Delayed rectifier and Ca2+-dependent K+ currents in human esophagus: roles in regulating muscle contraction

Gregory R. Wade1, Lisanne G. Laurier2,3, Harold G. Preiksaitis1,2,3, and Stephen M. Sims1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 at -70°C until isolation of RNA.

Electrophysiology. 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 MOmega , 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), and beta -actin genes (GenBank accession number M10278; Ref. 25) were used to design specific PCR primers (Primer Designer). Actin primers were designed to span an intron so that genomic DNA contamination of the samples could be assessed. Because of the close sequence homology between Kv1.2 and Kv1.5 and other members of the Kv channel family, nonhomologous regions were targeted for primer selection. For Kv1.2 the upstream primer was 5'-AGACCACGAGTGCTGTGAGA-3'; the downstream primer was 5'-GGAATAGGTGTGGAAGGTCA-3' (corresponding to nucleotides 81-618; predicted PCR product size of 538 bp). For Kv1.5 the upstream primer was 5'-GTGTAACGTCAAGGCCAAGAGCAAC-3'; the downstream primer was 5'-AGACAGAGGCTTGGAGACACAGGAA-3' (nucleotides 1909-2593; predicted size of 685 bp). For beta -actin the upstream primer was 5'-CACTCTTCCAGCCTTCCTTC-3'; the downstream primer was 5'-CTCGTCATACTCCTGCTTGC-3' (nucleotides 820-1133; predicted size of 314 bp).

PCR reactions were carried out for 40 cycles in a GeneAmp 2400 PCR thermal cycler (Perkin-Elmer, Norwalk, CT) using 2.5 mM MgCl2, 0.2 mM dNTPs, 100 µM of primers, and 0.2 µl of Taq DNA polymerase (Taq Gold, Perkin-Elmer). Each cycle was 0.5 min at 94°C, 0.5 min at 58°C, and 1 min at 72°C, followed by 7-min extension at 72°C. PCR products (13 µl) were analyzed by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining. Product identity was confirmed by restriction digest.

Muscle 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 10-6 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 1.   Outward currents in human esophageal smooth muscle cells. A: series of overlapping traces illustrate currents elicited by prolonged depolarization, in which outward current peaked and then decayed to a steady-state level. Sustained current exhibited increased noise at more positive voltages. On a faster time scale, delayed activation of initial current was apparent, with delay to peak reduced at more positive potentials (inset). Transient current is delayed rectifier K+ (KV) current and sustained current is Ca2+-dependent K+ (KCa) current. B: current-voltage relationship is plotted for peak outward and sustained currents, recorded using voltage protocols as in A. Sustained current was measured as the average over 200 ms, 4 s after the onset of depolarization. Values are means ± SE for currents recorded in 5 cells. C: voltage-dependent inactivation of KV was assessed using conditioning pulses of 5 s from -90 to 0 mV, followed by test pulses to 10 mV. Transient current was inactivated with depolarizing prepulses. D: summary of activation and inactivation of K+ currents. Voltage dependence of inactivation of KV current was determined as in C and is plotted as a fraction of peak current (I/Ipeak; black-down-triangle  indicates means ± SE, n = 5 longitudinal muscle cells). Conductance of KV was fitted with a Boltzmann relation, with half-maximal inactivation at -42 mV. Conductance of KCa () was determined as current at positive potentials when stepping from a holding potential of 0 mV. Conductance of KV () was determined from peak current recorded on depolarization from -90 mV to various potentials and corrected by subtraction of KCa. Conductance of KV was fitted with a Boltzmann relation with half-maximal activation at 4 mV and slope factor of 13 mV. Traces are from longitudinal muscle cells.

The transient current was apparent with step depolarization to potentials more positive than -40 mV, peaking within 100-200 ms at 0 mV (Fig. 1A, inset), then declining with persistent depolarization. The time course of decay varied among the cells studied, with longer decay apparent in some other cells (see Fig. 2). Based on delayed activation and inactivation, we refer to this current as a KV current. The sustained current was apparent at more positive potentials and accompanied by additional noise on the current traces, suggestive of the large-conductance KCa channel. The current-voltage relationship for the two outward currents indicated different voltage sensitivities (Fig. 1B). The transient current exhibited voltage-dependent inactivation, which was assessed by recording outward current at 10 mV after 5-s conditioning pulses (Fig. 1C), with half-maximal inactivation at -43 mV (Fig. 1D). The voltage dependence of activation of KV and KCa is plotted as conductance (Fig. 1D), assuming a K+ equilibrium potential of -84 mV. KCa conductance was determined from the mean outward current elicited by step depolarization of cells from a holding potential of 0 mV to potentials from 10 mV to 50 mV. (The persistent depolarization of cells to 0 mV for >2 min inactivated the transient current, permitting isolation of the KCa current; see Fig. 2B.) The conductance of KV was determined in the same cells from peak outward current elicited by step depolarization from a holding potential of -90 mV to potentials from -60 mV to 40 mV after subtraction of KCa current. The conductance of KV plotted in Fig. 1D was fit by a Boltzmann equation with half-maximal activation at 4 mV. The overlap of the inactivation and activation curves for the KV conductance suggests the presence of a "window current."

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.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 2.   KV and KCa currents in longitudinal and circular esophageal smooth muscle layers. A: KV current was elicited with depolarization from a holding potential (Vhold) of -60 mV to test potentials more positive than -20 mV in cells from longitudinal and circular layers, as indicated. B: persistent depolarization (>2 min) of same cells as in A to 0 mV inactivated KV, with remaining noisy current elicited by depolarization due to KCa.

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).


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 3.   Pharmacological separation of KV and KCa currents in human esophagus. A: depolarization- elicited KV and KCa currents (control). KV current was apparent at less positive potentials and declined with time. Further depolarization of cells revealed KCa current as noisy traces. Tetraethylammonium (TEA, 2 mM) reduced KCa, leaving the transient current. Concentration dependence of TEA is shown at right. Effect of TEA on KCa was evaluated by step depolarization to 50 mV from a holding potential of 0 mV. TEA caused half-maximal inhibition at ~0.1 mM TEA. B: 4-aminopyridine (4-AP, 5 mM) blocked KV, leaving only noninactivating, noisy KCa current. The concentration dependence of 4-AP blockade of KV was evaluated by step depolarization to 0 mV from a holding potential of -60 mV (right). 4-AP caused half-maximal inhibition of the peak current at ~3.3 mM 4-AP. Solid lines indicate best fit curves, with number of cells studied indicated in parentheses near each point. Traces in A are from circular and in B are from longitudinal muscle cells.

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).


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 4.   Charybdotoxin (ChTX) and iberiotoxin (IbTX) block KCa current. KCa was isolated by holding cells at 0 mV and stepping positively. A: both ChTX (300 nM, left) and IbTX (200 nM, right) reduced outward current compared with control, with concentration dependence shown in B. C: depolarization from -60 mV to 80 mV elicited both KV and KCa currents. ChTX (300 nM, left) and IbTX (100 nM, right) reduced a sustained component of outward current, with no effect on transient current. Traces are from longitudinal muscle cells.

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.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5.   Spontaneous transient outward currents (STOCs) in esophageal muscle. A: K+ channel blocker TEA (5 mM) reversibly abolished STOCs recorded at 0 mV, with recovery at right after 1-min wash. B: STOCs were also blocked by ChTX (100 nM), consistent with involvement of KCa channels. C: current-clamp recording from human esophageal cells revealing spontaneous transient hyperpolarizations. Periodic injection of current (10 pA) also hyperpolarized cell. TEA (4 mM) abolished spontaneous transient hyperpolarizations and depolarized cells.

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 beta -actin to monitor the fidelity of the PCR reaction and to screen for genomic DNA contamination of the sample.


View larger version (66K):
[in this window]
[in a new window]
 
Fig. 6.   Kv1.2 and Kv1.5 K+ channel mRNA expressed in circular and longitudinal layers of human esophageal smooth muscle. A: RNA was extracted from tissues as described in METHODS. After RT, cDNA was probed using primers specific for the coding regions of Kv1.2 and Kv1.5. PCR amplification revealed products of expected size for circular muscle (CM) and longitudinal muscle (LM), as visualized on an ethidium bromide-stained agarose gel. For control lanes, no cDNA was added to reaction mixture. For positive controls, mRNA was isolated from human heart and colon, tissues previously demonstrated to express Kv1.2 and Kv1.5. B: similar results were obtained using RNA from 3 different preparations of dispersed muscle cells (see methods), confirming localization of Kv1.2 and Kv1.5 message to esophageal smooth muscle.

To exclude the possibility that RNA transcripts originated from cells other than esophageal smooth muscle (see Ref. 30), we carried out RT-PCR using RNA isolated from purified esophageal smooth muscle cells. Transcripts from Kv1.2 and Kv1.5 were also identified in three different cell preparations (Fig. 6B).

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).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 7.   Effects of K+ channel blockers on nerve-mediated contraction of human esophagus. Muscle contraction was monitored during periodic field stimulation of muscle strip (0.5-ms pulses, 10 Hz, 5 s, at times indicated by upward-pointing arrows). A: 4-AP increased amplitude of nerve-mediated contractions and elicited oscillatory contractions. Reversibility of effects was apparent during periods of washout (wash). B: TEA caused a concentration-dependent increase in both amplitude and duration of nerve-evoked contractions. Effects of TEA were also reversible (wash). Responses illustrated are from strips isolated from circular muscle layer.

However, we could not exclude the possibility that these K+ channel blockers acted presynaptically to alter neurotransmitter release from endogenous nerve varicosities. To assess the direct effects of K+ channel blockers on muscle, atropine (1 µM) and TTX (1 µM) were included in the bathing solution to block neuronal action potentials and muscarinic receptor activation. Myogenic excitation was evoked with a single broad-pulse electrical stimulation (see methods). Under these conditions, 4-AP caused a concentration-dependent increase in the baseline tension but did not cause an increase in the peak amplitude of the electrically evoked contractions (Fig. 8A). As summarized in Table 1, 4-AP increased baseline tension in all strips tested from both circular and longitudinal muscles. Although the increased tension produced by 4-AP was greater in longitudinal muscle than in circular, the control electrically evoked responses were also greater, precluding direct comparison between the muscle layers. TEA increased the amplitude and duration of electrically evoked contractions and also caused a small increase in the baseline tension in both layers, although the change in resting tension was only half of that seen with 4-AP, (Fig. 8B and Table 1). To quantify the effect of TEA on the prolongation of contraction, we measured the area under the tension trace (standardized using the control response), revealing half-maximal augmentation of contraction with 1.9 mM TEA for circular muscle and 2.9 mM TEA for longitudinal muscle (Fig. 8C; values were not significantly different). This augmentation by TEA occurred in both longitudinal and circular muscle (Table 1) and suggested a role for TEA-sensitive channels in limiting depolarization of esophageal muscle. The precise identity of the channels will require blockade by more selective pharmacological agents. Blockade of L-type Ca2+ channels with nifedipine (10 µM) reduced the tension elicited by electrical stimulation under control conditions as well as in the presence of TEA (n = 4; Fig. 8D), consistent with a role for K+ channels in limiting depolarization and activation of voltage-dependent Ca2+ channels.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 8.   Myogenic contraction of human esophageal smooth muscle. Inclusion of atropine (1 µM) and TTX (1 µM) abolished nerve-mediated contractions and a single broad-pulse stimulus (500 ms, applied at times indicated by upward-pointing arrows) caused direct activation of smooth muscle. A: 4-AP increased resting tension with no consistent change in the twitch contraction in response to electrical stimulation. B: in contrast, TEA caused a concentration-dependent increase in amplitude and duration of contraction, with little change in resting tension. C: augmentation of myogenic contraction by TEA was quantified as change in area of contractile responses, standardized relative to control response. Solid lines indicate best fit by a logistic equation, revealing half-maximal augmentation at 1.9 mM for circular muscle () and 2.9 mM for longitudinal muscle (). Data represent mean responses from 8 circular and 6 longitudinal muscle strips. D: blockade of L-type Ca2+ channels with nifedipine (10 µM) abolished TEA-mediated augmentation of contraction.


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effects of K+ channel blockers on evoked and resting tension

We also noted that 4-AP and TEA caused spontaneous contractions in a portion of the esophageal strips, similar to that described previously in opossum esophagus (12, 13). 4-AP caused oscillatory contraction in five of six circular muscle strips and zero of seven longitudinal muscle strips, with "high-frequency" oscillatory contractile activity shown in Fig. 9A. TEA caused a lower frequency oscillatory contractile activity in five of eight circular muscle strips and only one of eight longitudinal muscle strips (Fig. 9B).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 9.   Examples of K+ channel blockers causing oscillatory contraction of circular esophageal muscle. A: 4-AP caused elevation of resting tension and high-frequency oscillatory contractions. B: TEA increased resting tension and caused low-frequency oscillatory contractions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adda, S., B. K. Fleischmann, B. D. Freedman, M. Yu, D. W. P. Hay, and M. I. Kotlikoff. Expression and function of voltage-dependent potassium channel genes in human airway smooth muscle. J. Biol. Chem. 271: 13239-13243, 1996[Abstract/Free Full Text].

2.   Akbarali, H. I. K+ currents in rabbit esophageal muscularis mucosae. Am. J. Physiol. 264 (Gastrointest. Liver Physiol. 27): G1001-G1007, 1993[Abstract/Free Full Text].

3.   Akbarali, H. I., N. Hatakeyama, Q. Wang, and R. K. Goyal. Transient outward current in opossum esophageal circular muscle. Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G979-G987, 1995[Abstract/Free Full Text].

4.   Boyle, J. P., M. Tomasic, and M. I. Kotlikoff. Delayed rectifier potassium channels in canine and porcine airway smooth muscle cells. J. Physiol. (Lond.) 447: 329-350, 1992[Abstract].

5.   Carl, A. Multiple components of delayed rectifier K+ current in canine colonic smooth muscle. J. Physiol. (Lond.) 484: 339-353, 1995[Abstract].

6.   Carl, A., N. G. McHale, N. G. Publicover, and K. M. Sanders. Participation of Ca2+-activated K+ channels in electrical activity of canine gastric smooth muscle. J. Physiol. (Lond.) 429: 205-221, 1990[Abstract].

7.   Cayabyab, F. S., and E. E. Daniel. K+ channel opening mediates hyperpolarizations by nitric oxide donors and IJPs in opossum esophagus. Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G831-G842, 1995[Abstract/Free Full Text].

8.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

9.   Curran, M. E., G. M. Landes, and M. T. Keating. Molecular cloning, characterization, and genomic localization of a human potassium channel gene. Genomics 12: 729-737, 1992[Medline].

10.   Fleischmann, B. K., R. J. Washabau, and M. I. Kotlikoff. Control of resting membrane potential by delayed rectifier potassium currents in ferret airway smooth muscle cells. J. Physiol. (Lond.) 469: 625-638, 1993[Abstract].

11.   Hart, P. J., K. E. Overturf, S. N. Russell, A. Carl, J. R. Hume, K. M. Sanders, and B. Horowitz. Cloning and expression of a Kv1.2 class delayed rectifier K+ channel from canine colonic smooth muscle. Proc. Natl. Acad. Sci. USA 90: 9659-9663, 1993[Abstract].

12.   Helm, J. F., S. L. Bro, W. J. Dodds, S. K. Sarna, and G. Hoffman. Myogenic mechanism for peristalsis in opossum smooth muscle esophagus. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G953-G959, 1992[Abstract/Free Full Text].

13.   Helm, J. F., S. L. Bro, W. J. Dodds, S. K. Sarna, G. Hoffman, and R. C. Arndorfer. Myogenic oscillatory mechanism for opossum esophageal smooth muscle contractions. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G377-G383, 1991[Abstract/Free Full Text].

14.   Hurley, B. R., H. G. Preiksaitis, and S. M. Sims. Characterization and regulation of large conductance Ca2+-dependent K+ channels in human esophageal smooth muscles. Am. J. Physiol. 276 (Gastrointest. Liver Physiol. 39): G843-G852, 1999[Abstract/Free Full Text].

15.   Jan, L. Y., and Y. N. Jan. Cloned potassium channels from eukaryotes and prokaryotes. Annu. Rev. Neurosci. 20: 91-123, 1997[Medline].

16.   Jury, J., K. R. Boev, and E. E. Daniel. Nitric oxide mediates outward potassium currents in opossum esophageal circular smooth muscle. Am. J. Physiol. 277 (Gastrointest. Liver Physiol. 40): G932-G938, 1999.

17.   Kirber, M. T., E. F. Etter, J. J. Singer, F. S. Fay, and J. V. Walsh, Jr. Ca++ sparks from protein kinase C dependent intracellular stores in esophageal circular smooth muscle cells (Abstract). Gastroenterology 112: A761, 1997.

18.   Koh, S. D., K. M. Sanders, and A. Carl. Regulation of smooth muscle delayed rectifier K+ channels by protein kinase A. Pflügers Arch. 432: 401-412, 1996[Medline].

19.   Kuriyama, H., K. Kitamura, T. Itoh, and R. Inoue. Physiological features of visceral smooth muscle cells, with special reference to receptors and ion channels. Physiol. Rev. 78: 811-920, 1998[Abstract/Free Full Text].

20.   McCobb, D. P., N. L. Fowler, T. Featherstone, C. J. Lingle, M. Saito, J. E. Krause, and L. Salkoff. A human calcium-activated potassium channel gene expressed in vascular smooth muscle. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H767-H777, 1995[Abstract/Free Full Text].

21.   Muraki, K., Y. Imaizumi, T. Kojima, T. Kawai, and M. Watanabe. Effects of tetraethylammonium and 4-aminopyridine on outward currents and excitability in canine tracheal smooth muscle cells. Br. J. Pharmacol. 100: 507-515, 1990[Abstract].

22.   Murray, J. A., E. F. Shibata, T. L. Buresh, H. Picken, B. W. O'Meara, and J. L. Conklin. Nitric oxide modulates a calcium-activated potassium current in muscle cells from opossum esophagus. Am. J. Physiol. 269 (Gastrointest. Liver Physiol. 32): G606-G612, 1995[Abstract/Free Full Text].

23.   Nelson, M. T., H. Cheng, M. Rubart, L. F. Santana, A. D. Bonev, H. J. Knot, and W. J. Lederer. Relaxation of arterial smooth muscle by calcium sparks. Science 270: 633-637, 1995[Abstract].

24.   Overturf, K. E., S. N. Russell, A. Carl, F. Vogalis, P. J. Hart, J. R. Hume, K. M. Sanders, and B. Horowitz. Cloning and characterization of a Kv1.5 delayed rectifier K+ channel from vascular and visceral smooth muscles. Am. J. Physiol. 267 (Cell Physiol. 36): C1231-C1238, 1994[Abstract/Free Full Text].

25.   Ponte, P., S. Y. Ng, J. Engel, P. Gunning, and L. Kedes. Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta -actin cDNA. Nucleic Acids Res. 12: 1687-1696, 1984[Abstract].

26.   Quayle, J. M., M. T. Nelson, and N. B. Standen. ATP-sensitive and inwardly rectifying potassium channels in smooth muscle. Physiol. Rev. 77: 1165-1232, 1997[Abstract/Free Full Text].

27.   Ramashwami, M., M. Gautam, A. A. Kamb, B. Rudy, M. A. Tanouye, and M. K. Mathew. Human potassium channel genes: molecular cloning and functional expression. Mol. Cell. Neurosci. 1: 214-223, 1990.

28.   Russell, S. N., K. E. Overturf, and B. Horowitz. Heterotetramer formation and charybdotoxin sensitivity of two K+ channels cloned from smooth muscle. Am. J. Physiol. 267 (Cell Physiol. 36): C1729-C1733, 1994[Abstract/Free Full Text].

29.   Sarna, S. K., E. E. Daniel, and W. E. Waterfall. Myogenic and neural control systems for esophageal motility. Gastroenterology 73: 1345-1352, 1977[Medline].

30.   Schmalz, F., J. Kinsella, S. D. Koh, F. Vogalis, A. Schneider, E. R. Flynn, J. L. Kenyon, and B. Horowitz. Molecular identification of a component of delayed rectifier current in gastrointestinal smooth muscles. Am. J. Physiol. 274 (Gastrointest. Liver Physiol. 37): G901-G911, 1998[Abstract/Free Full Text].

31.   Sims, S. M., Y. Jiao, and H. G. Preiksaitis. Regulation of intracellular calcium in human esophageal smooth muscles. Am. J. Physiol. 273 (Cell Physiol. 42): C1679-C1689, 1997[Abstract/Free Full Text].

32.   Sims, S. M., M. B. Vivaudou, C. Hillemeier, P. Biancani, J. V. Walsh, Jr., and J. J. Singer. Membrane currents and cholinergic regulation of K+ current in esophageal smooth muscle cells. Am. J. Physiol. 258 (Gastrointest. Liver Physiol. 21): G794-G802, 1990[Abstract/Free Full Text].

33.   Snetkov, V. A., S. J. Hirst, C. H. Twort, and J. P. Ward. Potassium currents in human freshly isolated bronchial smooth muscle cells. Br. J. Pharmacol. 115: 1117-1125, 1995[Abstract].

34.   Vogalis, F., and K. M. Sanders. Characterization of ionic currents of circular smooth muscle cells of the canine pyloric sphincter. J. Physiol. (Lond.) 436: 75-92, 1991[Abstract].

35.   Vogalis, F., T. Vincent, I. Qureshi, F. Schmalz, M. W. Ward, K. M. Sanders, and B. Horowitz. Cloning and expression of the large-conductance Ca2+-activated K+ channel from colonic smooth muscle. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G629-G639, 1996[Abstract/Free Full Text].

36.   Vogalis, F., M. Ward, and B. Horowitz. Suppression of two cloned smooth muscle-derived delayed rectifier potassium channels by cholinergic agonists and phorbol esters. Mol. Pharmacol. 48: 1015-1023, 1995[Abstract].

37.   Wade, G. R., H. G. Preiksaitis, and S. M. Sims. Ca2+ and K+ channels in human esophageal smooth muscle (Abstract). Biophys. J. 72: A353, 1997.


Am J Physiol Gastroint Liver Physiol 277(4):G885-G895
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society