CCK regulates nonselective cation channels in guinea pig gastric smooth muscle cells

Beibei Wang and Stephen M. Sims

Department of Physiology, The University of Western Ontario, London, Ontario, Canada N6A 5C1

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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.

acetylcholine; G proteins; muscarinic receptors; patch clamp

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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) (GTPgamma 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 MOmega , 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 MOmega . 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.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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 approx 70% of original length, with recovery to approx 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 approx 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 approx 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 approx 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.

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.

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.

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.

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 (approx 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 approx 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.

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.