA can be used to interfere with the mevalonate pathway that leads to the production of the farnesyl diphosphate (Crowell et al. 1991
). PA inhibits Ras-induced Ca2+-activated K+ channel expression in balb 3T3 and NIH 3T3 cells (Huang and Rane 1994
).
Inclusion of
-HFA (10 or 100 µM) in the culture medium did not affect control IBa (P > 0.05) yet prevented the increase in current seen in the presence of NGF (Table 1). Similar concentration-dependent effects were seen with PA (0.1 or 1 mM, Table 1). These results are consistent with the involvement of Ras in the induction of Ca2+ currents by NGF.
Blockade of the effect of NGF on Ca2+ channels by a MAPKK inhibitor
MAPKK, a downstream target of Ras, was also examined to see whether it is involved in NGF-mediated Ca2+ channel induction. PD98059 was used effectively to block the in vivo activation of MAPK activity induced by platelet-derived growth factor (Dudley et al. 1995
) and NGF (Pang et al. 1995
). The high degree of specificity of PD98059 in vitro and in vivo is supported by its failure to inhibit a wide variety of serine/threonine kinases and tyrosine kinases (Alessi et al. 1995
; Dudley et al. 1995
). Moreover, it does not prevent the in vivo activation of Raf or the activation of other MAPKK and MAPK homologs, such as c-jun kinase or p38 (Alessi et al. 1995
). PD98059 inhibits MAPKK activity in a manner that is not competitive with either substrate (MAPK) or ATP binding and has no effect on MAPK itself, suggesting that it most likely inhibits MAPKK through an allosteric mechanism (Dudley et al. 1995
). Furthermore, NGF-, EGF- and PDGF-receptor tyrosine autophosphorylation is completely insensitive to PD98059 pretreatment of cells (Lin et al. 1994
).
To evaluate its role in NGF-mediated Ca2+ channel induction, the cells were treated with 10 or 100 µM PD98059 alone or plus NGF. The increase in the Ca2+ channel current density attributed to NGF was reduced in a concentration-dependent manner (Table 1), suggesting that MAPKK was implicated in the regulation of Ca2+ channel currents by NGF. Some typical data records illustrating attenuation of the effect of NGF by PD 98059 are shown in Fig. 3.

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| FIG. 3.
PD98059, a specific mitogen-activated protein kinase kinase (MAPKK) inhibitor, blocked the effects of NGF on IBa density. Currents evoked by a series of steps in 10-mV increments from a holding potential of 80 to + 70 mV. Top: examples of IBa recorded after 6 days in culture in the presence or absence of PD98059 (100 µM). Bottom: examples of IBa recorded after 6 days in culture in the presence or 200 ng/ml NGF with or without PD98059 (100 µM).
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|
Extracellular Ca2+ is not required for the NGF response
Influx of extracellular Ca2+ regulates gene expression in response to synaptic activation in primary cultures of cortical neurons (Murphy et al. 1991
) and it also mediates induction of gene expression in response to membrane depolarization of PC12 cells (Morgan and Curran 1986
). Furthermore, Ca2+ influx stimulates MEK and MAPK via activation of Ras (Rosen et al. 1994
; Rosen and Greenberg 1996
).
To investigate whether extracellular Ca2+ was necessary for NGF-induced Ca2+ channel expression, we replaced the Ca2+ from the culture medium with Mg2+ (1.8 mM) and used 2 mM ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid to chelate any additional source of Ca2+. This did did not affect NGF-mediated increase of IBa density (Table 1), suggesting Ca2+ was required for neither the activation of NGF receptors (Birren et al. 1992
) nor the signal transduction mechanisms underlying the induction of Ca2+ channels by NGF.
 |
DISCUSSION |
NGF increases both N- and L-type Ca2+ channel currents and attenuates the inactivation of gBa in adult BFSG B-cells (Lei et al. 1997a
). Because almost all the steady-state inactivation of gBa is removed when these cells are held at
80 mV (Lei et al. 1997a
), the experiments described here, which were done using a holding potential of
80 mV, address the signal transduction mechanism(s) underlying the induction of N- and L-type Ca2+ currents by NGF and not those underlying attenuation of inactivation.
Biological actions of NGF can be ascribed to binding to TrkA or p75 or to a cooperative interaction between the two (Barbacid 1993
). The cooperative relationship varies from cell line to cell line, and the degree of cooperativity between p75 and TrkA is related to the relative abundance of these two receptors on the cell (Greene and Kaplan 1995
; Verdi et al. 1994
). These results are consistent with the involvement of Trk because treatment of the neurons with anti-Trk Ig-G mimicked the NGF response, and 20 µM genistein and 1 µM lavendustin A attenuated the NGF-induced increase in IBa density. Because the antibody we used does not distinguish among TrkA, TrkB, and TrkC, we have no firm evidence regarding which of these three receptors is involved. By analogy with other systems however TrkA is the most likely candidate (Meakin and Shooter 1992
).
Although the p75 antibody did not attenuate the effect of NGF, a possible cooperative role for the low-affinity neurotrophin receptor cannot be excluded. This is because the antibody was directed against rat p75, and it may be ineffective in our amphibian system. Another anomaly in the data that may be related to p75 involvement is the observation that NGF increased IBa density in the presence of high concentrations of tyrosine kinase inhibitors (Table 1). Although this effect is difficult to explain, it may relate to the observation that signaling via TrkA can impede signaling via p75 (Frade et al. 1996
; Tolkovsky 1997
). Thus profound inhibition of Trk by 100 µM genistein or 10 µM lavendustin A may enable signaling via p75. It would therefore be interesting to know whether NGF-induced enhancement of IBa in the presence of these high inhibitor concentrations can be blocked by the p75 antibody.
The downstream targets for TrkA-induced regulation of Ca2+ channel current are likely to include MAPKK and MAPK. This conclusion is based on positive results with PD98059. As will be discussed below, these kinases probably serve as downstream targets for Ras. Because the effect of NGF was not abrogated by two specific yet different types of PI3K inhibitor, wortmannin and LY294002, the possibility that PI3K participated in the NGF-mediated Ca2+ channel regulation through the production of phosphoinositides was excluded. Although PLC-
was implicated in signaling modulation of a voltage-activated K+ channel by peptide growth factor receptors expressed in Xenopus oocytes (Timpe and Fantl 1994
) and in the acute activation of Ca2+-dependent K+ channels in rat cortical neurons by NGF and NT-3 (Holm et al. 1997
), the lack of effect of U73122 and neomycin argue against the involvement of PLC-
in NGF-induced IBa enhancement in adult BFSG cells. It was not possible to test for the involvement of the SNT pathway (Kaplan and Stephens 1994
) in NGF-mediated Ca2+ channel induction because specific inhibitors for this pathway are not yet available.
The results with cordycepin and actinomycin D show that gene transcription is required for NGF-mediated increase in Ca2+ channel currents and imply that the interaction of MAPK within the nucleus leads to an increase in the synthesis of Ca2+ channel proteins. It remains to be determined whether differential transduction mechanisms are associated with regulation of N-type Ca2+ channels compared with L-type channels as both are increased by NGF (Lei et al. 1997a
).
The concentration-dependent attenuation of the effect of NGF by PA and
-HFA, which act through different biochemical mechanisms, implicates a farnesylation-dependent element in the transduction process. Covalent attachment of lipids is a near-universal mechanism controlling the membrane localization of
-subunits of heterotrimeric G-proteins and monomeric G-proteins. This may involve palmitoylation, myristoylation, or prenylation. The latter process involves covalent attachment of a geranylgeranyl or farnesyl group to the protein. Farnesylation is a characteristic modification of members of the Ras superfamily (Casey 1994
). In fact, PA and
-HFA were developed as putative anticancer drugs (Gibbs et al. 1993
; Kohl et al. 1993
; Pompliano et al. 1992
) because they inhibit Ras, and their selectivity derives from their ability to impede farnesylation. Their effectiveness in attenuating the actions of NGF in our experiments is therefore consistent with the involvement of Ras in the transduction process. This proposition is reinforced by the large body of nonpharmacological literature that implicated Ras in NGF actions in other cell types, including PC12 cells (Borasio et al. 1993
; Hagag et al. 1986
; Kremer et al. 1991
; Nobes and Tolkovsky 1995
; Nobes et al. 1996
; Pollock and Rane 1996
; Qui et al. 1991
).
The suggestion that Ras-mediated signaling appears necessary for NGF-induced Ca2+ channel enhancement in adult sympathetic neurons parallels that seen in differentiating PC12 cells where constitutive expression of the dominant negative Ras blocked the growth factor-induced increase in Ca2+ channel currents (Pollock and Rane 1996
). However, sustained activation of the p21Ras signaling pathway in the PC12 cell system failed to increase Ca2+ channel current densities, suggesting Ras signaling is necessary but not sufficient on its own to mediate Ca2+ channel induction by growth factors. It would be interesting to know therefore whether sustained activation of Ras would be sufficient to increase IBa in adult BFSG neurons. Coactivation the Ras and SNT pathways was implicated in NGF-promoted neuritogenesis (Peng et al. 1995
). If a second pathway is required for Ras-mediated IBa enhancement in BFSG B-cells, the SNT pathway (Kaplan and Stephens 1994
) would be a prime candidate.
Although Ca2+-influx was implicated in Ras activation (Rosen et al. 1994
; Rosen and Greenberg 1996
), this does not seem to be necessary for the effects NGF on IBa in adult BFSG neurons as the effect persisted in Ca2+-free media. Depolarization-induced Ca2+ influx will also activate Ras (Rosen et al. 1994
; Rosen and Greenberg 1996
), but we were unable to investigate the effect of chronic depolarization on our cultured cells because they failed to survive in medium that contained a high concentration of extracellular K+ (unpublished observations).
The Ras pathway was also implicated in the growth factor-mediated induction of K+ channel currents in fibroblast cell lines (Huang and Rane 1994
), but it is not required for growth factor-induced Na+ channel expression in differentiating PC12 cells (Fanger et al. 1993
; Pollock and Rane 1996
). This suggests that the developmental expression of different channel types is mediated by different regulatory pathways in response to growth factor stimulation. We found (unpublished data) that treatment of cultures of adult BFSG with NGF increases Na+ channel current density, whereas K+ channel currents are largely unaffected. It remains to be determined whether differential cellular mechanisms for ion channel maintenance are preserved into adulthood.