Functional and molecular analysis of L-type calcium channels in human esophagus and lower esophageal sphincter smooth muscle
Jason R. Kovac,1
Harold G. Preiksaitis,1,2 and
Stephen M. Sims1
Departments of 1Physiology and Pharmacology and 2Medicine, The University of Western Ontario, London, Ontario, Canada
Submitted 30 November 2004
; accepted in final form 12 July 2005
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ABSTRACT
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Excitation of human esophageal smooth muscle involves the release of Ca2+ from intracellular stores and influx. The lower esophageal sphincter (LES) shows the distinctive property of tonic contraction; however, the mechanisms by which this is maintained are incompletely understood. We examined Ca2+ channels in human esophageal muscle and investigated their contribution to LES tone. Functional effects were examined with tension recordings, currents were recorded with patch-clamp electrophysiology, channel expression was explored by RT-PCR, and intracellular Ca2+ concentration was monitored by fura-2 fluorescence. LES muscle strips developed tone that was abolished by the removal of extracellular Ca2+ and reduced by the application of the L-type Ca2+ channel blocker nifedipine (to 13 ± 6% of control) but was unaffected by the inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase by cyclopiazonic acid (CPA). Carbachol increased tension above basal tone, and this effect was attenuated by treatment with CPA and nifedipine. Voltage-dependent inward currents were studied using patch-clamp techniques and dissociated cells. Similar inward currents were observed in esophageal body (EB) and LES smooth muscle cells. The inward currents in both tissues were blocked by nifedipine, enhanced by Bay K8644, and transiently suppressed by acetylcholine. The molecular form of the Ca2+ channel was explored using RT-PCR, and similar splice variant combinations of the pore-forming
1C-subunit were identified in EB and LES. This is the first characterization of Ca2+ channels in human esophageal smooth muscle, and we establish that L-type Ca2+ channels play a critical role in maintaining LES tone.
patch clamp; Ca2+ current; muscle tone; polymerase chain reaction; Cav1.2
A PATHOPHYSIOLOGICAL ABNORMALITY contributing to gastroesophageal reflux disease (GERD) is failure of the lower esophageal sphincter (LES) smooth muscle to maintain tone. By remaining tonically contracted, the LES separates the esophagus from the gastric environment, protecting it from damage. The mechanisms underlying generation of this spontaneous tone in humans are incompletely understood, and discovery of how tone is maintained could lead to new drug targets in the treatment of GERD and spastic esophageal smooth muscle disorders such as achalasia (5).
The LES is a complex structure, and both myogenic and neurogenic mechanisms contribute to the tonic contraction (24). In contrast to LES smooth muscle, esophageal body (EB) smooth muscle develops little spontaneous tone and is normally phasically active during peristalsis. Several structural and biochemical specializations may account for the differences between these anatomically adjacent, yet physiologically distinct, smooth muscle types, including the regulation of intracellular Ca2+ concentration ([Ca2+]i) and different contractile protein isoforms (8, 31, 32). We (28) previously examined Ca2+ regulation in human EB smooth muscle and found that cholinergic excitation involved both the release of Ca2+ from intracellular stores and influx via L-type Ca2+ channels. Cholinergic regulation of human LES smooth muscle has not previously been studied.
Systemic administration of the dihydropyridine (DHP) L-type Ca2+ channel blocker nifedipine decreases LES pressure in healthy volunteers (16) and in achalasia patients (5, 35). These findings suggest a role for Ca2+ channels in the genesis and maintenance of LES tone in humans. However, pharmacological intervention is ineffective in some patients experiencing achalasia, suggesting that still other mechanisms may contribute to the development of tone (1). In vitro experiments on canine (27), feline (22), and opossum (41) models have indicated that LES tone involves nifedipine-sensitive Ca2+ influx. In contrast, Biancani and co-workers (2) reported that in feline LES muscle, intracellular Ca2+ stores were critical to the maintenance of LES tone. However, no studies to date have identified or characterized the Ca2+ channels involved in regulating human esophageal smooth muscle tone and contraction.
Alternative splicing of Ca2+ channel gene products can give rise to a functional diversity of channels with distinct ionic properties and activation/inactivation characteristics, regulated in large part through the pore-forming, DHP-binding
1-subunit (30). For example, the effectiveness of DHPs in cardiovascular diseases such as hypertension is due to a specific Ca2+ channel subunit splice variant in vascular smooth muscle (11). We considered whether expression of a specific combination of exons may give rise to the unique ability of the LES to generate tone. We therefore examined the expression of several splice variant combinations of the Ca2+ channel using RT-PCR and compared the channels in LES and EB. In conjunction with these molecular studies, we used functional, electrophysiological, and Ca2+ fluorescence studies to evaluate the roles for Ca2+ channels in human EB and LES smooth muscle.
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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 of Western Ontario Review Board for Health Sciences Research Involving Human Subjects and conformed to the Helsinki Declaration. Tissues were obtained from patients undergoing esophageal resection due to cancer, as described previously (24, 28). In total, muscle was obtained from 52 specimens and studied using the different approaches.
For isolation of smooth muscle cells (SMCs), segments of the esophagus were cut into strips (
2 mm wide, 10 mm long) and placed in 2.5 ml of dissociation solution consisting of 135 mM K+ solution (composition given below in Solutions) as described previously (28). Cells were studied within 8 h of dispersion.
Tissue bath studies.
Muscle strips were dissected from the circular muscle layer of the EB or the clasp portion of the LES, a region chosen because it develops greater spontaneous tension than sling muscle (24). Strips were mounted individually in water-jacketted tissue baths containing 10 ml Krebs bicarbonate solution continuously bubbled with 5% CO2-95% O2 at 37°C, as described previously. Acetylcholine (ACh) and carbachol (CCh) evoked similar responses. For quantification, tension responses were expressed either as a percentage of maximal relaxation obtained in each individual strip with sodium nitroprusside (SNP; 100 µM) or as a percentage of the control CCh-evoked increase in tension.
Electrophysiological recordings.
Dispersed cells were allowed to settle and adhere to the bottom of a perfusion chamber mounted on the stage of a Nikon inverted microscope and perfused with bathing solution at 13 ml/min. Cells selected for study appeared phase bright and contracted in response to ACh. Whole cell recordings were made in the perforated-patch configuration with electrode solution containing nystatin (250 µg/ml). All currents were recorded at room temperature (2124°C) with an Axopatch 200A amplifier (Axon Instruments; Foster City, CA) filtered at 1 kHz and sampled at 5 kHz using pCLAMP 6 software (Axon Instruments). To characterize inward currents, we blocked K+ currents using Cs+ electrode solution (see Solutions). Pipette resistance before seal formation ranged from 1 to 9 M
. Whole cell recording was initiated when access resistance had stabilized at <40 M
to allow series resistance compensation of up to 80% to be used. Capacitive currents were compensated on-line using amplifier circuitry and linear leakage corrected off-line as assessed at negative potentials. In some traces, uncancelled capacitive currents were compensated off-line or blanked.
Measurement of [Ca2+]i.
Cells were loaded by incubation with fura-2 AM (0.2 µM) at room temperature for 40 min. Cells were allowed to settle onto a glass coverslip that comprised the bottom of a perfusion chamber (
0.75 ml volume). The chamber was mounted on a Nikon inverted microscope, and bathing solution was perfused (13 ml/min, room temperature) during the experiment. The ratio of fluorescence emission at 510 nm with alternate excitation wavelengths of 345 and 380 nm was measured using a Deltascan system (Photon Technology; London, Ontario, Canada), as previously described (28). Responses illustrated are from single cells and are representative of responses elicited in multiple samples from two or more patients.
Solutions.
The Krebs solution used for the retrieval of tissues and contraction studies consisted of (in mM) 116 NaCl, 5 KCl, 2.5 CaCl2, 1.2 MgSO4, 2.2 NaH2PO4, 25 NaHCO3, and 10 D-glucose equilibrated with 5% CO2-95% O2 (pH 7.4). The Na+-HEPES bathing solution used for electrophysiological recordings and fluorescence studies contained (in mM) 130 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 20 HEPES, and 10 D-glucose (adjusted to pH 7.4 with NaOH). Ca2+-free solutions had the same composition as above except with the addition of varying amounts of EGTA and omission of CaCl2. In electrophysiological recordings, the CsCl recording electrode solution contained (in mM) 130 CsCl, 20 HEPES, 1 MgCl2, 10 tetraethylammonium chloride, 0.4 CaCl2, and 1 EGTA (adjusted to pH 7.2 with CsOH).
RT-PCR.
Total RNA was extracted from EB and LES tissue by phenol-chloroform extraction using frozen muscle samples. The integrity of the RNA was confirmed using agarose gel electrophoresis and ethidium bromide staining. Four micrograms of total RNA were reverse transcribed with random hexamers using a first-strand cDNA synthesis kit (Pharmacia Biotech; Madison, WI). PCR was performed in 50 µl of PCR buffer containing 2 mM MgCl2, 200 µM dNTPs, 0.1 nM each primer, 2 units Taq DNA polymerase (Quiagen; Valencia, CA), and 1015 µl cDNA reaction mixture. PCR was carried out in a GeneAmp 2400 PCR thermal cycler (Perkin-Elmer; Norwalk, CT) for 35 cycles with cycling parameters of 1 min at 94°C, 1 min at 54°C, 2 min at 72°C, and a final 10-min extension at 72°C. The PCR primers used to amplify cDNA are shown in Table 1. PCR primers for
-actin were used to confirm the fidelity of the PCR and to detect genomic DNA contamination. The amplified products were analyzed by electrophoresis on 1% agarose-Tris-acetic acid-EDTA [10 mM Tris (pH 7.5), 5.7% glacial acetic acid, and 1 mM EDTA] gels and visualized by ethidium bromide staining. Sequencing of PCR products was done at the Robarts Research Institute Core Molecular Biology Facility (London, Ontario, Canada).
Chemicals.
Chemicals were obtained from Sigma (St. Louis, MO), BDH (Toronto, Ontario, Canada), or Calbiochem (San Diego, CA) unless otherwise stated. Test substances were prepared from stock solutions in distilled water or DMSO, diluted into the appropriate bathing solution, and applied either by bath perfusion or pressure ejection from glass micropipettes (Picospritzer II, General Valve; Fairfield, NJ). Pipettes were positioned 25100 µm from cells with the concentration reported being that in the application pipette. Control studies carried out with vehicle alone had no effect.
Statistics.
Values are means ± SE with sample sizes (n) indicating the number of cells or muscle strips studied. All traces shown are representative of at least three experiments on muscle or cells from two or more esophageal specimens. For patch-clamp and Ca2+ fluorescence experiments, only one recording was obtained per cell. Comparisons were made using the Student's paired and unpaired t-tests, ANOVA, and Tukey's test, with P < 0.05 considered significant.
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RESULTS
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Roles for Ca2+ in maintaining LES tone and in cholinergic contraction.
We first investigated a role for extracellular Ca2+ and Ca2+ channels in the maintenance of tone in human LES using tissue bath studies. LES muscle strips developed spontaneous tension and relaxed in response to activation of intrinsic nerves by electrical field stimulation or in response to the nitric oxide donor SNP (100 µM; Fig. 1A), all features characteristic of LES smooth muscle (13, 24). Removal of Ca2+ from the bathing solution (plus the addition of 0.5 mM EGTA) abolished LES tone (reduced to 12 ± 1% of control, n = 7), with recovery upon the readdition of Ca2+ (Fig. 1B). Because previous studies have suggested that low-Ca2+ chelator concentrations failed to completely block Ca2+ influx, thus influencing tension development in canine LES smooth muscles (27), we varied EGTA concentrations (0.05 mM, n = 10; 0.1 mM, n = 11; and 0.2 mM, n = 6; data not shown) and found that basal tone was inhibited comparably under all conditions.

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Fig. 1. Lower esophageal sphincter (LES) smooth muscle tone and contraction is dependent on extracellular Ca2+. A: LES muscle strips characteristically developed spontaneous tension with stretch and relaxed to electrical field stimulation (EFS; 0.5 ms, 10 Hz, 4080 mV) or the nitric oxide donor sodium nitroprusside (SNP; 100 µM). B: removal of extracellular Ca2+ resulted in the gradual loss of tension and abolition of carbachol (CCh; 10 µM)-evoked contraction. Recovery of tone is shown at right after the readdition of Ca2+, followed by relaxation induced by SNP. C: nifedipine (10 µM) significantly reduced tone, indicating a role for L-type Ca2+ channels in the regulation of LES tone.
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To further examine a role for Ca2+ entry, we applied the DHP Ca2+ channel blocker nifedipine (10 µM), which significantly decreased LES tension (13 ± 6% of control, P < 0.01, n = 11; Figs. 1C and 2C). Application of vehicle alone had no effect in these or any further experiments. These data support a role for Ca2+ entry through L-type Ca2+ channels in the maintenance of LES tone.

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Fig. 2. Cholinergic excitation of LES smooth muscle involves influx and release of Ca2+ from stores. A: nifedipine (10 µM) inhibited LES tone and CCh-evoked contraction. B: LES tone persisted, but CCh-evoked contractions were attenuated in the presence of the sarco(endo)plasmic reticulum Ca2+-ATPase blocker cyclopiazonic acid (CPA; 10 µM). C: summary of the experiments shown in A and B with means ± SE presented as a percentage of basal tension (left) and of control CCh responses (right). Spontaneous tension was inhibited by nifedipine (n = 11) but unaffected by CPA (n = 6), indicating a critical role for L-type Ca2+ channels in the maintenance of tone. CCh-evoked contractions were reduced by nifedipine (n = 5) and CPA (n = 4), indicating the involvement of both Ca2+ influx and intracellular stores in contraction (*P < 0.05; **P < 0.01). D: similar experiment as in B except carried out using circular layer of esophageal body (EB) smooth muscle. Notably, CPA caused a slow development of tension and abolished subsequent CCh-evoked contraction, distinct from that observed in the LES.
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Because intracellular stores of Ca2+ are proposed to participate in maintenance of LES tone in feline LES (2, 22), we investigated the effects of sarco(endo)plasmic reticulum Ca2+-ATPase blockade with cyclopiazonic acid (CPA). CPA (10 µM) did not affect basal LES tone (n = 6; Fig. 2, B and C). As a positive control, we examined the effects of CPA on EB muscle, where CPA did cause an increase in tension (Fig. 2D). The lack of effect of CPA on LES tone, coupled with the marked sensitivity to Ca2+ channel blockers, supports the conclusion that differences in Ca2+ handling exist between LES and EB. In LES smooth muscle, CCh-evoked contractions were inhibited by perfusion with a Ca2+ -free solution (0.5 mM EGTA, 3 ± 3% of control, n = 4; Fig. 1B) or by the addition of nifedipine (5 ± 4% of control, n = 5; Fig. 2, A and C). In addition, CPA decreased CCh-evoked contractions (37 ± 12% of control, n = 4; Fig. 2, B and C). The actions of cholinergic agonists are mediated by muscarinic receptors, as indicated in previous studies using selective antagonists (17, 25). Although multiple muscarinic receptor subtypes are present, contraction of EB occurs predominantly through the M3 receptor subtype (25, 38), although the subtypes mediating contraction in LES have not previously been examined. Taken together, these results are consistent with cholinergic contraction of LES smooth muscle being dependent on Ca2+ influx and release from stores, whereas tone was dependent on influx.
L-type Ca2+ current in EB and LES SMCs.
In view of the demonstrated involvement of Ca2+ entry, we next used patch-clamp electrophysiology to characterize voltage-dependent Ca2+ currents in SMCs isolated from EB and LES. Enzymatic dissociation of SMCs yielded spindle-shaped cells that appeared phase bright and ranged in length from 50 to 150 µm. Cells isolated from EB and LES smooth muscle were of similar size with whole cell capacitance values of 61 ± 4 (n = 26) and 66 ± 7 pF (n = 22), respectively. We used the Cs+ electrode solution to block K+ currents (37) and allow resolution of inward currents. From a holding potential of 60 mV, depolarization elicited a rapid, transient inward current in both EB and LES SMCs (Fig. 3). The current-voltage relationship for EB SMCs revealed a peak inward current of 35 ± 7 pA at 0 mV (n = 13; Fig. 3B). An inward current with essentially the same kinetics and voltage dependence was observed in LES SMCs, with a peak inward current of 31 ± 7 pA at 0 mV (n = 10; Fig. 3C).

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Fig. 3. Voltage-dependent Ca2+ current in human EB and LES smooth muscle cells (SMCs). A: EB SMCs were held at 60 mV and depolarized to the potentials indicated. Depolarization induced a transient inward current that increased with positive potentials. Dotted lines indicate zero current level in this and subsequent records. In all current recordings, Cs+ electrode solution was used to block K+ currents. B: average current-voltage relationship for EB SMCs. Peak inward current was recorded at 0 mV (n = 13; means ± SE). C: LES SMCs exhibited a similar average current-voltage distribution with equivalent peak inward current at 0 mV (n = 10). Whole cell capacitance measurements revealed that EB and LES SMCs were of similar size.
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Additional similarities in the properties of the currents were noted when responses to DHP Ca2+ channel regulators were studied. The Ca2+ channel agonist Bay K8644 (10 µM) significantly increased voltage-dependent peak inward currents in both EB (51 ± 11 pA, n = 6, vs. control 31 ± 7 pA, n = 9, P < 0.01; Fig. 4B) and LES (48 ± 7 pA vs. control 24 ± 3 pA, n = 6, P < 0.01; Fig. 4D) SMCs. Furthermore, the L-type Ca2+ channel antagonist nifedipine essentially abolished peak inward current in both EB (3 ± 5 pA, n = 5, vs. control 31 ± 7 pA, n = 9, P < 0.01; Fig. 4B) and LES (1 ± 1 pA vs. control 25 ± 5 pA, n = 5, P < 0.01; Fig. 4D) SMCs.

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Fig. 4. L-type Ca2+ current in human EB and LES SMCs. A: representative traces show that depolarizing voltage steps (60 to 0 mV) elicited inward current in EB SMCs that was increased with the L-type Ca2+ channel agonist Bay K8644 (10 µM) and inhibited with nifedipine (10 µM). B: summary of data in A. Peak control current at 0 mV (n = 9) was enhanced with Bay K8644 (n = 6) and inhibited with nifedipine (n = 5). C: representative traces from LES SMCs showing Bay K8644 and nifedipine effects similar to those observed in EB. D: summary of data obtained in C. Peak inward current at 0 mV was significantly enhanced with Bay K8644 (n = 6) and inhibited with nifedipine (n = 5) (*P < 0.05; **P < 0.01).
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To study the physiological regulation of Ca2+ currents, we recorded responses to ACh, which has previously been shown to cause elevation of [Ca2+]i and contraction of human esophageal muscle cells (28). Cells were held under voltage clamp and periodically depolarized from 60 to 0 mV to elicit Ca2+ current. Stimulation with ACh caused a marked inhibition of the Ca2+ current in both EB (Fig. 5, A and B) and LES (Fig. 5C) SMCs. As shown in Fig. 5D, peak inward current was reduced to 7 ± 3 pA from a control level of 35 ± 7 pA (n = 9) in EB and to 7 ± 1 pA from a control level of 39 ± 8 pA in LES (n = 8) SMCs. Inhibition of the Ca2+ current was rapidly reversible and consistent with the time course for Ca2+ inhibition of L-type Ca2+ channels previously established in other smooth muscles (37). Evidence to support the agonist-induced rise of [Ca2+]i in LES cells is shown below, which could account for the acute Ca2+ current inhibition. In addition to the inhibition of the Ca2+ current, ACh often elicited an inward nonselective cation current (Fig. 5A; J. R. Kovac. H. G. Preiksaitis, and S. M. Sims, unpublished observations). Thus, in addition to their voltage-activation range and pharmacological sensitivity, Ca2+ currents in EB and LES smooth muscle are subject to similar physiological regulation.

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Fig. 5. Acetylcholine (ACh) inhibits L-type Ca2+ current in human EB and LES SMCs. A: representative trace from an EB SMC held at 60 mV and periodically stepped to 0 mV to elicit Ca2+ current. ACh (10 µM) elicited a small inward current and caused acute and transient inhibition of Ca2+ current, with recovery evident on washout (break in recording represents 5 min). Sections are amplified for display in the traces (i, control; ii, ACh; iii, washout) shown in B. C: representative traces from LES SMCs show similar inhibition of Ca2+ current in response to ACh. D: summary of data shown in B and C. Peak inward current at 0 mV was significantly inhibited by ACh in both EB (n = 9) and LES (n = 8) SMCs (**P < 0.01).
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Molecular characteristics of L-type Ca2+ channels.
We used RT-PCR to probe for transcripts encoding the pore-forming
1C-subunit of L-type Ca2+ channels, the subunit responsible for conferring Ca2+ and DHP sensitivity (Fig. 6) (30). Alternative splicing of this subunit influences voltage dependence and current kinetics (42) and exhibits tissue specificity (19). We hypothesized that differences in channel expression might be evident between phasic EB and tonic LES smooth muscle, accounting for the unique physiological characteristics of smooth muscles (4). Primers were designed based on the gene sequence encoding the smooth muscle
1C-subunit (Cav1.2), with the explicit aim of resolving the presence of three splice combinations. We examined the exons coding for membrane-spanning regions IS-6 (exon 8/8a), IIIS-2 (exons 21/22 plus 23), and IVS-3 (exons 31/32 plus 33) (19, 30, 40) (Table 1 and Fig. 6). Cav1.2[
1C(IIIS-2)] was identified as a 275-bp band shown by direct sequencing of four independent samples to contain exon 21 but not exon 22, plus exon 23. The variant Cav1.2[
1C(IVS-3)] was detected as a 234-bp band that contained exons 32 and 33 but not exon 31 (Fig. 7). Moreover, the IS-6 region was identified in both EB and LES smooth muscle as coded for by exon 8, not exon 8a (sequenced in 3 independent samples). Although not quantitative, the presence of exons 32 and 33 (but not exon 31) and exon 8 (but not exon 8a) indicates the presence of the smooth muscle variant of the
1C-subunit portion of the L-type Ca2+ channel. This identifies the variant in esophageal muscle as Cav1.2b, and distinguishes it from the cardiac isoform, Cav1.2a. The identification of a single PCR product at 314 bp for
-actin messenger RNA (Fig. 7) ruled out contamination by genomic DNA. Consistent with the electrophysiological similarities described above, we found no differences in Ca2+ channel
1C-subunit splice combinations between EB and LES smooth muscle.
[Ca2+]i in EB and LES SMCs.
We used the fluorescent indicator dye fura-2 to investigate [Ca2+]i in EB and LES SMCs, testing the hypothesis that chronically elevated [Ca2+]i in LES SMCs might account for tonic contraction (10). Although we acknowledge that the Ca2+ measurements are semiquantitative, we did not detect differences in basal [Ca2+]i between EB (120 ± 17 nM, n = 7) and LES (100 ± 5 nM, n = 15) SMCs (Fig. 8B). Moreover, ACh caused an elevation of [Ca2+]i accompanied by contraction of LES SMCs (Fig. 8A) similar to that observed previously in EB SMCs (28). Cholinergic stimulation resulted in similar [Ca2+]i increases (EB: 317 ± 37 nM, n = 7; LES: 302 ± 27 nM, n = 15; Fig. 8B). Nifedipine caused a modest, but consistent, decrease in basal [Ca2+]i levels in both EB (19 ± 3 nM, n = 4) and LES (17 ± 5 nM, n = 7) SMCs, suggesting a small contribution of L-type Ca2+ channels to the regulation of basal [Ca2+]i (Fig. 8B). Nifedipine caused only a slight reduction in the peak rise of [Ca2+]i elicited by ACh (Fig. 8B), although there was a consistent decrease in a later, sustained rise of Ca2+. These findings are consistent with agonist-activated release of Ca2+ from stores as well as a component due to Ca2+ influx.

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Fig. 8. Cytosolic free Ca2+ concentration ([Ca2+]i) in EB and LES SMCs. A: ACh (10 µM), applied for the time indicated by the solid bar, evoked an elevation of [Ca2+]i that was unaffected by nifedipine (10 µM). Nifedipine alone induced a modest, but consistent, decrease in basal [Ca2+]i (break in recording represents 5-min washout). B: summary of data shown in A. EB and LES SMCs exhibited similar basal (left) and peak ACh-evoked (middle) [Ca2+]i levels (EB, n = 7; LES, n = 15 in both data sets). Peak [Ca2+]i evoked by ACh was unaffected by nifedipine (right; EB, n = 7; LES, n = 8).
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DISCUSSION
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The contribution of Ca2+ to LES smooth muscle tone was examined using a combination of functional studies of contraction in intact muscle strips, freshly dispersed cells to characterize ionic currents and [Ca2+]i responses, and RT-PCR to study the transcriptional expression of L-type Ca2+ channels. In smooth muscle strips, removal of bath Ca2+ or application of nifedipine essentially abolished LES tone, indicating a critical role for Ca2+ entry in tonic LES contraction. Patch-clamp recordings characterized a DHP-sensitive, voltage-dependent, inward Ca2+ current in EB and LES smooth muscle. The L-type Ca2+ channel
1C-subunit was identified, and similar splice combinations were observed between EB and LES smooth muscles.
The present study provides the first direct in vitro evidence of the essential role for L-type Ca2+ channels in maintaining tone in human LES. Previous in vivo studies showed that Ca2+ channel blockade with nifedipine decreased LES tone in healthy volunteers (16). Indeed, Ca2+ channel blockers may predispose individuals to GERD and have been used, with moderate success, in the treatment of spastic esophageal disorders such as achalasia, a condition in which the LES fails to relax (1, 5).
In agreement with our findings, early pioneering in vitro studies by Tottrup and co-workers (34) on human esophageal muscle showed that removal of extracellular Ca2+ attenuated LES tone. More recently, a role for Ca2+ influx via L-type Ca2+ channels was demonstrated in several animal models, including canine (27), feline (22), and opossum (41) LES. Salapatek and co-workers (27) hypothesized that low EGTA concentrations may fail to completely block Ca2+ influx resulting in a residual contraction. We found similar inhibition by all concentrations of EGTA tested (0.050.5 mM), suggesting the presence of a small nifedipine-insensitive component to LES contraction. This small residual component may represent a Ca2+-independent contraction (7) or the release of Ca2+ from other sites, including intracellular stores. Given the pharmacological profiles of the Ca2+ currents observed, it does not appear that a DHP-insensitive Ca2+ channel contributes to human esophageal LES tone, such as that reported in some vascular smooth muscles (20).
[Ca2+]i stores are proposed to participate in the maintenance of tone in the feline LES (2, 22). We found that blockade of sarco(endo)plasmic reticulum Ca2+-ATPase with CPA inhibited agonist-induced contraction but had no effect on LES tone, indicating a minimal role for intracellular stores in tonic contraction of human LES. In contrast, in parallel studies of EB, we found that CPA did elicit a gradual rise in tension, as would be expected for elevation of [Ca2+]i. These findings are in contrast to those in the feline LES, where application of CPA resulted in a significant increase in tone (2, 22). Thus there appear to be fundamental differences between human LES and that of animal models, emphasizing the importance of the present study.
One mechanism proposed to account for the unique ability of the LES to maintain tone is distinct Ca2+ channel expression allowing continuous Ca2+ influx at resting potentials (10). Support for this hypothesis came from work showing the resting membrane potential of LES (40 to 50 mV) to be more positive than EB SMC (50 to 60 mV) (9, 18, 41). These findings raise the possibility of a persistent "window current" in human esophageal muscle. However, recording of window Ca2+ current is difficult to achieve electrophysiologically. In a thorough and technically demanding analysis using combined patch and fluorescence recordings of tracheal SMCs, window Ca2+ current was estimated to be <0.5 pA (12). We could not resolve such a small current in our patch-clamp recordings, especially as we used physiological bath Ca2+ levels. Changes of [Ca2+]i may be a more sensitive indicator of Ca2+ influx (12), and we did observe a reduction of basal [Ca2+]i with the addition of L-type blockers (Fig. 8).
Patch-clamp electrophysiology revealed inward currents with similar kinetics, voltage dependence, and DHP sensitivities in EB and LES smooth muscles. Ca2+ currents with similar properties and pharmacology to those in the present study have been reported in feline LES smooth muscle (21). However, other sources of Ca2+ entry exist in esophageal muscle, including nonselective cation channels of the transient receptor potential C (TRPC) family (39). It remains to be determined whether TRPC channels contribute to Ca2+ influx and the maintenance of tone in LES muscle.
To further assess the contribution of L-type Ca2+ channels in EB and LES smooth muscle, we examined the expression pattern of L-type Ca2+ channel
1C-subunit splice combinations. The
1C-subunit defines the Ca2+ conduction pore and voltage sensor and confers DHP sensitivity (29). Indeed, the effectiveness of DHPs in cardiovascular disease is due to their influence on specific splice variants of the Ca2+ channel
1C-subunit in vascular smooth muscle (11). Expression is regulated through alternative splicing that generates Ca2+ channels with distinct gating, pharmacology, and activation/inactivation characteristics (19, 40). As such, we hypothesized that the functional diversity observed between tonic and phasic esophageal muscles could be the result of alternatively spliced Ca2+ channel transcripts. We examined the IS-6, IIIS-2, and IVS-3 variants because they have been identified in smooth muscle and confer sensitivity to DHPs (4, 26, 29, 42). Previous studies of the cat esophagus have demonstrated evidence for the
1C-subunit, based on an antibody to a conserved region common to L-type channels in many tissues, including cardiac and brain cells (21). We advanced this basic finding by examining several regions contributing to functional diversity and pharmacological sensitivity.
Other Ca2+ channel subunits (i.e.,
1,
2
,
, and
) influence channel activity; however, they were not examined in the present study. Our findings of similar variants between the EB and LES did not account for the fundamental differences in the physiology of the muscles. This, combined with the similar profile of membrane currents, suggests other factors must account for the physiological specialization.
Another mechanism proposed to explain the maintenance of tone is that LES exhibits chronically increased [Ca2+]i, above the threshold to elicit contraction (10). Anatomically, LES SMCs exhibit larger cellular diameters, increased mitochondrial content, and a more developed sarcoplasmic reticulum compared with EB SMCs (8, 31, 32). In our in vitro studies, basal [Ca2+]i levels were similar among EB and LES muscles, suggesting that these theories do not apply in the human esophagus under these conditions. It is possible that contraction and tone may occur under constant [Ca2+]i levels through increased Ca2+ sensitivity, or sensitization, of the smooth muscle contractile apparatus. For example, downregulation of myosin light chain phosphatase (MLCP) activity either directly via Rho kinase phosphorylation of the MLCP targeting subunit or indirectly via Rho kinase and PKC phosphorylation of a MLCP inhibitor (6). Whether such a mechanism applies in human LES muscle remains to be determined.
Although the LES can autonomously generate tone, under physiological conditions it is also regulated by inhibitory and excitatory neurotransmitters (3). ACh is the primary excitatory neurotransmitter of the gastrointestinal tract and evokes contraction of human EB (28). A cholinergic contribution to LES contraction has been described (2, 22, 27); however, the sources of Ca2+ remain controversial. We provide evidence that cholinergic contractions are abolished on removal of extracellular Ca2+ or the addition of nifedipine, supporting a key role for influx in human LES tone.
We also report that cholinergic stimulation causes a transient inhibition of Ca2+ current in both EB and LES muscles, similar to that observed in guinea pig ileal, gastric, and tracheal muscles (36, 37). PKC-mediated suppression of Ca2+ current has been suggested (23); however, others have found that the initial suppression of Ca2+ influx was due to the release of Ca2+ from stores (36, 37). This negative feedback controls [Ca2+]i levels and may be an intrinsic mechanism by which EB and LES smooth muscles regulate contraction. ACh evoked essentially identical elevations in [Ca2+]i that were largely unaffected by nifedipine in both EB and LES SMCs. These results suggest that cholinergic regulation of contraction involves release from intracellular stores. However, we found that cholinergic contractions are chiefly dependent on Ca2+ influx with only a small contribution from intracellular stores. Several reasons may account for this apparent discrepancy: 1) dispersion of cells may alter [Ca2+]i regulation (33); 2) ACh-evoked contraction may result in cross-talk between PKC-dependent and -independent pathways, resulting in other second messengers subsequently influencing tone and contraction (15); and 3) the existence of discrete [Ca2+]i stores that are inositol (1,4,5)trisphosphate insensitive and cannot be depleted by CPA (14).
In summary, these data provide the first identification and characterization of L-type Ca2+ channels in human esophageal smooth muscles. We establish a contribution for Ca2+ influx in the maintenance of LES tone and demonstrate the expression of smooth muscle-specific
1C-subunit splice combinations in EB and LES smooth muscles.
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GRANTS
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This study was supported by Canadian Institutes of Health Research (CIHR) Grants MOP10019 and MOP12608. J. R. Kovac was supported by a Postgraduate Fellowship from the Natural Sciences and Engineering Research Council and a CIHR MD/PhD Studentship. H. G. Preiksaitis was supported by an Ontario Ministry of Health Career Scientist Award.
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ACKNOWLEDGMENTS
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We are grateful to T. Chrones, H. Prokopiw, and C. Liu for technical assistance; T. Karkanis and G. Wade for preliminary studies; N. Panupinthu for advice regarding RT-PCR studies; and Drs. R. I. Inculet, R. A. Malthaner, C. Rajgopal, and S. E. Carroll for providing the esophagectomy specimens.
Portions of this work have been previously presented in abstract form at the Canadian Digestive Diseases Week, 2003.
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FOOTNOTES
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Address for reprint requests and other correspondence: S. M. Sims, Dept. of Physiology and Pharmacology, Univ. of Western Ontario, London, Ontario, Canada N6A 5C1 (e-mail: stephen.sims{at}schulich.uwo.ca)
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.
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REFERENCES
|
---|
- Bassotti G and Annese V. Review article: pharmacological options in achalasia. Aliment Pharmacol Ther 13: 13911396, 1999.[CrossRef][ISI][Medline]
- Biancani P, Hillemeier C, Bitar KN, and Makhlouf GM. Contraction mediated by Ca2+ influx in esophageal muscle and by Ca2+ release in the LES. Am J Physiol Gastrointest Liver Physiol 253: G760G766, 1987.[Abstract/Free Full Text]
- Biancani P, Sohn UD, Rich HG, Harnett KM, and Behar J. Signal transduction pathways in esophageal and lower esophageal sphincter circular muscle. Am J Med 103: 23S28S, 1997.[CrossRef][ISI][Medline]
- Bielefeldt K. Molecular diversity of voltage-sensitive calcium channels in smooth muscle cells. J Lab Clin Med 133: 469477, 1999.[CrossRef][ISI][Medline]
- Bortolotti M and Labo G. Clinical and manometric effects of nifedipine in patients with esophageal achalasia. Gastroenterology 80: 3944, 1981.[ISI][Medline]
- Brozovich FV. Myosin light chain phosphatase: it gets around. Circ Res 90: 500502, 2002.[Free Full Text]
- Cao W, Harnett KM, Behar J, and Biancani P. Group I secreted PLA2 in the maintenance of human lower esophageal sphincter tone. Gastroenterology 119: 12431252, 2000.[ISI][Medline]
- Christensen J and Roberts RL. Differences between esophageal body and lower esophageal sphincter in mitochondria of smooth muscle in opossum. Gastroenterology 85: 650656, 1983.[ISI][Medline]
- Crist J, Surprenant A, and Goyal RK. Intracellular studies of electrical membrane properties of opossum esophageal circular smooth muscle. Gastroenterology 92: 987992, 1987.[ISI][Medline]
- Daniel EE. Lower esophagus: structure and function. In: Sphincters: Normal FunctionChanges in Disease, edited by Daniel EE, Tomita T, Tsuchida S, and Watanabe M. Boca Raton, FL: CRC, 1992, p. 4966.
- De Leeuw PW and Birkenhager WH. The effects of calcium channel blockers on cardiovascular outcomes: a review of randomised controlled trials. Blood Press 11: 7178, 2002.[CrossRef][ISI][Medline]
- Fleischmann BK, Murray RK, and Kotlikoff MI. Voltage window for sustained elevation of cytosolic calcium in smooth muscle cells. Proc Natl Acad Sci USA 91: 1191411918, 1994.[Abstract/Free Full Text]
- Fox JA and Daniel EE. Role of calcium in genesis of lower esophageal tone and other active contractions. Am J Physiol Endocrinol Metab Gastrointest Physiol 237: E163E171, 1979.[Abstract/Free Full Text]
- Golovina VA and Blaustein MP. Spatially and functionally distinct Ca2+ stores in sarcoplasmic and endoplasmic reticulum. Science 275: 16431648, 1997.[Abstract/Free Full Text]
- Hillemeier C, Bitar KN, Sohn U, and Biancani P. Protein kinase C mediates spontaneous tone in the cat lower esophageal sphincter. J Pharmacol Exp Ther 277: 144149, 1996.[Abstract]
- Hongo M, Traube M, and McCallum RW. Comparison of effects of nifedipine, propantheline bromide, and the combination on esophageal motor function in normal volunteers. Dig Dis Sci 29: 300304, 1984.[CrossRef][ISI][Medline]
- Hurley BR, Preiksaitis HG, and Sims SM. Characterization and regulation of Ca2+-dependent K+ channels in human esophageal smooth muscle. Am J Physiol Gastrointest Liver Physiol 276: G843G852, 1999.[Abstract/Free Full Text]
- Kannan MS, Jager LP, and Daniel EE. Electrical properties of smooth muscle cell membrane of opossum esophagus. Am J Physiol Gastrointest Liver Physiol 248: G342G346, 1985.[Abstract/Free Full Text]
- Liao P, Yu D, Lu S, Tang Z, Liang MC, Zeng S, Lin W, and Soong TW. Smooth muscle-selective alternatively spliced exon generates functional variation in Cav1.2 calcium channels. J Biol Chem 279: 5032950335, 2004.[Abstract/Free Full Text]
- Morita H, Cousins H, Onoue H, Ito Y, and Inoue R. Predominant distribution of nifedipine-insensitive, high voltage-activated Ca2+ channels in the terminal mesenteric artery of guinea pig. Circ Res 85: 596605, 1999.[Abstract/Free Full Text]
- Muinuddin A, Kang Y, Gaisano HY, and Diamant NE. Regional differences in L-type Ca2+ channel expression in feline lower esophageal sphincter. Am J Physiol Gastrointest Liver Physiol 287: G772G781, 2004.[Abstract/Free Full Text]
- Muinuddin A, Neshatian L, Gaisano HY, and Diamant NE. Calcium source diversity in feline lower esophageal sphincter circular and sling muscle. Am J Physiol Gastrointest Liver Physiol 286: G271G277, 2004.[Abstract/Free Full Text]
- Ozaki H, Zhang L, Buxton IL, Sanders KM, and Publicover NG. Negative-feedback regulation of excitation-contraction coupling in gastric smooth muscle. Am J Physiol Cell Physiol 263: C1160C1171, 1992.[Abstract/Free Full Text]
- Preiksaitis HG and Diamant NE. Regional differences in cholinergic activity of muscle fibers from the human gastroesophageal junction. Am J Physiol Gastrointest Liver Physiol 272: G1321G1327, 1997.[Abstract/Free Full Text]
- Preiksaitis HG, Krysiak PS, Chrones T, Rajgopal V, and Laurier LG. Pharmacological and molecular characterization of muscarinic receptor subtypes in human esophageal smooth muscle. J Pharmacol Exp Ther 295: 879888, 2000.[Abstract/Free Full Text]
- Rich A, Kenyon JL, Hume JR, Overturf K, Horowitz B, and Sanders KM. Dihydropyridine-sensitive calcium channels expressed in canine colonic smooth muscle cells. Am J Physiol Cell Physiol 264: C745C754, 1993.[Abstract/Free Full Text]
- Salapatek AM, Lam A, and Daniel EE. Calcium source diversity in canine lower esophageal sphincter muscle. J Pharmacol Exp Ther 287: 98106, 1998.[Abstract/Free Full Text]
- Sims SM, Jiao Y, and Preiksaitis HG. Regulation of intracellular calcium in human esophageal smooth muscles. Am J Physiol Cell Physiol 273: C1679C1689, 1997.[Abstract/Free Full Text]
- Soldatov NM. Genomic structure of human L-type Ca2+ channel. Genomics 22: 7787, 1994.[CrossRef][ISI][Medline]
- Soldatov NM, Bouron A, and Reuter H. Different voltage-dependent inhibition by dihydropyridines of human Ca2+ channel splice variants. J Biol Chem 270: 1054010543, 1995.[Abstract/Free Full Text]
- Szymanski PT, Chacko TK, Rovner AS, and Goyal RK. Differences in contractile protein content and isoforms in phasic and tonic smooth muscles. Am J Physiol Cell Physiol 275: C684C692, 1998.[Abstract]
- Szymanski PT, Szymanska G, and Goyal RK. Differences in calmodulin and calmodulin-binding proteins in phasic and tonic smooth muscles. Am J Physiol Cell Physiol 282: C94C104, 2002.[Abstract/Free Full Text]
- Taggart MJ. Smooth muscle excitation-contraction coupling: a role for caveolae and caveolins? News Physiol Sci 16: 6165, 2001.[ISI][Medline]
- Tottrup A, Forman A, Uldbjerg N, Funch-Jensen P, and Andersson KE. Mechanical properties of isolated human esophageal smooth muscle. Am J Physiol Gastrointest Liver Physiol 258: G338G343, 1990.[Abstract/Free Full Text]
- Traube M, Hongo M, Magyar L, and McCallum RW. Effects of nifedipine in achalasia and in patients with high-amplitude peristaltic esophageal contractions. JAMA 252: 17331736, 1984.[Abstract]
- Unno T, Komori S, and Ohashi H. Inhibitory effect of muscarinic receptor activation on Ca2+ channel current in smooth muscle cells of guinea-pig ileum. J Physiol 484: 567581, 1995.[Abstract]
- Wade GR, Barbera J, and Sims SM. Cholinergic inhibition of Ca2+ current in guinea-pig gastric and tracheal smooth muscle cells. J Physiol 491: 307319, 1996.[Abstract]
- Wang J, Krysiak PS, Laurier LG, Sims SM, and Preiksaitis HG. Human esophageal smooth muscle cells express muscarinic receptor subtypes M1 through M5. Am J Physiol Gastrointest Liver Physiol 279: G1059G1069, 2000.[Abstract/Free Full Text]
- Wang J, Laurier LG, Sims SM, and Preiksaitis HG. Enhanced capacitative calcium entry and TRPC channel gene expression in human LES smooth muscle. Am J Physiol Gastrointest Liver Physiol 284: G1074G1083, 2003.[Abstract/Free Full Text]
- Welling A, Ludwig A, Zimmer S, Klugbauer N, Flockerzi V, and Hofmann F. Alternatively spliced IS6 segments of the alpha 1C gene determine the tissue-specific dihydropyridine sensitivity of cardiac and vascular smooth muscle L-type Ca2+ channels. Circ Res 81: 526532, 1997.[Abstract/Free Full Text]
- Zhang Y, Miller DV, and Paterson WG. Opposing roles of K+ and Cl currents in maintenance of opossum lower esophageal sphincter tone. Am J Physiol Gastrointest Liver Physiol 279: G1226G1234, 2000.[Abstract/Free Full Text]
- Zuhlke RD, Bouron A, Soldatov NM, and Reuter H. Ca2+ channel sensitivity towards the blocker isradipine is affected by alternative splicing of the human alpha1C subunit gene. FEBS Lett 427: 220224, 1998.[CrossRef][ISI][Medline]
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