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The Journal of Neuroscience, 2000, 20:RC105:1-5
RAPID COMMUNICATION
Bradykinin, But Not Muscarinic, Inhibition of M-Current in Rat
Sympathetic Ganglion Neurons Involves Phospholipase C- 4
Jane E.
Haley,
Fe C.
Abogadie,
Jose M.
Fernandez-Fernandez,
Mariza
Dayrell,
Yvonne
Vallis,
Noel J.
Buckley, and
David A.
Brown
Wellcome Laboratory for Molecular Pharmacology, Department of
Pharmacology, University College London, London, WC1E 6BT, United
Kingdom
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ABSTRACT |
Rat superior cervical ganglion (SCG) neurons express low-threshold
noninactivating M-type potassium channels
(IK(M)), which can be inhibited by
activation of M1 muscarinic receptors (M1 mAChR) and bradykinin (BK) B2 receptors.
Inhibition by the M1 mAChR agonist oxotremorine
methiodide (Oxo-M) is mediated, at least in part, by the pertussis
toxin-insensitive G-protein G q (Caulfield et al., 1994 ;
Haley et al., 1998a), whereas BK inhibition involves Gq
and/or G11 (Jones et al., 1995). Gq and
G11 can stimulate phospholipase C- (PLC-), raising
the possibility that PLC is involved in
IK(M) inhibition by Oxo-M and BK. RT-PCR and antibody staining confirmed the presence of PLC-1, -2, -3, and
-4 in rat SCG. We have tested the role of two PLC isoforms (PLC-1
and PLC-4) using antisense-expression constructs. Antisense constructs, consisting of the cytomegalovirus promoter driving antisense cRNA corresponding to the 3'-untranslated regions of PLC-1
and PLC-4, were injected into the nucleus of dissociated SCG
neurons. Injected cells showed reduced antibody staining for the
relevant PLC- isoform when compared to uninjected cells 48 hr later.
BK inhibition of IK(M) was significantly
reduced 48 hr after injection of the PLC-4, but not the PLC-1,
antisense-encoding plasmid. Neither PLC- antisense altered
M1 mAChR inhibition by Oxo-M. These data support the
conclusion of Cruzblanca et al. (1998) that BK, but not M1
mAChR, inhibition of IK(M) involves PLC and
extends this finding by indicating that PLC-4 is involved.
Key words:
M-current; muscarinic receptor; bradykinin; phospholipase
C-; antisense; superior cervical ganglion neuron
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INTRODUCTION |
The
M-type potassium current (IK(M)) is a
noninactivating potassium current present in various peripheral and
central neurons, including rat superior cervical ganglion (SCG) neurons
(for review, see Brown, 1988). It is activated in the subthreshold
range for action potentials and increases with membrane depolarization
and may, therefore, be involved in controlling cell excitability, because inhibition of this current results in depolarization and increased action potential discharge.
IK(M) in SCG can be inhibited by
stimulating various receptors including the M1
muscarinic receptor (M1 mAChR; Marrion et al.,
1989; Bernheim et al., 1992; Hamilton et al., 1997) and bradykinin (BK)
B2 receptor (Jones et al., 1995), both of which
couple via Bordetella pertussis toxin (PTX)-insensitive GTP-binding proteins (G-proteins). We have previously
demonstrated that the subunit of Gq mediates
inhibition by M1 mAChR agonists (Haley et al.,
1998a) and that Gq and/or
G11 is required for inhibition by BK (Jones et
al., 1995). Because both Gq and
G11 are known to stimulate PLC- (Singer et
al., 1997), we have now used antisense directed at two PLC- isoforms
(PLC-1 and PLC-4) to deplete the cells of these enzymes and so
determine whether either is required for inhibition of
IK(M) by M1
mAChR agonists or BK.
Some of this data has been previously presented in abstract form (Haley
et al., 1998b)
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MATERIALS AND METHODS |
DNA plasmids. The constructs used in this study
were made by PCR cloning using standard molecular techniques (Abogadie
et al., 1997). Primers that were deemed specific for each target PLC- isoform were used in PCR, and the products were
TA-cloned into pCR3 or pCR3.1 (Invitrogen, San Diego, CA).
Antisense orientation was confirmed by sequencing. The clones are as
follows, in 5' to 3' orientation [nucleotide (nt); plus and minus
signs indicating downstream and upstream, respectively, of the stop
codon]: PLC-1 antisense (clone 239-8), nt +22 to +172; PLC-2
antisense (clone F128-5), nt  536 to 309; PLC-3 antisense (clone
E92-18), nt 93 to +67; PLC-4 antisense (C58-13), nt +5 to
+386.
Cell culture. Sympathetic neurons were isolated from SCG of
15- to 19-d-old Sprague Dawley rats and cultured using standard procedures as described previously (Delmas et al., 1998).
Microinjection. DNA plasmids, purified using maxiprep
columns (Qiagen, Hilden, Germany), were diluted to 400 µg/ml in
calcium- and glucose-free Krebs' solution (290 mOsm/l; pH 7.3)
containing 0.5% FITC-dextran (70,000 MW) and pressure injected into
the nucleus of SCG neurons 2 d in culture using an Eppendorf
microinjector (Hamburg, Germany). Cells were maintained in culture for
a further 2 d, and a survival rate of 75-85% was obtained.
Electrophysiology. IK(M)
was measured from SCG neurons cultured for 5 d, using the
amphotericin-B perforated-patch technique (Horn and Marty, 1988; Rae et
al., 1991). Patch electrodes (1.5-4 M ) were filled by dipping the
tip for 40 sec into filtered internal solution that comprised (in
mM): potassium acetate 80, KCl 30, HEPES 40, MgCl2 3 (adjusted to pH 7.3-7.4 with KOH, and
280 mOsmol/l with K acetate). The pipette was then back-filled with
internal solution containing 0.07-0.1 mg/ml amphotericin-B. High
resistance seals (>2 G) were initially achieved, and after
amphotericin-B permeabilization, access resistances were generally <25
M. SCG neurons were perfused at 5-10 ml/min at 32°C with an
external solution consisting of (in mM): NaCl
120, KCl 3, HEPES 5, NaHCO3 23, glucose 11, MgCl2 1.2, CaCl2 2.5, and
tetrodotoxin (TTX) 0.0005, pH 7.4. Cells were voltage-clamped at
approximately 25 mV to preactivate
IK(M) using a switching amplifier
(Axoclamp-2A; Axon Instruments, Foster City, CA; switching frequencies
3-5 kHz, filter 0.1 kHz). IK(M) was
measured from the slow deactivation relaxation after a 1 sec jump to a
command potential of approximately 55 mV (Haley et al., 1998a), and
inhibition was measured as the fractional reduction in the amplitude of
this deactivation relaxation in response to either cumulatively
increasing concentrations of oxotremorine methiodide (Oxo-M; Research
Biochemicals, Natick, MA) or a single application of 1 nM BK (Bachem, Torrance, CA) (see Fig. 2). Data were collected and analyzed using pClamp6 software (Axon Instruments) and expressed as mean ± SEM. Statistical analysis of the Oxo-M dose-response curves used two-way ANOVA comparing all treatments across all concentrations of agonist. If a significant effect of
treatment was found overall, further analysis was performed using
two-way ANOVA to determine which treatment groups contributed to this
significance. The bradykinin data were analyzed using Student's
t test with Welch's correction. p values < 0.05 were considered significant.
Reverse transcription PCR. RNA was extracted from rat SCGs
using RNAzol B (Biogenesis Ltd.) and reverse-transcribed using oligo-dT
and mouse murine leukemia virus reverse transcriptase (Promega,
Madison, WI). The oligonucleotide primers used in the PCR were those
that were deemed least conserved among the different PLC isoforms to
ensure specificity in the amplification. The primers are as follows
("u" denotes sequence in the 3' untranslated region, "s"
denotes sense primer, and "a" denotes antisense primer): PLC-1
u22 s/u199a; PLC-2 3111 s/3319a; PLC-3 3564 s/u45a; PLC-4 u5
s/u366a. Cycling conditions were 95°C for 5 min and then 35 cycles of
95°C for 30 sec, 58°C for 30 sec, and 72°C for 1 min followed by
a final extension step of 72°C for 10 min.
Immunocytochemistry. SCG neurons, cultured and injected as
described above, were fixed in acetone and stained using specific polyclonal antibodies against PLC-1 (sc-205), PLC-2 (sc-206), PLC-3 (sc-403), and PLC-4 (sc-404) (Santa Cruz Biotechnology, Santa Cruz, CA) diluted either 1:1000 or 1:500. Specificity of the
antibodies was confirmed by pre-absorbing the antibody with 6- to
10-fold excess (by weight) of the relevant immunogenic peptides (also
from Santa Cruz Biotechnology). All dishes of SCG neurons recorded in
the electrophysiology experiments were subsequently fixed and stained.
The alkaline phosphatase substrate system used was
5-bromo-4-chloro-3-indoxyl phosphate and nitroblue tetrazolium chloride
(BCIP/NBT) (Dako, Carpinteria, CA). Because the purple-blue product
was too dark to quantitate photometrically, we have assessed whether
there was an overall qualitative reduction in staining by comparing
each injected cell with its nearest uninjected neighbor and determining
(by eye) whether the level of staining was equal to or less than that
of the uninjected cell. Using this method we have estimated the
proportion of cells with a visible reduction in staining (regardless of
the magnitude of this reduction) 48 hr after injection of the antisense plasmid.
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RESULTS |
PLC- isoforms expressed in SCG
RT-PCR demonstrated the presence of mRNA for all four isoforms of
PLC- (1, 2, 3, and 4) in rat SCG (Fig.
1A) while the protein for all isoforms was detected immunocytochemically in cultured SCG
neurons. Intranuclear injection of antisense against PLC-1 and
PLC-4 resulted in a reduction in the level of staining for the
relevant enzyme 48 hr later (Fig. 1B,C). The PLC-1
antisense was highly effective and clearly reduced PLC-1 staining in
37 of 53 neurons (70%; n = 7 dishes of cells) without
altering levels of PLC-2 (1 of 12 cells showed reduced staining;
8%; n = 3 dishes), PLC-3 (1 of 12 cells; 8%;
n = 3 dishes), or PLC-4 (0 of 13 cells; n = 2 dishes). The PLC-4 antisense was less
effective but still reduced visible PLC-4 staining in 12 of 32 cells
(38%; n = 10 dishes of cells). Although the PLC-4
antisense was designed to specifically target the PLC-4 isoform, it
also reduced levels of PLC-1 (11 of 21 cells; 52%;
n = 6 dishes) but did not alter PLC-3 staining (1 of
9 cells; 11%; n = 3 dishes) or PLC-2 staining (1 of
21 cells; 5%; n = 2 dishes). Antisense-encoding
plasmids were also designed against the remaining PLC- isoforms, but
neither the PLC-2 antisense (2 of 39 cells; 5%; n = 5 dishes) nor the PLC-3 antisense (1 of 9 cells; 11%;
n = 5 dishes) reduced staining of its respective
protein. Electrophysiological data with these ineffective antisense
clones is not shown although, as expected, they did not alter
IK(M) modulation by Oxo-M or BK,
confirming that injection of DNA plasmids per se does not alter
modulation of IK(M).
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Figure 1.
A, RT-PCR indicating the presence
of mRNA for PLC-1, PLC-2, PLC-3, and PLC-4 in rat SCG
neurons. Lanes labeled M are size markers; lane
B is a blank where no template was included in the PCR
reaction. B, SCG neuron injected with antisense against
PLC-1 (along with a fluorescent marker; left
panel) and stained for PLC-1 (right
panel). C, SCG neuron injected with
PLC-4 antisense and stained for PLC-4.
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Effect of PLC- antisense plasmids on
IK(M) inhibition by a muscarinic agonist and
by bradykinin
Intranuclear injection of antisense against PLC-1 or PLC-4
did not alter the amplitude of IK(M).
Mean values (in picoamperes per picofarad) ± SEM were:
uninjected cells, 2.91 ± 0.41 (n = 8); PLC-1
antisense, 2.39 ± 0.52 (n = 10); and PLC-4
antisense, 2.33 ± 0.36 (n = 10). The resting
membrane potential was not altered in cells with reduced PLC-1 or
-4 levels (uninjected: 58.1 ± 2.0 mV, n = 10; PLC-1 antisense: 60.3 ± 1.9 mV, n = 13;
PLC-4 antisense: 58.8 ± 1.5 mV, n = 11).
Neither antisense affected M1 mAChR-mediated
inhibition of IK(M) tested 48 hr later
(when a reduction in the levels of these enzymes was observed). The
dose-response curves to the muscarinic agonist Oxo-M in PLC-1 or
PLC-4-depleted cells were not significantly different from one
another or from that in uninjected neurons: IC50
values and Hill slopes were 0.4 µM and 1.1 for
uninjected neurons, 0.8 µM and 1.0 for PLC-1
antisense-injected neurons, and 0.5 µM and 1.1 for PLC-4 antisense-injected cells (Figs. 2, 3). In
contrast, inhibition by BK was reduced in cells injected with the
PLC-4, but not PLC-1 antisense-expressing plasmid: 1 nM BK produced 28.5 ± 6.8% inhibition in
uninjected cells (n = 8), 30.8 ± 7.9% in
PLC-1 antisense-injected neurons (n = 10), but only
12.5 ± 2.0% inhibition in PLC-4 antisense-injected cells (n = 10; p < 0.05 compared with
uninjected or PLC-1 antisense-injected neurons) (Fig.
4).
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Figure 2.
Time course of normalized
IK(M) amplitude during cumulatively
increasing concentrations of Oxo-M and a single application of
bradykinin, as indicated, for neurons injected with PLC-1 and
PLC-4 antisense plasmids. IK(M) was
recorded every 5 sec (30 sec during wash-out). Each Oxo-M concentration
was applied for 1 min, and bradykinin was applied for 1.5 min. Oxo-M
and bradykinin were applied to the same neurons, and wash-out of Oxo-M
continued during the break in the x-axis.
IK(M) amplitudes were renormalized before
bradykinin application to eliminate any differences in recovery from
the Oxo-M application.
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Figure 3.
PLC-1 and PLC-4 antisense do not alter
inhibition of IK(M) by Oxo-M. Left
panel, Dose-response curves (mean ± SEM, plus best fit
curve) for Oxo-M inhibition of IK(M) in
antisense plasmid-injected SCG neurons. There is no significant
difference between neurons injected with PLC-1 antisense
(n = 11), PLC-4 antisense (n = 10), and the uninjected neurons (n = 10).
Right panel, Typical IK(M)
deactivation relaxations elicited by a 30 mV step for 1 sec from a
holding potential of 25 mV, in the absence and presence of 1 and 10 µM Oxo-M for cells injected with either PLC-1 or
PLC-4 antisense plasmids (as indicated). The dotted
lines represent 0 pA, and the calibration bars
indicate 250 pA.
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Figure 4.
Inhibition of IK(M) by
bradykinin is attenuated in neurons injected with PLC-4 antisense.
Left panel, Mean data ± SEM for inhibition of
IK(M) by 1 nM BK. Neurons
injected with PLC-4 antisense produced significantly less inhibition
compared to either uninjected or PLC-1 antisense plasmid-injected
neurons (*p < 0.05). Right panel,
Typical IK(M) waveforms after a 1 sec step
in the presence and absence of 1 nM bradykinin with either
PLC-1 or PLC-4 antisense plasmid-injected neurons (as indicated).
The dotted lines represent 0 pA, and the
calibration bars represent 250 pA.
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DISCUSSION |
The principal inference to be drawn from these experiments is that
activation of PLC-4 probably contributes to the inhibition of
IK(M) produced by stimulating BK
receptors, but not to that produced by activating
M1 muscarinic acetylcholine receptors. This
differentiation accords with, and amplifies, previous conclusions of
Cruzblanca et al. (1998) regarding the differential participation of
the PLC pathway in M-current inhibition after stimulation of these two
G protein-coupled receptors.
RT-PCR clearly demonstrated the presence of mRNA for PLC-1, -2,
-3, and 4 in rat SCG. Staining of cultured SCG neurons using
specific antibodies also confirmed the presence of these proteins.
Using antisense-encoding DNA plasmids we were able to reduce the levels
of PLC-1 and PLC-4 in SCG neurons. Although both PLC-1 and
PLC-4 have been shown to exist as two splice variants (Bahk et al.,
1994; Kim et al., 1998), the antisense sequences we have used should
target both variants of each enzyme. Indeed, the PLC-1 antisense
reduced PLC-1 staining in both the cytosol and the nucleus of
cultured SCG neurons (Fig. 1B), suggesting that
levels of both splice variants were reduced because, in C6Bu-1 cells,
PLC-1a has been shown to be present mainly in the cytosol, whereas
PLC-1b was present in the nucleus (Bahk et al., 1998). As we found
previously with antisense against Gq and
G11 (Haley et al., 1998a), not all the
antisense sequences designed were effective, and the constructs driving
expression of antisense PLC-2 and antisense PLC-3 were unable to
reduce staining of their target proteins.
Although the PLC-1 antisense resulted in a robust reduction of
PLC-1 levels (Fig. 1B), there was no reduction in
either Oxo-M or BK inhibition of
IK(M). By contrast, the PLC-4
antisense reduced BK inhibition of
IK(M) but left Oxo-M inhibition
unaltered. This difference between BK and Oxo-M is unlikely to be
caused by the fact that Oxo-M is a more "efficacious" inhibitor of
IK(M) (in the sense that it produced a
larger maximum inhibition), because the response to even a low
concentration (0.3 µM) of Oxo-M that matched
that produced by BK was unaffected by the PLC-4 antisense (Fig. 3).
Also, the concomitant reduction in PLC-1 staining produced by the
PLC-4 antisense is unlikely to be responsible for the loss of BK
inhibition because the PLC-1 antisense produced a greater and more
consistent reduction in PLC-1 protein levels yet did not alter this
response. At the very least, therefore, it is reasonable to conclude
that BK inhibition of IK(M) requires PLC-4 activation to a demonstrably greater extent than Oxo-M inhibition. In this respect, our results are in harmony with the conclusions of Cruzblanca et al. (1998) that inhibition of
IK(M) by BK, but not by Oxo-M,
involves PLC activation.
In the absence of positive controls, we cannot make any firm conclusion
from the negative effect of PLC-1 antisense for example, it is
possible that sufficient PLC-1 protein remained after antisense depletion to continue to drive IK(M)
inhibition. Likewise, because antisense constructs against PLC-2 or
PLC-3 did not reduce the levels of the cognate proteins, we cannot
make any conclusions regarding the roles of these two isoforms in
IK(M) inhibition. Nevertheless, a
potential selective involvement of PLC-4 in the action of BK would
provide an interesting correlation with the results of our previous
experiments using overexpressed  and constitutively active
-subunits (Haley et al., 1998a), showing that it is the subunit
of Gq or G11, not the
subunit, that mediates inhibition of
IK(M), because, of the four isoforms
of PLC, it is only PLC-4 that is solely activated by the -subunit and not by -subunits (Singer et al., 1997).
At first sight, our data (and also those of Cruzblanca et al., 1998)
would seem to be in conflict with those of Hildebrandt et al. (1997),
who found that an inhibitor of PLC did not alter BK inhibition of the
IK(M)-like current in NG108 cells.
Recent advances in our understanding of the channels underlying the
IK(M), however, provide a possible
explanation for this discrepancy. It is now thought that the
IK(M)-like current in NG108 cells
actually comprises two separate currents: the first has similarities
with IK(M) in SCG and probably results
from KCNQ2/3 channel activity (cf. Wang et al., 1998), whereas the
second, slower current, is pharmacologically distinct and probably
results from merg channel activity (Selyanko et al., 1999).
The "SCG-like" (KCNQ) IK(M) in
NG108 cells is sensitive to blockade by TEA (Selyanko et al., 1999):
because the experiments presented in Hildebrandt et al. (1997) were all
performed in the presence of TEA, they were probably examining BK
inhibition of the merg based
IK(M)-like current, which may be
inhibited by a different mechanism from
IK(M) in SCG.
IK(M) channels in SCG neurons can be
inhibited by high concentrations of intracellular
Ca2+ (Selyanko and Brown, 1996). Because
BK can increase intracellular Ca2+ levels
in rat SCG neurons (Cruzblanca et al., 1998; del Rio et al., 1999), it
is possible that BK may inhibit IK(M)
by stimulating PLC-4 to generate IP3 and
thereby mobilizing Ca2+ from internal
stores (Cruzblanca et al., 1998). We attempted to determine whether BK
induced Ca2+ rises in SCG require PLC-4
activation but found that the Ca2+ rises
we detected were small and very variable, making assessment of any
antisense effect impossible (our unpublished data).
M1 mAChR mediated inhibition of
IK(M) is unlikely to involve
Ca2+ as a messenger because agonists at
this receptor do not appear to raise internal
Ca2+ levels in SCG (Cruzblanca et al.,
1998). Even when modest rises in internal
Ca2+ levels have been observed (e.g.,
under conditions where the cells are depolarized) these rises are
not correlated with inhibition of
IK(M) (del Rio et al., 1999).
Furthermore, buffering of intracellular Ca2+ by the
Ca2+ chelator BAPTA attenuates BK
inhibition of IK(M), but leaves Oxo-M
inhibition intact (Cruzblanca et al., 1998).
Thus, both the experiments of Cruzblanca et al. (1998) and those
described in the present paper lead to the conclusion that the
inhibition of M-current in rat SCG neurons produced by stimulating muscarinic or bradykinin receptors proceeds via different intracellular pathways. Because both receptors can couple to the same family of
G-proteins and because both are intrinsically capable of inducing inositol phosphate production within these neurons (del Rio et al.,
1999), the reason for this divergence in M channel signaling is not yet
clear. Nevertheless, it highlights the point that M channels may be
regulated by more than one mechanism.
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FOOTNOTES |
Received May 23, 2000; revised Aug. 8, 2000; accepted Aug. 10, 2000.
This work was supported by the Wellcome Trust and the United Kingdom
Medical Research Council. We thank Dr. Patrick Delmas for helpful discussions.
Correspondence should be addressed to Prof. David Brown, Department of
Pharmacology, University College London, Gower Street, London WC1E 6BT,
UK. E-mail: d.a.brown{at}ucl.ac.uk.
Ms. Vallis's present address: Laboratory for Molecular Biology,
Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, UK.
Dr. Buckley's present address: School of Biochemistry and Molecular
Biology, University of Leeds, Leeds LS2 9DT, UK.
This article is published in
The Journal of Neuroscience, Rapid Communications Section,
which publishes brief, peer-reviewed papers online, not in print. Rapid
Communications are posted online approximately one month earlier than
they would appear if printed. They are listed in the Table of Contents
of the next open issue of JNeurosci. Cite this article as:
JNeurosci, 2000, 20:RC105 (1-5). The
publication date is the date of posting online at
www.jneurosci.org.
| |
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Copyright © 2000 Society for Neuroscience 0270-6474/00/$05.00/0
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ABSTRACT
INTRODUCTION
