1 Departments of Physiology and 2 Medicine, The University of Western Ontario, and 3 Lawson Research Institute, St. Joseph's Health Centre, London, Ontario, Canada N6A 5C1
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ABSTRACT |
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We have examined K+ channels and their function in human esophageal smooth muscle using perforated patch recording, RT-PCR to identify channel mRNA, and muscle contraction to study the effects of channel blockers. Depolarization revealed at least two types of currents: a 4-aminopyridine (4-AP)-sensitive transient delayed rectifier K+ (KV) and a Ca2+-dependent K+ (KCa) current. KCa current was active at positive potentials and was blocked by tetraethylammonium (TEA), iberiotoxin, and charybdotoxin but was insensitive to 4-AP. The mRNA encoding the gene products of Kv1.2 and Kv1.5 was identified in muscle and dissociated cells, consistent with these channel types contributing to KV current. 4-AP increased resting tension of muscle strips, suggesting a role for KV in setting the membrane potential. TEA, but not 4-AP, augmented the amplitude and duration of electrically evoked contraction, effects that were abolished by nifedipine. Here we provide the first description of macroscopic K+ currents in human esophagus. KV channels participate in regulation of resting tension, whereas the KCa channel limits depolarization and contraction during excitation.
patch clamp; reverse transcriptase-polymerase chain reaction; contraction; Kv1.2; Kv1.5
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INTRODUCTION |
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A VARIETY OF INWARDLY RECTIFYING, voltage-, Ca2+-, and ATP-dependent K+ channels play key roles in controlling the excitability of smooth muscles (19, 26). In visceral smooth muscles, including gastrointestinal (34) and airway (10, 21), studies support an important role for channels of the delayed rectifier family in the regulation of resting membrane potential. Studies of spontaneous transient outward currents suggest the large-conductance Ca2+-dependent K+ (KCa) channel can also participate in the regulation of membrane potential and excitability of smooth muscle (23).
K+ channels are encoded by a superfamily of K+ channel genes (15). Recent studies have determined the molecular identity of some voltage-dependent K+ channels from smooth muscle, including a number of delayed rectifier channel types from the Shaker family. For example, mRNA for Kv1.1, Kv1.2, and Kv1.5 has been identified in visceral and vascular smooth muscles (1, 11, 24). Additionally, a voltage-dependent channel from the shab family (Kv2.2) was recently cloned and expressed from colonic smooth muscle (30). KCa channels have been cloned from vascular and visceral smooth muscles (20, 35).
Studies of esophageal muscle from a number of animal models have identified several types of K+ channels. Transient delayed rectifier K+ (KV) and KCa currents have been observed in circular muscle from opossum esophagus and rabbit esophageal muscularis mucosae (2, 3, 16). Blockade of a transient outward K+ current in opossum esophageal muscle cells with 4-aminopyridine (4-AP) caused depolarization, leading Akbarali and co-workers (3) to conclude that this current contributes to setting the resting membrane potential. In early studies of opossum esophagus, tetraethylammonium (TEA), a blocker of KCa currents, was shown to cause development of myogenic contractions (12, 13, 29). At the time of those studies, the range of K+ channel types expressed in esophageal muscle had not yet been described, and still little is known of their role in human esophagus.
The objectives of this study were to characterize K+ currents in cells from the body of human esophagus and to examine their functional role in regulation of contraction. We have identified two types of K+ currents, a KV current and a KCa current. mRNAs encoding the Kv1.2 and Kv1.5 genes were identified. Functional studies of contraction of human smooth muscle strips revealed that K+ channels serve distinct roles in the regulation of esophageal contraction. Portions of this work have been presented in abstract form (37).
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METHODS |
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Tissue retrieval and isolation of cells. Tissue collection was carried out in accordance with the guidelines of the University Review Board for Research Involving Human Subjects and conformed to the Helsinki Declaration. Patient consent was obtained for removal of tissues. Tissues were obtained from 30 patients undergoing esophageal resection because of cancer. On resection, specimens were immediately cooled on ice. A sample of the entire thickness of the muscularis propria (~1 cm2) was removed from a disease-free region of the distal third of the esophagus (referred to as the esophageal body) and placed in ice-cold, oxygenated Krebs solution (see Solutions) for transport to the laboratory. Portions of muscle were dissected for preparation of dispersed cells and others for tissue strip studies (see Muscle contraction).
Muscle cells were dispersed as previously described (31). Briefly, segments of esophagus (~1 mm wide by 1 cm length) were placed in 2.5 ml of dissociation solution consisting of (in mM) 5 NaCl, 135 KCl, 0.5 CaCl2, 1 MgCl2, 20 HEPES, and 10 D-glucose, plus 0.2 mg/ml collagenase (Sigma blend type F), 2 mg/ml BSA (ICN Biomedicals), 2.5 mg/ml papain, 0.4 mg/ml 1,4-dithio-L-threitol, 10 mM taurine, and 0.5 mM EDTA, adjusted to pH 7.0 with NaOH. Tissues were stored in dissociation solution at 4°C overnight. The following day, tissues were warmed to room temperature and then placed in a gently shaking water bath at 31°C for 60 min. Tissues were rinsed with warm Ringer solution (31°C) and dispersed by gentle trituration with fire-polished pipettes. Cells used for RNA isolation (see RT-PCR) were filtered through a 210-µm mesh and washed with ~4 ml of sterile ice-cold PBS by repeated centrifugation (1,000 rpm for 5 min at 4°C). The cell pellet was resuspended in 250 µl of medium 199, layered on Percoll, and sedimented at 30,000 g for 15 min. The cells formed a clearly visible band that was collected and stored atElectrophysiology.
Dispersed cells were allowed to settle and adhere to the bottom of a
1-ml perfusion chamber mounted on the stage of a Nikon inverted
microscope and were perfused with bathing solution at 1-3 ml/min.
Electrophysiological studies were performed at room temperature
(22-25°C) within 8 h of dispersion. Whole cell recordings of
current employed the nystatin perforated-patch technique using an
Axopatch 1D amplifier (Axon Instruments, Foster City, CA). Recording
was initiated when the access resistance had stabilized at <20 M,
and series resistance compensation up to 80% was often used. Currents
were filtered at 500 Hz and sampled at 2 kHz. Capacitive currents were
compensated online using amplifier circuitry and linear leakage
corrected as assessed at negative potentials.
Solutions. Electrophysiological studies were performed with cells bathed in Ringer solution containing (in mM) 130 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 20 HEPES, and 10 D-glucose, adjusted to pH 7.4 with NaOH. The recording electrode solution contained (in mM) 30 KCl, 100 potassium aspartate, 10 NaCl, 20 HEPES, 1 MgCl2, 1 EGTA, and 0.4 CaCl2, adjusted to pH 7.2 with NaOH. Electrodes were filled at the tip with filtered solution and then back-filled with solution containing 250 µg/ml nystatin. Krebs bicarbonate solution contained (in mM) 116 NaCl, 5 KCl, 2.2 NaH2PO4, 25 NaHCO3, 1.2 MgSO4, 2.5 CaCl2, and 10 D-glucose, equilibrated with 5% CO2-95% O2.
RT-PCR. Total RNA was isolated from human esophageal longitudinal and circular muscle layers or from dispersed cells using the method of Chomczynski and Sacchi (8), with polyinosinic acid (20 µg) as a carrier. RNA samples were run out on 1% Tris-acetic acid-EDTA agarose to verify integrity. Two micrograms of total RNA from each sample were reverse transcribed for 60 min at 42°C using random hexamers and Superscript RNase H- (GIBCO BRL, Gaithersburg, MD). The cDNA was diluted 2.5 times, and 5-8 µl were used in each 50-µl PCR reaction.
The cDNA coding sequences for human Kv1.2 (GenBank accession number L02752; Ref. 27), Kv1.5 (GenBank accession number M83254; Ref. 9), andMuscle contraction.
Muscle strips were prepared as previously described
(31). Briefly, muscle strips (~2 × 10 mm) were dissected from
circular and longitudinal layers of the tissue, ensuring that cells
were oriented along the long axis. Strips were mounted in
water-jacketed tissue baths containing 10 ml of Krebs bicarbonate
solution and bubbled with 5%
CO2-95%
O2 at 37°C. One end of
each strip was attached to a Grass FT03 isometric force transducer
coupled to a Grass 79E chart recorder (Grass Instruments, Quincy, MA).
After 1-h equilibration, the tension of each strip was adjusted so that maximum tension was achieved with
106 M carbachol. Electrical
stimulation was applied via two platinum wire electrodes, encircling
the muscles 1 cm apart. To activate intrinsic nerves, 0.5-ms square
waves in 5-s trains were applied at 10 Hz and 40-60 V. Smooth
muscle cells are not significantly activated by 0.5-ms pulses because
of their longer time constant. Hence, to study myogenic responses,
single square-wave pulses of 500-ms duration were applied after
nerve-mediated responses were blocked by 1 µM TTX and 1 µM
atropine. Stimulus parameters were adjusted to give maximal contraction.
Chemicals. Unless otherwise stated, chemicals were from Sigma or BDH (Toronto, ON). Drugs were prepared from stock solutions in distilled water and diluted into the appropriate bathing solution. In the electrophysiology studies, drugs were applied focally to cells (Picospritzer II; General Valve, Fairfield, NJ) with the concentration reported being that in the application pipette. For the tissue strip studies, stock solutions of drugs were added directly to the bath to achieve the concentration reported.
Statistics. Values are the means ± SE. Comparisons were made using the Student's t-test, with P < 0.05 considered significant. Traces are representative of at least three experiments on muscle from two or more preparations.
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RESULTS |
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Freshly isolated human esophageal muscle cells were phase-dense, spindle-shaped, and from 50-150 µm in length. Cells isolated from circular or longitudinal layers were of similar size, with capacitance of 69 ± 5 pF in circular muscle cells (n = 11) and 61 ± 4 pF (n = 49) in longitudinal muscle cells, values that were not significantly different. Electrophysiological recording was carried out on 112 cells from 30 specimens.
Outward currents in freshly isolated human esophageal smooth muscle
cells.
When studied under voltage clamp, depolarization of cells with step
voltage commands elicited outward current that peaked and then declined
with time (Fig.
1A).
At more positive potentials, a sustained, noisier component became
apparent, suggesting the presence of at least two distinct
conductances. Outward current was abolished when CsCl was
substituted for KCl in the electrode solution (data not shown) and was
sensitive to several K+ channel
blockers, leading us to conclude that the outward currents were due to
opening of K+-selective channels.
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KCa and KV
currents from longitudinal and circular muscle cells.
We compared KV and
KCa currents from cells of the
longitudinal (Fig. 2,
left) and circular
(right) layers. The mean current density calculated from the peak
KV current elicited by step
depolarization from 60 mV to 0 mV was 2.7 ± 0.6 pA/pF for
longitudinal cells and 2.9 ± 0.4 pA/pF for circular cells
(n = 7), values that are not
significantly different. KCa
current was isolated in longitudinal (Fig
2B,
left) and circular
(right) cells by holding cells at 0 mV to inactivate KV currents. Mean
current density at 50 mV was 3 ± 0.5 pA/pF for longitudinal cells
(n = 8), less than that recorded in
circular cells (5.7 ± 0.9 pA/pF; n = 10, P < 0.05), a factor that may
contribute to functional differences between layers noted below.
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Pharmacological isolation of KV and
KCa currents.
K+ channel blockers were used to
distinguish components of the outward current. Step depolarization of
cells elicited a current with both transient and sustained components
(Fig.
3A). TEA
(2 mM) reduced the current noise and a steady component of the current. However, it is notable that the inactivating component of the current
was not affected, consistent with selective blockade of KCa current revealing the
transient current. In contrast, 4-AP (5 mM) reduced the inactivating
component of the current, whereas the noisy noninactivating
KCa current persisted (Fig.
3B). The concentration-dependent
blockade by TEA was determined from the amplitude of
KCa currents elicited by
depolarization to 50 mV from 0 mV (Fig.
3A,
right).
KCa was half-maximally blocked at 0.1 mM TEA as determined from the best fit of a logistic equation. The
concentration-dependent blockade of
KV current by 4-AP was determined
for currents elicited at 0 mV (to minimize contributions from
KCa), revealing half-maximal
inhibition at 3.3 mM (Fig. 3B, right).
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Selective reduction of KCa current by
peptide blockers charybdotoxin and iberiotoxin.
The sensitivity of esophageal K+
channels to the peptide channel blockers charybdotoxin (ChTX) and
iberiotoxin (IbTX) was investigated. When the sustained current was
studied in isolation (depolarization from holding potential of 0 mV),
both ChTX (300 nM) and IbTX (200 nM) reduced the amplitude and the
noise on the current trace (Fig. 4A),
consistent with the presence of the large-conductance
Ca2+ dependent
K+ channel. In contrast to that
seen with TEA, high concentrations of these peptide blockers blocked
the outward current incompletely (Fig.
4B). This finding raises the
possibility of additional K+
currents in these cells that are TEA sensitive and ChTX and IbTX insensitive. As expected, the transient current was insensitive to
IbTX. Notably, ChTX did not block the transient component of the
outward current (Fig. 4C),
indicating that the KV current in
human esophagus cannot be due solely to the gene products of Kv1.2,
which is blocked at low concentrations of ChTX (28).
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Spontaneous transient outward currents in human esophageal muscle.
Studies of KCa current presented
to this point revealed the current only at positive potentials.
However, we also found evidence for activation of
KCa currents at less positive
potentials, with spontaneous transient outward currents (STOCs)
apparent in 21 of the 112 cells studied. STOCs were abolished by TEA
(2-10 mM, n = 4) and ChTX (100 nM, n = 3; Fig.
5, A and
B). When studied in current clamp,
spontaneous transient hyperpolarizations were observed at negative
resting membrane potentials (n = 3;
Fig. 5C), likely due to STOCs.
Treatment of cells with TEA resulted in blockade of the spontaneous
events and depolarization (Fig. 5C,
right), consistent
with the idea that summation of STOCs can contribute a steady
hyperpolarizing influence on the membrane potential.
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RT-PCR identification of KV
channels.
Several families of KV channels
have been identified in gastrointestinal smooth muscle, including
Kv1.1, Kv1.2, and Kv1.5 (1, 11, 24). The
KV current in human esophagus was
insensitive to ChTX, suggesting that Kv1.2 alone does not account for
the delayed rectifier. Heteromultimers of Kv1.2 and Kv1.5 channels show
reduced sensitivity to ChTX (28), perhaps accounting for the
insensitivity of native delayed rectifier in esophageal cells. We
considered the possibility that coexpression of an insensitive delayed
rectifier channel contributed to ChTX insensitivity. RT-PCR amplification of RNA from circular and longitudinal layers of human
esophageal smooth muscle with primers specific for Kv1.2 and Kv1.5
yielded the predicted 538-bp and 685-bp products (Fig. 6). Similar results were obtained using
three different specimens of intact muscle tissue. Rat brain, human
heart, and human colon, tissues previously found to express Kv1.2 and
Kv1.5, were used as positive controls (11, 24). The cDNA samples were
also tested with primers for human -actin to monitor the fidelity of
the PCR reaction and to screen for genomic DNA contamination of the
sample.
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Functional effects of
K+ channel
blockers.
We investigated the effects of K+
channel blockers on contraction of muscle strips. Electrical
stimulation of circular muscle (see
METHODS) elicited contraction of
muscle strips (Fig.
7A, control). TTX (1 µM, n = 6; data not
shown) blocked this response, confirming the nerve-mediated
(neurogenic) basis of the excitation of the muscle. 4-AP (0.5-2
mM) caused a reversible increase in the peak contractile force of these
neurogenic contractions and also produced spontaneous oscillatory
contractions (Fig. 7A). Similarly,
TEA (1-4 mM) caused a reversible, concentration-dependent increase
in the amplitude, as well as prolongation, of neurogenic contractions
(Fig. 7B).
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DISCUSSION |
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Little is known of the expression and function of K+ channels in human esophageal muscle. With the use of freshly isolated smooth muscle cells, we identified KV and KCa currents. Functional studies of the effects of KV and KCa current blockers on human smooth muscle strips reveal distinct roles for these currents in the regulation of contraction. The KV current appears to play a dominant role in regulating resting tension of esophageal muscle, whereas KCa current largely limits contraction associated with excitation.
The transient current was identified as a delayed rectifier based on a
number of properties. This current was apparent with depolarization to
potentials more positive than 40 mV, greater in amplitude at more
positive membrane potentials, and delayed in both its onset and time to
peak. Plots of the voltage dependence of conductance and inactivation
overlapped, giving rise to a window current over a physiological range
of potentials. The transient current was blocked in a
concentration-dependent manner by 4-AP, with half-maximal inhibition at
3.3 mM, and was insensitive to TEA at 2 mM and lower concentrations.
This delayed rectifier was less sensitive to 4-AP than the cloned Kv1.2
channel, which was half-maximally inhibited at 74 µM 4-AP (11). This
difference may reflect differences between native and expressed
channels, or, as considered below, the
KV current we observed may be due to heteromultimeric channels. KV
currents with similar voltage-dependent characteristics and
pharmacological sensitivity have been characterized in a variety of
other smooth muscles, including airway (4, 10, 33) and gastrointestinal
muscles (5, 34). A similar K+
current was reported for opossum esophageal smooth muscle (3).
Kv1.2 transcripts have been found in several types of smooth muscles (1, 11). RT-PCR analysis of RNA isolated from human esophageal muscle revealed Kv1.2 in these studies. Currents from cloned Kv1.2 channels are sensitive to ChTX (27), in contrast to our findings for the KV currents in human esophagus. Coexpression of Kv1.2 with Kv1.5 channels gives rise to ChTX-insensitive heteromultimeric K+ currents (28). Indeed, as found in other smooth muscles (24), mRNA for Kv1.5 was also identified in human esophageal muscle, providing a possible explanation for the ChTX insensitivity of the KV currents here. More recent studies found evidence for Kv2.2 in several types of smooth muscles (30), raising the possibility that this too may be expressed in the esophagus. Studies of the quinine sensitivity of KV currents will help to resolve the presence of Kv2.2 and other delayed rectifiers in human esophageal tissues.
In addition to KV currents, we also identified a sustained outward current with characteristics of KCa. The current activated at positive voltages and was blocked in a concentration-dependent manner by TEA and the selective KCa peptide antagonist IbTX. These characteristics are consistent with the presence of large-conductance Ca2+-dependent K+ channels, which have been characterized at the single channel level in these cells (14). Both IbTX and ChTX failed to completely inhibit the sustained outward current, suggesting the presence of an additional conductance, possibly sensitive to blockade by TEA, that has not yet been characterized in these cells. KCa currents are identified in most smooth muscles and have been cloned and expressed from vascular and colonic muscles (20, 36). Although in voltage-clamp recordings macroscopic KCa currents were not active at resting potentials, STOCs and their corresponding transient hyperpolarizations were observed in some esophageal cells. These observations, combined with functional studies of the effects of TEA on contraction of muscle strips (discussed below), suggest that KCa can participate, in some cases, in the regulation of resting membrane potential, as described in other cell types (6). Elevation of Ca2+ concentration in restricted regions (Ca2+ sparks) due to release of Ca2+ from intracellular stores underlies STOCs in vascular and esophageal smooth muscle (17, 23). We have previously described the contribution of Ca2+ stores in cholinergic excitation of these muscles (31), as well as regulation of KCa channels (14). TEA-sensitive STOCs have previously been suggested to participate in the control of resting membrane potential in cat esophageal smooth muscle cells (32).
Functional roles for KV currents were investigated in muscle strips using channel blockers. We observed that 4-AP consistently caused an increase in resting tension, accompanied in some tissues by spontaneous oscillations. Although 4-AP also augmented nerve-mediated contraction, little effect was observed on myogenic contractions when nerve-mediated responses were blocked with TTX and atropine. Thus the multiple effects of 4-AP suggest that it acts to block K+ channels both pre- and postsynaptically. In any case, the increase in basal tension recorded under conditions in which nerve-mediated responses were blocked are most consistent with a role for KV currents in setting the resting membrane potential of human esophagus. Activity of K+ channels would limit opening of voltage-dependent Ca2+ channels and Ca2+ influx, which have previously been shown to contribute to excitation of these muscles (31). KV channels control resting membrane and muscle tone in a variety of muscles, including opossum esophagus (3) and airway muscles (1, 10, 21).
TEA-sensitive currents had distinct functional roles in regulating the contraction of human esophagus. Most notably, TEA caused marked augmentation of electrically evoked contractions, increasing both their peak tension and duration. The effects of TEA were concentration dependent, blocked by nifedipine, and not affected by blockade of nerves in the tissues. Together, these findings suggest a role for KCa channels in limiting excitation and contraction, although we cannot at present eliminate the possibility that TEA also blocks another component of the K+ current. Elevation of Ca2+ will initiate contraction, as well as activation of KCa channels, causing outward K+ current and hastening repolarization of the membrane, thereby limiting Ca2+ entry and contraction. Consistent with this model, KCa is activated during muscarinic excitation of human esophageal cells (14).
There are a number of limitations to these studies. We note that the concentration of TEA required to augment contraction was higher than that required to block KCa currents in single cells, raising the possibility that, at higher concentrations, TEA is blocking other channels as well. However, the fact that TEA in the majority of muscle strips did not alter baseline tension suggests that either it does not block KV channels to a large extent or that they are not active over the range of potentials found in resting muscle. We do not presently understand the reasons why a different concentration dependence of blockers was observed for reduction of currents in single cells compared with effects on contraction of tissues. It is possible that the blockers have limited accessibility in the intact tissues compared with cells. Also, because intact tissues exist as a syncytium by virtue of gap junctions, it may be necessary to achieve close to complete blockade of channels (requiring higher concentrations) to observe comparable effects on contraction of tissues.
Blockade of KCa with TEA in some strips did result in small increases in resting tension and spontaneous oscillatory contractions. This occurred most frequently in longitudinal muscle, indicating that KCa channels may in some cases participate in regulating the resting membrane potential and resting tension. Although macroscopic KCa currents were not apparent at resting potentials in unstimulated muscles, STOCs can contribute to setting the resting membrane potential (23, 32). KCa channels have previously been implicated in the control of esophageal smooth muscle. Although peristalsis is primarily a nerve-mediated phenomenon, TEA induces waves of contraction that propagate in both directions in an intact esophagus, even with intrinsic nerves blocked (12, 29). Although differences between circular and longitudinal layers have been noted in some smooth muscles, there were no qualitative differences between layers in esophageal muscle. We base this on the characteristics of cell capacitance, mRNA expression of delayed rectifiers, and functional effects on contraction of muscle strips. However, circular muscle did have a larger KCa current density, and muscle strips from this layer exhibited oscillatory contractions in response to K+ channel blockers, although the relationship between these two variables is not presently understood.
In summary, we have identified KV and KCa currents in human esophageal muscle and find they serve distinct physiological roles. K+ channels in many tissues are targets for modulation by inhibitory as well as excitatory factors. Agents that cause relaxation of esophageal muscle activate K+ currents, contributing to hyperpolarization. For example, nitric oxide activates the KCa current in opossum esophagus, contributing to inhibitory junction potentials (7, 22), and KV channels are activated by cAMP-dependent pathways (18). In addition, K+ currents are targets for suppression by excitatory pathways. Cholinergic excitation suppresses STOCs in esophageal muscle (32) and inhibits some cloned smooth muscle KV currents (36). Further studies are required to characterize the physiological regulation of K+ currents in human esophagus.
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ACKNOWLEDGEMENTS |
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We thank Yang Jiao for help in preparation of cells, Tom Chrones for advice regarding muscle strips, Tom Karkanis for help with blocker studies, and Drs. R. I. Inculet, R. A. Malthaner, C. Rajgopal, and S. E. Carrol for providing the esophagectomy specimens.
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FOOTNOTES |
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This work was supported by grants from the Medical Research Council of Canada and the PSI Foundation. H. G. Preiksaitis was supported by an Ontario Ministry of Health Career Scientist award, and S. M. Sims was supported by a Medical Research Council Scientist award.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. M. Sims, Dept. of Physiology, The Univ. of Western Ontario, London, Ontario, Canada N6A 5C1 (E-mail: sims{at}physiology.uwo.ca)
Received 8 March 1999; accepted in final form 19 July 1999.
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