Departments of 1 Physiology and Pharmacology and 2 Medicine, University of Western Ontario, and 3 Lawson Health Research Institute, London, Ontario, Canada N6A 5C1
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Smooth muscle contraction is critical to
peristalsis in the human esophagus, yet the nature of the channels
mediating excitation remains to be elucidated. The objective of this
study was to characterize the inward currents in human esophageal
smooth muscle cells (HESMCs). Esophageal tissue was isolated from
patients undergoing surgery for cancer and grown in primary culture,
and currents were recorded using patch-clamp electrophysiology.
Depolarization elicited inward current activating positive to 40 mV
and peaking at 0 mV and consisting of transient and sustained
components. The transient current was half activated at
16 mV and
half inactivated at
67 mV. The transient current was abolished by
removal of bath Na+ or application of TTX (IC50
~20 nM), whereas it persisted in the absence of bath Ca2+
or the presence of Cd2+. These data provide evidence that
cultured HESMCs express voltage-dependent Na+ channels.
RT-PCR revealed mRNA transcripts for Nax, the
"atypical" Na+ channel isoform, as well as
Nav1.4. These studies provide the first evidence of
Nav1.4 in smooth muscle and contribute to a model of
excitation in HESMCs.
sodium channel; esophagus; smooth muscle; electrophysiology; Nav1.4.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SMOOTH MUSCLE COMPRISES the muscularis externa in the distal third of the human esophagus and is critical for peristalsis (18). At the level of the individual smooth muscle cell (SMC), depolarization leads to the opening of voltage-dependent Ca2+ channels, and the subsequent rise in cytosolic Ca2+ initiates contraction (31). Ion channels, therefore, influence esophageal motility by controlling membrane potential. Little is known, however, about the nature and regulation of the channels mediating excitation in human esophageal smooth muscle.
Efforts to understand the mechanisms of excitation in human esophageal
SMCs (HESMCs) have focused on identifying the ion channels and
signaling pathways. Previous studies have identified several classes of
K+ current (32). Delayed rectifier
K+ (Kv) current serves to regulate resting
tension, whereas Ca2+-activated K+
(KCa) current limits depolarization during contraction. The
excitatory inward currents in the human esophagus, however, have not
been investigated. Studies of esophagus from other species have
revealed an L-type Ca2+ current (3) and a
Ca2+-activated Cl current (34).
The depolarization mediated by inward Cl
current may lead
to the activation of other voltage-dependent currents, thereby
initiating action potentials.
Although the rising phase of the smooth muscle action potential was originally suggested to be due solely to Ca2+ current, it is now clear that Na+ currents also contribute to excitation in some, although not all, smooth muscles (23). Voltage-dependent Na+ currents have been recorded in a number of smooth muscle tissues, including arterial (10), lymphatic (19), uterine (36, 37), and vas deferens smooth muscle (6). In the gastrointestinal tract, Na+ current has been observed in the ileum (30), large intestine (35), and fundus (25). Zholos et al. (38) recently provided electrophysiological evidence of a TTX-sensitive Na+ current in human intestinal smooth muscle. The observed differences in activation and inactivation ranges of these currents have led to the suggestion that considerable heterogeneity may exist among smooth muscle Na+ channels (7). Thus it is critical to study channel properties in specific tissues.
Despite these reports of Na+ currents recorded in smooth muscle, little is known regarding the molecular identity of smooth muscle Na+ channels. Na+ channels are encoded by a single gene family (17), and the most recent nomenclature is based on the convention adopted for other voltage-gated channels. The first isoform shown to exist in smooth muscle was Nax (22), formerly considered the "atypical" Nav2 subfamily but subsequently reclassified (17). In humans, Nax is found in heart and uterus (15), although because of the heterogenous composition of whole tissues, with multiple cell types present, it is unclear whether the channel is specifically expressed in SMCs. Notably, Nax has not been demonstrated to mediate voltage-dependent inward current, despite attempts to express it in heterologous expression systems, leading to the suggestion that it may be a pseudogene (4, 17). The skeletal muscle Nav1.4 (16) has not been previously identified in smooth muscle, whereas a recent report identified mRNA encoding SCNA5 (cardiac Nav1.5 channel) (14) in human jejunal muscle (20).
The overall objective of these studies was to characterize the inward
currents in human esophageal smooth muscle. To address limited
availability of fresh tissues, we have developed a culture cell system
that has proven valuable for studying esophageal physiology (33). Immunocytochemistry experiments performed on these
cultures reveal the presence of smooth muscle -actin, but not
markers for endothelial cells, neuroglial cells, or interstitial cells of Cajal (33). Thus the culture system provides an
opportunity to examine the molecular identity of smooth muscle ion
channels without contamination from other cell types. We present
evidence that cultured HESMCs exhibit a TTX-sensitive,
voltage-dependent Na+ current and that this current
contributes to cellular excitation. Furthermore, we have used RT-PCR to
examine the molecular identity of smooth muscle Na+
channels and find evidence for multiple Na+ channel
isoforms. In addition to the atypical Nax isoform,
we report the presence of the Nav1.4 skeletal muscle
isoform for the first time in any smooth muscle. These data suggest a
putative function for Na+ channels in esophageal motility
and have possible implications for our understanding of esophageal development.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue retrieval and cell culture.
Retrieval of human esophageal tissue and subsequent cell culture were
performed as previously described (33), in accordance with
the guidelines of the University of Western Ontario Review Board for
Research Involving Human Subjects. Tissues were obtained from a
disease-free region of the distal esophagus from patients undergoing
surgery for cancer. The circular muscle layer was dissected and
dispersed by incubation with 1.7 mg/ml collagenase, 0.5 mg/ml elastase,
and 1 mg/ml bovine serum albumin in Hanks' balanced salt solution for
45 min at 37°C. Dispersed cells were grown in primary culture in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin at 37°C
in a humidified atmosphere of 5% CO2 in air. In some
cases, dissected tissue samples were rapidly frozen and stored at
70°C for subsequent RNA extraction (see RNA extraction and
RT-PCR).
Electrophysiology.
For electrophysiological recording, cells were lifted from culture
dishes using 0.25% trypsin-0.1 mM EDTA (GIBCO) and allowed to settle
on the glass bottom of a perfusion chamber. The chamber had a volume of
1 ml and was placed on the stage of a Nikon inverted phase-contrast
microscope. Patch-clamp recordings were performed at room temperature
using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA).
Experiments using K+ in the patch pipette (see
Solutions) were performed using the nystatin
perforated-patch technique. To block K+ currents and allow
characterization of inward current, we used Cs+ electrode
solution (see Solutions) and recorded in the whole cell
configuration. Pipette resistance in solution ranged from 3 from 6 M, with up to 80% series resistance compensation applied. In some
traces, capacitive and leak currents were compensated off-line, as
assessed by subthreshold pulses. Uncancelled capacitive transients have
been blanked in the current traces.
Solutions. During electrophysiological recordings, cells were perfused with Ringer solution containing (in mM) 130 NaCl, 5 KCl, 20 HEPES, 10 D-glucose, 1 CaCl2, and 1 MgCl2 and adjusted to pH 7.4 with NaOH. K+ electrode solution contained (in mM) 140 KCl, 20 HEPES, 1 EGTA, 1 MgCl2, and 0.4 CaCl2 and was adjusted to pH 7.2 with KOH. Cs+ electrode solution contained (in mM) 117 CsCl, 13 NaCl, 1 EGTA, 20 HEPES, 1 MgCl2, 10 tetraethylammonium chloride, and 0.4 CaCl2 and was adjusted to pH 7.2 with CsOH.
RNA extraction and RT-PCR.
HESMCs were grown for 10-15 days, and total RNA was isolated by
acid guanidinium thiocyanate-phenol-chloroform extraction. For
controls, RNA was also isolated from human brain and heart. We also
obtained RNA from intact tissues of the proximal esophagus, consisting
of striated muscle, and of the distal esophagus, consisting of smooth
muscle. RNA integrity was verified by agarose gel electrophoresis using
ethidium bromide staining. Total RNA (4 µg) was reverse transcribed
using random hexamers and Superscript RT (Life Technologies, Gaithersburg, MD). Three micrograms of cDNA reaction mixture were used
in each PCR. The PCR oligonucleotide primers used to amplify cDNA were
designed on the basis of human sequences and are listed in Table
1. PCR was performed in a 50-µl
reaction containing PCR buffer, 2 mM MgCl2, 200 µM dNTPs,
primer at 0.1 nM each, and 2 U of Taq DNA polymerase
(Qiagen, Valencia, CA). PCR was carried out in a GeneAmp 2400 PCR
thermal cycler (Perkin-Elmer, Norwalk, CT) for 28-33 cycles. Each
cycle was timed for 1 min at 94°C, 1.5 min at 55°C, 2 min at
72°C, and a final 10-min extension at 72°C. PCR primers for
-actin were used to detect genomic DNA contamination. Amplified
products (10 µl) were analyzed by electrophoresis on 1% agarose-TAE
gels (10 mM Tris, pH 7.5, 5.7% glacial acetic acid bromide stain, and
1 mM EDTA) and visualized by ethidium bromide staining. PCR product
identity was confirmed by direct sequencing (Robarts Research Institute
Core Molecular Biology Facility, London, ON, Canada).
|
Drugs. Unless otherwise stated, all drugs were obtained from Sigma (St. Louis, MO) or Calbiochem (San Diego, CA). Drugs were prepared as concentrated stock solutions and diluted into bathing solution just before use. During electrophysiological recording, test solutions were applied focally by pressure ejection (Picospritzer II, General Valve, Fairfield, NJ) from a micropipette. Concentrations reported are those in the application pipette.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Characterization of ionic currents in HESMCs. Electrophysiological experiments were performed on a total of 152 HESMCs that had been derived from 21 patients and grown in primary culture for 7-15 days. We first investigated the outward K+ currents to establish that the electrophysiological properties were consistent with previous reports. For these studies, voltage-clamp recording was carried out using the nystatin perforated-patch configuration with electrode solution containing 140 mM K+ (see METHODS).
Depolarization from
|
Depolarization elicits a mixture of inward and outward currents.
In addition to the outward K+ currents, depolarization also
elicited a transient, inward current (Fig.
2A), which became the focus of
the present studies (4 of 6 cells studied with K+ electrode
solution and with a prepulse to 100 mV). The current was voltage
dependent and activated and inactivated rapidly; thus shorter voltage
steps were used to examine its properties. The inward current first
became apparent with voltage jumps to
20 mV, peaked near 10 mV, and
then declined in amplitude at more positive potentials (Fig.
2B). The outward current activated with depolarization as
shown above, although the decline shown above was not evident on this
rapid time scale. On the basis of the time course and kinetics of the
inward current, we examined its sensitivity to the Na+
channel blocker TTX. TTX (1 µM) selectively blocked the transient inward component, leaving outward K+ currents (Fig.
2C). A small persistent, inward current was also evident at
negative potentials, which may be an L-type Ca2+ current
(see below). The TTX-sensitive component was isolated by subtraction of
current traces in the presence of blocker from control traces (Fig.
2D). These results suggest the presence of a
voltage-dependent Na+ current in HESMCs. We proceeded to
characterize the inward current by investigating its voltage-dependent
properties, ion permeation, and pharmacological regulation.
|
Voltage-dependent properties of the inward current.
K+ in the electrode solution was replaced with 117 mM
Cs+ and 13 mM Na+ (see METHODS),
thereby eliminating the outward K+ currents and
establishing a theoretical Na+ equilibrium potential of +60
mV. Many of the experiments were performed in the whole cell
configuration to minimize access resistance. This configuration was
also used to minimize the contribution of L-type Ca2+
current, which is subject to rundown, allowing us to examine the
TTX-sensitive current in isolation. Cells were held at 60 mV and step
depolarized to various potentials after a hyperpolarizing prepulse to
maximize available current (Fig.
3A). The resulting inward
current displayed the rapid activation and inactivation described
earlier, and a persistent component was also evident at several
potentials. For the purposes of these studies, we have defined these
currents as the transient and sustained components, respectively. An
examination of the current-voltage relationship for the peak inward
current demonstrated activation commencing at approximately
40 mV and
peak current occurring at ~0 mV (Fig. 3B;
n = 15). The voltage dependence of steady-state
inactivation was determined using a two-step protocol with a 5-s
conditioning pulse followed by a test pulse to 0 mV (Fig.
4A; n = 15).
To quantify the voltage dependence of activation, conductance was
calculated from the current-voltage relationship as the chord
conductance at each point and normalized to peak conductance to produce
an activation curve (Fig. 4B). When conductance was fitted
with a Boltzmann relation, the half-maximal activation occurred at
16 mV and half-maximal inactivation occurred at
67 mV. The average activation and inactivation values are summarized in Fig. 4B
and are consistent with a voltage-dependent Na+ current.
|
|
Na+ serves as charge carrier for the
transient inward current.
Given that the inward current appeared unaffected by rundown in the
whole cell configuration, we considered it unlikely to be carried by
Ca2+. Blockade of the current by TTX was suggestive of a
Na+ current, although the involvement of other ions could
not be excluded. We therefore used ion-substitution experiments to
resolve the identity of the current. Currents were recorded in solution where Na+ was replaced with the large cation
N-methyl-D-glucamine in an equimolar fashion.
Removal of bath Na+ reversibly abolished the inward current
(Fig. 5; n = 5),
indicating that Na+ is the charge carrier. Further
experiments were performed to assess the contribution of
Ca2+. Application of the L-type Ca2+ channel
blocker nifedipine at 10 µM (Fig.
6A; n = 10)
caused a slight reduction of the sustained component but failed to
abolish the transient inward current. Similarly, the transient current persisted after withdrawal of bath Ca2+ in solution
containing 130 mM Na+, again accompanied by a slight
reduction of the persistent component (Fig. 6B;
n = 3). These data suggest that although L-type
Ca2+ channels may partially mediate the sustained current,
Ca2+ does not contribute to the rapidly inactivating inward
current. The transient current also remained in the presence of 1 mM
Cd2+, which blocks voltage-dependent Ca2+
channels and TTX-resistant Na+ channels (Fig.
6C).
|
|
TTX sensitivity of the Na+ channel.
The TTX sensitivity of the inward component shown in Fig. 2C
provided evidence that the Na+ current was carried through
specific Na+ channels. Given the considerable variation
known to exist in the TTX sensitivity of Na+ channels, the
concentration dependence of TTX blockade in HESMCs was established.
Concentration dependence was examined by measuring percentage of peak
current at 0 mV in various concentrations of TTX (Fig.
7A), with the average values
showing half-maximal blockade at ~20 nM (Fig. 7B;
n 4 at each concentration). At 1 µM, TTX completely
abolished the inward current (Fig. 7C; n = 20). Interestingly, an examination of the difference curve under these
conditions revealed that the transient and sustained components were
TTX sensitive, suggesting that Na+ channels also partially
account for the sustained current (Fig. 7D).
|
Na+ channels contribute to the
esophageal action potential.
The role of Na+ channels in promoting cellular excitation
in many tissues is well established (2). We wished to
determine the contribution of these channels to the action potential in HESMCs. Using K+ electrode solution and a series of
depolarizing current steps, we recorded from cells in the current-clamp
configuration. The magnitude of each current step was increased until
action potentials became apparent, in addition to the passive charging
of the membrane. The rapid phase of depolarization was inhibited by 1 µM TTX, with recovery on washout (Fig.
8; n = 5), demonstrating
a role for Na+ channels in excitation.
|
HESMCs express multiple Na+ channel
isoforms.
To determine the molecular isoform of Na+ channels in
HESMCs, we extracted mRNA as described in METHODS and used
RT-PCR to probe for mRNA transcripts of several Na+ channel
isoforms. Primers were designed for the coding regions of
Nav1.4 (skeletal muscle isoform), Nav1.5
(cardiac muscle isoform), Nav1.2 (neuronal isoform), and
Nax (heart and uterine tissue) and are listed in Table 1
with predicted product sizes. Tissues known to express each of these
isoforms were used as positive controls. Striated muscle from the
proximal esophagus was used as a control for Nav1.4
(16), whereas human heart was used as a control for
Nav1.5 and Nax (14, 15), and human
brain was used as a control for Nav1.2 (1).
PCR was carried out in the absence or presence of cDNA from these
respective tissues. The results are shown in Fig.
9. Primer sets for -actin, designed to
span one or more introns, were used to amplify the same cDNA. In all
cases, a single transcript was detected, excluding the possibility of
genomic DNA contamination in our samples. The identity of amplified
products was confirmed by direct sequencing. These findings are
summarized in Table 2.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have identified a voltage-dependent Na+ current in cultured esophageal smooth muscle cells. Using patch-clamp electrophysiology, we characterized this current on the basis of its biophysical properties and pharmacological sensitivity. Furthermore, we used RT-PCR to identify the specific Na+ channel subtypes. Esophageal smooth muscle tissue expresses the Nav1.4 isoform, characteristic of skeletal muscle, and the Nax isoform, found in heart and uterus. To our knowledge, these studies represent the first report of Nav1.4 expression in any smooth muscle. We propose that these channels contribute to esophageal excitation and may have implications for understanding esophageal muscle development.
Given the heterogeneity known to exist among smooth muscle
Na+ currents (7), attention was paid to the
biophysical and pharmacological properties of the current identified
here. The rapid activation and inactivation kinetics, as well as the
voltage activation range, are characteristic of the fast
Na+ current found in many neuronal and muscle tissues
(8, 17). However, because L-type Ca2+ currents
have been reported in esophageal SMCs (3), we first considered the possibility that this current was carried by
Ca2+. We did not anticipate a large contribution by
Ca2+ to the inward current, inasmuch as Ca2+
current rundown is well established in the whole cell configuration. Although the sustained inward current was slightly reduced by application of nifedipine or withdrawal of bath Ca2+, the
transient current persisted under these conditions. The abolition of
the inward current in the absence of bath Na+ provides
evidence that cultured HESMCs exhibit a voltage-dependent Na+ current. Because half-maximal inactivation occurs at
67 mV, it is likely that this current is largely inactivated in
resting cells in vivo. Examination of the activation and inactivation curves reveals a small window current over a physiological range of
membrane potentials. It remains to be established whether this window
current contributes to esophageal excitability. Although these
biophysical studies support the presence of an Na+ current,
the pharmacological characterization provides evidence that the current
is carried through specific Na+ channels. The TTX
sensitivity of the current (IC50 ~20 nM), combined with
its insensitivity to Cd2+, indicates that cultured HESMCs
express channels similar to those found in skeletal muscle and neurons,
but not those found in cardiac muscle (2). This finding is
consistent with the characteristics of some Na+ channels
found in the smooth muscle of humans (28, 35, 38) and
animals (6, 25, 30). In contrast, a recent report of voltage-dependent Na+ channels in human jejunal SMCs found
them to be relatively resistant to blockade by TTX (20).
We observed that the sustained component of the current also displayed sensitivity to TTX, suggesting that the Na+ current consists of two components. Sustained Na+ currents have been identified in a number of cell types. In neurons, they enhance rhythmicity and facilitate repetitive firing of action potentials (5). Other studies have provided evidence that transient and sustained Na+ currents are mediated by the same population of Na+ channels (5, 24). Interestingly, Gage and co-workers (13) demonstrated that sustained Na+ currents also occur in mammalian skeletal muscle. Expression studies in oocytes showed that, much like neuronal Na+ channels, transient and sustained currents can be mediated by a single population of skeletal muscle Na+ channels (9). Therefore, although sustained currents have been observed in several smooth muscle systems (10, 28), their presence cannot be used to distinguish between neuronal and skeletal muscle channel isoforms.
Our culture system is useful for determining the molecular identity of
the Na+ channels. Previous studies have used RT-PCR to
identify mRNA for Na+ channels in tissues containing smooth
muscle (15), but multiple cell types are present in whole
tissues. Previous studies from this laboratory demonstrate that these
cultured cells show positive staining for smooth muscle-specific
-actin and do not stain for markers of endothelial cells, neuroglial
and Schwann cells, or interstitial cells of Cajal (33). We
recognize that cells in culture can exhibit some properties that differ
from their tissue of origin. We addressed this possibility in several
ways. Only cells that had been maintained in primary culture were used,
reducing the chance that an unrepresentative subpopulation might
dominate the culture. It has been demonstrated previously that cultured and freshly dissociated HESMCs express all five muscarinic subtypes and
respond to ACh with a rise in cytosolic Ca2+ (27,
29). In the present studies, we show that these cultured HESMCs
exhibit two distinct K+ currents, each with biophysical and
pharmacological properties characteristic of the Kv and
KCa currents described in freshly isolated cells
(32). In combination with the molecular and immunologic studies described earlier (33), this finding further
supports the use of our culture system as a model for investigating
esophageal physiology.
We used RT-PCR to show the presence of mRNA encoding voltage-dependent Na+ channels in fresh esophageal smooth muscle tissue. Our findings indicate that HESMCs express mRNA for the Nax isoform, previously the only isoform reported in cultured and whole esophageal smooth muscle (16, 22). However, previous attempts to demonstrate functional Na+ currents from Nax have not been successful (12, 17). On the basis of the structure of this channel, Akopian et al. (4) argued that Nax is unlikely to be a functional voltage-gated Na+ channel. Hence, we consider it unlikely that the Na+ currents recorded here are mediated by Nax. Our investigation focused on the two remaining muscle isoforms: the skeletal muscle Nav1.4 isoform and the cardiac Nav1.5 isoform.
The presence of mRNA encoding Nav1.4 in cultured HESMCs and fresh esophageal tissue is consistent with the pharmacological sensitivity of the whole cell Na+ current we recorded. The cells being studied were from the smooth muscle portion of the esophagus, and we confirmed the presence of Nav1.4 in intact muscle tissue from the distal esophagus, which contains smooth but not striated muscle (18). Accordingly, we also identified Nav1.4 in striated muscle isolated from the proximal esophagus of humans. The electrophysiological evidence of a whole cell TTX-sensitive Na+ current, combined with the expression of mRNA for Nav1.4 in cultured HESMCs, suggests that the Nav1.4 expressed in esophageal tissue is specifically present in smooth muscle. Although we did not identify transcripts for Nav1.5 in the cultured HESMCs, evidence for expression of this cardiac isoform was recently found in human jejunal muscle (20), consistent with some heterogeneity of channel expression in different muscles.
The presence of skeletal muscle Na+ channels in esophageal smooth muscle presents possible implications in organ development. Several studies have demonstrated that muscle cells in the mammalian esophagus undergo a process known as transdifferentiation in the normal course of development (26). In the fetal mouse, individual esophageal SMCs are suggested to convert from the smooth to a skeletal muscle phenotype (26). This transition is accompanied by the appearance of cells expressing markers of both muscle phenotypes. In contrast to the adult mouse esophagus, the distal muscularis externa of humans is composed of smooth muscle (18). Thus our finding of Nav1.4 expression in human esophageal smooth muscle indicates that these cells express characteristics of skeletal muscle, perhaps reflecting a fundamental developmental process.
Because Na+ channels are involved in the rising phase of the action potential in several tissues (2), we suspected that the Na+ current in HESMCs served to promote cellular excitation. We have provided evidence that Na+ channels contribute to action potentials in these cells, similar to the effect seen in lymphatic SMCs (19). Earlier recordings of action potentials in human gastrointestinal smooth muscle revealed that Na+ and Ca2+ act as charge carriers to mediate action potentials (11), providing precedent for the findings here. Depolarization elicited by Na+ channel activity could lead to the opening of voltage-dependent Ca2+ channels and a subsequent rise in cytosolic Ca2+.
In summary, these studies have identified inward Na+ current in cultured human esophageal smooth muscle. We used RT-PCR to demonstrate the expression of mRNA encoding multiple Na+ channel isoforms in HESMCs, including the first report of Nav1.4 expression in smooth muscle. These data contribute to a developing model of excitation in HESMCs and may have implications in our understanding of esophageal development and motor function.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank T. Chrones for technical assistance and Drs. R. Inculet, R. Malthananer, and C. Rajgopal for providing esophagectomy specimens.
![]() |
FOOTNOTES |
---|
These studies were supported by grants from the Canadian Institutes of
Health Research and the Canadian Association of Gastroenterology. M. A. Deshpande was supported by an Ontario Graduate
ScholarshipScience and Technology, J. Wang by a Canadian Institutes
of Health Research Postdoctoral Fellowship, and H. G. Preiksaitis
by an Ontario Ministry of Health Career Scientist Award.
Address for reprint requests and other correspondence: S. M. Sims, Dept. of Physiology and Pharmacology, University of Western Ontario, London, ON, Canada N6A 5C1 (E-mail: stephen.sims{at}fmd.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.
May 29, 2002;10.1152/ajpcell.00359.2001
Received 27 July 2001; accepted in final form 20 May 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ahmed, CM,
Ware DH,
Lee SC,
Patten CD,
Ferrer-Montiel AV,
Schinder AF,
McPherson JD,
Wagner-McPherson CB,
Wasmuth JJ,
Evans GA,
Primary structure, chromosomal localization, and functional expression of a voltage-gated sodium channel from human brain.
Proc Natl Acad Sci USA
89:
8220-8224,
1992[Abstract].
2.
Aidley, D.
The Physiology of Excitable Cells. Cambridge, UK: Cambridge University Press, 1989.
3.
Akbarali, HI,
and
Goyal RK.
Effect of sodium nitroprusside on Ca2+ currents in opossum esophageal circular muscle cells.
Am J Physiol Gastrointest Liver Physiol
266:
G1036-G1042,
1994
4.
Akopian, AN,
Souslova V,
Sivilotti L,
and
Wood JN.
Structure and distribution of a broadly expressed atypical sodium channel.
FEBS Lett
400:
183-187,
1997[ISI][Medline].
5.
Alzheimer, C,
Schwindt PC,
and
Crill WE.
Modal gating of Na+ channels as a mechanism of persistent Na+ current in pyramidal neurons from rat and cat sensorimotor cortex.
J Neurosci
13:
660-673,
1993[Abstract].
6.
Belevych, AE,
Zima AV,
Vladimirova IA,
Hirata H,
Jurkiewicz A,
Jurkiewicz NH,
and
Shuba MF.
TTX-sensitive Na+ and nifedipine-sensitive Ca2+ channels in rat vas deferens smooth muscle cells.
Biochim Biophys Acta
1419:
343-352,
1999[ISI][Medline].
7.
Bolton, TB,
Prestwich SA,
Zholos AV,
and
Gordienko DV.
Excitation-contraction coupling in gastrointestinal and other smooth muscles.
Annu Rev Physiol
61:
85-115,
1999[ISI][Medline].
8.
Catterall, WA.
From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.
Neuron
26:
13-25,
2000[ISI][Medline].
9.
Chang, SY,
Satin J,
and
Fozzard HA.
Modal behavior of the µ1 Na+ channel and effects of coexpression of the 1-subunit.
Biophys J
70:
2581-2592,
1996[Abstract].
10.
Cox, RH,
Zhou Z,
and
Tulenko TN.
Voltage-gated sodium channels in human aortic smooth muscle cells.
J Vasc Res
35:
310-317,
1998[ISI][Medline].
11.
El-Sharkawy, TY,
Morgan KG,
and
Szurszewski JH.
Intracellular electrical activity of canine and human gastric smooth muscle.
J Physiol
279:
291-307,
1978[Abstract].
12.
Felipe, A,
Knittle TJ,
Doyle KL,
and
Tamkun MM.
Primary structure and differential expression during development and pregnancy of a novel voltage-gated sodium channel in the mouse.
J Biol Chem
269:
30125-30131,
1994
13.
Gage, PW,
Lamb GD,
and
Wakefield BT.
Transient and persistent sodium currents in normal and denervated mammalian skeletal muscle.
J Physiol
418:
427-439,
1989[Abstract].
14.
Gellens, ME,
George AL, Jr,
Chen LQ,
Chahine M,
Horn R,
Barchi RL,
and
Kallen RG.
Primary structure and functional expression of the human cardiac tetrodotoxin-insensitive voltage-dependent sodium channel.
Proc Natl Acad Sci USA
89:
554-558,
1992[Abstract].
15.
George, AL,
Knittle TJ,
and
Tamkun MM.
Molecular cloning of an atypical voltage-gated sodium channel expressed in human heart and uterus: evidence for a distinct gene family.
Proc Natl Acad Sci USA
89:
4893-4897,
1992[Abstract].
16.
George, AL, Jr,
Komisarof J,
Kallen RG,
and
Barchi RL.
Primary structure of the adult human skeletal muscle voltage-dependent sodium channel.
Ann Neurol
31:
131-137,
1992[ISI][Medline].
17.
Goldin, A.
Resurgence of sodium channel research.
Annu Rev Physiol
63:
871-894,
2001[ISI][Medline].
18.
Goyal, RK,
and
Sivarao DV.
Functional anatomy and physiology of swallowing and esophageal motility.
In: The Esophagus (3rd ed.), edited by Castell DO,
and Richter JE.. Philadelphia, PA: Lippincott Williams & Wilkins, 1999.
19.
Hollywood, MA,
Cotton KD,
Thornbury KD,
and
McHale NG.
Tetrodotoxin-sensitive sodium current in sheep lymphatic smooth muscle.
J Physiol
503:
13-20,
1997[Abstract].
20.
Holm, AN,
Rich A,
Miller SM,
Strege P,
Ou Y,
Gibbons S,
Sarr MG,
Szurszewski JH,
Rae JL,
and
Farrugia G.
Sodium current in human jejunal circular smooth muscle cells.
Gastroenterology
122:
178-187,
2002[ISI][Medline].
21.
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:
G843-G852,
1999
22.
Knittle, TJ,
Doyle KL,
and
Tamkun MM.
Immunolocalization of the mNav2.3 Na+ channel in mouse heart: upregulation in myometrium during pregnancy.
Am J Physiol Cell Physiol
270:
C688-C696,
1996
23.
Kuriyama, H,
Kitamura K,
Itoh T,
and
Inoue R.
Physiological features of visceral smooth muscle cells, with special reference to receptors and ion channels.
Physiol Rev
78:
811-920,
1998
24.
Moorman, JR,
Kirsch GE,
VanDongen AM,
Joho RH,
and
Brown AM.
Fast and slow gating of sodium channels encoded by a single mRNA.
Neuron
4:
243-252,
1990[ISI][Medline].
25.
Muraki, K,
Imaizumi Y,
and
Watanabe M.
Sodium currents in smooth muscle cells freshly isolated from stomach fundus of the rat and ureter of the guinea-pig.
J Physiol
442:
351-375,
1991[Abstract].
26.
Patapoutian, A,
Wold BJ,
and
Wagner RA.
Evidence for developmentally programmed transdifferentiation in mouse esophageal muscle.
Science
270:
1818-1821,
1995[Abstract].
27.
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:
879-888,
2000
28.
Quignard, JF,
Ryckwaert F,
Albat B,
Nargeot J,
and
Richard S.
A novel tetrodotoxin-sensitive Na+ current in cultured human coronary myocytes.
Circ Res
80:
377-382,
1997[ISI][Medline].
29.
Sims, SM,
Jiao Y,
and
Preiksaitis HG.
Regulation of intracellular calcium in human esophageal smooth muscles.
Am J Physiol Cell Physiol
273:
C1679-C1689,
1997
30.
Smirnov, SV,
Zholos AV,
and
Shuba MF.
Potential-dependent inward currents in single isolated smooth muscle cells of the rat ileum.
J Physiol
454:
549-571,
1992[Abstract].
31.
Somlyo, AP,
and
Somlyo AV.
Signal transduction and regulation in smooth muscle.
Nature
372:
231-236,
1994[ISI][Medline].
32.
Wade, GR,
Laurier LG,
Preiksaitis HG,
and
Sims SM.
Delayed rectifier and Ca2+-dependent K+ currents in human esophagus: roles in regulating muscle contraction.
Am J Physiol Gastrointest Liver Physiol
277:
G885-G895,
1999
33.
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:
G1059-G1069,
2000
34.
Wang, Q,
Akbarali HI,
Hatakeyama N,
and
Goyal RK.
Caffeine- and carbachol-induced Cl and cation currents in single opossum esophageal circular muscle cells.
Am J Physiol Cell Physiol
271:
C1725-C1734,
1996
35.
Xiong, Z,
Sperelakis N,
Noffsinger A,
and
Fenoglio-Preiser C.
Fast Na+ current in circular smooth muscle cells of the large intestine.
Pflügers Arch
423:
485-491,
1993[ISI][Medline].
36.
Yoshino, M,
Wang SY,
and
Kao CY.
Sodium and calcium inward currents in freshly dissociated smooth myocytes of rat uterus.
J Gen Physiol
110:
565-577,
1997
37.
Young, RC,
and
Herndon-Smith L.
Characterization of sodium channels in cultured human uterine smooth muscle cells.
Am J Obstet Gynecol
164:
175-181,
1991[ISI][Medline].
38.
Zholos, AV,
Fenech CJ,
Prestwich SA,
and
Bolton TB.
Membrane currents in cultured human intestinal smooth muscle cells.
J Physiol
528:
521-537,
2000
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |