Gastrointestinal Diseases Research Unit and Departments of Medicine and Physiology, Queen's University, Kingston, Ontario, Canada K7L 5G2
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ABSTRACT |
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The
ionic basis underlying the maintenance of myogenic tone of lower
esophageal sphincter circular muscle (LES) was investigated in opossum
with the use of standard isometric tension and conventional intracellular microelectrode recordings in vitro. In tension recording studies, nifedipine (1 µM) reduced basal tone to 27.7 ± 3.8%
of control. The K+ channel blockers tetraethylammonium
(TEA, 2 mM), charybdotoxin (100 nM), and 4-aminopyridine (4-AP, 2 mM)
enhanced resting tone, whereas apamin and glibenclamide were without
affect. Cl channel blockers DIDS (500 µM) and
5-nitro-2-(3-phenylpropylamino)-benzoic acid (500 µM), as well as
niflumic acid (0.1-300 µM), decreased basal tone, but tamoxifen
was without effect. Intracellular microelectrode recordings revealed
ongoing, spontaneous, spike-like action potentials (APs). Nifedipine
abolished APs and depolarized resting membrane potential (RMP). Both
TEA and 4-AP significantly depolarized RMP and augmented APs, whereas
niflumic acid dose-dependently hyperpolarized RMP and abolished APs.
These data suggest that, in the opossum, basal tone is associated with
continuous APs and that K+ and Ca2+-activated
Cl
channels have important opposing roles in the genesis
of LES tone.
smooth muscle; spontaneous action potentials; myogenic tone; niflumic acid; nifedipine.
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INTRODUCTION |
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TONIC CONTRACTION IS THE KEY feature of circular smooth muscle of the lower esophageal sphincter (LES) and results in a pressure barrier at the gastroesophageal junction that prevents the reflux of gastric contents. Basal tone of the LES is primarily myogenic in origin but can be modulated by both neural and hormonal factors (36). Pharmacological manipulation of myogenic LES tone has obvious therapeutic importance but has been difficult to achieve, largely because the ionic mechanisms underlying LES tone remain poorly understood.
Daniel et al. (17) reported that the resting membrane
potential (RMP) of opossum LES circular muscle was about 40 mV, which was relatively more positive than that of the adjacent esophageal body
(about
50 mV) (14). It was proposed that this
more positive RMP resulted in continuous activation of
voltage-sensitive Ca2+ channels, and thus constant
Ca2+ entry into the cell, which helped maintain basal LES
tone. The ionic mechanisms underlying the RMP of the LES are unclear,
but changes in several ionic conductances could theoretically
contribute. Since the RMP is due to a major contribution of
K+ channels in smooth muscle (7), less
activity of K+ channels could be associated with the more
positive membrane potential of LES circular muscle. In addition, one or
more other channels that carry inward currents, such as nonselective
cation channels (5) and Cl
channels, could
contribute to the more positive RMP.
It is estimated that the reversal potential of the
Cl channel is between
30 mV and
20 mV (1,
2). Activation of Cl
channels would allow outward
movement of Cl
ions to carry an inward current, resulting
in the depolarization of RMP (31). Recent studies from a
number of different laboratories have provided evidence that
Cl
channels are involved in tonic contraction of certain
vascular (33) and gut (34) smooth muscles and
in maintenance of RMP in tracheal smooth muscle (49).
Furthermore, it has been reported that DIDS- and indanyloxyacetic
acid-94 sensitive volume-regulated channels are functionally
and molecularly expressed in canine vascular and colonic smooth muscle
cells (18, 46).
On the basis of the above observations, we hypothesize that a balance
of K+ and Cl channel activity might be one of
the ionic mechanisms responsible for maintenance of LES basal tone.
This possibility was investigated with the use of conventional
isometric tension and intracellular microelectrode recordings in LES
circular smooth muscle of the opossum.
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METHODS |
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Tissue Preparation
The protocols were approved by the Animal Care Committee of Queen's University. Opossums (Didelphis virginiana) of either sex, weighing between 2.5 and 5 kg, were anesthetized by tail vein injection of phenobarbital sodium (40 mg/kg). The chest and abdominal cavities were then exposed via midline incisions, and the lower part of the esophagus and the short segment of attached stomach were removed and placed in preoxygenated Krebs solution. The opossum was then killed by intracardiac injection of phenobarbital sodium. The lower part of the esophagus and esophagogastric junction were opened longitudinally and pinned out with the mucosa side up in a dissecting dish. With the use of a binocular microscope, the mucosa and connective tissue layers were carefully removed by sharp dissection. The LES was visible as a distinct thickening of circular muscle in the resultant tissue, located just on the gastric side of the squamocolumnar junction (35, 36). A strip of LES (with attached longitudinal muscle) of ~3 × 15 mm was cut for tension studies, and a sheet of LES of ~2 × 3 × 15 mm was excised for intracellular recordings. In 10 animals, similar sheets of esophageal body circular smooth muscle from ~2 cm above the LES were also excised and prepared for intracellular recordings.Isometric Tension Recordings
Standard isometric tension recordings were used to study the mechanical responses. A strip was hung in a water-jacketed tissue bath containing 10 ml Krebs solution gassed with 5% CO2-95% O2 at 35°C. One end of a strip was fixed to a hook at the base of the tissue bath, and the other was tied, using a fine silk thread, to a Grass FT03 isometric force transducer that coupled to the Windaq data acquisition system (DATAQ Instruments). Signals were sampled at 100 Hz and stored in a Pentium computer for subsequent analysis. Strips were initially stretched to 140% of the unloaded length and equilibrated for at least 1 h. This degree of stretch has previously been shown to result in optimal responses (37, 45).Electrical Recordings
Conventional intracellular microelectrode recording techniques were employed to study electrical properties of LES circular smooth muscle. The sheet of LES tissue was pinned on the silicon-coated bottom of a 2-ml electrophysiological recording chamber mounted on the stage of an Olympus IX-70 inverted microscope (Olympus). The chamber was continuously perfused at 1.6 ml/min with prewarmed and preoxygenated Krebs solution and maintained at 35°C. Tissue was allowed to equilibrate for 2 h before the experiment. Glass microelectrodes were pulled using a vertical microelectrode puller (Sutter Instrument) and filled with 3 M KCl. Microelectrode resistance was 50-70 MSolutions and Drugs
The modified Krebs solution contained (mM): 118.07 NaCl, 25 NaHCO3, 11.10 D(+)-glucose, 4.69 KCl, 2.52 CaCl2, 1 MgSO4, and 1.01 NaH2PO4. Niflumic acid was purchased from ICN Biochemicals, TTX, charybdotoxin, and apamin were from Research Biochemicals, and all other materials were from Sigma. Niflumic acid, DIDS, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), and glibenclamide were dissolved in DMSO as stock solutions. Tamoxifen and nifedipine were prepared in ethyl alcohol (100%), and others were prepared in distilled water. These were diluted to final concentrations with Krebs solution. In cumulative dose-response experiments of niflumic acid, final concentration of DMSO in Krebs solution was no more than 1%. The Krebs solution containing diluted drugs was fully bubbled with 5% CO2-95% O2 to restore pH before application.Statistical Analysis
Data are means ± SE. For tension recording studies n refers to the number of animals, whereas for intracellular recording studies n refers to the number of cells impaled. Pre- and postdrug comparisons were made with the paired Student's t-test, and a P value of <0.05 was considered statistically significant. ![]() |
RESULTS |
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Isometric Tension Studies
Mechanical activity.
After stretch to 140% of resting length, LES strips slowly developed
tone that reached a steady state of 52.9 ± 5.5%
(n = 20) of KCl-induced (60 mM) maximal contraction in
1-1.5 h. The application of atropine (3 µM) plus guanethidine (3 µM) did not significantly alter the basal tone, but TTX (1 µM)
increased tone by 26.1 ± 11.1% over control (n = 5, P < 0.05). Twenty-five percent of strips showed
spontaneous phasic contractions, superimposed on basal tone, with a
frequency of 3.6 ± 0.5/min and amplitude of 13.5 ± 2.9% of
basal tone. Nifedipine (1 µM), an L-type Ca2+ channel
blocker, abolished the spontaneous phasic contractions and diminished
basal tone to 27.7 ± 3.8% of control (n = 4)
(Fig. 1B).
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Effect of K+ and Cl
channel blockers on LES basal tone.
4-Aminopyridine (4-AP; 2 mM), a transient K+ channel
blocker, increased resting tone to 83.1 ± 6.5% over basal values
(n = 4, P < 0.05), whereas glibenclamide
(10 µM), an ATP-sensitive K+ channel blocker, did not
significantly alter basal tone (n = 4).
Tetraethylammonium (TEA; 2 mM) and charybdotoxin (100 nM), large-conductance K+ channel blockers, increased basal tone
over control by 32.9 ± 15.5 and 18.42 ± 5.9%, respectively
(n = 4, P < 0.05). However, apamin (300 nM), a small-conductance Ca2+-activated K+
channel blocker, increased the basal tone by 9.4 ± 5.7%
over control (n = 6, P > 0.05) (Fig.
1A). DIDS (500 µM) and NPPB (500 µM), nonspecific
Cl
channel blockers, deceased basal tone to 75.0 ± 7.4 and 67.3 ± 10.8%, respectively, of control
(n = 4). Niflumic acid, a putative selective
Ca2+-activated Cl
channel blocker, relaxed
LES in a dose-dependent manner with an IC50 of 1.23 ± 0.33 µM (n = 9) (Fig.
2). The maximally effective concentration
of niflumic acid (300 µM) decreased tone to 29.4 ± 5.1% of
control (n = 9), which was not different from the
effect induced by a maximally effective concentration of nifedipine (1 µm) (P > 0.05). Tamoxifen (10 µM), a
volume-activated Cl
channel blocker, increased basal tone
by 12.1 ± 6.0% over control (n = 4, P > 0.05) (Fig. 1B).
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Studies to exclude a nonspecific effect of niflumic acid.
Although niflumic acid has been demonstrated to selectively block
Ca2+-activated Cl channels (31),
nonspecific effects of this agent have also been reported
(24). Niflumic acid (10 and 300 µM) had no effect on KCl
(60 mM)-induced LES contraction, excluding an effect of niflumic acid
on contractile proteins or voltage-sensitive Ca2+ channels.
In addition, the niflumic acid-induced LES relaxation was unaffected by
preincubating the strips with either 2 mM TEA (n = 3)
or 10 µM glibenclamide (n = 3), excluding activation
of either large-conductance Ca2+-activated or ATP-sensitive
K+ channels, respectively, as the mechanism of the niflumic
acid-induced LES relaxation.
Intracellular Recording Studies
Electrical activity.
The electrical properties of LES circular smooth muscle are summarized
in Table 1. The intracellular recordings
revealed the existence of ongoing, spontaneous, spike-like APs. Two
patterns of APs were observed (Fig.
3B): APs occurring on a stable
RMP baseline and APs superimposed on the plateau as well as the valley of slow wave-like potentials. Slow wave-like potentials were recorded in only 2 of 24 cells and had a frequency of 3.5 ± 0.78/min (Fig. 3B). Nifedipine (1 µM) abolished APs and depolarized RMP
from 41.3 ± 1.3 to
34.4 ± 3.4 mV (n = 3, P < 0.05) (Fig. 4),
indicating that Ca2+ entry through L-type Ca2+
channels is responsible for the APs.
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Effects of K+ channel and
Cl channel blockers on electrical properties.
Table 1 summarizes the effects of K+ and Cl
channel blockers on electrical properties of LES circular smooth muscle
cells. Both TEA (2 mM, n = 6) and 4-AP (2 mM,
n = 4) significantly depolarized RMP and increased
amplitude, maximal rate of upstroke, and frequency of APs (Fig.
5). TEA, but not 4-AP, also significantly
shortened the lower 1/3-amplitude duration of APs. Time course studies
of niflumic acid (300 µM) demonstrated that it gradually
hyperpolarized RMP from
40.6 ± 2.3 mV to
48.1 ± 2.2 mV
(n = 5, P < 0.05), decreased the amplitude
and maximal rise rate of upstroke of APs, and eventually abolished the
ongoing APs after ~5 min (Fig. 6). The
studies of the cumulative dose response of niflumic acid yielded an
IC50 for hyperpolarization of 19.6 ± 2.2 µM and for
inhibition of maximal rise rate of upstroke of 81.7 ± 15.9 µM
(n = 5) (Fig.
7B).
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DISCUSSION |
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The major novel findings of the current study are that
1) compared with circular smooth muscle from the esophageal
body, LES circular smooth muscle displays ongoing, spontaneous,
spike-like APs and has a more positive RMP; 2) resting tone
of the opossum LES is increased by K+ channel blockers and
decreased by Cl channel blockers, and this corresponds to
the opposing effects of these agents on RMP and spontaneous APs; and
3) changes in LES tone appear to correlate better with
alterations in spontaneous APs than with RMP, in that niflumic acid and
nifedipine, which both profoundly decrease resting LES tone, markedly
inhibit spontaneous APs yet have opposing effects on RMP.
It is well established that the pressure barrier between esophagus and stomach, which prevents gastroesophageal reflux, is due to tonic myogenic contraction of the LES (36). The physiological mechanisms underlying this myogenic tone are poorly understood. For years it was known that the LES was unique physiologically but not anatomically. However, more recent studies have clearly defined a number of anatomic features of this muscle that provide clues to its function. Compared with circular smooth muscle from the esophageal body, LES cells are larger, form fewer gap junction contacts with adjacent cells, and display an irregular cell surface with prominent evaginations (16, 40). Furthermore, mitochondria and smooth endoplasmic reticulum are larger in LES muscle cells, possibly reflecting their greater metabolic activity (9).
The LES also displays different contractile properties relative to circular smooth muscle from the esophageal body (43). Biancani and colleagues (6) reported that LES tone in the cat was only partially reduced in a Ca2+-free physiological solution or in the presence of L-type Ca2+ blockade, suggesting that tonic LES contraction is primarily dependent on intracellular Ca2+ stores. They subsequently demonstrated a unique signal transduction mechanism for maintenance of LES tone that was different from that used for phasic, acetylcholine-induced contraction (41, 42, 47). Salapatek et al. (38) recently reported that resting tone of the dog LES is dependent on continuous entry of Ca2+ through L-type Ca2+ channels, but this was from an extracellular site not readily equilibrated with the external solution in that Ca2+ chelators were required to demonstrate an effect. They speculated that this extracellular Ca2+ store may be closely associated with the plasmalemma, at or near caveolae.
Our experiments in the opossum are consistent with a major role for extracellular Ca2+ in the maintenance of LES tone, because the L-type Ca2+ channel blocker nifedipine caused marked reduction in resting tone of LES muscle strips. However, in the presence of maximally effective concentrations of nifedipine or niflumic acid, some residual tone remained. It is unclear whether this is due to active muscle contraction via a mechanism independent of extracellular Ca2+ or is due to passive forces alone.
Electrophysiological properties of the LES are also distinct in that its RMP appears to be relatively more positive compared with the esophageal body (13, 17, 21, 22), although this has not been found by all investigators (10, 11). Such a relatively positive RMP has been hypothesized to contribute to myogenic tone via activation of voltage-gated Ca2+ channels, which in turn leads to entry of extracellular Ca2+. Studies performed in vivo using extracellular electrodes have described "spike-dependent" and "spike-independent" resting LES tone (3). The sphincter region in this study was characterized by continuous spike activity at rest that disappeared following the administration of isoproterenol or following neurogenic LES relaxation. However, there was evidence of resting tone that appeared independent of the continuous spike activity. In this in vivo study, however, it was not possible to determine whether the continuous smooth muscle spike activity was coming from the LES circular smooth muscle layer, which is responsible for LES resting pressures, or the external longitudinal muscle layer.
Our findings generally support the previous electrophysiological studies. First, we confirmed a relatively more positive RMP of LES circular smooth muscle. We also were able to record continuous and spontaneous APs from all cells, which was abolished by blockade of L-type Ca2+ channels. This is similar to the studies performed in vivo but is unlike previous reports of LES intracellular recordings, in which spontaneous APs were not recorded (11, 17, 21). The reasons for this difference are unclear; however, we suspect that it relates to technical aspects of the intracellular recordings. Because it is difficult to impale and maintain intracellular recordings in actively contracting smooth muscle, many investigators delay attempts at impalement for a period of time to allow the muscle to become relatively quiescent. This might result in recordings being made at a time when spontaneous APs have fatigued. In our studies, impalements were performed after a relatively short equilibration period when the muscle still displayed spontaneous contractions.
It is interesting that nifedipine produced a marked decrease in LES tone yet resulted in relative depolarization of membrane potential. This is consistent with a more important role for spontaneous APs, as opposed to RMP per se, in the generation of resting LES tone. Depolarization of smooth muscle membrane potential by nifedipine has been reported previously (30). The exact mechanism whereby this occurs is unclear, but two mechanisms have been proposed: 1) direct blockade of voltage-dependent K+ channels (4, 44, 48) or 2) secondary blockade of Ca2+-activated K+ channels. After blockade of L-type Ca2+ channels by nifedipine, the decreased entry of Ca2+ into the intracellular compartment may result in less activity of Ca2+-activated K+ channels (32).
It is well established that K+ channels play an important
role in the maintenance of RMP in visceral smooth muscles (7, 27,
39). Our results demonstrated the actions of K+
channels in the LES in that selective blockade of either
large-conductance Ca2+-activated K+ channels or
transient K+ channels significantly depolarized LES RMP and
increased tone. In addition, delayed-rectifier K+ channels
may contribute to LES RMP, because TEA and 4-AP at the tested
concentration (2 mM) may also block this kind of K+
channel. A functional role for Cl channels in visceral
smooth muscles is less certain. It was reported that Cl
channels mediated the norepinephrine-induced constriction of vascular
smooth muscle (12, 28, 29) and also appeared to be
involved in myogenic tone of cerebral arteries (33) and
tracheal smooth muscle (20). Evidence also exists
implicating activation of Cl
channels in agonist-induced
contraction of colonic smooth muscle (26), and closing of
Cl
channels might be the mechanism whereby certain
inhibitory neurotransmitters cause hyperpolarization of esophageal
(14) and ileal (15) smooth muscle. Our
studies are the first to suggest a role for Ca2+-activated
Cl
channels in the maintenance of myogenic tone in a
gastrointestinal smooth muscle.
The appropriate interpretation of our results rests on the selectivity
of the Cl channel blockers used. Both DIDS and NPPB are
known to be nonselective Cl
channel blockers; therefore,
it is possible that their effect on resting LES tone could be due to an
action on something other than the Cl
channel
(25). Niflumic acid, on the other hand, is believed to be
a relatively selective blocker of the Ca2+-activated
Cl
channels. Some studies have suggested that this drug
may also have nonselective effects (24). For instance, it
has been shown that in certain tissues niflumic acid may activate
large-conductance Ca2+-activated K+ channels
(19) and ATP-sensitive K+ channels
(24) in vascular smooth muscle. However, such actions could not explain the results in our studies. First, glibenclamide had
no effect on resting LES tone, suggesting that ATP-sensitive K+ channels are, at least, not active in this tissue in the
resting state. Furthermore, neither TEA nor glibenclamide at
concentrations that are known to block large-conductance and
ATP-sensitive K+ channels, respectively, affected the
niflumic acid-induced relaxation of LES circular smooth muscle strips.
More recent studies by Kato et al. (23) suggested that
niflumic acid relaxed pulmonary arteries preconstricted by endothelin-1 via an effect that was independent of Cl channel
blockade. This effect was slowly reversible and seemed unrelated to the
activation of large-conductance Ca2+-activated or
ATP-sensitive K+ channels. Furthermore, in this model,
niflumic acid also relaxed pulmonary arteries preconstricted by the
Ca2+ ionophore A-23187, leading the authors to suggest that
the niflumic acid effect may be via Ca2+-dependent
contractile processes. Such mechanisms cannot explain the LES relaxant
effect of niflumic acid in our experiments because 1) the
effect of niflumic acid was rapidly reversible in our experiments; 2) niflumic acid had no effect on KCl-induced LES
contraction, indicating that it was not affecting LES tone via a
nonspecific effect of voltage-gated Ca2+ channels or
contractile proteins; 3) nifedipine depolarized RMP and
relaxed LES, but niflumic acid hyperpolarized RMP and relaxed LES; and
4) maximal relaxation induced by niflumic acid was
comparable to the maximal relaxation induced by nifedipine, suggesting
that niflumic acid might inhibit LES tone by closing L-type
Ca2+ channels via membrane hyperpolarization.
Finally, Cao et al. (8) have recently reported that
maintenance of LES tone in the cat was related to release of
arachidonic acid via spontaneous activity of phospholipase
A2. This in turn led to formation of PGF2
and thromboxane A2 via a cyclooxygenase pathway. These
mediators then activated G proteins, which led to protein kinase
C-dependent muscle contraction. The nonsteroidal anti-inflammatory
drugs ASA and indomethacin, by blocking cyclooxygenase and hence
PGF2
and thromboxane A2 generation,
decreased LES tone in the cat. It is unlikely that the effect of
niflumic acid seen in our experiments is due to inhibition of
cyclooxygenase. First, the onset and reversal of the LES relaxant
effect of niflumic acid was rapid, whereas the effect of indomethacin
described in the cat LES has a much slower time course. In our opossum
LES strips, addition of indomethacin (3-10 µM) had inconsistent
effects on LES tone (unpublished observations), and if relaxation was observed it was very slow to develop compared with niflumic acid. Furthermore, niflumic acid caused prompt membrane hyperpolarization in
LES circular smooth muscle, an effect that could not be explained by
inhibition of cyclooxygenase and decreased prostaglandin or thromboxane
generation. However, it nevertheless is possible that second messengers
derived from the cyclooxygenase pathway may be involved in activation
of Ca2+-activated Cl
channels.
In summary, our studies demonstrate for the first time that resting
tone of the opossum LES is dependent on ongoing, spontaneous, and
spike-like APs. Furthermore, the data suggest that a balance between
K+ channels and Ca2+-activated Cl
channels sets RMP of LES circular smooth muscle at a more positive level. This in turn activates L-type Ca2+ channels, leading
to ongoing spontaneous APs and the generation of resting tone.
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ACKNOWLEDGEMENTS |
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We thank Dr. E. E. Daniel for reviewing this work and for providing constructive comments.
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FOOTNOTES |
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This work was supported by Medical Research Council of Canada Grant MT-9978.
Address for reprint requests and other correspondence: W. G. Paterson, Gastrointestinal Diseases Research Unit, Hotel Dieu Hospital, 166 Brock St., Kingston, ON, Canada K7L 5G2 (E-mail: patersow{at}hdh.kari.net).
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.
Received 13 March 2000; accepted in final form 18 July 2000.
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