Departments of 1 Anatomy and Neurobiology and 4 Pharmacology, The University of Vermont, Burlington, Vermont 05405; 2 Department of Physiology, University of Extremadura, 10071 Cáceres, Spain; and 3 Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557
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ABSTRACT |
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The current study was undertaken to test the existence and possible role of ether-a-go-go-related gene 1 (ERG1) protein K+ channels in gallbladder smooth muscle (GBSM). Transcripts encoding ERG1 were detected in human, mouse, and guinea pig GBSM, and ERG1 immunoreactivity was observed in GBSM cells. In intracellular voltage recordings, addition of E-4031 (100 nM-1 µM) or cisapride (100 nM-2 µM) caused concentration-dependent excitation of guinea pig GBSM that was not affected by 500 nM TTX + 5 µM atropine, and E-4031 also depolarized the resting membrane potential. In muscle strip studies, E-4031 either induced phasic contractions or significantly increased the amplitude of phasic contractions in spontaneously active tissues (P = 0.001). E-4031 also potentiated bethanechol-induced contractions. In conclusion, ERG1 channels are expressed in the GBSM, where they play a role in excitation-contraction coupling probably by contributing to repolarization of the plateau phase of the action potential and to the resting membrane potential.
biliary motility; rapidly activated delayed rectifier; E-4031; cisapride
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INTRODUCTION |
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GALLBLADDER DISEASE IS
TYPICALLY accompanied by decreased gallbladder motility
(18). However, the currents that underlie gallbladder
smooth muscle (GBSM) activity remain subjects of investigation. Intracellular voltage recordings from intact guinea pig GBSM have revealed characteristic spontaneous action potentials that occur with a
frequency of ~0.3 Hz (27). These myogenic action
potentials have four distinct components: from a resting membrane
potential of 40 to
50 mV, the cells exhibit a rapidly depolarizing
and repolarizing spike; this is followed immediately by a more slowly developing and declining plateau (27). The spike results
from rapid activation and inactivation of L-type voltage-dependent calcium channels, whereas 4-aminopyridine-sensitive voltage-dependent K+ channels (Kv channels) contribute to
repolarization of both the spike and the plateau (27).
Nevertheless, repolarization still occurs despite Kv
channel blockade, thus indicating that other conductances play an
important role in repolarizing the plateau of these action potentials.
Although cardiac muscle is histologically and physiologically distinct from smooth muscle, this tissue also displays action potentials with characteristic spike and plateau phases (16). Repolarization of the plateau in cardiac cells involves at least two conductances, rapidly activating (IKr) and slowly activating delayed rectifier K+ currents (13). Molecular studies have revealed that IKr involves the product of the ether-a-go-go-related gene (erg1) (21). The ERG1 protein is a member of the Kv family of K+ channels whose unique gating properties can allow it to pass more current during repolarization than depolarization (19). Heterologous expression of ERG1 together with the accessory protein, Min-K-related protein (also called KCNE2), results in currents almost identical to native IKr (1, 24) Moreover, both IKr and ERG1 currents are blocked by similar concentrations of the methanesulfonanilide drug E-4031 (10, 28). A growing number of other drugs has also been found to block these currents. For example, the gastrointestinal prokinetic drug cisapride can block ERG1 channels (14) in addition to its action as an agonist of 5-HT4 receptors (3, 12). Cisapride has also been studied in vivo and ex vivo as a potential prokinetic drug in gallbladder (8, 23). However, most such work has interpreted its action to be via 5-HT4 receptors.
ERG1 has also been suggested to function in the regulation of smooth muscle activity in the rat stomach and in the circular muscle of the opossum esophagus (2, 17). We set out to determine whether ERG1 channels are expressed in GBSM, whether inhibition of these channels alters spontaneous electrical activity of GBSM, and whether blockade of ERG1 channels affects basal tone and/or receptor-activated contractions of gallbladder muscle strips.
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METHODS |
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Tissue preparation. Adult guinea pigs of either sex were anesthetized with isoflurane and killed by exsanguination. This method has been reviewed and approved by the Institutional Animal Care and Use Committee. The abdominal cavity was opened, and the gallbladder was removed and transferred to cold, oxygenated (95% O2-5%CO2) Krebs solution (in mM: 121 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, and 8 glucose) as previously described (27). Briefly, under a dissecting microscope, the gallbladder was opened from the cystic duct to the base, then stretched and pinned mucosa-side up on a Sylgard 184 elastomer (Dow Corning, Midland, MI)-coated dish, and the mucosa was gently removed. These preparations of GBSM were then used for intracellular recording, immunohistochemistry, or RNA isolation as described below or further processed to isolate smooth muscle cells for RNA isolation. For cell isolation, the tissue was rinsed in dispersion medium (in mM: 10 HEPES, 10 glucose, 6 KCl, 55 NaCl, 80 Na-glutamate, and 2 MgCl2), cut into smaller pieces, and incubated with agitation for 34 min at 37°C in dispersion medium containing 1 mg/ml papain and 1 mg/ml dithioerythritol. Tissues were then transferred to dispersion medium containing 1 mg/ml collagenase and 0.1 mM CaCl2 for 10 min at 37°C with agitation. Pieces of tissue were triturated with a blunt-tipped pipette, and the resulting suspension was placed on the stage of an inverted microscope where ~100-150 isolated gallbladder smooth muscle cells were collected.
Adult mice of either sex were anesthetized with CO2 and exsanguinated, as approved by the Institutional Animal Care and Use Committee. The mucosa was removed, and RNA was immediately extracted from the muscularis layer, as described below. Normal human gallbladder specimens were obtained from liver transplant donors at the Royal Victoria Hospital of the McGill University Medical Center. This protocol was approved by the Institutional Review Board for Human Research. The muscularis was dissected from 0.5-mm2 specimens, placed in RNAlater (Ambion, Austin, TX), and stored at 4°C for up to 2 wk before RNA extraction.RT-PCR. RNA from GBSM tissues or isolated GBSM cells was purified with Tri-Reagent (Sigma, St. Louis, MO) using the manufacturer's protocol. For isolated cells, 240 µg polyinosinic acid (Sigma-Aldrich, St. Louis, MO) was also added before RNA purification as a carrier to visualize the precipitate. Reverse transcription was performed with Superscript II (Invitrogen, Carlsbad, CA). Primers were designed spanning 242- to 251-bp segments of ERG1 from guinea pig, mouse, and human {guinea pig, accession no. U75211, nucleotides (nt) 29-48 [5'-tggtcatctacacggctgtc-3'] and nt 279- 260 [5'-gatgagaaaccagcccttga-3']; mouse, accession no. AF012868, nt 1583-1602 [5'-tggtcatctacacggcagtc-3'] and nt 1832-1813 [5'-atgagaaaccagcccttgaa-3']; human, accession no. U04270, nt 1433-1452 [5'-tggtcatctacacggctgtc-3'] and nt 1674-1655 [5'-ccagcccttgaagtagtgga-3']}. The thermal cycler program for PCR amplification consisted of 1 min each at 96°C, 61°C, and 72°C for 35-50 cycles and a final extension at 72°C for 10 min. Products were resolved on 1% agarose gels and visualized with 1 µg/ml ethidium bromide.
Immunohistochemistry. Tissues for immunohistochemistry were fixed with 0.1 M sodium phosphate buffer containing 2% paraformaldehyde and 0.2% picric acid overnight at 4°C. After tissues were washed with PBS, they were preincubated for 1 h at room temperature with PBS + 0.1% Triton X-100 + 4% normal goat serum (PBS-Triton-NGS). They were then transferred to PBS-Triton-NGS with rabbit anti-ERG1 (1:50; Sigma) and mouse anti-PGP9.5 (13C4, UltraClone, Wellow, Isle of Wight, UK) or without primary antiserum (control) overnight at 4°C. Tissues were then rinsed in PBS-Triton (3×, 5 min each) and transferred to PBS-Triton-NGS + Cy3-conjugated goat anti-rabbit and fluorescein isothiocyanate-conjugated goat anti-PGP9.5 (1:400 each, Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 h at room temperature. After 3 more rinses (PBS, 5 min each), tissues were rinsed briefly in water and mounted on a slide with Citifluor (Citifluor, London, UK).
Intracellular recording.
GBSM preparations were pinned out in a Sylgard-lined recording chamber
with recirculating, oxygenated Krebs solution (10-12 ml/min) and
placed on the stage of an inverted microscope (Nikon Diaphot). Smooth
muscle bundles were visualized at ×200 with Hoffman Modulation
Contrast optics (Modulation Optics, Greenvale, NY). Temperature was
maintained at 35-37°C. Wortmannin (500 nM) was added to inhibit
tissue contractions without altering electrical activity
(4). Glass microelectrodes were filled with 2 M KCl and
had resistances from 60 to 110 M. Transmembrane potential was
measured with an Axoclamp 2A amplifier (Axon Instruments, Union City,
CA) and was recorded using MacLab hardware and software (AD
Instruments, Castle Hills, Australia). All drugs were added directly to
the bathing solution.
Muscle strip studies. GBSM preparations were typically dissected into four strips each. Strips were placed vertically in a 10-ml organ bath filled with oxygenated (95% O2-5% CO2) nutrient solution (in mM: 113 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, and 11.5 glucose) maintained at 37°C. Isometric contractions were measured using force displacement transducers interfaced with a Macintosh computer using MacLab hardware and software. Strips were placed under an initial resting tension equivalent to a 1.5-g load and allowed to equilibrate for 1 h with solution changes every 20 min. The effects of E-4031 were studied by addition of the drug to the organ bath in the presence of 1 µM TTX and 10 µM atropine to minimize potential neural actions of the drug. For experiments with bethanechol, atropine was not present and tension measurements were initially recorded at successively higher concentrations of bethanechol. The bethanechol was then washed out of the tissue before addition of either 500 nM or 1 µM E-4031, and tension measurements were repeated with each concentration of bethanechol.
Drugs. Stock solutions of wortmannin (1 mM; Sigma-Aldrich) and cisapride (1 mM; Research Diagnostics, Flanders, NJ) were prepared in dimethylsulfoxide. TTX (0.5 mM; Sigma-Aldrich) was dissolved in ethanol. Stock solutions of E-4031 (1 mM; Sigma-Aldrich), atropine (0.2 mM; Sigma-Aldrich), diltiazem (50 mM; Sigma-Aldrich), and bethanechol (1 mM; Sigma-Aldrich) were prepared in water.
Data analysis. Bursts were defined as excitatory events with extra spikes superimposed on the plateau and/or closely spaced action potentials that failed to remain repolarized for at least 0.5 s (see below). Duration of bursts was measured at the membrane potential halfway between the previous resting potential and the first plateau potential. Events were considered to be part of a single burst when the membrane potential did not remain below the halfway point for at least 0.5 s. Measurements of spike frequency were not possible, because the spikes often became less pronounced during prolonged bursts. Action potential frequency was not a useful description, because it was increased when bursts consisted of closely spaced action potentials and reduced when the plateau phase was prolonged. The measurements at a given drug concentration began 1 min after addition of the drug for a 4- to 5-min interval before addition of the next highest concentration. Least-squares nonlinear regression analysis of bursting data was used to create sigmoidal dose-response curves and calculate EC50 values with Prism v. 3.0a for Macintosh GraphPad Software (La Jolla, CA). Basal tension values were expressed in milliNewtons. Bethanechol-induced contractions were expressed as percentages of the maximal response. Each concentration-response curve was analyzed to calculate the EC50 by using GraphPad Prism software. Data are expressed as means ± SE, and statistical comparisons were made with ANOVA or two-tailed t-test, as appropriate.
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RESULTS |
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ERG1 expression in guinea pig GBSM.
RT-PCR was used to test for expression of erg1 RNA using primers that
span a 251-bp segment of the guinea pig erg1 gene from the
S1 to the S3 transmembrane domains. Primers that span
similar-sized segments of the comparable region in human and mouse
erg1 were also used to confirm its expression in other
species. Expression of erg1 was found in guinea pig heart
and the smooth muscle layers of the gallbladder as well as in human and
mouse gallbladder (Fig. 1A).
The localization of the transcripts in smooth muscle cells from guinea
pig gallbladder was confirmed by RT-PCR of RNA extracted from isolated
GBSM cells (Fig. 1A).
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Effects of ERG1 blockers on the electrical activity of GBSM.
Intracellular recordings were made from 21 GBSM cells in 21 preparations, with a mean resting membrane potential of 44 ± 1 mV. The properties of GBSM cells that were impaled in this study were
comparable with those reported in previous studies (27). Each spontaneous action potential comprised four phases: a rapid depolarization upstroke, a transient repolarization downstroke, a
plateau phase, and a complete repolarization back to the resting membrane potential. Spontaneous action potentials typically occurred at
a frequency of 0.3-0.4 Hz, although in some cells the normal low-frequency pattern of individual action potentials was occasionally disrupted by high-frequency "bursts" of action potentials (see below).
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Effect of E-4031 on contractility of GBSM strips.
Under resting tone conditions, E-4031 had excitatory effects on guinea
pig gallbladder strip contractility that were concentration dependent.
Application of E-4031 resulted in an increase in resting basal tone
(E-4031 100 nM: 1.24 ± 0.23 mN; 500 nM: 3.12 ± 0.59 nM; 1 µM: 4.06 ± 0.74 mN) and in the amplitude of phasic activity when it was present (E-4031 100 nM: 2.70 ± 0.61 vs. 3.63 ± 0.69 mN, 39.7% increase; P < 0.001, n = 7; E-4031 500 nM: 2.16 ± 0.62 vs. 3.86 ± 1.05 mN, 84.3%;
P < 0.05, n = 5; E-4031 1 µM:
1.64 ± 0.51 vs. 3.20 ± 0.72 mN, 112.9%; P < 0.01, n = 5; Fig. 5).
As demonstrated in Fig. 5, the increase in the amplitude of phasic contractions was associated with a decrease in their frequency. In some
strips, E-4031 induced phasic contractions when they were not present
under basal conditions (data not shown).
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DISCUSSION |
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The current study was conducted to determine whether ERG channels are expressed in GBSM and, if so, whether they contribute to the electrical and contractile activity of the gallbladder muscularis. We have shown that erg1 transcripts are expressed in GBSM and that GBSM bundles are ERG1-immunoreactive but blood vessels, ganglia, and nerve fibers are not. Moreover, pharmacological blockade of ERG1 channels caused excitatory "bursting" of action potentials in GBSM by delaying the repolarization of the plateau phase of the action potential. This is reminiscent of the excitatory afterdepolarizations that ERG1 blockers can cause in cardiac muscle (21, 22). In fact, current spread through extensive cell coupling is thought to suppress early afterdepolarizations in cardiac cells (20, 21, 26), and so the action potential bursting observed in GBSM may reflect an exaggerated response, reflecting more limited cell coupling in this tissue. It is also likely that ERG channels contribute to the resting membrane potential in GBSM, because application of the ERG-channel blocker E-4031 led to a depolarization of smooth muscle cells. As also reported for cardiac myocytes (7, 25), E-4031 both increased basal contractility of gallbladder muscle strips and enhanced their contractile response to receptor activation. Together, these data indicate an important role for ERG1 channels in regulation of excitation-contraction coupling in GBSM and suggest that ERG1 channels may be potential targets of receptor-activated events in these tissues.
In the current study, the EC50 values for the excitatory effects of ERG-channel blockers on intact GBSM cells were in the 0.5 to 1.0 µM range. These values are substantially higher than the values reported for their inhibition of IKr in dispersed cardiac muscle cells or heterologously expressed ERG1 in cultured cells (~10 nM) (10, 28). This may reflect more limited access of these blockers to ERG channels in intact tissues. Alternatively, it is also possible that the molecular composition of ERG1 channels in GBSM varies from that in the heart, thus rendering GBSM less sensitive to these drugs. Indeed, splice variants of ERG1 may give rise to channels with different properties (9, 11). It is also possible that as yet unidentified subunits of ERG1 channels may differentially regulate their function. In fact, controversy still exists as to whether all of the subunits that underlie IKr have been identified (24). Further studies will be required to distinguish between these possibilities.
Despite the induction of action potential bursting by both E-4031 and cisapride, these reagents did not act identically on GBSM. For example, whereas increasing concentrations of E-4031 continued to enhance both the percentage of bursting time and maximal burst time within the concentration range used, the maximal burst time induced by cisapride reached a plateau value of substantially shorter duration. This seems likely to reflect the broader range of targets for cisapride, because it is known to act as an agonist of 5-HT4 receptors to facilitate neurotransmitter release (3, 6, 12) and may have other, as yet uncharacterized, activity. Indeed, previous studies have often reported conflicting data regarding the effects of cisapride on gallbladder motility in vivo (23). Furthermore, at least one previous study has shown complex mechanical effects of cisapride on guinea pig GBSM strips that appear to involve both cholinergic and noncholinergic pathways (8). Complex electrophysiological responses of GBSM to cisapride were detected in experiments designed to test its effects on the resting membrane potential in the absence of action potentials (E. Parr and G. M. Mawe, unpublished data). These effects included transient depolarizations and hyperpolarizations that cannot easily be explained on the basis of ERG1 channel blockade alone and may have been junction potentials elicited by enhanced neurotransmitter release. Nevertheless, the excitatory effects of ERG blockers reported herein are unlikely to reflect neurogenic activity because the effects were not inhibited by addition of TTX and atropine. Furthermore, in those instances in which 5-HT4 receptors have been reported on smooth muscle, agonists act to relax the muscle (5, 15). In any event, the present study suggests that ERG1-channel blockade by cisapride is one more factor that affects the complex responses of the gallbladder.
The distribution of ERG channels in gastrointestinal smooth muscle has not been completely resolved. Recently, it has been reported that ERG1 expression in the rat gastrointestinal tract is primarily restricted to the stomach (17). Interestingly, those investigators also found that E-4031 depolarized isolated smooth muscle cells from the rat stomach, supporting a role for ERG1 channels in the maintenance of smooth muscle resting membrane potential. This also parallels the E-4031- and cisapride-evoked depolarizations of esophageal smooth muscle (2), and the present study indicates that this is also likely to be the case for guinea pig GBSM. On the other hand, this contrasts with cardiac preparations, in which E-4031 does not markedly alter the resting potential (25), perhaps due to the more negative resting potential in those tissues. Nevertheless, the gradual deactivation of ERG1 channels allows some current to persist following repolarization, and the decay of ERG1 currents during this interval has also been suggested to serve a potential role in the subsequent repolarization (28). Thus ERG1 might also contribute to pacing of action potentials in GBSM.
In conclusion, intact GBSM consistently generates rhythmic spontaneous action potentials that involve the openings and closings of at least two different types of ion channels, including dihydropyridine-sensitive Ca2+ channels and 4-aminopyridine-sensitive K+ channels. The findings reported here indicate that another class of K+ channel, the ERG1 K+ channel, is present in GBSM and contributes to the contour and rhythmicity of the action potential. When ERG1 channels are pharmacologically suppressed in GBSM, the membrane is depolarized by bursts of action potentials; these changes would lead to elevated Ca2+ entry into the cells. Consistent with this, ERG1-channel blockade led to increased contractility of gallbladder muscle strips. These findings support the view that ERG1 channels contribute to a steady-state modulation of GBSM electrical and contractile activities.
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ACKNOWLEDGEMENTS |
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We thank L. Ellis and Drs. B. Manning, D. Linden, and T. Firth of the Univ. of Vermont for technical assistance, and we are also thankful to Drs. J. Tchervenkov, A. Di Carlo, and G. Tzimas of McGill Univ. for providing human gallbladder tissue for these studies.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grant NS-26995 and McyT Grant SAF-2001-0295.
Address for reprint requests and other correspondence: G. M. Mawe, D403A Given Bldg., Dept. of Anatomy and Neurobiology, The Univ. of Vermont, Burlington, VT 05405 (E-mail: garymawe{at}uvm.edu).
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.
First published November 13, 2002;10.1152/ajpgi.00325.2002
Received 6 August 2002; accepted in final form 30 October 2002.
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