Departments of 1 Medicine and 2 Physiology and Pharmacology, The University of Western Ontario, and 3 The Lawson Health Research Institute, St. Joseph's Health Centre, London, Ontario N6A 4V2, Canada
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transient receptor potential channel (TRPC) genes encode Ca2+-permeable channels mediating capacitative Ca2+ entry (CCE), which maintains intracellular Ca2+ stores. We compared TRPC gene expression and CCE in human esophageal body (EB) and lower esophageal sphincter (LES), because these smooth muscles have distinct contractile functions that are likely associated with different Ca2+ regulatory mechanisms. Circular layer smooth muscle cells were grown in primary culture. Transcriptional expression of TRPC genes was compared by semiquantitative RT-PCR. CCE was measured by fura 2 Ca2+ fluorescence after blockade of sarcoplasmic reticulum Ca2+-ATPase with thapsigargin. mRNA for TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 was identified in EB and LES. TRPC3 and TRPC4 were more abundant in LES than EB. Basal concentration of free intracellular Ca2+ ([Ca2+]i) was similar in cells from LES (138 ± 8 nmol/l) and EB (110 ± 6 nmol/l) and increased with ACh (10 µmol/l; 650 ± 28 and 590 ± 21 nmol/l, respectively). With zero Ca2+ in bath, thapsigargin (2 µmol/l) increased [Ca2+]i more in LES (550 ± 22 nmol/l) than EB (250 ± 15 nmol/l, P < 0.001). Subsequent external application of 1 mmol/l Ca2+ increased [Ca2+]i more in LES (585 ± 35 nmol/l) than EB (295 ± 21 nmol/l, P < 0.001), indicating enhanced CCE in LES. This demonstrates CCE and TRPC transcriptional expression in human esophageal smooth muscle. In LES cells, enhanced CCE and expression of TRPC3 and TRPC4 may contribute to the physiological characteristics that distinguish LES from EB.
esophagus; sphincter; store-operated Ca2+ influx
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FAILURE OF LOWER ESOPHAGEAL sphincter (LES) smooth muscle to maintain tonic contraction is one factor that contributes to chronic gastroesophageal reflux. Early studies of the cat LES demonstrated that spontaneous tonic contraction is supported mainly by release of Ca2+ from intracellular stores (3), and we have demonstrated that cholinergic activation of human esophageal smooth muscle also relies in part on release of Ca2+ from intracellular stores (23). Elevation of Ca2+ in the cytosol is matched by activation of transporters and pumps on the plasma membrane and sarcoplasmic reticulum (SR) (14). Ca2+ is returned to intracellular stores, but some is also lost to the extracellular space. Thus an efficient and highly coupled process for ensuring that intracellular Ca2+ stores remain filled would be essential to maintain LES tonic contraction.
Release of Ca2+ from intracellular stores is coupled to activation of store-operated channels (SOC) that allow influx of Ca2+ to replenish these stores. The mechanisms involved are not completely understood, but an intimate relationship between the content of Ca2+ stores and SOCs at the plasma membrane has been proposed, termed by Putney as capacitative Ca2+ entry (CCE) (20). Recent attention has focused on a family of mammalian genes that encode Ca2+-permeable channels as putative mediators of CCE (7, 19, 32). These channels are referred to as TRPC channels, since they resemble the transient receptor potential channel of Drosophila melanogaster. At least seven mammalian forms of the TRPC gene have been identified thus far, and several putative functions have been attributed to them, including that of receptor-operated channels (ROC) and SOCs. Both the process of CCE and expression of TRPC genes have been demonstrated in multiple tissues and species, supporting their importance as physiological regulators (7). Expression of TRPC genes has been demonstrated in several vascular and gastrointestinal smooth muscles, where they have been associated with regulation of cellular proliferation, cellular excitation through activation of nonselective cation current, and CCE (10, 13, 29). To date the expression and function of TRPC channels have not been studied in esophageal smooth muscle.
We previously demonstrated that both Ca2+ influx through L-type Ca2+ channels and Ca2+ release from intracellular stores are important in cholinergic excitation of human esophageal smooth muscle (23). Furthermore, L-type Ca2+ channels did not serve an essential role in refilling of depleted stores, indicating that other Ca2+ influx pathways must exist. We hypothesize that TRPC channels are expressed in human esophageal smooth muscle cells (SMCs) and that functional CCE contributes to Ca2+ homeostasis. Moreover, we propose that TRPC channel expression and CCE may differ between esophageal body (EB), which is phasically active, and LES, which displays tonic contraction, since these distinct contractile functions are likely to be associated with distinct Ca2+ regulatory mechanisms.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue retrieval and SMC culture.
Muscle samples were obtained from disease-free portions of human
esophagus being removed because of cancer, as previously described
(18, 23, 30). Smooth muscle samples were obtained from the
circular muscle layer of the middle of the distal third of the EB and
from the clasp portion of the LES, as previously described in detail
(18). The correct identity of LES smooth muscle was
confirmed on the basis of the ability of this muscle to develop
spontaneous, myogenic tone and relax with activation of intrinsic
nerves (18). Portions of the muscle were frozen on dry ice
and stored at 70°C for RNA extraction. Primary cultures of cells
derived from the EB and LES were established as previously described
(30). Briefly, cells were isolated by digestion with collagenase (1.7 mg/ml), elastase (0.5 mg/ml), and 1 mg/ml BSA and
plated at a density of ~400 cells/cm2 onto 13-mm
coverslips (for fluorescence microscopy) or directly in 10-cm petri
dishes (for RNA extraction) in DMEM (GIBCO-BRL). Cells were grown in a
humidified atmosphere of 5% CO2 in air at 37°C with 10%
fetal bovine serum.
RNA isolation and RT-PCR.
Total RNA was extracted from EB and LES by phenol-chloroform extraction
using either frozen muscle samples or cultured cells grown to near
confluence (days 10-14), as described previously (30). Agarose gel electrophoresis and ethidium
bromide staining verified the integrity of the RNA. Four micrograms of
total RNA were reverse transcribed with random hexamers using a
first-strand cDNA synthesis kit (Pharmacia Biotech, Madison, WI).
Typically, 3-5 µl of the cDNA reaction mixture was used in each
PCR reaction performed in a 50-µl volume containing PCR buffer, 2 mmol/l MgCl2, 200 µmol/l dNTPs, 0.1 nmol/l of each
primer, and 2 units of Taq DNA polymerase (Quiagen,
Valencia, CA). PCR was carried out in a GeneAmp 2400 PCR thermal cycler
(Perkin-Elmer, Norwalk, CT) for 28-33 cycles. Cycling parameters
were 94°C for 1 min, 52-60°C for 1 min, and 72°C for 2 min,
followed by a final extension at 72°C for 10 min. To ensure that we
were working within the linear phase of each amplification reaction,
aliquots of individual PCR reactions were removed at 2- to 3-cycle
intervals, electrophoresed on 1% agarose gels, and stained with
ethidium bromide. We also examined the effect of different amounts of
input cDNA on PCR product accumulation. The PCR oligonucleotide primers
used to amplify cDNA are listed in Table
1. PCR primers for -actin were used to
confirm fidelity of the PCR reaction and to detect genomic DNA
contamination. The amplified products (10 µl) were analyzed by
electrophoresis on 1% agarose-Tris-acetic acid-EDTA gels and visualized by ethidium bromide staining. Sequencing of PCR products for
verification was done in the Robarts Research Institute Core Molecular
Biology Facility (London, ON, Canada).
|
Measurement of intracellular Ca2+. Free intracellular Ca2+ concentration ([Ca2+]i) was determined in cultured cells grown on coverslips using fura 2 as previously described (23). Cells were loaded by incubation in serum-free DMEM containing 0.2 µmol/l fura 2-AM (30 min, 25°C) then transferred to fresh DMEM for 30 min at 37°C for dye cleavage. Coverslips were then transferred to a 0.75-ml chamber mounted on a Nikon inverted microscope and superfused at 2-5 ml/min at room temperature. Individual cells were illuminated with alternating 345- and 380-nm light using a Deltascan system (Photon Technology International, London, ON, Canada), with emission detected by a photomultiplier at 510 nm. Following correction for background fluorescence, [Ca2+]i was calculated as described previously (23, 30). The superfusion buffer had the following composition (in mmol/l): 130 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 20 HEPES, and 10 D-glucose, adjusted to pH 7.4. Ca2+-free buffer contained 0.5 mmol/l EGTA to chelate residual Ca2+.
Drugs and materials. Fura 2-AM (Molecular Probes) was prepared in dimethylsulfoxide. All drugs used were obtained from Sigma (St. Louis, MO). Other reagents were obtained from VWR (Mississauga, ON, Canada) or as indicated above. Drugs were prepared as concentrated stock solutions and diluted into the appropriate bathing solution before the addition to cells.
Statistics. Data are expressed as means ± SE. The number of cells tested is designated by an n. All experiments were repeated with at least three different esophageal specimens. Statistical analyses were performed using Student's t-test. Differences were considered to be significant when P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
EB and LES SMCs in culture.
Dispersed SMCs from EB and LES showed signs of growth after 2-3
days in culture and displayed similar growth characteristics, morphology, and -actin immunostaining (Figs. 1 and
2). These primary cultures are composed
of >96% SMCs without contamination by other cell types, based on the
absence of significant immunostaining for markers for endothelial
cells, nerve cells, and interstitial cells of Cajal, as previously
described (30). Individual SMCs were phase bright with a
larger central area containing the nucleus and were spindle shaped
(Fig. 1). At confluence, cells assumed a typical hill-and-valley
appearance (Fig. 1), as described for other smooth muscles
(5). Immunostaining for
-smooth muscle actin revealed
positive staining in virtually all cells seen as intracellular strands
(Fig. 2).
|
|
RT-PCR identifies TRPC channel mRNA in LES and EB.
PCR primers were designed to amplify sequences specific for six of
seven mammalian TRPC channels that have been cloned to date (7,
17, 19, 32). TRPC2 is a pseudogene in the human and
was not examined in this study (28). mRNA for five TRPC channels was detected in cultured cells from both the EB and LES, including TRPC1, TRPC3, TRPC4,
TRPC5, and TRPC6 (Fig.
3). The PCR products were of the expected
size, and in each case the identity of the PCR product was confirmed by
direct sequencing. The presence of a single band for -actin in each
sample rules out genomic DNA contamination. mRNA for TRPC7,
which was recently cloned from mouse brain (17), was
readily detected in rat brain and human brain and heart (Fig. 3) but
could not be identified in any of four esophageal specimens tested.
Primer sets for all of the TRPC genes except TRPC7
were based on the known human gene sequences. In the case of
TRPC7, the primers were based on the mouse sequence (17), but the gene product was homologous to the human
sequence that has been identified subsequently (GenBank accession no.
AJ272034).
|
|
|
|
EB and LES SMCs show CCE.
TRPC channels have been implicated in a number of cellular functions
including CCE, the putative mechanism for refilling of Ca2+
stores following store depletion (7, 19, 32). To explore the functional correlate of the difference in TRPC channel expression in LES and EB cells, we next compared CCE in these cell types. CCE was
quantified using an approach previously applied in a number of other
cell types (2, 10). The application of ACh produced a
transient increase in [Ca2+]i (Fig.
7), which has been previously shown to be
due largely to [Ca2+]i release from
intracellular stores (30). The application of thapsigargin
(TG), an SR-Ca2+-ATPase inhibitor, in the absence of
extracellular Ca2+, resulted in an elevation of
[Ca2+]i, reflecting the emptying of
intracellular stores. Under these conditions, a Ca2+ entry
pathway is activated, as illustrated by the transient rise in
[Ca2+]i seen on subsequent application of
external Ca2+ (Fig. 7, A and B). This
Ca2+ influx was not observed in control experiments when TG
application was omitted, indicating that depletion of intracellular
Ca2+ stores is essential for activating this
Ca2+ influx pathway (Fig. 7B). Furthermore,
Ca2+ influx was reversibly blocked by 10 µmol/l
La3+ (Fig. 7C) but not by the L-type
voltage-gated Ca2+ channel blocker nifedipine (1 µmol/l;
Fig. 7A). A similar transient rise in
[Ca2+]i on application of external
Ca2+ following TG was also observed in freshly isolated
cells (data not shown). In addition, we previously showed that blockade
of the SR-Ca2+-ATPase in intact muscle strips in the
presence of extracellular Ca2+ results in prolonged
contraction, consistent with Ca2+ influx (23).
Together, these findings are characteristic of the presence of SOCs
that mediate CCE (2).
|
Comparison of CCE in LES and EB SMCs.
Despite the fact that a defining characteristic of LES smooth muscle is
the development of sustained tonic contraction, resting [Ca2+]i was not significantly different in
SMCs from the LES (138 ± 8 nmol/l, n = 21)
compared with EB (110 ± 6 nmol/l, n = 18) (Fig. 8). Furthermore, the application of ACh
elicited a transient increase in [Ca2+]i of
similar amplitude in both cell types (650 ± 28 nmol/l and 590 ± 21 nmol/l, respectively) (Fig. 8). However, the
TG-associated increases in [Ca2+]i were about
twofold greater in LES SMCs (550 ± 22 nmol/l, n = 21) compared with EB SMCs (250 ± 15 nmol/l, n = 18, P < 0.001) (Fig. 8D). CCE, as measured
by the peak increase in [Ca2+]i on
application of external Ca2+, was also significantly
greater in LES (585 ± 35 nmol/l, n = 21) than in
EB SMCs (295 ± 21 nmol/l, n = 19, P < 0.001) when assessed by application of 1 mmol/l
Ca2+ and measuring the peak increase in
[Ca2+]i (Fig. 8, C and
D). This difference is maintained when the results are
normalized to the peak transient [Ca2+]i
elicited by ACh, minimizing possible effects of differences in cell
size (Fig. 8E).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have identified the expression of mRNA species encoding five of the seven known mammalian TRPC channel genes in human EB and LES smooth muscles. Using semiquantitative RT-PCR, we demonstrated enrichment of mRNA for TRPC3 and TRPC4 in muscle from the LES compared with muscle from the EB. We have demonstrated that CCE can be activated in these muscles by store depletion and that this pathway is more prominent in LES SMCs. These differences in the Ca2+ homeostatic mechanism may contribute to the characteristic property of LES smooth muscle to develop and maintain spontaneous tonic contraction.
Our previous studies have demonstrated that both Ca2+ influx and release from intracellular Ca2+ stores contribute to cholinergic excitation in human esophageal smooth muscle (23). We showed that cyclopiazonic acid, which acts similarly to TG and blocks the SR-Ca2+-ATPase, produces a sustained contraction of esophageal smooth muscle, consistent with participation of intracellular Ca2+ stores and activation of a Ca2+ influx pathway. The present data confirms the presence of CCE as a functional Ca2+ entry pathway with characteristic features similar to those described in several other cell types. CCE was activated by inhibition of the SR-Ca2+-ATPase, reversibly blocked by La3+ but insensitive to the L-type Ca2+ channel inhibitor nifedipine (2). Application of extracellular Ca2+ without prior depletion of intracellular stores did not produce a transient rise in [Ca2+]i, indicating that Ca2+ store depletion is required for CCE activation.
To date, seven members of the TRPC family of genes have been identified, with recent evidence for the existence of splice variants of TRPC4 and TRPC7 (7, 17, 29, 32). These channels can be categorized into four groups based on structural and functional similarities as follows: TRPC1; TRPC2; TRPC3, -6, -7; and TRPC4, -5 (7). There remains some controversy over the regulation of each TRPC subtype and whether they function as ROCs, SOCs, or both. TRPC2 is a pseudogene in the human and has not been shown to form a functional channel (28). Of the remaining channels, TRPC1, TRPC4, and TRPC5 can be activated by store depletion and thus are suggested to mediate CCE (7). In the present study, CCE was activated by store depletion following blockade of the SR-Ca2+-ATPase and thus did not involve agonist-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] or diacylglycerol (DAG) generation. Since TRPC4 can be activated by store depletion without stimulation by Ins(1,4,5)P3 or DAG, our finding that mRNA for TRPC4 is more abundant in LES compared with EB can be correlated with enhanced CCE in SMCs derived from LES.
Recent evidence suggests that TRPC4 may also function as an ROC that
can be activated independently of store depletion in some conditions
(22). The signaling pathway is not clearly defined, but
others have shown that TRPC4 can bind and be activated by Ca2+-calmodulin (25, 27). Ioune et al.
(13) recently provided evidence that TRPC6 forms a ROC
that mediates -adrenoceptor activation of a nonselective cation
current in vascular SMCs. A similar receptor-activated nonselective
cation current that can cause smooth muscle depolarization has been
demonstrated in opossum (1) and human (Sims SM and Preiksaitis HG, unpublished observations) esophageal smooth muscles and
represents an additional candidate function for TRPC4, TRPC6, or other
TRPC subtypes.
LES smooth muscle also showed greater transcriptional expression of TRPC3. Whether this may contribute to enhanced CCE as well is not known, since we did not study CCE during agonist stimulation, conditions that would result in accumulation of Ins(1,4,5)P3 or DAG, which activate TRPC3 (7, 32). Additional possible roles for TRPC3 mediating other Ca2+ entry pathways, such as ROCs mentioned above, will need to be considered. Moreover, emerging evidence suggests that the functional TRPC channel is a tetramer formed by four subunits and that these channels may exist as heteromultimers (15, 24, 31). For example, coassembly of TRPC1 and TRPC3 or TRPC1 and TRPC5 to form novel cation channels has been demonstrated in HEK-293 cells (15, 24), and TRPC1 and TRPC5 have similar distribution in the hippocampus (24). This raises the possibility that the physiologically active forms of these channels in intact tissues, including LES or EB smooth muscles, may result from a combination of TRPC subtypes.
The pattern of TRPC channel subtype mRNA expression in human esophageal smooth muscle found in the present study differs from that of other smooth muscle types. Walker et al. (29), recently showed that TRPC4, TRPC6, and TRPC7 were the predominant subtypes expressed in canine and murine smooth muscles. In contrast, we could not detect mRNA expression for TRPC7 in either cultured cells or tissue of the human esophagus, although our methods readily identified the gene product in rat brain and human brain and heart. We did not examine other human gastrointestinal smooth muscles for the expression of TRPC7. In dog and mouse, TRPC4 and TRPC7 showed a substantial quantitative variation in gene expression between different gastrointestinal smooth muscles (29), but the functional correlate of these differences has not been studied. On the other hand, TRPC3 mRNA was detected in EB and LES, whereas none was identified in canine or murine gastric, jejunal, or colonic smooth muscles (29) or in the rat or canine pulmonary artery (16, 29). The functional implications of these striking tissue-dependent differences in TRPC subtype expression remain to be elucidated. It should recognized that these comparisons are based on detection of mRNA in these tissues and not functional channel proteins. Further studies using subtype-specific antibodies or blocking drugs are limited by the lack of general availability of such reagents. Moreover, EB and LES cells in culture assume a proliferative noncontractile phenotype similar to other SMC cultures (5, 30), raising the possibility that changes in TRPC channel expression may be related to cell phenotype. The fact that we identified a similar pattern of TRPC mRNA expression in whole tissues from the EB and LES indicates that this is unlikely.
LES muscle is characterized by its ability to maintain spontaneous myogenic tension, whereas the adjacent muscle of the EB contracts in response to nerve activation during peristalsis. The unique features of LES smooth muscle that impart its characteristic behavior are incompletely understood. The regional differences in TRPC channel expression and CCE we identified in the present study add to the growing body of information characterizing the distinct physiological properties of LES and EB smooth muscles. For example, compared with EB, LES SMCs are larger, have a more irregular cell surface, a more depolarized resting membrane potential, greater SR density, more plentiful mitochondria, different contractile protein composition, and lower levels of cytochrome c oxidase (8, 12). The central role of Ca2+ in smooth muscle contraction provides a logical focus for exploring differences in muscle function. The findings of the present study provide additional evidence that fundamentally important differences exist between these muscle types in the regulation of cell Ca2+.
The relative importance of intracellular Ca2+ stores or influx from the extracellular space in the characteristic phasic contraction of EB smooth muscle during peristalsis vs. the sustained muscular contraction of LES remains controversial. Biancani et al. (3, 4) showed that LES tonic contraction in the cat was dependent on continuous low-level Ca2+ release from intracellular stores. Such a mechanism would require an efficient pathway for replenishing these stores. In several other species, removing extracellular Ca2+ or blocking influx reduces LES tone (9, 21, 26), although it remains uncertain whether this represents a direct contribution of Ca2+ influx to [Ca2+]i or an indirect effect on intracellular Ca2+ stores. Influx of Ca2+ via L-type channels has been shown to contribute only partially to refilling of intracellular Ca2+ stores in the canine LES (21) and not at all in human esophageal smooth muscle (23). Previously, we demonstrated that removal of extracellular Na+ had no significant effect on basal Ca2+ levels or the response of [Ca2+]i to ACh stimulation in human esophageal cells, indicating that Na+/Ca2+ exchange pathways did not contribute significantly to Ca2+ homeostasis in this tissue. Finally, enhanced CCE in LES cells was also reflected by enhanced Mn2+ entry. Mn2+ is a poor substrate for the SR-Ca2+-ATPase and the Na+/Ca2+ exchanger (11). Together, these observations suggest that the maintenance and refilling of intracellular Ca2+ stores requires additional influx pathways, most likely CCE.
In addition to enrichment in transcriptional expression of TRPC subtypes and enhanced CCE in smooth muscle from the LES, the acute increase in [Ca2+]i following blockade of the SR-Ca2+-ATPase with TG was greater in cells derived from the LES compared with EB. We interpret this finding as indicating the presence of larger releasable Ca2+ stores in the LES smooth muscle. This finding may be relevant to previous studies in the opossum, which have shown more abundant SR in the LES compared with EB (6).
In summary, the present study demonstrates the expression of mRNA encoding multiple TRPC channel subtypes in smooth muscle of the human EB and LES. mRNA for TRPC3 and TRPC4 are enriched in smooth muscle from the LES, which also shows enhanced CCE. These differences may contribute to the unique physiological properties of these two smooth muscle types.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. R. Inculet, R. Malthaner, and C. Rajgopal for providing esophagectomy specimens and to Tom Chrones for help in preparing the manuscript.
![]() |
FOOTNOTES |
---|
This work was supported by grants from The Canadian Institutes of Health Research (MT1009 and MOP 12608). J. Wang was a Canadian Institutes of Health Research Postdoctoral Fellow, and H. G. Preiksaitis is an Ontario Ministry of Health Career Scientist.
Address for reprint requests and other correspondence: H. G. Preiksaitis, Dept. of Medicine, St. Joseph's Health Centre, 268 Grosvenor St., London, Ontario N6A 4V2, Canada (E-mail: haroldp{at}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.
10.1152/ajpgi.00227.2002
Received 12 June 2002; accepted in final form 13 January 2003.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Akbarali, HI,
Hatakeyama N,
Wang Q,
and
Goyal RK.
Transient outward current in opossum esophageal circular muscle.
Am J Physiol Gastrointest Liver Physiol
268:
G979-G987,
1995
2.
Arnon, A,
Hamlyn JM,
and
Blaustein MP.
Na+ entry via store-operated channels modulates Ca2+ signaling in arterial myocytes.
Am J Physiol Cell Physiol
278:
C163-C173,
2000
3.
Biancani, P,
Harnett KM,
Sohn UD,
Rhim BY,
Behar J,
Hillemeier C,
and
Bitar KN.
Differential signal transduction pathways in cat lower esophageal sphincter tone and response to ACh.
Am J Physiol Gastrointest Liver Physiol
266:
G767-G774,
1994
4.
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:
G760-G766,
1987
5.
Chamley-Campbell, J,
Campbell GR,
and
Ross R.
The smooth muscle cell in culture.
Physiol Rev
59:
1-61,
1979
6.
Christensen, J,
and
Roberts RL.
Differences between esophageal body and lower esophageal sphincter in mitochondria of smooth muscle in opossum.
Gastroenterology
85:
650-656,
1983[ISI][Medline].
7.
Clapham, DE,
Runnels LW,
and
Strubing C.
The TRP ion channel family.
Nat Rev Neurosci
2:
387-396,
2001[ISI][Medline].
8.
Daniel, EE.
Lower esophagus: structure and function.
In: Sphincters: Normal FunctionChanges in Diseases, edited by Daniel EE,
Tomita T,
Tsuchida S,
and Watanabe M.. Boca Raton, FL: CRC, 1992, p. 49-66.
9.
Fox, JA,
and
Daniel EE.
Role of Ca2+ in genesis of lower esophageal sphincter tone and other active contractions.
Am J Physiol Endocrinol Metab Gastrointest Physiol
237:
E163-E171,
1979
10.
Golovina, VA,
Platoshyn O,
Bailey CL,
Wang J,
Limsuwan A,
Sweeney M,
Rubin LJ,
and
Yuan JX.
Upregulated TRP and enhanced capacitative Ca2+ entry in human pulmonary artery myocytes during proliferation.
Am J Physiol Heart Circ Physiol
280:
H746-H755,
2001
11.
Golovina, VA.
Cell proliferation is associated with enhanced capacitative Ca2+ entry in human arterial myocytes.
Am J Physiol Cell Physiol
277:
C343-C349,
1999
12.
Goyal, RK,
and
Sivarao DV.
Functional anatomy and physiology of swallowing and esophageal motility. The Esophagus (3rd ed.), edited by Castel DO.. Baltimore, MD: Lippincott Williams & Wilkins, 1999, p. 1-43.
13.
Inoue, R,
Okada T,
Onoue H,
Hara Y,
Shimizu S,
Naitoh S,
Ito Y,
and
Mori Y.
The transient receptor potential protein homologue TRP6 is the essential component of vascular alpha1-adrenoceptor-activated Ca2+-permeable cation channel.
Circ Res
88:
325-332,
2000[ISI].
14.
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
15.
Lintschinger, B,
Balzer-Geldsetzer M,
Baskaran T,
Graier WF,
Romanin C,
Zhu MX,
and
Groschner K.
Coassembly of TRP1 and TRP3 proteins generates diacylglycerol- and Ca2+-sensitive cation channels.
J Biol Chem
275:
27799-27805,
2000
16.
McDaniel, SS,
Platoshyn O,
Wang J,
Yu Y,
Sweeney M,
Krick S,
Rubin LJ,
and
Yuan JX.
Capacitative Ca2+ entry in agonist-induced pulmonary vasoconstriction.
Am J Physiol Lung Cell Mol Physiol
280:
L870-L880,
2001
17.
Okada, T,
Inoue R,
Yamazaki K,
Maeda A,
Kurosaki T,
Yamakuni T,
Tanaka I,
Shimizu S,
Ikenaka K,
Imoto K,
and
Mori Y.
Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. Ca2+-permeable cation channel that is constitutively activated and enhanced by stimulation of G protein-coupled receptor.
J Biol Chem
274:
27359-27370,
1999
18.
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:
G1321-G1327,
1997
19.
Putney, JW, Jr.
TRP, inositol 1,4,5-trisphosphate receptors, and capacitative calcium entry.
Proc Natl Acad Sci USA
96:
14669-14671,
1999
20.
Putney, JW, Jr.
Muscarinic, -adrenergic and peptide receptors regulate the same calcium influx sites in the parotid gland.
J Physiol
268:
139-149,
1977[Abstract].
21.
Salapatek, AM,
Lam A,
and
Daniel EE.
Calcium source diversity in canine lower esophageal sphincter muscle.
J Pharmacol Exp Ther
287:
98-106,
1998
22.
Schaefer, M,
Plant TD,
Obukhov AG,
Hofmann T,
Gudermann T,
and
Schultz G.
Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5.
J Biol Chem
275:
17517-17526,
2000
23.
Sims, SM,
Jiao Y,
and
Preiksaitis HG.
Regulation of intracellular calcium in human esophageal smooth muscle.
Am J Physiol Cell Physiol
273:
C1679-C1689,
1997
24.
Strubing, C,
Krapivinsky G,
Krapivinsky L,
and
Clapham DE.
TRPC1 and TRPC5 form a novel cation channel in mammalian brain.
Neuron
29:
645-655,
2001[ISI][Medline].
25.
Tang, Y,
Tang J,
Chen Z,
Trost C,
Flockerzi V,
Li M,
Ramesh V,
and
Zhu MX.
Association of mammalian TRP4 and phospholipase C isozymes with a PDZ domain-containing protein, NHERF.
J Biol Chem
275:
37559-37564,
2000
26.
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:
G338-G343,
1990
27.
Trost, C,
Bergs C,
Himmerkus N,
and
Flockerzi V.
The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins.
Biochem J
355:
663-670,
2001[ISI][Medline].
28.
Vannier, B,
Peyton M,
Boulay G,
Brown D,
Qin N,
Jiang M,
Zhu X,
and
Birnbaumer L.
Mouse TRP2, the homologue of the human TRPC2 pseudogene, encodes mTRP2, a store depletion-activated capacitative Ca2+ entry channel.
Proc Natl Acad Sci USA
96:
2060-2064,
1999
29.
Walker, RL,
Hume JR,
and
Horowitz B.
Differential expression and alternative splicing of TRP channel genes in smooth muscles.
Am J Physiol Cell Physiol
280:
C1184-C1192,
2001
30.
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
31.
Xu, XZ,
Li HS,
Guggino WB,
and
Montell C.
Coassembly of TRP and TRPL produces a distinct store-operated conductance.
Cell
89:
1155-1164,
1997[ISI][Medline].
32.
Zhu, X,
Jiang M,
Peyton M,
Boulay G,
Hurst R,
Stefani E,
and
Birnbaumer L.
TRP, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry.
Cell
85:
661-671,
1996[ISI][Medline].