Mechanisms of modulation of neuronal nicotinic receptors by
substance P and OAG
Tomio
Andoh,
Hideki
Itoh,
Itaru
Watanabe,
Toshio
Sasaki, and
Tomoko
Higashi
Department of Anesthesiology, Yokohama City University School
of Medicine, Kanazawa-ku, Yokohama 236-0004, Japan
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ABSTRACT |
Substance P is known to modulate neuronal nicotinic
acetylcholine receptors (nAChRs) in the sympathetic nervous system.
There are two conflicting proposals for the mechanism of this effect, an indirect action mediated by protein kinase C (PKC) and a direct interaction with receptor subunits. We studied the mechanisms of this
effect in PC-12 cells. Substance P enhanced the decay of the
nicotine-induced whole cell current. This effect was fast in its onset
and was not antagonized by guanosine
5'-O-(2-thiodiphosphate), a G protein blocker, or
staurosporine, a nonselective PKC blocker. Staurosporine failed to
reverse the inhibition by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic diacylglycerol analog known to activate PKC. The
inhibitory effects of the peptide and OAG were preserved in excised
patches, but substance P applied to the extra patch membrane was
ineffective in the cell-attached patch configuration. We conclude that
substance P modulates neuronal nAChRs most likely by direct interactions with the receptors but independently from activation of
PKC or G proteins and that PKC does not participate in modulation by OAG.
ion channel; neuropeptide; diacylglycerol; PC-12 cells; 1-oleoyl-2-acetyl-sn-glycerol
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INTRODUCTION |
SUBSTANCE P HAS BEEN
KNOWN to modulate neuronal nicotinic acetylcholine receptors
(neuronal nAChRs) in a number of preparations such as sympathetic
(33) and parasympathetic (19) neurons, adrenal chromaffin cells (6), and PC-12 cells
derived from rat pheochromocytoma cells (27, 28, 32).
Substance P enhances the agonist-induced desensitization of nAChRs in
these cells without the involvement of neurokinin receptors
(28). This modulation is considered to be physiologically
relevant, since the substance P-containing fibers have been identified
in sympathetic ganglia and adrenal medulla (33, 35).
Simmons et al. (29) have shown that substance P acts via
an intracellular second messenger system, since nAChRs, monitored by
cell-attached patches, are modulated by the peptide bath applied to the
extra patch membrane in chick sympathetic neurons. They suggested that
protein kinase C (PKC) is a likely candidate to mediate the effect of
substance P because modulation of nAChR desensitization was blocked by
staurosporine, and activators of PKC mimicked the substance P-induced
effects. This proposal is further supported by the findings that
substance P stimulates phosphatidylinositol metabolism and leads to an
activation of PKC in other systems (25).
However, other studies suggest that substance P modulates nAChRs by a
direct interaction with the receptor subunits (4, 30, 31).
Stafford et al. (30) demonstrated that two domains of the
-subunit control the affinity of substance P to neuronal nAChRs,
suggesting a direct interaction. In addition, a line of evidence
indicates that substance P directly binds to Torpedo nicotinic receptors (4).
Besides substance P, neuronal nAChRs are modulated by other
neuropeptides such as calcitonin gene-related peptide (CGRP) and dynorphins (10, 24). CGRP has been found to inhibit the
current mediated by neuronal nAChRs by two distinct pathways in rat
adrenal chromaffin cells: fast block, most likely by direct effects,
and slow inhibition through G protein-coupled receptor activation (10). We recently reported that dynorphin A also inhibits
neuronal nAChRs by a direct interaction leading to a reduction of
channel open time in PC-12 cells (15, 24). These
observations prompted us to clarify whether substance P directly
inhibits neuronal nAChRs or whether second messengers mediate this
effect. We found that modulation of nicotinic receptors by substance P
does not require diffusible cytoplasmic factors in PC-12 cells. We also
found that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic
diacylglycerol analog known to activate PKC (5, 23),
inhibits neuronal nAChR-mediated current without the involvement of PKC activation.
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MATERIALS AND METHODS |
Cell preparation.
PC-12 cells were cultured as previously described (1). For
the experiments, cells were plated on collagen- and
poly-L-lysine-coated coverslips, and they were used after
treatment with 100 ng/ml of 2.5S nerve growth factor (NGF; Takara
Shuzo, Shiga, Japan) for 4-6 days.
Current recording.
Nicotine-evoked currents were recorded at room temperature in either
whole cell, outside-out, or cell-attached configurations by using
AXOPATCH 200A (11). Heat-polished patch pipettes had a tip
resistance of 2-7 M
when filled with an intracellular solution. Cells on the coverslips were placed in a recording bath with an approximate volume of 1.5 ml and continuously perfused at the rate of 2 ml/min with an external solution. Current responses were evoked by
application of nicotine using a rapid application technique described
as the "Y-tube" method (21). The tip of the Y-tube was
positioned about 300 µm from the recorded cell. This method enabled
the complete exchange of the external solution surrounding the cell
within 50-150 ms, as estimated by recording the liquid junction
current produced at an open patch pipette. The series resistance ranged
from 8 to 23 M
and was not compensated.
Recording solutions and materials.
The extracellular perfusion solution consisted of (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 10 HEPES, and 11.1 glucose (pH was adjusted to 7.4 with NaOH). For whole cell current
recordings, the intracellular pipette solution contained (in mM) 150 CsCl, 10 HEPES, 5 EGTA, 2 ATP-Mg, and 0.3 GTP-Na2 (pH 7.3 with CsOH). GTP-Na2 was replaced with 2 mM guanosine
5'-O-(2-thiodiphosphate) (GDP
S) for blockade of
GTP-binding proteins (G protein) in some experiments. Staurosporine was
added to the standard pipette solution at a final concentration of 0.1 µM for blockade of PKC in other cases. The measurements started >15
min after the whole cell mode was established in these experiments. For
outside-out patch recordings, ATP-Mg, and GTP-Na2 were
omitted in the intracellular solution. For cell-attached patch
recordings, the perfusion solution consisted of (in mM) 140 NaCl, 2.7 KCl, 10 CaCl2, 1.0 MgCl2, and 10 HEPES (pH was
adjusted to 7.4 with NaOH). The pipette solution was the same as the
perfusion solution except that the concentration of CaCl2
was lowered to 1.0 mM, and 5 µM nicotine was included. (
)-Nicotine was purchased from Wako (Osaka, Japan); ATP-Mg, GTP-Na2,
GDP
S, and OAG were from Sigma (St. Louis, MO); and substance P was
from Research Biochemicals. OAG was freshly suspended with the external solution by sonication to make 85 µM solution immediately before use.
Whole cell recording.
In whole cell current recording, NGF-treated PC-12 cells were voltage
clamped at
60 mV, and 30 µM nicotine with or without 10 µM
substance P was applied for 5 s. The recordings were made in
isolated cells without connection to the surrounding cells, and each
application was separated by 5 min. The currents were low-pass filtered
at 1 kHz and digitized at 5 kHz. For preincubation with substance P,
the external solution containing the peptide was perfused at the rate
of 5 ml/min for 5 min before rapid application. Cells were perfused
with the plain external solution at the same rate for 5 min to wash out
the drugs from the bath after measurement. We measured the peak and the
nondesensitized current, which was defined as the average of the
last 50 points during 5-s agonist application. Because
nicotine-induced currents slightly declined with each application of
nicotine, the response in the presence of substance P was normalized to
the average of the elicited currents before and after the peptide
application. The relative current in the presence of substance P was
compared with control, which is the second response normalized to the
average of the first and third responses in three successive nicotine
applications with an interval of 5 min. Desensitization was evaluated
by calculating the percentage decay of the current (%current decay)
during agonist application, defined by the following equation
(33)
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We also studied the effects of OAG, an activator of PKC, on the
nicotine-induced current in the same way as substance P. Because the
effect of OAG was not fully reversible, the current in the presence of
OAG was normalized to the response immediately before OAG. The second
response, normalized to the first response among three successive
responses induced by nicotine, served as control in these cases.
Outside-out patch recordings.
Single channel recordings were obtained from outside-out patches
excised from NGF-treated cells. Nicotine (3 µM) in the absence and
presence of 10 µM substance P or 10 µM OAG was applied for 30 s at the membrane potential of
60 mV, and each application was
separated by 2 min. Substance P or OAG was applied without preincubation. The signals were filtered at 1 kHz (Bessel) and digitized at 5 kHz. The pCLAMP software package (Axon Instruments) was
used to obtain all points amplitude histograms and the mean currents.
Opening and closing of the channels were detected using 50% threshold
criterion. Events <1 ms were ignored. The mean currents were obtained
by integrating the current records during agonist application (30 s)
and dividing the integral by the time of agonist application (30 s).
Cell-attached patch recordings.
Single channel currents were also recorded in the cell-attached
configuration using NGF-treated cells. Nicotine (5 µM) was included
in the pipette solution, and the command potential was kept at 0 mV.
After the control response was recorded, 10 µM substance P was
applied to the extra patch membrane for 2.5 min. Channel activity was
recorded until 3 min after the end of the application. Single channel
currents were analyzed in the same way as outside-out patch recordings,
and the mean currents were measured for the following three periods of
30 s: the control period immediately before the application of
substance P, the last 30 s during the application (2 min after the
beginning of the application), and 5 min after the beginning of the
application. We also recorded nicotine-induced single channel currents
and measured the mean currents, applying plain external solution
instead of substance P, to see time-dependent changes.
Statistical analysis.
Data are expressed as means ± SE. Statistical analysis was made
by unpaired t-test or analysis of variance, followed by the Bonferroni multiple-comparison test, to estimate the significance when
appropriate. P < 0.05 was considered to be significant.
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RESULTS |
Inhibition of whole cell current by substance P.
Nicotine elicited inward currents that decayed rapidly during nicotine
application due to desensitization at the membrane potential of
60
mV. Substance P alone produced no current responses in tested cells at
10 µM. Substance P (10 µM) depressed the whole cell current
elicited by 30 µM nicotine reversibly and augmented the current decay
when it was coapplied with nicotine after preincubation (Fig.
1A). The magnitude of
inhibition was much greater for the nondesensitized current than that
for the peak current (Fig. 2, A and B). When substance P was coapplied with
nicotine without preincubation, it similarly inhibited the
nicotine-induced current (Fig. 1B). However, the magnitude
of inhibition of the peak current was significantly smaller than that
caused by preincubation plus coapplication, whereas the inhibitory
effects on the nondesensitized current were not influenced by the
presence or absence of preincubation (Fig. 2, A and
B). When nicotine alone was applied after a 5-min preincubation of substance P, inhibition of the nondesensitized current
was much weaker than that caused by preincubation plus coapplication,
while the magnitude of the peak current inhibition did not change
(Figs. 1C and 2, A and B). These
results indicate that the onset of inhibition by substance P is fast
because preincubation is not required for the suppression of the
nondesensitized current, and they also suggest that channel opening is
required for substance P to exert its full effects.

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Fig. 1.
Inhibition of nicotine-induced whole cell current by
substance P. Records shown are evoked currents in single PC-12 cells to
5-s pulses of 30 µM nicotine before, during, and after application of
substance P. A: 10 µM substance P was coapplied with
nicotine after a 5-min preincubation. B: substance P and
nicotine were coapplied without pretreatment of substance P. Substance
P depressed the nicotine-induced current and accelerated current decay
similarly in these conditions, resulting in greater inhibition of the
nondesensitized current than the peak current. C: nicotine
alone was applied after a 5-min preincubation of substance P. Reduction
of nondesensitized current by preincubation alone was smaller than that
by coapplication. All these effects of substance P were reversible.
Holding potential was 60 mV. Nicotine was applied as indicated by
thick horizontal bars with an interval of 5 min. Substance P was
applied as indicated by dotted lines.
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Fig. 2.
Summarized data for effects of substance P on the peak
(A) and nondesensitized (B) component of the
nicotine-induced current. The current in the presence of 10 µM
substance P was normalized to the average of control responses before
and after application of substance P. In control, nicotine alone was
applied successively three times, and the second response was compared
with the average of the first and third responses. Substance P was
coapplied with 30 µM nicotine after a 5-min preincubation except for
preincubation ( ) and preincubation alone. In the preincubation ( )
group, substance P and nicotine were coapplied without pretreatment. In
the preincubation alone group, nicotine alone was applied after
preincubation of substance P. In the guanosine
5'-O-(2-thiodiphosphate) (GDP S) and staurosporine groups,
substance P and nicotine were coapplied after preincubation, while
GDP S and staurosporine were included in the pipette solution at
final concentrations of 2 mM and 0.1 µM. Relative peak and
nondesensitized currents were 1.0 ± 0.02 and 1.01 ± 0.03 for control (n = 8), 0.54 ± 0.05 and 0.01 ± 0.01 for preincubation (+; n = 8), 0.77 ± 0.05 and 0.03 ± 0.02 for preincubation ( ; n = 4),
0.72 ± 0.06 and 0.64 ± 0.07 for preincubation alone
(n = 7), 0.41 ± 0.06 and 0.01 ± 0.004 for
GDP S (n = 5), and 0.67 ± 0.06 and 0.03 ± 0.01 for staurosporine (n = 6). Each column represents
the mean and SE. *P < 0.05, compared with control.
**P < 0.01, significantly different from control.
#P < 0.05, significantly different from
preincubation (+). ##P < 0.01, significantly different from preincubation (+).
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We then studied the possibility of the involvement of G proteins and
PKC in the inhibitory effects of substance P. Inclusion of GDP
S or
staurosporine to the intracellular solution did not significantly
affect the magnitudes of inhibition caused by 10 µM substance P (Fig.
2, A and B). Substance P accelerated the current
decay during nicotine application, resulting in an increase in
%current decay. This effect was not antagonized by GDP
S or staurosporine (Table 1). Staurosporine
failed to reverse the substance P-induced effect, even at 1.0 µM
(n = 3, data not shown). These results indicate that G
proteins or PKC is not involved in the modulation of neuronal
nAChRs by substance P.
Effects of substance P on single channel currents.
To further investigate the mechanism of the inhibition, we studied the
effects of substance P on the single channel current in outside-out
patches and the cell-attached mode. In the outside-out configuration,
the patches were exposed to 3 µM nicotine, a concentration that
results in little desensitization, and 10 µM substance P was applied
without preincubation. First, nicotine was applied for 30 s, and
then nicotine with or without substance P was applied again after a
2-min interval. As illustrated in Fig.
3A, substance P consistently
inhibited the nicotine-induced single channel activity. The amplitude
histogram showed that substance P did not decrease the amplitude of the
unitary current (Fig. 3B). Although the mean current of the
second response normalized to that of the first response was close to
1.0 when nicotine alone was applied twice, the relative mean current
was significantly reduced when substance P was coapplied with nicotine
(Fig. 3C). Open probability was also reduced by the peptide,
and the onset of the action seemed within a few seconds (Fig.
3D). The relative mean current in the presence of substance
P was comparable to the relative nondesensitized current without
preincubation but in the presence of substance P, suggesting that the
magnitude of inhibition observed in excised patches is similar to that
in whole cell recordings. These results indicate that the inhibitory
effects of substance P do not require diffusible intracellular
components. The channel activity did not last long enough to obtain a
sufficient number of events for further kinetic analysis.

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Fig. 3.
Suppression of single channel activity by substance
P in excised patches. A: top 2 traces show the
successive responses elicited by 3 µM nicotine in a 2-min interval.
Bottom 2 traces depict the responses from another patch that
was first exposed to nicotine and then to nicotine + 10 µM
substance P (Sub P) after a 2-min interval. B: amplitude
histogram of single channel current in absence or presence of substance
P. Substance P (10 µM) reduced the number of events without changing
amplitude of unitary current. Similar changes in amplitude histogram
were observed in 5 other patches. C: summary of effects of
substance P on mean current. When nicotine alone was applied twice, the
mean current of the second response was 115.2 ± 16% of that of
the first response (Nicotine). Substance P (10 µM) significantly
decreased the relative mean current of the second responses to 11 ± 3% (Substance P). The number of experiments was 6 for nicotine and
substance P. **P < 0.01, significantly different from
nicotine. D: fast onset of inhibition of single channel
activity by substance P. Top 2 traces are a representative continuous
recording of the first response to nicotine alone. Recordings show a
period of 20 s, starting from 1 s after the beginning of the
application. Bottom 2 traces depict recording from the same patch
exposed to nicotine + substance P for same period. E:
time course of changes in open probability with or without substance P. Data were obtained from another patch, different from the one shown in
D. Substance P greatly reduced open probability of the
channel, and a full effect was observed within a few seconds. Open
probability of the channel was reduced from 0.098 ± 0.03 for
nicotine alone (Control) to 0.012 ± 0.002 for nicotine with
substance P (Substance P) when data from 6 patches were averaged.
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On the contrary, the peptide did not significantly inhibit
nicotine-induced currents when applied to the extra patch membrane in
cell-attached recordings (Fig.
4A). The mean currents at 2 min and 5 min after the application of substance P or the plain external solution were normalized to those of the control period. The
relative mean currents slightly decreased after the application of
substance P; however, the changes did not reach statistical significance (Fig. 4B). There was no significant difference
in the relative mean currents between the cells that received substance P and those exposed to the plain external solution. These findings also
indicate that the roles of diffusible intracellular messengers are not
important in the effects of substance P.

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Fig. 4.
Effects of substance P on single channel current in
cell-attached mode. Recordings were made with pipettes containing 5 µM nicotine before, during, and after 10 µM substance P or plain
external solution was applied outside pipettes. A: traces
shown are recorded immediately before application of peptide (Control),
2 min after beginning the application (2 min after substance P), and 5 min after beginning the application (5 min after substance P).
B: mean currents were measured for 3 periods of 30 s in
2 groups; one received substance P (Substance P, n = 5), and the other was exposed to plain external solution (External,
n = 5). Mean currents at 2 and 5 min after application
were normalized to those of the control period. Relative mean currents
at 2 min were 85.9 ± 26.8 and 69.8 ± 19.2% for external
and substance P group, and they were 89.9 ± 20.6 and 74.5 ± 21.4% for external and substance P group at 5 min. Relative mean
currents slightly decreased after application of substance P; however,
the changes did not reach statistical significance for either group.
There was no significant difference in relative mean currents between 2 groups.
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Inhibition of neuronal nAChRs by OAG.
OAG (10 µM) also reduced the nicotine-induced whole cell current and
accelerated the current decay when it was coapplied with nicotine after
preincubation (Fig. 5). The peak and
nondesensitized currents were depressed to 64.4 ± 3.7 and
25.4 ± 2.3% of the current immediately before OAG. The
inhibition was not reversible after a 5- or 10-min washout. After a
5-min washout, the peak and nondesensitized currents remained 53.5 ± 6.0 and 32.6 ± 5.0% of the response immediately before OAG,
while the peak and nondesensitized components of the third response
accounted for 91.4 ± 3.1 and 77.6 ± 2.2% of the first
response during three repeated nicotine applications. Recovery was
significantly depressed after OAG. Inhibitory effects of 10 µM OAG
were significantly decreased when preincubation was omitted. There was
no significant difference in the magnitude of inhibition by 85 and 10 µM OAG (Fig. 6, A and
B). Preincubation of 85 µM OAG alone was as effective as
preincubation plus coapplication (data not shown). These findings
suggest that OAG similarly inhibits the nicotine-induced current and
that the action is slow in its onset and is long lasting, but it does
not require channel activation.

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Fig. 5.
Inhibition of the nicotine-induced whole cell current by
1-oleoyl-2-acetyl-sn-glycerol (OAG). Records shown are
currents evoked by 30 µM nicotine before, during, and after OAG
application. Measurements were performed in the same way as Fig.
1A. OAG (10 µM) was coapplied with nicotine after a 5-min
preincubation of OAG. Nicotine was applied as indicated by thick
horizontal bars with an interval of 5 min. OAG was applied as indicated
by a thin bar. OAG depressed the nicotine-induced current and
accelerated current decay. Effects were not reversible after a 5-min
washout.
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Fig. 6.
Summarized data for effects of OAG on the peak
(A) and nondesensitized (B) component of the
nicotine-induced current. Current in the presence of OAG was normalized
to the current immediately before application of OAG. In control,
nicotine alone was applied successively 3 times, and amplitude of the
second response was compared with that of the first response. OAG (10 or 85 µM) was coapplied with 30 µM nicotine after a 5-min
preincubation except for OAG 10 pre . OAG (10 µM) and nicotine were
coapplied without preincubation in OAG 10 pre . Staurosporine (0.1 µM) was added to pipette solution in OAG 10 + stauro and OAG
85 + stauro. Relative peak and nondesensitized currents were
0.95 ± 0.02 and 0.9 ± 0.01 for control (n = 8), 0.98 ± 0.05 and 0.57 ± 0.05 for OAG 10 pre (n = 8), 0.64 ± 0.04 and 0.25 ± 0.02 for
OAG 10 pre + (n = 6), 0.77 ± 0.07 and
0.33 ± 0.05 for OAG 10 + stauro (n = 8),
0.64 ± 0.07 and 0.16 ± 0.04 for OAG 85 (n = 8), and 0.42 ± 0.05 and 0.12 ± 0.02 for OAG 85 + stauro (n = 4). Each column represents the mean and SE.
*P < 0.05, significantly different from control.
**P < 0.01, significantly different from control.
##P < 0.01, significantly different from
OAG 10 pre +. Stauro, staurosporine.
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Staurosporine included in the pipette solution did not significantly
alter inhibitory effects of 10 or 85 µM OAG (Fig. 6, A and
B). Enhancement of current decay during nicotine application by 10 or 85 µM OAG was also not affected by staurosporine (Table 2). These results indicate that
inhibition of neuronal nAChRs by OAG does not involve PKC activation.
Effects of 10 µM OAG on single channel currents were studied in
outside-out patches in the same protocol as that used for substance P. OAG strongly depressed the nicotine-induced single channel activity
upon coapplication without preincubation (Fig. 7A). The inhibition seemed to
develop gradually during the first several seconds of coapplication,
whereas the channel activity was maintained when nicotine alone was
applied. OAG coapplied with nicotine reduced open probability and the
mean current without changing the amplitude of the unitary current
(Fig. 7, B-D). The time course of changes in open
probability showed a slow onset of the action of OAG. When data from
all studied patches were combined, open probability was 0.189 for the
first 10 s and 0.124 for the later 20 s during application of
nicotine alone. In the presence of OAG, it was reduced to 0.059 for the
first 10 s and 0.04 for the later 20 s. There was a
significant difference in open probability between the early and later
phases of the response in the presence of OAG, while this was not the
case for control. Therefore, blockade by OAG was significantly enhanced
in the later 20 s compared with the first 10 s. The magnitude
of reduction of the mean current in excised patches was comparable with
that of inhibition of nondesensitized current in whole cell recordings. Preservation of the inhibitory effects of OAG in outside-out patches also indicates that diffusible cytoplasmic factors are not responsible for the inhibition.

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Fig. 7.
OAG depressed nicotine-induced single channel activity in
outside-out patches. A: representative examples of single
channel activity with or without OAG. Top 2 traces are a continuous
recording of the first response to 3 µM nicotine alone, showing a
period of 20 s, starting from 1 s after beginning the
application. Bottom 2 traces depict recording from same patch exposed
to nicotine + 10 µM OAG after a 2-min interval. Onset of
inhibition by OAG seemed slower than that by substance P. B:
amplitude histogram of single channel current in absence or presence of
OAG. OAG (10 µM) reduced number of events without changing amplitude
of unitary current. Similar changes in amplitude histogram were
observed in 4 other patches. C: summary of effects of OAG on
mean current. Nicotine alone was applied twice with an interval of 2 min in nicotine group, while the patches were first exposed to nicotine
and then to nicotine + OAG after a 2-min interval in OAG group.
Mean current of the second response was normalized to that of the first
response. Relative mean currents were 115.2 ± 16% for nicotine
group and 15.4 ± 3.6% for OAG group (n = 6 and 5 for nicotine and OAG). **P < 0.01, significantly
different from nicotine. D: time course of changes in open
probability with or without OAG. Data were obtained from another patch
different from the one shown in A. Depression of open
probability gradually advanced during the first 10 s. Open
probability of the channel was reduced from 0.258 ± 0.068 for
nicotine alone (control) to 0.045 ± 0.018 for nicotine with OAG
(OAG) when data from all studied patches were averaged.
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DISCUSSION |
The results of the present study demonstrate that substance P and
OAG both inhibit the neuronal nAChR-mediated current and augment the
current decay during agonist application. They also demonstrate that
these effects did not require activation of diffusible cytoplasmic
factors, including PKC, in PC-12 cells. Lack of the involvement of PKC
is inconsistent with earlier studies for both substance P and OAG
(9, 29). Staurosporine included in the pipette solution
failed to reverse the effects of both compounds in our study. It might
be possible that the entire intracellular fluid was not effectively
dialyzed with the pipette solution containing staurosporine because of
the size and shape of differentiated PC-12 cells. However, this is
unlikely because sufficient time elapsed before the measurements were
started. Moreover, the finding that the inhibitory effects of substance
P and OAG were well preserved in excised patches strongly opposes the
involvement of diffusible cytoplasmic factors. As for substance P, the
mechanism independent from a second messenger system was also supported
by the rapid onset of the effect and the finding that the peptide was
ineffective when applied to the extra patch cell membrane in
cell-attached recordings.
Our findings indicate that inhibition by substance P does not involve
cytoplasmic second messengers or G proteins in PC-12 cells. How, then,
does this peptide modulate neuronal nAChRs? The rapid onset of the
effect favors a direct action on neuronal nAChRs. This notion is
consistent with the findings of the study reported by Stafford et al.
(30). They showed that the affinity of substance P is
strongly influenced by two domains of the
4-subunit of
rat nAChRs, an extracellular NH2-terminal domain and
another domain containing the M1, M2, and M3 regions using chimeric
-subunit coexpressed with the
3-subunit in
Xenopus oocytes. Binding of substance P to the channel is
also indicated by affinity labeling of
125I-p-benzoylphenylalanine substance P to
the M2 region of the
-subunit from Torpedo
(4). On the contrary, cytoplasmic second messengers, including PKC, are suggested to be involved in modulation by substance P in embryonic chick sympathetic neurons (29). Unlike our
results, channel opening is not important for inhibitory effects of
substance P on chick neuronal nAChRs because a significant inhibition
was observed by pretreatment of the peptide, followed by application of
nicotine alone after washout of substance P in the same preparation (33). Moreover, our results of cell-attached recordings
were inconsistent with those obtained in chick sympathetic neurons (29). What factors explain these discrepancies? The
concentration of substance P studied in our experiment was 10 µM,
which is comparable with that mainly studied in chick neurons (20 µM). Therefore, differences in the concentrations of the peptide
would not account for the discrepancies. Differences in species and
types of cells may explain the discrepancies in a part. Although the
exact subunit combinations of nAChRs are not identified in PC-12 cells
or chick sympathetic neurons,
3
4-containing receptors are likely to be expressed dominantly in both types of cells. However, amino acid sequence identity is not as high for the
4-subunit as
for the
3-subunit (74% for
4- vs. 83%
for
3-subunit), and there are large differences in
pharmacology between rat and chick clones (7, 20, 26). It
may be possible that substance P has lower affinity to nAChRs in chick
sympathetic neurons than to those in PC-12 cells and that indirect
actions are dominant in chick preparations. There may be differences in
the extent of modulation by protein kinases among species and types of neurons.
PKC activators, including OAG, have been shown to enhance the current
decay of neuronal nAChR-mediated current in chick sympathetic neurons
(9); however, evidence supporting the idea that PKC activation mediates the effect is circumstantial: a slow onset of the
effect and similarity in effective concentrations for modulation of
nicotinic receptors and PKC activation. As for phorbol esters, lack of
changes in the current decay by an inactive analog also supported the
idea. Again, this idea was not supported by the results of our study.
Lack of reversal of the effects of OAG by staurosporine and
preservation of the effects in cell-free excised patches strongly
oppose the involvement of cytosol PKC. Although we did not obtain a
dose-response relationship for the effects of OAG, nicotinic receptors
in PC-12 cells seemed more sensitive to OAG than those in chick
sympathetic neurons because 10 µM OAG was as effective as 85 µM OAG
in our study, while 20 µM OAG exerted only a small effect in chick
neurons (9). Inhibition of the nondesensitized current was
not reversible after a 5-min washout in PC-12 cells, whereas it was
partially reversible in chick neurons (9). The onset of
the effect of OAG seemed to be slower than that of substance P in the
present study, since inhibition of the nondesensitized current was
significantly enhanced by preincubation in whole cell recordings, and a
reduction in open probability gradually increased during the first
10 s in excised patch recordings, while those were not the cases
for substance P. The slow onset and recovery of the effect of OAG
suggest indirect paths other than diffusible cytoplasmic factors, such
as modulation through G proteins or changes in membrane lipid
environment. We cannot exclude the possibility of slow channel block,
as suggested in the earlier study (9). Diacylglycerol has
been shown to perturb phospholipid bilayers and to modulate function of
certain membrane proteins independent of PKC activation (2, 8,
17, 36). The effect of OAG on nicotinic receptors may represent
another example of effects of diacylglycerol on membrane proteins not mediated by PKC activation.
It has been shown that some types of protein kinases and phosphatases
can be closely associated with membrane ion channels and can remain
bound when the patch is excised (3, 34). It might be
possible that substance P and OAG activate PKC tethered to the excised
patch, resulting in modulation of neuronal nAChRs. However, there has
been no report suggesting this type of association between nAChRs and
PKC, only for other types of ion channels such as
Ca2+-activated potassium channels and
Ca2+-sensitive nonspecific cation channels (3,
34). If activation of PKC plays an important role in the
inhibitory effects of substance P, the peptide applied to the extra
patch membrane should have exhibited its effect through activating
cytosol PKC. Therefore, the involvement of PKC in excised patch
recordings is very unlikely, at least for substance P.
Phosphorylation of nAChRs has been considered to regulate the rate of
desensitization of nicotinic receptors (14, 18). As for
phosphorylation of muscle receptors by protein kinase A and protein
tyrosine kinase, this hypothesis was directly supported by the
experiments using cell-free reconstituted channels (12, 13). However, consequences of phosphorylation by PKC have not been directly examined, but merely suggested, by indirect evidence that
PKC activators enhance desensitization of muscle type and neuronal type
nAChRs (9). A recent study has shown that 4
-phorbol 12,13-dibutyrate, a kind of phorbol ester known to activate PKC, directly modulates Torpedo nAChRs independently of PKC
activation (22). Moreover, it has recently been revealed
that activation or depression of PKC does not influence the onset of
desensitization of neuronal nAChRs but regulates the rate of recovery
from desensitization in rat chromaffin cells (16).
Therefore, it is not surprising that modulation by OAG or substance P
does not involve PKC activation.
When one takes into account the earlier studies, it appears that three
neuropeptides, substance P, CGRP, and dynorphin A, modulate neuronal
nAChRs, most likely by direct interactions with receptor subunits in
rat chromaffin cells and a related cell line (10, 15). It
may be interesting to explore whether a common region of receptor
subunits is responsible for the effects of these peptides and whether
there is any synergetic or antagonistic interactions among these peptides.
In summary, we have studied mechanisms of modulation of neuronal nAChRs
by substance P and OAG, which have been considered to exert their
effects through activation of PKC. We found that both compounds inhibit
the nicotine-induced current independent from diffusible cytoplasmic
factors in PC-12 cells.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: T. Andoh, Dept. of Anesthesiology, Yokohama City Univ. School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan (E-mail:
tandoh{at}med.yokohama-cu.ac.jp).
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 26 January 2001; accepted in final form 20 August 2001.
 |
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