Neuropeptide Y inhibition of calcium channels in PC-12
pheochromocytoma cells
Laura A.
McCullough,
Terrance M.
Egan, and
Thomas C.
Westfall
Department of Pharmacological and Physiological Science, Saint
Louis University Health Sciences Center, St. Louis, Missouri 63104
 |
ABSTRACT |
We previously
demonstrated, using rat PC-12 pheochromocytoma cells differentiated to
a sympathetic neuronal phenotype with nerve growth factor (NGF), that
neuropeptide Y (NPY) inhibits catecholamine synthesis as well as
release. Inquiry into the mechanisms of these inhibitions implicated
distinct pathways involving reduction of
Ca2+ influx through
voltage-activated Ca2+ channels.
In the present investigation the effects of NPY on whole cell
Ba2+ currents were examined to
obtain direct evidence supporting the mechanisms suggested by those
studies. NPY was found to inhibit the voltage-activated
Ba2+ current in NGF-differentiated
PC-12 cells in a reversible fashion with an
EC50 of 13 nM. This inhibition was
pertussis toxin sensitive and resulted from NPY modulation of L- and
N-type Ca2+ channels. The
inhibition of L-type channels was not seen with <1 nM free
intracellular Ca2+ or when protein
kinase C (PKC) was inhibited by chelerythrine or PKC-(19
31).
Furthermore, the effect of NPY on L-type channels was mimicked by the
PKC activator phorbol 12-myristate 13-acetate. These studies
demonstrate that, in addition to inhibition of N-type Ca2+ channels, in
NGF-differentiated PC-12 cells NPY inhibits L-type Ca2+ channels via an intracellular
Ca2+- and PKC-dependent pathway.
nerve growth factor;
-conotoxin GVIA; nifedipine; pertussis
toxin
 |
INTRODUCTION |
NEUROPEPTIDE Y (NPY) is a 36-amino acid peptide
neurotransmitter that is colocalized and coreleased with catecholamines
in the central and peripheral nervous systems. Peripherally, NPY acts
to inhibit the release of catecholamines from sympathetic neurons (45),
cultured superior cervical ganglion (SCG) cells (36), and PC-12 cells
differentiated to a sympathetic neuronal phenotype with nerve growth
factor (NGF) (6, 8). The mechanism of this action has been widely
investigated, suggesting that the inhibition of catecholamine release
involves the Y2-receptor subtype and inhibition of N-type voltage-gated
Ca2+ channels (6, 36, 44). N-type
Ca2+ current inhibition by NPY has
been directly demonstrated in several systems, including SCG (11, 37),
dorsal root ganglion (3), neuroblastoma (32), and nodose ganglion (47)
neurons. This inhibition is pertussis toxin (PTX) sensitive (20, 32,
47) and is likely produced by the voltage-sensitive direct interaction of G protein subunits with the channel, a mechanism utilized by multiple inhibitory neurotransmitters (18).
We recently characterized a novel effect of NPY in NGF-differentiated
PC-12 cells in addition to its inhibition of catecholamine release, a
PTX-sensitive inhibition of catecholamine synthesis. In contrast to its
effects on release, the inhibition of synthesis is mediated by
inhibition of L-type Ca2+ channels
through Y3-receptor activation
(30, 31). Furthermore, the inhibition of synthesis is mimicked and
occluded by the protein kinase C (PKC) activator phorbol 12-myristate
13-acetate (PMA) and attenuated by the selective PKC antagonist
chelerythrine, suggesting that the reduction of L-type channel current
by NPY is through the action of PKC (31).
In the present study we use electrophysiological techniques to
definitively demonstrate the NPY-induced modulation of
Ca2+ channels in
NGF-differentiated PC-12 cells that was suggested by our previous
studies on catecholamine synthesis and release. Specifically, we show
that NPY produces PTX-sensitive inhibition of L- as well as N-type
Ca2+ channels in these cells and
provide evidence that the modulation of L-type channels involves an
intracellular Ca2+- and
PKC-dependent pathway. This study is the first to establish inhibition
of neuronal L-type channels by NPY and to investigate the mechanism of
this inhibition. In addition, in combination with previous studies, our
work suggests that inhibition of different Ca2+ channel subtypes by NPY
results in distinct physiological effects.
 |
MATERIALS AND METHODS |
Cell culture.
Stock cultures of rat PC-12 pheochromocytoma cells
(passages 19-30) were obtained
from Dr. Steven Sabol (National Institutes of Health, Bethesda, MD) and
grown in DMEM supplemented with 2 mM glutamine, 1 mM pyruvate, 5% FCS,
10% heat-inactivated horse serum, 100 U/ml penicillin, 100 µg/ml
streptomycin, and 0.25 µg/ml fungizone at 37°C in a humidified
atmosphere containing 5% CO2 in
air. Cells were passaged once per week with medium changes every
2-3 days. For electrophysiological studies, cells were plated onto
35-mm plates (Falcon Primaria) at a density of 2-2.5 × 105 cells/plate and differentiated
with 50 ng/ml NGF for 4-7 days.
Whole cell patch-clamp recordings.
Voltage-clamp recordings were obtained using the whole cell patch-clamp
technique (13). Borosilicate glass patch pipettes were coated with
Sylgard (Dow Corning) and fire polished. The pipettes were filled with
a solution containing 120 mM
N-methyl-D-glucamine aspartate, 1 mM MgCl2, 20 mM
tetraethylammonium chloride, 10 mM HEPES, 10 mM EGTA, 0-4.3 mM
CaCl2, 4 mM Mg-ATP, and 1 mM
Na-GTP (pH 7.4). Pipette resistances averaged 3-6
M
. Immediately before each experiment, PC-12 cells were
dissociated and suspended in buffer containing 144 mM NaCl, 5.4 mM KCl,
5 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4). An aliquot of the cell suspension was transferred to
a polycarbonate-and-glass recording chamber positioned on the stage of
an inverted microscope equipped with Hoffman-modulation optics.
Currents through voltage-activated
Ca2+ channels were measured using
Ba2+ as the charge carrier in an
external solution containing 144 mM NaCl, 10 mM CsCl, 10 mM
BaCl2, 1 mM
MgCl2, 10 mM HEPES, 10 mM glucose,
1 µM tetrodotoxin, and 300 µM anthracene-9-carboxylate (pH 7.4).
Solutions containing the drugs of interest were applied by manually
moving the cell, attached to the patch pipette, into the line of flow
of solution exiting one of an array of six inlet tubes. This setup
allows very rapid solution exchange and short duration (1-3 min)
of drug application. Whole cell currents were recorded with an AxoPatch
200A amplifier (Axon Instruments) and digitized at 5 kHz with 16-bit
accuracy with use of a MacADIOS II/16 board and Superscope II software
(GW Instruments). Capacitive transients were canceled, and membrane
currents were leak subtracted using a P/4 regimen. Voltage-activated
Ba2+ currents were evoked by
stepping the voltage every 20 s to 0 mV for 40 ms from a holding
potential of
80 mV. For analysis of tail currents, the voltage
was stepped to
40 mV for 40 ms after the step to 0 mV.
Current-voltage curves were obtained using command voltage ramps, which
changed the membrane voltage from
80 to +60 mV at a rate of 1.2 V/s.
Data analysis and statistics.
Raw data were analyzed off-line with a Macintosh computer and IGOR
software (Wavemetrics). Control and drug-modulated currents were
compared by determining the total charge entry (pC) during the 40-ms
depolarizing pulse through integration of the respective currents. Tail
current amplitudes were measured at a point 5 ms after a repolarizing
step from 0 to
40 mV, as described by Jones and Jacobs (23).
Free Ca2+ concentrations were
calculated using CHELATOR (41). Values are means ± SE. Statistical
significance was determined using Student's two-tailed
t-test or one-way ANOVA followed by
Student-Newman-Keuls multiple comparisons test as appropriate.
P
0.05 was considered significant.
Materials.
NPY was purchased from Peninsula Laboratories (Belmont, CA) and
American Peptide (Sunnyvale, CA). NGF was purchased from Collaborator Biomedical Products (Bedford, MA). Fetal bovine serum and horse serum
were purchased from JRH Biosciences (Lenexa, KS). DMEM and penicillin,
streptomycin, and fungizone were purchased from GIBCO BRL (Grand
Island, NY). Chelerythrine chloride was purchased from RBI (Natick,
MA). PKC-(19
31) was purchased from BIOMOL (Plymouth Meeting, PA).
Nifedipine,
-conotoxin GVIA (CgTX), PTX, PMA, and all other agents
were purchased from Sigma Chemical (St. Louis, MO).
 |
RESULTS |
Unless otherwise specified, all experiments were done using an
intracellular solution containing 10 mM EGTA and 4.3 mM
CaCl2 to give a calculated free
intracellular Ca2+ concentration
([Ca2+]i)
of 100 nM (41), which is close to the resting
[Ca2+]i
in these cells (6). Currents through voltage-activated
Ca2+ channels were measured using
an extracellular solution containing Ba2+ as the charge carrier.
NPY inhibits voltage-activated
Ba2+ current in
NGF-differentiated PC-12 cells.
Our hypothesis is that the NPY-induced inhibition of catecholamine
release and synthesis is associated with inhibition of Ca2+ influx through voltage-gated
channels. To test this hypothesis, we first determined the effect of
NPY on whole cell Ba2+ current.
Figure 1 shows an example of
the inhibition of voltage-activated Ba2+ current by NPY. This
inhibition was often accompanied by an increase in the time to peak of
the current (Fig. 1A), which most
likely reflected a change in channel activation kinetics, and was not associated with a shift in the current-voltage relationship (Fig. 1B). An identical pattern of kinetic
slowing often accompanies G protein-mediated inhibition of
Ca2+ current in neuronal tissues
(18). The concentration-response curve for NPY-induced (0.1-300
nM) inhibition exhibited an EC50 of 13 nM and an average maximal effect of 35% inhibition (Fig. 1C).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 1.
Neuropeptide Y (NPY) inhibits voltage-activated
Ba2+ current in nerve growth
factor-differentiated PC-12 cells. A:
inhibition by 100 nM NPY of current elicited by a 40-ms depolarizing
pulse to 0 mV from a holding potential of 80 mV.
B: inhibition by 100 nM NPY of current
elicited by a voltage ramp from 80 to +60 mV at a rate of 1.2 V/s. C: cumulative
concentration-response relationship for NPY inhibition of
Ba2+ current. Percent inhibition
was determined here and in subsequent figures by comparing control and
drug-modulated total charge entry during depolarizing pulse as
described in MATERIALS AND METHODS.
Values are means ± SE from 3-6 cells. Curve was fit using
dose-response fitting routine of Origin (Microcal Software). Calculated
EC50 for inhibition by NPY was 13 nM.
|
|
NPY inhibits L- and N-type
Ca2+ channels.
Inhibition of neurotransmitter release by NPY has previously been
associated with inhibition of N-type
Ca2+ channels in several neuronal
systems (7, 44, 47). In contrast, our studies on the mechanism of NPY
inhibition of catecholamine synthesis suggested that inhibition of
L-type Ca2+ channels was involved.
Therefore, the next series of studies was designed to demonstrate this
implied inhibition of L-type channels in addition to inhibition of
N-type Ca2+ channels.
The Ca2+ current in
NGF-differentiated PC-12 cells is carried primarily by N- and L-type
Ca2+ channels. The N-type channel
blocker CgTX (500 nM) irreversibly inhibited the total current by 42 ± 4% whereas the L-type channel blocker nifedipine (1 µM)
produced a reversible inhibition of 24 ± 4%. The combination of
CgTX and nifedipine blocked the total current by 57 ± 10%. NPY
(100 nM) was found to produce additional, although reduced,
Ba2+ current inhibition after
blockade of L-type channels by nifedipine or N-type channels by CgTX
(Fig. 2), suggesting that NPY causes inhibition of both Ca2+ channel
subtypes.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
NPY inhibits L- and N-type Ca2+
channels. A: sample traces
demonstrating that 100 nM NPY produces further inhibition of current
remaining after blockade of L-type
Ca2+ channels with 1 µM
nifedipine (Nif) or N-type Ca2+
channels with 500 nM -conotoxin GVIA (CgTX).
B: summary of results from 5-7
cells showing percent inhibition (mean ± SE) by nifedipine or CgTX
alone (control) and with NPY (+NPY). * Significantly different
from control, P < 0.05.
|
|
The actions of NPY in NGF-differentiated PC-12 cells, including
inhibition of catecholamine synthesis and release and inhibition of
cAMP accumulation, are PTX sensitive (6, 8, 30). In the present study
we tested the PTX sensitivity of the observed inhibition of N- and
L-type Ca2+ channels by incubating
cells in PTX (50 ng/ml) for 18 h. NPY no longer produced significant
Ba2+ current inhibition after PTX
pretreatment (Fig. 3), demonstrating the
involvement of Gi or
Go protein subtypes in N- and
L-type channel inhibition, consistent with our previous findings.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
NPY inhibition of Ca2+ channels is
pertussis toxin (PTX) sensitive. Cells were incubated with PTX (50 ng/ml) for 18 h. A: sample traces
obtained from a PTX-pretreated cell demonstrating that NPY (100 nM)
does not produce significant current inhibition.
B: summary of results showing percent
inhibition (mean ± SE) by NPY without ( PTX,
n = 24) and with PTX pretreatment
(+PTX, n = 5). * Significantly
different from PTX, P < 0.05.
|
|
Mechanism of L-type channel inhibition.
In many systems, neurotransmitter inhibition of L-type
Ca2+ channels is lost when free
intracellular Ca2+ is buffered to
very low levels with the Ca2+
chelators EGTA or
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (14, 22, 24, 28, 40). This finding suggests that
Ca2+ is required in the pathway
responsible for inhibition of these channels. Using pipette solutions
containing 10 mM EGTA with 4.3 mM
CaCl2 (~100 nM free
intracellular Ca2+) or 10 mM
EGTA without CaCl2 (<1 nM free
intracellular Ca2+), we tested
for the presence of such a mechanism in our observed NPY-induced
inhibition of L-type channels (41).
With ~100 nM free intracellular
Ca2+, nifedipine applied in the
presence of NPY had little effect, indicating that NPY had inhibited a
significant portion of the current carried by L-type
Ca2+ channels (Fig.
4, A and
B). In contrast, with <1 nM free
intracellular Ca2+, nifedipine
inhibited Ba2+ current to the same
degree in the absence or presence of NPY, indicating that NPY no longer
produced inhibition of L-type channels (Fig. 4,
C and
D). It is noteworthy, however, that
NPY still inhibited a portion of the total current at <1 nM free
intracellular Ca2+ (Fig.
4C), indicating that intracellular
Ca2+ is not required for the
inhibition of N-type channels.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 4.
Buffering free intracellular Ca2+
concentration
([Ca2+]i)
to <1 nM prevents NPY-induced inhibition of L-type
Ca2+ channels.
A and
C: peak current as a function of time
during application of 1 µM nifedipine alone or in presence of 100 nM
NPY with ~100 nM (A) or <1 nM
(C) free intracellular
Ca2+.
Insets: sample traces obtained during each of
the test conditions. B and
D: summaries of results from 4-5
cells showing nifedipine-sensitive (L-type) component of total charge
entry (mean ± SE) in absence ( NPY) or presence (+NPY) of NPY
with ~100 nM (B) or <1 nM free
intracellular Ca2+
(D). * Significantly different
from NPY, P < 0.05.
|
|
To more directly demonstrate NPY-induced modulation of L-type
Ca2+ channels, we tested its
effect on the slow tail current induced by the L-type channel agonist
BAY K 8644. This drug prolonged tail current deactivation in every cell
tested, increasing by more than fourfold the amount of current measured
5 ms after a repolarizing step from 0 to
40 mV (Fig.
5A).
This enhanced portion of the tail current selectively represents
current through L-type Ca2+
channels (23). With ~100 nM free intracellular
Ca2+, NPY reversibly inhibited the
BAY K 8644-enhanced slow tail current (Fig.
5B) by an average of ~50% (Fig.
5C). However, with <1 nM free
intracellular Ca2+, NPY had no
significant effect on BAY K 8644-enhanced tail currents (Fig.
5C). These results demonstrate that
the inhibition of L-type Ca2+
channels by NPY is mediated by an intracellular
Ca2+-dependent mechanism.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 5.
NPY produces Ca2+-dependent
inhibition of BAY K 8644-enhanced tail currents.
A: BAY K 8644 (1 µM) slows
deactivation of tail currents elicited by a repolarizing step from 0 to
40 mV. B: NPY (300 nM) reduces
BAY K 8644-induced slow tail current (~100 nM free intracellular
Ca2+). Series resistance
compensation was 60-80%. , Time at which tail currents were
measured. C: summary of results
obtained in presence of 1 µM BAY K 8644 from 3 cells with ~100 nM
(+Ca2+) and <1 nM free
intracellular Ca2+
( Ca2+) showing tail
current inhibition by 300 nM NPY (mean ± SE) as measured 5 ms after
repolarizing step. * Significantly different from
+Ca2+,
P < 0.05.
|
|
The Ca2+-dependent second
messenger responsible for the NPY-induced inhibition of L-type channels
suggested by our previous studies on catecholamine synthesis is PKC,
since this effect of NPY was mimicked and occluded by the PKC activator
PMA and prevented by the selective PKC inhibitor chelerythrine (31).
Indeed, several groups have reported that PKC activation by phorbol
esters inhibits Ca2+ influx into
PC-12 cells (5, 9, 15, 27, 33). We used two selective inhibitors of
PKC, chelerythrine (17) and the pseudosubstrate peptide PKC-(19
31)
(21) to test for the involvement of this enzyme. Preincubation (>15
min) of cells with chelerythrine or intracellular dialysis (>10 min)
with PKC-(19
31) prevented the subsequent NPY-induced inhibition of
L-type channels, as demonstrated by equality of the
nifedipine-sensitive (L-type) current in the absence and presence of
NPY (Fig. 6). In addition, application of
the PKC agonist PMA produced inhibition of the total
Ba2+ current (Fig.
7A),
which was not seen after treatment with chelerythrine or PKC-(19
31)
(Fig. 7, B and
C). Furthermore, PMA did not cause additional current inhibition in the presence of nifedipine (Fig. 7D), suggesting that the inhibition
is limited to L-type channels in agreement with the results of others
(5).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 6.
Protein kinase C (PKC) inhibitors chelerythrine and PKC-(19 31)
prevent inhibition of L-type Ca2+
channels by NPY. A:
nifedipine-sensitive (L-type) subtraction currents in absence
( NPY) and presence (+NPY) of 100 nM NPY in control and PKC
inhibitor-treated cells. Traces were obtained by subtraction of
currents recorded before and after nifedipine application. Scale bars,
50 pA, 10 ms. B: summary of results
showing percentage of control nifedipine-sensitive (L-type) component
of total charge entry remaining in presence of NPY (mean ± SE)
without PKC inhibitors (n = 5), after
preincubation (>15 min) with 10 µM chelerythrine
(n = 4), or after intracellular
dialysis (>10 min) with 50 µM PKC-(19 31)
(n = 5). * Significantly
different from NPY alone, P < 0.05.
|
|

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 7.
Activation of PKC by phorbol 12-myristate 13-acetate (PMA) produces
inhibition of L-type Ca2+
channels. A, B, and
C: plots of peak current as a function
of time during application and washout of 1 µM PMA in control,
chelerythrine-pretreated, and PKC-(19 31)-dialyzed cells,
respectively. Insets: sample traces
obtained in absence and presence of PMA. Scale bars, 100 pA, 20 ms.
D: summary of results from 4-5
cells showing percent inhibition by PMA (means ± SE) in absence and
presence of 1 µM nifedipine. * Significantly different from PMA
alone, P < 0.05.
|
|
 |
DISCUSSION |
The results of this study show that NPY produces PTX-sensitive
inhibition of L- and N-type voltage-activated
Ca2+ channels in
NGF-differentiated PC-12 cells. Although NPY-mediated inhibition of
N-type channels is a well-accepted concept (20, 32, 47), the present
study is the first to definitively demonstrate NPY-mediated inhibition
of neuronal L-type channels and to investigate the mechanism of this
effect. Furthermore, these results provide strong support for our
previous hypotheses on the pathways of NPY-induced modulation of
catecholamine neurotransmission: modulation of release through
inhibition of N-type channels and modulation of synthesis through
inhibition of L-type channels (31).
The inhibition of L-type channels by NPY was lost after the removal of
CaCl2 from the intracellular
recording solution. This, in combination with the presence of 10 mM
EGTA, reduces the calculated free
[Ca2+]i
to <1 nM. Although many enzymes are
Ca2+ dependent, the results of our
previous studies, in which the NPY-induced inhibition of catecholamine
synthesis was mimicked and occluded by PMA and prevented by
chelerythrine, suggested that PKC was the most likely mediator of
L-type channel inhibition (31). In the present study we used the
selective PKC inhibitors chelerythrine and PKC-(19
31), both of which
prevented the inhibitory effect of NPY on L-type
Ca2+ channels. In addition, the
PKC activator PMA mimicked the NPY-induced inhibition of L-type
channels. The results of these experiments, in conjunction with our
previous studies (31), provide strong evidence that NPY inhibits L-type
Ca2+ channels in
NGF-differentiated PC-12 cells through the action of PKC. L-type
channel and catecholamine synthesis inhibition by NPY are PTX
sensitive, consistent with the observation that the stimulation of
phospholipase C by several other neurotransmitters is transduced
through 
-subunits of
Gi/Go
(PTX-sensitive) rather than Gq
(PTX-insensitive) proteins (10).
Previous investigations into the role of PKC in
Ca2+ channel modulation have
produced conflicting results. Although PKC activation has been reported
to inhibit Ca2+ channels in
adrenal chromaffin cells (38, 42) and several other cell types (4, 14,
16, 25, 39), PKC activation enhances
Ca2+ channel currents in other
systems (43, 48, 49). However, the evidence supporting
Ca2+ channel inhibition by PKC in
our model system, PC-12 cells, is consistent and compelling. Indeed,
several independent investigators, using
45Ca2+
influx and
[Ca2+]i-imaging
techniques, demonstrated that PKC activation inhibits Ca2+ channels in PC-12 cells (9,
15, 27, 33). In addition, an electrophysiological study has been
performed that confirms that PKC activation by PMA inhibits L-type, but
not N-type, Ca2+ channels in
NGF-differentiated PC-12 cells (5). Therefore, our hypothesis of
PKC-mediated inhibition of L-type channels in PC-12 cells is well
supported by our previous and present experiments as well as by the
work of other investigators. The explanation for the complex actions of
PKC on Ca2+ channels is unclear,
but it may involve different subtypes or forms of the channels or,
alternatively, the actions of additional mediators, kinases,
phosphatases, or G proteins that are present at various levels in
different cell types.
In our present as well as previous studies, we have used
NGF-differentiated PC-12 cells as a sympathetic neuronal model. In rat
SCG neurons, NPY also inhibits
Ca2+ channels (11, 37). As opposed
to its effects in NGF-differentiated PC-12 cells, in SCG neurons NPY
did not produce further inhibition after CgTX application (11) and did
not inhibit tail currents prolonged by the L-type channel agonist
(+)-(S)-202-791 (37), suggesting
that its effects were limited to inhibition of N-type channels.
However, the recording conditions used in the SCG studies (high
intracellular BAPTA or EGTA, no added
Ca2+) may not be optimal for the
observation of Ca2+-dependent
L-type channel inhibition by NPY.
Catecholamine synthesis and release are two separate but related
processes that influence the level of catecholaminergic
neurotransmission in the central and peripheral nervous systems. Our
studies have revealed that NPY, which is coreleased with
catecholamines, can act to regulate both of these processes. In
addition, NPY appears to utilize different mechanisms to modulate the
two processes, with inhibition of N-type
Ca2+ channels coupled to
inhibition of catecholamine release and inhibition of L-type
Ca2+ channels coupled to
inhibition of catecholamine synthesis. Because different NPY receptor
subtypes are responsible for inhibition of catecholamine release
(Y2) and synthesis
(Y3), it is likely that
different NPY receptor subtypes are responsible for the inhibition of
the different Ca2+ channel
subtypes. We recently performed experiments which suggest that this is
indeed the case (29).
The importance of N-type Ca2+
channels in the stimulation of neuronal transmitter release has been
well demonstrated (19, 34), and numerous neurotransmitters in addition
to NPY have been found to inhibit transmitter release through
inhibition of this channel subtype (18). In contrast, in adrenal
chromaffin cells, L-type Ca2+
channels play a dominant role in secretion (1, 26). Although the
function of L-type channels in neurons is less well characterized, depolarization-induced stimulation of transmitter synthesis (31) and
gene transcription (2, 12, 35) are processes that depend on L-type
channel activity. Therefore, the demonstration that NPY and other
transmitters can modulate L- as well as N-type
Ca2+ channels suggests that they
can induce long- and short-term alterations in neuronal function.
In summary, this investigation is the first to establish NPY-induced
inhibition of neuronal L-type voltage-gated
Ca2+ channels in addition to
inhibition of N-type channels. The finding that the reduction of L-type
channel current is PTX sensitive and mediated through an intracellular
Ca2+- and PKC-dependent pathway is
in agreement with our investigations into the mechanism of
catecholamine synthesis inhibition by NPY. This study, in combination
with our previous work, provides strong evidence supporting the
hypothesis that NPY acts through distinct pathways to modulate
catecholamine synthesis and release and thereby regulate sympathetic
neuronal function.
 |
ACKNOWLEDGEMENTS |
We thank Drs. V. A. Chiappinelli and K.-W. P. Yoon for critical
reading of the manuscript.
 |
FOOTNOTES |
This study was supported by National Institutes of Health Grants
HL-26319, HL-60260, and 5-T32-GM-08306 (to T. C. Westfall) and HL-56236
(to T. M. Egan).
Address for reprint requests: L. A. McCullough, Dept. of
Pharmacological and Physiological Science, Saint Louis University
Health Sciences Center, 1402 South Grand Blvd., St. Louis, MO 63104.
Received 9 October 1997; accepted in final form 22 January 1998.
 |
REFERENCES |
1.
Artalejo, C. R.,
M. E. Adams,
and
A. P. Fox.
Three types of Ca2+ channel trigger secretion with different efficacies in chromaffin cells.
Nature
367:
72-76,
1994[Medline].
2.
Bading, H.,
D. D. Ginty,
and
M. E. Greenberg.
Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways.
Science
260:
181-186,
1993[Medline].
3.
Bleakman, D.,
W. F. Colmers,
A. Fournier,
and
R. J. Miller.
Neuropeptide Y inhibits Ca2+ influx into cultured dorsal root ganglion neurones of the rat via a Y2 receptor.
Br. J. Phamacol.
103:
1781-1789,
1991[Abstract].
4.
Boland, L. M.,
A. C. Allen,
and
R. Dingledine.
Inhibition by bradykinin of voltage-activated barium current in a rat dorsal root ganglion cell line: role of protein kinase C.
J. Neurosci.
11:
1140-1149,
1991[Abstract].
5.
Bouron, A.,
and
B. F. X. Reber.
Differential modulation of pharmacologically distinct components of Ca2+ currents by protein kinase C activators and phosphatase inhibitors in nerve-growth-factor-differentiated rat pheochromocytoma (PC12) cells.
Pflügers Arch.
427:
510-516,
1994[Medline].
6.
Chen, X.,
and
T. C. Westfall.
Modulation of intracellular calcium transients and dopamine release by neuropeptide Y in PC-12 cells.
Am. J. Physiol.
266 (Cell Physiol. 35):
C784-C793,
1994[Abstract/Free Full Text].
7.
Colmers, W. F.,
and
D. Bleakman.
Effects of neuropeptide Y on the electrical properties of neurons.
Trends Neurosci.
17:
373-379,
1994[Medline].
8.
DiMaggio, D. A.,
J. M. Farah,
and
T. C. Westfall.
Effects of differentiation on neuropeptide-Y receptors and responses in rat pheochromocytoma cells.
Endocrinology
134:
719-727,
1994[Abstract].
9.
Di Virgilio, F.,
T. Pozzan,
C. B. Wollheim,
L. M. Vincentini,
and
J. Meldolesi.
Tumor promoter phorbol myristate acetate inhibits Ca2+ influx through voltage-gated Ca2+ channels in two secretory cell lines, PC12 and RINm5F.
J. Biol. Chem.
261:
32-35,
1986[Abstract/Free Full Text].
10.
Exton, J. H.
Regulation of phosphoinositide phospholipases by hormones, neurotransmitters, and other agonists linked to G proteins.
Annu. Rev. Pharmacol. Toxicol.
36:
481-509,
1996[Medline].
11.
Foucart, S.,
D. Bleakman,
V. P. Bindokas,
and
R. J. Miller.
Neuropeptide Y and pancreatic polypeptide reduce calcium currents in acutely dissociated neurons from adult rat superior cervical ganglia.
J. Pharmacol. Exp. Ther.
265:
903-909,
1993[Abstract].
12.
Greenberg, M. E.,
E. B. Ziff,
and
L. A. Greene.
Stimulation of neuronal acetylcholine receptors induces rapid gene transcription.
Science
234:
80-83,
1986[Medline].
13.
Hamill, O. P.,
A. Marty,
E. Neher,
B. Sakmann,
and
F. J. Sigworth.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch.
391:
85-100,
1981[Medline].
14.
Hammond, C.,
D. Paupardin-Tritsch,
A. C. Nairn,
P. Greengard,
and
H. M. Gerschenfeld.
Cholecystokinin induces a decrease in Ca2+ current in snail neurons that appears to be mediated by protein kinase C.
Nature
325:
809-811,
1987[Medline].
15.
Harris, K. M.,
S. Kongsamut,
and
R. J. Miller.
Protein kinase C mediated regulation of calcium channels in PC-12 pheochromocytoma cells.
Biochem. Biophys. Res. Commun.
134:
1298-1305,
1986[Medline].
16.
Haymes, A. A.,
Y. W. Kwan,
J. P. Arena,
R. S. Kass,
and
P. M. Hinkle.
Activation of protein kinase C reduces L-type calcium channel activity of GH3 pituitary cells.
Am. J. Physiol.
262 (Cell Physiol. 31):
C1211-C1219,
1992[Abstract/Free Full Text].
17.
Herbert, J. M.,
J. M. Augereau,
J. Gleye,
and
J. P. Maffrand.
Chelerythrine is a potent and specific inhibitor of protein kinase C.
Biochem. Biophys. Res. Commun.
172:
993-999,
1990[Medline].
18.
Hille, B.
Modulation of ion-channel function by G-protein-coupled receptors.
Trends Neurosci.
17:
531-536,
1994[Medline].
19.
Hirning, L. D.,
A. P. Fox,
E. W. McCleskey,
B. M. Olivera,
S. A. Thayer,
R. J. Miller,
and
R. W. Tsien.
Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons.
Science
239:
57-61,
1988[Medline].
20.
Hirning, L. D.,
A. P. Fox,
and
R. J. Miller.
Inhibition of calcium currents in cultured myenteric neurons by neuropeptide Y: evidence for direct receptor/channel coupling.
Brain Res.
532:
120-130,
1990[Medline].
21.
House, C.,
and
B. E. Kemp.
Protein kinase C contains a pseudosubstrate prototope in its regulatory domain.
Science
238:
1726-1728,
1987[Medline].
22.
Howe, A. R.,
and
D. J. Surmeier.
Muscarinic receptors modulate N-, P-, and L-type Ca2+ currents in rat striatal neurons through parallel pathways.
J. Neurosci.
15:
458-469,
1995[Abstract].
23.
Jones, S. W.,
and
L. S. Jacobs.
Dihydropyridine actions on calcium currents of frog sympathetic neurons.
J. Neurosci.
10:
2261-2267,
1990[Abstract].
24.
Kramer, R. H.,
L. K. Kaczmarek,
and
E. S. Levitan.
Neuropeptide inhibition of voltage-gated calcium channels mediated by mobilization of intracellular calcium.
Neuron
6:
557-563,
1991[Medline].
25.
Linden, D. J.,
and
A. Routtenberg.
Cis-fatty acids, which activate protein kinase C, attenuate Na+ and Ca2+ currents in mouse neuroblastoma cells.
J. Physiol. (Lond.)
419:
95-119,
1989[Abstract].
26.
López, M. G.,
A. Albillos,
M. T. de la Fuente,
R. Borges,
L. Gandía,
E. Carbone,
A. G. García,
and
A. R. Artalejo.
Localized L-type calcium channels control exocytosis in cat chromaffin cells.
Pflügers Arch.
427:
427-354,
1994.
27.
MacNicol, M.,
and
H. Schulman.
Cross-talk between protein kinase C and multifunctional Ca2+/calmodulin-dependent protein kinase.
J. Biol. Chem.
267:
12197-12201,
1992[Abstract/Free Full Text].
28.
Mathie, A.,
L. Bernheim,
and
B. Hille.
Inhibition of N- and L-type calcium channels by muscarinic receptor activation in rat sympathetic neurons.
Neuron
8:
907-914,
1992[Medline].
29.
McCullough, L. A., T. M. Egan, and T. C. Westfall. Neuropeptide Y receptors involved in calcium
channel regulation in PC12 cells. Regul. Pept. In
press.
30.
McCullough, L. A.,
and
T. C. Westfall.
Neuropeptide Y inhibits depolarization-stimulated catecholamine synthesis in rat pheochromocytoma cells.
Eur. J. Pharmacol.
287:
271-277,
1995[Medline].
31.
McCullough, L. A.,
and
T. C. Westfall.
Mechanism of catecholamine synthesis inhibition by neuropeptide Y: role of Ca2+ channels and protein kinases.
J. Neurochem.
67:
1090-1099,
1996[Medline].
32.
McDonald, R. L.,
P. F. T. Vaughan,
A. G. Beck-Sickinger,
and
C. Peers.
Inhibition of Ca2+ channel currents in human neuroblastoma (SH-SY5Y) cells by neuropeptide Y and a novel cyclic neuropeptide Y analogue.
Neuropharmacology
34:
1507-1514,
1995[Medline].
33.
Messing, R. O.,
C. L. Carpenter,
and
D. A. Greenberg.
Inhibition of calcium flux and calcium channel antagonist binding in the PC12 neural cell line by phorbol esters and protein kinase C.
Biochem. Biophys. Res. Commun.
136:
1049-1056,
1986[Medline].
34.
Miller, R. J.
Receptor-mediated regulation of calcium channels and neurotransmitter release.
FASEB J.
4:
3291-3299,
1990[Abstract/Free Full Text].
35.
Morgan, J. I.,
and
T. Curran.
Role of ion flux in the control of c-fos expression.
Nature
322:
552-555,
1986[Medline].
36.
Oellerich, W. F.,
D. D. Schwartz,
and
K. U. Malik.
Neuropeptide Y inhibits adrenergic transmitter release in cultured rat superior cervical ganglion cells by restricting the availability of calcium through a pertussis toxin-sensitive mechanism.
Neuroscience
60:
495-502,
1994[Medline].
37.
Plummer, M. R.,
A. Rittenhouse,
M. Kanevsky,
and
P. Hess.
Neurotransmitter modulation of calcium channels in rat sympathetic neurons.
J. Neurosci.
11:
2339-2348,
1991[Abstract].
38.
Pruss, R. M.,
and
K. A. Stauderman.
Voltage-regulated calcium channels involved in the regulation of enkephalin synthesis are blocked by phorbol ester treatment.
J. Biol. Chem.
263:
13173-13178,
1988[Abstract/Free Full Text].
39.
Rane, S. G.,
and
K. Dunlap.
Kinase C activator 1,2-oleoylacetylglycerol attenuates voltage-dependent calcium current in sensory neurons.
Proc. Natl. Acad. Sci. USA
83:
184-188,
1986[Abstract].
40.
Sayer, R. J.,
P. C. Schwindt,
and
W. E. Crill.
Metabotropic glutamate receptor-mediated suppression of L-type calcium current in acutely isolated neocortical neurons.
J. Neurophysiol.
68:
833-842,
1992[Abstract/Free Full Text].
41.
Schoenmakers, T. J. M.,
G. J. Visser,
G. Flik,
and
A. P. R. Theuvenet.
CHELATOR: an improved method for computing metal ion concentrations in physiological solutions.
Biotechniques
12:
870-879,
1992[Medline].
42.
Sena, C. M.,
A. R. Tomé,
R. M. Santos,
and
L. M. Rosário.
Protein kinase C activator inhibits voltage-sensitive Ca2+ channels and catecholamine secretion in adrenal chromaffin cells.
FEBS Lett.
359:
137-141,
1995[Medline].
43.
Swartz, K. J.
Modulation of Ca2+ channels by protein kinase C in rat central and peripheral neurons: disruption of G protein-mediated inhibition.
Neuron
11:
305-320,
1993[Medline].
44.
Toth, P. T.,
V. P. Bindokas,
D. Bleakman,
W. F. Colmers,
and
R. J. Miller.
Mechanism of presynaptic inhibition by neuropeptide Y at sympathetic nerve terminals.
Nature
364:
635-639,
1993[Medline].
45.
Westfall, T. C.,
X. Chen,
A. Ciarleglio,
K. Henderson,
K. Del Valle,
M. Curfman-Falvey,
and
L. Naes.
In vitro effects of neuropeptide Y at the vascular neuroeffector junction.
Ann. NY Acad. Sci.
611:
145-155,
1990[Medline].
46.
Wiley, J. W.,
R. A. Gross,
Y. Lu,
and
R. L. MacDonald.
Neuropeptide Y reduces calcium current and inhibits acetylcholine release in nodose neurons via a pertussis toxin-sensitive mechanism.
J. Neurophysiol.
63:
1499-1507,
1990[Abstract/Free Full Text].
47.
Wiley, J. W.,
R. A. Gross,
and
R. L. MacDonald.
Agonists for neuropeptide Y receptor subtypes NPY-1 and NPY-2 have opposite actions on rat nodose neuron calcium currents.
J. Neurophysiol.
70:
324-330,
1993[Abstract/Free Full Text].
48.
Yang, J.,
and
R. W. Tsien.
Enhancement of N- and L-type calcium channel currents by protein kinase C in frog sympathetic neurons.
Neuron
10:
127-136,
1993[Medline].
49.
Zhu, Y.,
and
S. R. Ikeda.
Modulation of Ca2+-channel currents by protein kinase C in adult rat sympathetic neurons.
J. Neurophysiol.
72:
1549-1560,
1994[Abstract/Free Full Text].
AJP Cell Physiol 274(5):C1290-C1297
0363-6143/98 $5.00
Copyright © 1998 the American Physiological Society