Department of Physiology, The University of Western Ontario, London,
Ontario, Canada N6A 5C1
CCK has widespread effects in the
gastrointestinal tract, stimulating pancreatic secretion and
contraction of smooth muscles. The cellular mechanisms by
which CCK causes smooth muscle contraction are poorly understood. We
investigated the effects of CCK on guinea pig gastric smooth muscle
cells using patch-clamp techniques. CCK caused contraction of cells
accompanied by inward current. The conductance activated by CCK was
nonselective for cations and showed little voltage dependence. Because
ACh also activates nonselective cation current, we examined
interactions between CCK and ACh. When CCK activated inward current,
ACh caused no further effect. When CCK failed to activate current,
subsequent ACh-activated current was larger and no longer exhibited its
characteristic voltage dependence. Intracellular dialysis with
guanosine
5'-O-(3-thiotriphosphate) caused
similar changes in the voltage dependence of the ACh-activated current,
suggesting a role for G proteins in regulation of the current.
Activation of nonselective cation current would depolarize muscle and
may contribute to the excitation mediated by CCK in tissues. These
findings provide evidence that multiple types of receptors converge to
regulate nonselective cation current.
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INTRODUCTION |
IN THE GASTROINTESTINAL TRACT, CCK has widespread
effects, acting through G protein-coupled receptors to stimulate
pancreatic secretion, cause contraction of gallbladder and gastric
smooth muscles, and inhibit gastric emptying (1, 7, 19, 28, 31).
Previous studies revealed that CCK caused contraction and increased the
frequency and duration of action potentials of antral smooth muscle
(21). The stimulatory effect was due to a direct effect of CCK on the
smooth muscle, since the effect of CCK was insensitive to atropine or
TTX and CCK caused contraction of isolated gastric smooth muscle cells
(2, 5, 7). However, the cellular mechanisms leading to regulation of
membrane potential in smooth muscle remain uncertain.
The action of CCK on ion channels is well described in pancreatic
acinar cells, in which CCK causes elevation of cytosolic free
intracellular Ca2+ concentration
([Ca2+]i)
and opening of Ca2+-activated
Cl
and nonselective cation
channels (26). Nonselective cation channels are present in many smooth
muscles and are activated in response to a variety of stimuli,
including neurotransmitters such as ACh or peptides (18, 24). Inward
current through nonselective cation channels causes depolarization of
the cell membrane, resulting in opening of voltage-dependent
Ca2+ channels and
Ca2+ influx, which can initiate
contraction (14, 20). Ca2+ also
facilitates opening of muscarinic nonselective cation channels, although receptor occupancy appears to be essential for channel opening
(3, 9, 11, 23, 27, 29).
Although CCK is known to cause contraction of smooth muscles, its
effects on ion channels are not well understood. We have used
patch-clamp recording to investigate the effects of CCK on ionic
currents and interactions between CCK and ACh. We provide evidence that
CCK can elicit nonselective cation current and can also alter the
voltage dependence of ACh-activated nonselective cation current. Thus
multiple receptor types converge to regulate the properties of
nonselective cation current in gastric smooth muscle.
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METHODS |
Isolation of gastric smooth muscle cells.
Smooth muscle cells were isolated from the guinea pig corpus and
antrum, using methods similar to those described previously (24, 27),
but with several modifications. Guinea pigs (300-350 g, either
sex) were killed by decapitation or by being stunned and then bled. The
stomach was removed and dissected, and strips of muscle were placed
into 2.5 ml of dissociation solution (see below for composition)
containing collagenase (0.4 mg/ml; Sigma Blend type F), papain (1 mg/ml; type IV), and
1,4-dithio-L-threitol (0.1 mg/ml) from Sigma Chemical (St. Louis, MO) and bovine albumin (1.2 mg/ml) from ICN Biomedicals (Cleveland, OH). Muscle strips were kept in
enzyme solution for 45 min at room temperature and then placed into a
gently shaking water bath at 31°C for 40-60 min, after which
cells were dispersed by trituration with fire-polished Pasteur
pipettes. Cells were studied within 5 h of dispersion.
Solutions and drugs.
Physiological saline solution (PSS) contained (in mM) 130 NaCl, 5 KCl,
1 CaCl2, 1 MgCl2, 20 HEPES, and 10 D-glucose, pH 7.4. In
experiments in which Na+ was
reduced in the bathing solution, we replaced
Na+ with
N-methyl-D-glucamine+
(NMG), with osmolarity maintained at 280-290 mosM and pH adjusted to 7.4 with NMG base. In cases in which
Ca2+ was increased in the bath
solution to 5 mM, NaCl was reduced to 125 mM to maintain osmolarity.
The dissociation solution contained (in mM) 135 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 0.25 EGTA, 10 D-glucose, and 10 L-taurine, pH set to 7.0 with
NaOH. The electrode solution contained (in mM) 130 CsCl (or KCl), 1 MgCl2, 20 HEPES, 1 EGTA, 10 tetraethylammonium, and 0.4 CaCl2
(estimated free Ca2+ of 100 nM),
pH set to 7.2 with CsOH (or KOH).
Low-Cl
electrode solution
was prepared by replacing CsCl with cesium glutamate.
The following stock solutions were prepared and kept frozen: sulfated
CCK-8 (100 µM; fragment 26-33) from Bachem Bioscience, guanosine
5'-O-(3-thiotriphosphate)
(GTP
S) (250 mM; tetralithium salt) from Calbiochem, and ACh (20 mM),
proglumide (10 mM), nystatin (100 µg/ml in dimethyl sulfoxide), ATP
(100 mM; magnesium salt), and GTP (100 mM; sodium salt) from Sigma
Chemical.
Electrophysiological recording.
All studies were performed at room temperature (22-25°C).
Recording of membrane current employed the nystatin perforated-patch or
whole cell patch configuration. Cells were allowed to settle and adhere
to the glass bottom of a recording chamber (bath vol, 0.75 ml) mounted
on the stage of an inverted microscope. The chamber was perfused with
PSS (1-3 ml/min). PSS was the solution used for all of the
following studies except where noted. For perforated-patch recording,
electrodes were filled at the tip with nystatin-free solution, then
backfilled with solution containing 250 ng/ml nystatin. Current
recording was initiated after the access resistance had stabilized at
<40 M
, whereupon >70% series resistance compensation was used.
For the whole cell configuration, 1 mM ATP and 1 mM GTP were added in
electrode solution. Recording electrodes had tip resistances of
2-5 M
. Recording of whole cell currents was initiated 1-3
min after cell membrane rupture, to allow time for diffusion of
compounds into cells. Membrane currents were recorded using an
Axopatch-1D or 200A patch-clamp amplifier (Axon Instruments, Foster
City, CA) interfaced to a computer using pClamp software. Current
signals were filtered at 500 Hz and recorded at 2 kHz, then stored on
disk. Cell capacitance and series resistance were compensated using the
amplifier circuitry.
Junction potentials between recording electrode and bath solutions were
measured using 3 M KCl reference electrodes (22). Liquid junction
potentials between the electrode and PSS were
2 mV for 130 CsCl
or KCl electrode solution and
10 mV for
low-Cl
solution (containing
43 mM Cl
). Voltage values
have been corrected for the liquid junction potentials. Values are
shown as means ± SD. All results shown are representative of
findings from at least three cells. Electrophysiological experiments
were carried out with cells from corpus and antrum (58 cells from
corpus and 97 cells from antrum). Electrophysiological responses for
corpus and antrum cells were essentially the same, so we have not
distinguished between the types of cells in the results.
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RESULTS |
CCK caused contraction of gastric smooth muscle cells.
Freshly dispersed guinea pig gastric smooth muscle cells were spindle
shaped, ranged in length from 50 to 150 µm, and showed bright-phase
regions around the cell periphery when viewed with phase-contrast
optics. A representative contraction is shown in Fig.
1A,
where CCK (50 nM) caused the cell to shorten to
70% of original
length, with recovery to
96% after washout of CCK for 5 min.
Maximum shortening occurred within 15-30 s of CCK application. CCK
caused contraction of cells essentially in an "all or none" manner (with the criteria for contraction being a reduction of cell
length >5%), so the concentration dependence was quantified as the
percentage of cells contracting in response to CCK at concentrations between 0.01 and 50 nM. Testing 40-50 bright-phase cells for each concentration of CCK (individual cells tested only once), we found that
the maximal percentage of cells contracting to high concentrations of
CCK (1, 10, and 50 nM) was 58%, and the concentration for half-maximal responses was
1 pM, based on the best fit of a sigmoidal curve to
the data (not shown). This value is close to that reported previously
for guinea pig gastric cells (7). Addition of the CCK receptor
antagonist proglumide (1 µM) to the bathing solution reduced the
maximum percentage of cells contracting to 22%, and the half-maximal
concentration of CCK was increased to 100 pM, consistent with the
contraction resulting from activation of CCK receptors.

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Fig. 1.
CCK elicits contraction and inward current in guinea pig gastric smooth
muscle cells. A: a series of video
images shows a typical cell before stimulation
(left) and 20 s after application of
CCK, when the cell was maximally contracted to 70% of its original
length (middle). CCK (50 nM) was
applied by pressure ejection from an application pipette (seen at left
in middle). Recovery to 96% of
initial length occurred after washout of CCK
(right, after 7 min). CCK-8S, sulfated
CCK-8. B: CCK (50 nM, applied for time
indicated by bar) elicited an inward current under voltage clamp using
the perforated-patch configuration, from a holding potential of
62 mV. Initiation of inward current correlated with onset of
cell contraction. Partial recovery occurred during washout, during
which time the cell relaxed. During the experiments, cells were
perfused with physiological saline solution (PSS) and recording
electrode contained CsCl to eliminate
K+ currents.
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Ionic currents were first recorded using the nystatin perforated-patch
technique. When cells were held at
62 mV, CCK (50 nM in
application pipette) induced inward current that partially recovered
during washout (Fig. 1B). Similar
current responses were seen in 11 cells where potential was held
steadily at
62 mV, with mean ± SD of peak inward current of
587 ± 116 pA.
Voltage-ramp commands (from
102 to +18 mV over 1 s) were used to
study the voltage dependence and reversal potential of the CCK-induced
current. As described above, CCK elicited inward current with a delay
of several seconds (Fig.
2A),
with recovery after washout (Fig. 2A,
right). Similar responses were
recorded in 48 cells with CsCl electrode solution and 5 cells with KCl
electrode solution, with approximately one-half of the cells studied
responding to CCK. Control experiments were carried out by applying
vehicle solution to the same cell, which caused no change in membrane currents (Fig. 2B,
n = 4). CCK activated a linear current
(ICCK) that
reversed direction at
1 ± 2 mV (Fig.
2C, n = 48). Most experiments were performed with CsCl electrode solution to
block contaminating K+ currents.
When K+ electrode solution was
used, the CCK-activated current showed similar features and reversed
direction close to 0 mV. These results are consistent with the
CCK-activated channel passing K+
as well as Cs+. The reversal
potential for CCK-activated current is close to the equilibrium
potential for Cl
(ECl), but
between the equilibrium potentials for
Na+
(ENa) and
K+
(EK),
suggesting that CCK might activate nonselective cation and/or
Cl
currents. To
characterize the activation range of the conductance elicited by CCK
(gCCK), we used the
equation gCCK = ICCK/(Vm
Vrev),
where Vm is
membrane potential and
Vrev is the
reversal potential of the current activated by the agonist. The chord
conductance activated by CCK was relatively constant over a range of
voltages (Fig. 2D), distinct from
that of the ACh-activated conductance, which increases sigmoidally with
depolarization (Refs. 9, 24, and see following).

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Fig. 2.
Voltage dependence of CCK-activated current. From a holding potential
of 62 mV, voltage ramps shifted membrane potential from
102 to +18 mV over 1 s. A: CCK
elicited inward currents with a latency of 5-20 s, with recovery
on washout of CCK (right). Currents
were averaged from traces indicated by braces.
B: control experiment was carried out
by applying vehicle (PSS) to the same cell, which did not cause change
of current. Cells were bathed in PSS, and recording electrodes were
filled with CsCl electrode solution.
C: CCK-activated current from
A, plotted as a function of voltage,
varied linearly with voltage, and the reversal potential was close to 0 mV. D: CCK-activated conductance was
weakly dependent on voltage. Traces were recorded using perforated
patch.
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Ion substitution was used to investigate the selectivity of the
CCK-activated current. When
ECl was shifted
from 0 to
30 mV using 43 mM intracellular
Cl
concentration, the
reversal potential shifted slightly from
1 ± 2 mV
(n = 48) to
6 ± 1 mV
(n = 11; Student's
t-test,
P > 0.05, not
significant). Thus
Cl
did not appear to
participate in the CCK-activated current. We next tested involvement of
Na+, replacing NaCl with NMG in
the bathing solution, whereupon the reversal potential of CCK-activated
current shifted from
6 ± 1 to
25 ± 6 mV in
Na+-free bathing solution (Fig.
3, n = 11). This shift of the reversal potential was significant (paired
Student's t-test,
P < 0.01) and reversible on
restoration of Na+ to the bath
solution. This suggested that CCK-activated current involved influx of
Na+ due to opening of nonselective
cation channels. To compare the degree of involvement of
Na+ in CCK and ACh-activated
cation current, we tested the ionic selectivity of ACh-activated
current under similar conditions. The reversal potential for
ACh-activated current was
3 ± 9 mV under control conditions
and shifted to
41 ± 7 mV (5 cells tested; paired Student's
t-test,
P < 0.01, data not shown) with
removal of extracellular Na+. The
reversal potential of the ACh-activated current in
low-Na+ solution was more negative
than the CCK-activated current (P < 0.05, Student's t-test). Assuming
that only a single channel type was activated in each case, the
permeability for CCK-activated current was calculated to be
Na+:K+:Cs+
of 1:1.1:1.3. When Na+ was
replaced with NMG, the permeability was
Na+:Cs+:NMDG
of 1:1.3:0.5. For ACh, the permeability was
Na+:Cs+:NMG
of 1:1.2:0.1. The difference observed between currents activated by CCK
and ACh may be due to involvement of distinct channels activated by CCK
or because CCK-activated current has different permeability for
cations.

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Fig. 3.
Extracellular Na+ concentration
influences reversal potential
(Vrev) of
CCK-activated current. Left:
current-voltage relationship of CCK-activated current for cell bathed
in PSS, where the current reversed direction close to 0 mV. On
replacement of bathing solution with
Na+-free solution
(Na+ was replaced with
N-methyl-D-glucamine+),
the reversal potential of CCK-activated current was shifted negative
(all traces from the same cell). The average reversal potential
(n = 11) shifted from 6 ± 1 mV (in PSS) to 25 ± 6 mV (in
Na+-free bathing solution). This
effect was reversible, with reversal potential returning to control
level on restoration of extracellular
Na+ concentration in the bath (not
shown). Low-Cl (43 mM Cl)
Cs electrode solution was used, and traces were recorded using
perforated patch.
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To study interactions between ACh and CCK, we examined the effects of
both agonists applied sequentially to the same cell. In cells in which
CCK activated inward cation current (as described above), subsequent
stimulation with ACh caused no further changes in currents (24 cells).
In other cells, described below, CCK did not activate detectable
membrane current or cause contraction, but the subsequent response to
ACh was altered. A sequence of responses is shown in Fig.
4 for a representative cell, where voltage-ramp commands were periodically applied to assess the voltage
dependence of the evoked currents. Initially, CCK was applied for 45 s
and caused little change in current for this cell. However, subsequent
stimulation with ACh elicited a large inward cation current (Fig.
4A, representative of 15 cells tested in this way). The current-voltage relationships for the ACh-evoked current were determined as the difference between control current and
that elicited by ACh, for the times indicated (Fig.
4A,
braces). Notably, prestimulation
with CCK resulted in ACh-evoked current that was nearly linear at
negative potentials (Fig. 4B,
trace 1). In contrast, the ACh-evoked
current seen after recovery from CCK (Fig.
4A,
brace
2) was nonlinear (Fig.
4B,
trace
2), which is typical of control
responses (10, 18, 25). CCK prestimulation did not alter the reversal
potential of the ACh-evoked current (Fig.
4B, 0 ± 7 vs.
1 ± 2 mV,
current in 15 CCK-treated cells vs. control ACh-evoked currents in 13 cells, respectively) or reduce the inward current at positive
potentials. In all cases, cell contraction accompanied the
development of inward current.

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Fig. 4.
CCK alters voltage dependence of ACh-activated nonselective cation
current. A: example of a cell in which
CCK (10 nM, 45 s) failed to induce detectable membrane current.
However, subsequent stimulation with ACh (20 µM) induced inward
current with reduced voltage sensitivity, apparent in the
current-voltage relationship in B.
ACh-activated current (trace 1) was determined as the difference
between control current (average of 3 ramps indicated by the unlabeled
brace in A) and current activated by
ACh after CCK pretreatment (brace 1). After several minutes of
recovery and washout, ACh elicited a smaller inward current with
typical voltage dependence (trace 2 in
B, from ramps indicated by
brace 2). Both currents reversed direction
close to 0 mV. C: with CCK
pretreatment, the typical sigmoid conductance activated by ACh showed
less voltage dependence. The recording electrode contained cesium
glutamate and 43 mM Cl, and bath solution contained 5 mM
Ca2+. Traces were recorded using
perforated patch.
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These features suggested that CCK pretreatment did not elicit a new
current but rather altered the voltage dependence of the ACh-activated
channels. When conductance was plotted as a function of voltage, the
control response showed the typical sigmoidal increase with
depolarization, whereas prestimulation with CCK resulted in the
ACh-activated conductance showing reduced dependence on voltage (Fig.
4C). The maximal conductance was
also larger after CCK pretreatment, which, combined with the reduced
voltage sensitivity, led to greater current at the holding potential
(Fig. 4A).
The average values of ACh-evoked currents and conductance (normalized
for capacitance of the cells to account for differences in cell size)
are presented in Fig. 5. The control data
represent responses of cells exposed only to ACh, where inward current
was reduced at negative voltages and the conductance was voltage
sensitive (
25% of the maximal conductance active at
100 mV;
Fig. 5, n = 11). The effects of CCK on
the ACh-evoked current were graded, so that stimulation of cells with
CCK for 20-30 s (n = 3) caused a
small increase in the ACh-evoked conductance, which still exhibited voltage dependence. Longer stimulation with CCK (45-60 s,
n = 8) caused a greater increase in
the conductance elicited by ACh combined with reduced voltage
sensitivity, so that
50% of the conductance remained active at
100 mV (Fig. 5B). In all
cases, we quantified only the first response to ACh, to allow for
suitable comparison among groups of cells. To test for reversibility,
we allowed a 5-min interval between CCK and ACh (rather than the brief,
10- to 20-s intervals used in the experiments described above), after
which ACh elicited typical nonlinear current-voltage relationships,
indicating reversibility of the response to CCK.

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Fig. 5.
Average values of ACh-evoked currents and conductance.
A: control data represent responses of
cells exposed only to ACh, where inward current was elicited that
reversed direction close to 0 mV and was reduced at negative voltages
(n = 11). CCK prestimulation increased
the amplitude of ACh-activated current with little change of the
reversal potential. B: conductance in
control cells was voltage sensitive, with only 25% of the maximal
conductance active at 100 mV. The effects of CCK on the
ACh-evoked current were graded, so that stimulation of cells with CCK
for 20-30 s (n = 3) caused a
small increase in the ACh-evoked conductance, which still exhibited
considerable voltage dependence. Longer stimulation with CCK
(45-60 s, n = 8) caused
a greater increase in the maximal conductance elicited by ACh, combined
with reduced voltage sensitivity, so that 50% of the conductance
remained active at 100 mV. Values of conductance were normalized for
capacitance of the cells. Data are from cells studied using
perforated-patch configuration.
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For control experiments, we prestimulated cells for 60 s with saline
solution followed by ACh. Under this condition, the ACh-evoked cation
current showed the typical voltage sensitivity (6 cells). Similarly,
when ACh was applied to the same cell repeatedly, the ACh-evoked
current was always voltage sensitive. We also considered the
possibility that the voltage sensitivity was related to the size or
time course of the current. Inspection of ACh-activated currents in
control cells revealed that the voltage sensitivity did not vary
greatly with the amplitude or the time course of the current (6 cells).
The
N-methyl-D-aspartate-activated
nonselective cation current in neurons shows similar voltage
dependence, due to Mg2+ blockade
(9). When nonselective cation current was elicited by ACh in
Mg2+-free solution, the current
exhibited the typical nonlinear shape, excluding involvement of
Mg2+ in determining the voltage
sensitivity (5 cells, not shown).
Previous studies by Zholos and Bolton (32) demonstrated that G protein
activation influences the voltage sensitivity of the muscarinic
nonselective cation current in longitudinal ileum. Using
similar strategies, we investigated whether the concentration of ACh
influenced the properties of nonselective current in gastric smooth
muscle. A representative experiment is depicted in Fig. 6, in which 2 µM ACh was first applied,
evoking cation current with a nonlinear current-voltage relationship
(Fig. 6, A and
B). Subsequent stimulation with 50 µM ACh then elicited a larger inward current (Fig.
6A), which reversed direction at
close to the same potential (Fig.
6B). However, the current activated
by a higher concentration of ACh had a more linear current-voltage
relationship (Fig. 6B), and the
conductance showed little sensitivity to voltage (Fig.
6C). Thus muscarinic regulation of
the nonselective cation channel in gastric muscle resembles that
described earlier for ileal muscle (32). The results of Fig. 6 are
representative of observations in 22 cells where a higher concentration
was applied from 5 to 20 s after the first stimulation. In other
instances (10 cells), initial stimulation with 1-10 µM ACh
caused cells to become unresponsive to subsequent stimulation with ACh,
suggesting desensitization of receptor signaling.