1Laboratory of Developmental Neurobiology and 2Section on Growth Factors, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; 3William T. Gossett Neurology Labs, Henry Ford Health Sciences Center, Detroit 48202; and 4John D. Dingell Veterans Administration Medical Center, Detroit, Michigan 48201
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ABSTRACT |
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Jia, Min,
Minxu Li,
Xu-Wen Liu,
Hao Jiang,
Phillip G. Nelson, and
Gordon Guroff.
Voltage-Sensitive Calcium Currents Are Acutely Increased by Nerve
Growth Factor in PC12 Cells.
J. Neurophysiol. 82: 2847-2852, 1999.
Whole cell calcium currents were
recorded from PC12 cells with the perforated patch technique. Currents
were evoked by step depolarization from a holding potential of 90 mV.
Nerve growth factor (NGF) increased calcium currents through L-type
calcium channels by >75% within 3-5 min. This increase was inhibited
by K-252a, by nifedipine, and by inhibition or down-regulation of kinase C. Brain-derived neurotrophic factor (BDNF) also increased calcium current, but to a smaller extent. Thus increases in calcium current can be linked to activation of either the high- or the low-affinity nerve growth factor receptor. Increases in presynaptic calcium uptake appear to be a crucial element in the short-term actions
of the neurotrophins on neurotransmitter release leading to long-term
potentiation. Also, the control of calcium uptake is likely to be an
important factor in the long-term actions of the neurotrophins on
neuronal survival and neuronal protection. The present data indicate
that the PC12 cell may be a useful model for studying the effect of the
neurotrophins on calcium uptake.
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INTRODUCTION |
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The neurotrophin family includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins 3, 4/5, 6, and 7. Two classes of neurotrophin receptors have been identified, the p140trk family, which exhibits specificity in the binding of the neurotrophins, and p75NGFR, which binds all the neurotrophins relatively equally.
The long-term effects of the neurotrophins on neuronal survival and
neuronal protection have been well studied in many types of neurons.
Neurotrophins act to support neuronal survival during development and
allow neurons to withstand damage on environmental insult. Both
neuronal survival (Collins et al. 1991) and neuronal protection (Cheng and Mattson 1991
) appear to require
appropriate intracellular calcium levels. Increasing evidence indicates
that neurotrophins are also involved, short-term, in various forms of
synaptic plasticity (Lohof et al. 1993
; Stoop and
Poo 1996
), phenomena that seem to underlie such fundamental
properties of the nervous system as learning and memory
(Berninger and Poo 1996
). Here, also, alterations in
intracellular calcium levels appear to be pivotal. It has been shown,
then, that neurotrophins can alter intracellular levels of calcium, and
this action may be crucial to both their long-term and their short-term functions.
Experiments using radiolabeled
45Ca2+ have demonstrated
(Nikodijevic and Guroff 1991) that NGF produces an
increase in calcium uptake into PC12 cells. Such stimulation of calcium
uptake involves a phosphorylation reaction (Nikodijevic and
Guroff 1992
) and protein kinase C (Dickens et al.
1997
). Recent studies indicate that both p140trk and p75NGFR will
support increased calcium uptake into the cells (Jiang et al.
1997
).
The effects of the neurotrophins on synaptic efficacy may involve such
changes in calcium uptake, and an increase in intracellular calcium
levels has been shown to accompany the increase of transmitter output
produced by BDNF at the Xenopus neuromuscular junction (Stoop and Poo 1996). Indeed, several studies have shown
that both spontaneous and evoked transmitter release can be increased by increasing presynaptic calcium ion concentration (Zucker
1989
).
The ion channels, if any, responsible for neurotrophin-dependent calcium uptake are unknown. We have addressed the possibility that voltage-sensitive calcium channels (VSCC) might be involved. We find that calcium currents through L-type VSCC are rapidly increased on NGF treatment of PC12 cells and that this effect is initiated by NGF binding to either p140trk or p75NGFR receptors. This is a plausible mechanism for the rapid effects of the neurotrophins in increasing transmitter output at responsive synapses, which leads to long-term potentiation and also may be involved in alterations in intracellular calcium related to neuronal survival and neuronal protection under various conditions.
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METHODS |
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PC12 cells were plated on collagen-polylysine-coated 35-mm
dishes in DMEM containing 7.5% fetal bovine serum, 7.5% donor horse serum, 100 µg/ml streptomycin, and 100 units/ml penicillin (Life Technologies) at 37°C with 6% serum. Nystatin-perforated whole cell
patch-clamp techniques were applied (Horn and Marty
1988). Whole cell currents under voltage-clamp mode were
recorded and analyzed with an Axo-patch 1B amplifier and ITC-16
computer interface and Macintosh computer. Junction potential and
series coupling resistance were compensated for, and this compensation
did not change during the period of drug application. The Synapse
software from Instrutech Corporation and Synergistic Research Systems
was used for data acquisition and analysis. Patch pipettes were
fire-polished, and the resistances of the patch pipettes were between
2.5 and 5.0 M
in our recording solution. Cell membrane capacitances
were measured by integrating the capacitive transient at a 5 mV
hyperpolarizing voltage step. Voltage-gated calcium currents were
evoked by depolarization pulses from a holding potential of
90 mV.
The currents were filtered at 500 Hz. The recording bath solution
contained (in mM) 135 TEACl, 1 4-aminopyridine, 10 HEPES, 10 CaCl2, 2 MgCl2, and 0.001 tetrodotoxin (pH 7.3). The pipette contained (in mM) 125 CsCl, 20 TEACl, 10 HEPES, 5 EGTA, and 2 MgCl2 (pH 7.3).
Calcium (10 mM) was used to increase calcium currents.
For statistical purposes the maximal voltage-sensitive inward current was determined for each cell with and without the addition of NGF (or other agents as indicated). The mean and standard deviation of those maximal currents was used in determining the statistical significance of differences between the various experimental conditions. The voltage at which the inward current was maximal was around +20 mV.
For the determination of TrkB mRNA expression, total RNA was isolated
from PC12 cells using RNA STAT-60 (Tel Test, Friendswood, TX). The TrkB
Primer Pair (Hutton et al. 1992) was purchased from Promega (Madison, WI). RT-PCR reactions were carried out using the
Superscript One-step RT-PCR system (Life Technologies, Bethesda, MD)
for one cycle of 50°C for 30 min and 94°C for 2 min, 35 cycles of
94°C for 1 min, 56°C for 1 min, and 72°C for 45 s. Total RNA from rat glioma C6 cells and primers for specific isoforms of the
1D
subunit of the calcium channel (Liu et al. 1996
)
(forward: 5'-GGAGAGGAGGGCAAACGAAACACTAGC-3',
1892-1918; reverse: 5'-CGTACACACCGGAACACAGAGACGC-3' 2364-2388, Genebank No. M57682) were used as a positive control. The
RT-PCR products were resolved on 4% agarose gels.
The down-regulation of protein kinase C in PC12 cells was accomplished
by treating the cells with 1 µM phorbol 12-myristate 13-acetate
(PMA) for 18 h. The down-regulation was estimated by immunoblot analysis of equal amounts of cell lysate protein with anti-PKC antibody (Santa Cruz Biotechnology). Protein content was
evaluated using a protein detection kit (Pierce).
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RESULTS |
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To address the question of the types of calcium channels that
might be involved in synaptic plasticity and the increased calcium uptake produced by neurotrophin treatment, we have used whole cell
patch-clamping in this study. As shown in Fig.
1, NGF (200 ng/ml) elicited an increase
(77%) in voltage-sensitive calcium currents at a membrane potention of
around +20 mV. This stimulation was substantially inhibited by
preincubation of the cells with K-252a (200 nM; Fig. 1C).
K-252a is a kinase inhibitor that specifically prevents the actions of
NGF on PC12 cells (Koizumi et al. 1988) by inhibiting
the p140trk tyrosine kinase (Berg et al.
1992
). This indicates that the increased uptake is, at least in
part, supported by p140trk.
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The time course of this augmentation (Fig.
2), a maximal effect seen 3-5 min after
NGF addition, is comparable with that shown in the activation of
synaptic currents by BDNF in the Xenopus neuromuscular
junction preparation (Lohof et al. 1993; Stoop
and Poo 1996
). These data are also temporally consistent with
those obtained in 45Ca2+
experiments (Nikodijevic and Guroff 1991
). Also
consistent are the present experiments in which we find that K-252a, by
itself, stimulates voltage-sensitive inward calcium currents (Fig.
1C), as it does
45Ca2+ uptake
(Nikodijevic and Guroff 1992
; Nikodijevic et al.
1995
).
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BDNF (200 ng/ml) also induced a small, but significant (24%) increase
in voltage-sensitive inward calcium currents (Fig.
3). Because BDNF binds only to
p75NGFR on PC12 cells, this result is consistent
with that from previous studies (Jiang et al. 1997)
using 45Ca2+ that suggested
that NGF-induced increases in calcium uptake can be mediated by both
p140trk and p75NGFR. These
data are supported by RT-PCR analysis of the PC12 cells used in this
study, which indicates that TrkB, the specific receptor for BDNF, is
not present (Fig. 4), leaving
p75NGFR the only receptor to which BDNF
might bind.
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The characteristics of the current elicited by NGF,
high-voltage-activated and long-lasting, suggests the participation of L-type calcium channels. This suggestion is supported by the
observation that nifedipine, an inhibitor of L-type calcium channels,
inhibits the action of NGF on calcium currents (Fig.
5A). Also consistent with the
involvement of L-channels is the finding that, in current-voltage relationship curves, the amplitudes of the currents from a holding potential of 40 mV and from a holding potential of
90 mV were almost identical (Fig. 5B). Finally, these data are
consistent with observations on 3T3 cells transfected with NGF
receptors (Jiang et al. 1999
) showing inhibition
of NGF-dependent increases in calcium levels by nifedipine. It should
be noted, however, that the concentration of nifedipine needed for
complete inhibition of the effect of NGF (10 µM) is relatively high,
so the final identification of the NGF-stimulated channel as L-type
must await molecular characterization.
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Protein kinase C is activated by treatment of PC12 cells with NGF
(Hama et al. 1986; Kondratyev et al.
1990
), and the involvement of kinase C has been shown in
several of the actions of NGF on its target cells (Balbi and
Allen 1994
; Perrone-Bizzozero et al. 1993
).
Protein kinase C
, specifically, has been implicated in the
NGF-stimulated uptake of 45Ca2+ into PC12 cells
(Dickens et al. 1997
). Figure
6A shows that chronic treatment of PC12 cells with 1 µM PMA, which has been shown to down-regulate protein kinase C in PC12 cells, largely prevented the
NGF-induced increase in calcium current. Such treatment did not
markedly alter basal calcium uptake by the cells. Support for these
data were found in experiments with Go6976, a specific inhibitor of the
calcium-dependent isoforms of kinase C (Fig. 6A), which
largely inhibited the action of NGF on calcium current, and with PMA,
which, on an acute basis activates kinase C and which stimulated
calcium current (Fig. 6A). Previous reports from this
laboratory (Zheng et al. 1996
) indicate that protein
kinase C
is the only classical form of kinase C in these PC12 cells, and the present data show that it is down-regulated by 24-h treatment with PMA (Fig. 6B). These data suggest that
phosphorylation of an L-type calcium channel by protein kinase C
is
necessary for at least a large part of the NGF-induced increase in
calcium current.
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DISCUSSION |
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The data presented here indicate that NGF acutely increases
voltage-sensitive calcium currents in PC12 cells, that both
p140trk and p75NGFR can
support such increases, and that phosphorylation by protein kinase C
is at least part of the mechanism by which these increases occur.
Because calcium influx is a critical step in the synaptic transmission
process, the effects of NGF demonstrated here could serve as a basis
for the increased synaptic efficacy produced by neurotrophins in a
number of systems. The increase in peak voltage-sensitive calcium
currents produced by NGF averaged 77% and that produced by BDNF
averaged 24%. If such a change occurred in a presynaptic terminal, it
could have a major effect on transmitter output, because of the
proportional relationship between calcium current and transmitter
release (Llinas et al. 1976
). It needs to be noted that
neither Lei et al. (1997)
, using adult frog sympathetic ganglion cells, nor Li et al. (1998)
, with cultured
embryonic hippocampal neurons, observed the acute neurotrophin-induced
changes in calcium currents seen here.
The dual effects of K-252a, inhibiting the actions of NGF, but having
actions comparable with those of NGF when administered alone, reflects
its dual role as both antagonist and partial agonist of
p140trk. Because its actions on the uptake of
45Ca2+ are
transient (Nikodijevic and Guroff 1991), it may be that
its actions on calcium currents are also transient, allowing a return to baseline during the 30-min preincuation before the addition of NGF.
When added alone and evaluated directly, however, the stimulation by
K-252a is revealed. The ability of K-252a to inhibit the calcium
currents stimulated by NGF, but to stimulate calcium currents itself is
consistent with its ability to inhibit the neurotrophic actions of NGF
(Doherty and Walsh 1989
; Koizumi et al.
1988
; Matsuda and Fukada 1988
), but to exhibit
neurotrophic actions of its own (Borasio 1990
;
Cheng et al. 1994
; Glicksman et al.
1993
).
The question of the possible relationship between the increase in
voltage-sensitive calcium currents demonstrated here, and the increased
45Ca2+ influx shown in
previous studies should be addressed. The resting potential of the PC12
cells used in the present work was usually about 60 mV, and at that
potential there was no difference in calcium current under voltage
clamp between the control and the NGF-treated cells. There was a small
difference at clamping potentials more positive than
40 mV, and it is
possible that a portion of the NGF-sensitive channels would be
activated even in the resting state. Alternatively, it may be that at
least a part of the population of PC12 cells, perhaps those in a
specific phase of the cell cycle, has a resting potential more positive
than the average, and those cells account for the increases in
45Ca2+ influx that NGF
produces. Finally, it is possible that NGF treatment itself changes the
resting potential of the cells. It is equally possible, however, that
the present observation that NGF produces an increase in the activity
of voltage-sensitive Ca2+ channels and the
increase in intracellular calcium shown to result from NGF action in
earlier studies represent two separate effects of this trophic molecule.
Recent data obtained with cultures of dissociated hippocampal neurons
from fetal rats (Li et al. 1998) indicate that the
neurotrophin-induced increase in synaptic currents in that system
depends on mobilization of intracellular calcium and only indirectly on
increases in calcium uptake. A similar reliance on mobilization of
intracellular calcium stores was observed in studies of
neurotrophin-induced neurotrophin release (Canossa et al.
1997
). However, more recent data (Kruttgen et al.
1998
) has shown that activation of either
p140trk or p75NGFR will
support neurotrophin-induced neurotrophin release. Because experiments
with 3T3 cells transfected with NGF receptors show that
p75NGFR does not mediate mobilization of
intracellular calcium but does mediate uptake of calcium from the
extracellular compartment, uptake of extracellular calcium would seem a
likely candidate for the support of at least some
neurotrophin-dependent release processes.
Whether, in fact, these results from PC12 cells provide a useful model
for neuronal synapses remains to be determined, but several
observations suggest parallels between the two experimental situations.
The stimulation by NGF in PC12 cells requires the participation of a
Trk receptor, as does the stimulation by NT-4 in the Xenopus
neuromuscular junction (Wang and Poo 1997). The stimulation by NGF in PC12 cells takes between 3 and 5 min, as does the
stimulation by BDNF in the Xenopus preparation (Stoop and Poo 1996
) and the stimulation by NGF in molluscan neurons (Wildering et al. 1995
). Finally, the stimulation by NGF
in PC12 cells is linked to the release of neurotransmitter
(Nikodijevic et al. 1990
), as is the stimulation by BDNF
in the Xenopus system (Lohof et al. 1993
).
Thus the characteristics of the NGF-induced increase in calcium
currents in PC12 cells would seem to be a plausible model for synaptic
events leading to long-term potentiation and other forms of synaptic
plasticity. It seems equally reasonable, in the light of recent data on
the role of L-channnel activation in the survival of cerebellar granule
neurons in culture (Blair et al. 1999
), to regard
NGF-induced L-channel activation in PC12 cells as a model for studying
the changes in calcium levels underlying neuronal survival.
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FOOTNOTES |
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Address for reprint requests: P. G. Nelson, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bldg. 49, Rm. 5A38, 49 Convent Dr., MSC 4480, Bethesda, MD 20892-4480.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 March 1999; accepted in final form 17 August 1999.
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REFERENCES |
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