Enhanced capacitative calcium entry and TRPC channel gene expression in human LES smooth muscle

Jian Wang1,2,3, Lisanne G. Laurier1,3, Stephen M. Sims2, and Harold G. Preiksaitis1,2,3

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

Immunocytochemistry of cultured cells was carried out as previously described (30). Mouse monoclonal anti-alpha -smooth muscle actin (1:50; Boehringer) was used with Cy3-linked anti-mouse secondary antibody (1:100; Jackson Labs, West Grove, PA). TO-PRO-1 dimeric cyanine dye (Molecular Probes, Eugene, OR) was used to stain nuclei. Primary antibody was omitted in controls. Coverslips were mounted on slides with FluoroGuard Antifade (Bio-Rad, Hercules, CA). Cells were examined and photographed by using an Olympus IMT-2 inverted microscope using phase-contrast optics or a Zeiss Axioscope fluorescence microscope equipped with a Sony DXC-950 3CCD color video camera for capture of immunocytochemistry images. Only primary cultures or intact whole muscle pieces were used in the present study.

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 beta -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).

                              
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Table 1.   PCR Primers

To assess the relative levels of TRPC transcripts, multiplex PCR was carried out in the presence of commercially available 18S RNA competimers (Ambion, Austin, TX) following the manufacturer's recommendations. For these experiments, 5 µl of cDNA was used in each PCR reaction along with specific TRPC gene primers. The 18S RNA primers were added to the PCR mix after 5 cycles of amplification, and the reaction was terminated after 28 cycles. PCR products were quantified by capillary electrophoresis using a Beckman P/ACE 2100 CE system equipped with laser-induced fluorescence detection, with peak height used to determine the relative ratio of the expression of TRPC and 18S RNA.

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -smooth muscle actin revealed positive staining in virtually all cells seen as intracellular strands (Fig. 2).


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Fig. 1.   Phase contrast microscopy of human lower esophageal sphincter (LES) and esophageal body (EB) smooth muscle cells (SMCs) cultured for 5 days, 8 days, and at confluence (14 days). LES and EB cells showed similar morphology and growth characteristics. Calibration bar at bottom right refers to all micrographs.



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Fig. 2.   Fluorescence immunocytochemical identification of alpha -smooth muscle actin in cultured cells. Smooth muscle-specific alpha -actin antibody identifies alpha -actin (shown in red) in cultured cells derived from LES and EB. Cells shown were grown in primary culture for 10 days. The nuclear marker TO-PRO-1 is shown in green, revealing that virtually all SMCs contained the smooth muscle-specific marker.

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 beta -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).


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Fig. 3.   Transcriptional expression detected for transient receptor potential channel genes TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 but not TRPC7 in human LES and EB SMCs. RT-PCR was carried out on cDNA samples from LES and EB using specific primers for TRPC and 18S RNAs (see Table 1). For each PCR reaction, primers for 18S RNA were added after 5 cycles of amplification. RT-PCR carried out with primers for mouse TRPC7 did not detect mRNA transcripts in LES or EB, although mRNA for TRPC7 was detected in rat brain and human heart and brain (right). PCR products of the expected sizes were obtained (Table 1), and their identities were confirmed by sequencing. The beta -actin sequence amplified spans a 206-bp intron, so the finding of a single band of 661 bp verifies that genomic DNA was not present. Ladder shows molecular weight markers.

Our initial assessment of TRPC subtypes in EB and LES revealed specific differences between LES and EB. mRNA for TRPC3 and TRPC4 appeared to be more abundant in LES compared with EB cells, whereas TRPC1, TRPC5, and TRPC6 showed similar levels of transcription in both tissues (Fig. 3). To investigate this in more detail, semiquantitative RT-PCR was carried out using primers for 18S RNA (competimers) in each PCR reaction as a reference standard. Samples of the PCR reaction mixture were removed at 2- to 3-cycle intervals, subjected to electrophoresis on agarose gels, and stained with ethidium bromide (Fig. 4A). Samples from these reactions were subjected to capillary electrophoresis, and amplification curves were constructed to verify linearity of the PCR reactions with respect to cycle number (Fig. 4B) using relative fluorescence intensity as a guide to the amount of PCR product. Serial dilution of the template cDNA yielded similar results (Fig. 4, C and D).


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Fig. 4.   Effect of cycle length and cDNA concentration on TRPC amplicon accumulation. RT-PCR was carried out using primers for 18S RNA and TRPC1, TRPC3, and TRPC4 using cDNA samples from cultured LES and EB SMCs. PCR amplicon accumulated in a nearly linear manner with the number of PCR cycles (A and B) and the amount of input cDNA (C and D). On the basis of these data, we used 5 µl cDNA and 28 cycles of amplification for subsequent semiquantitative RT-PCR. Straight lines are best-fits to the data, with solid lines for filled symbols and dashed lines for open symbols.

Based on these results, the amount of input cDNA (5 µl) and cycles of amplification (28 cycles) was selected for semiquantitative PCR. Figure 5 shows these results for TRPC1, TRPC3, and TRPC4 in EB and LES SMCs obtained from four separate esophageal specimens. Both ethidium bromide-stained agarose gels (Fig. 5, left) and quantification by capillary electrophoresis (Fig. 5, right) showed greater levels of TRPC3 and TRPC4 mRNA in SMCs from the LES compared with EB, whereas TRPC1 mRNA expression was not different. To address the possibility that these differences in TRPC expression might be unique to SMCs in culture, we next carried out similar experiments using intact tissues from EB and LES, where the pattern of TRPC mRNA expression was confirmed (Fig. 6). These data validate the primary culture model and confirm the physiological relevance of this observation.


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Fig. 5.   LES and EB show quantitative differences in TRPC subtype transcriptional expression. A: cDNA from cultured SMCs derived from 4 independent esophageal LES and paired EB specimens (S1-S4) was amplified with primers for TRPC1, TRPC3, TRPC4, and 18S RNA using reaction conditions described in Fig. 4 and in MATERIALS AND METHODS. PCR products were electrophoresed on agarose gels and stained with ethidium bromide (left). Aliquots of each reaction were subjected to capillary electrophoresis, peak heights were obtained, and ratios of TRPC/18S RNA mRNA expression were calculated. B: relative differences in the amounts of TRPC1, TRPC3, and TRPC4 in LES and EB (means ± SE; *P < 0.001).



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Fig. 6.   Intact EB and LES muscle shows a pattern of TRPC mRNA expression similar to cultured cells. RNA was extracted from intact muscle specimens from EB and LES, reverse-transcribed, and subjected to RT-PCR using primers specific for each TRPC channel and 18S RNA. PCR products of the expected size are seen for TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and 18S RNA. TRPC7 mRNA was not detected in LES or EB smooth muscle but was present in rat brain (positive control). Results shown are for LES and EB tissues from a single esophageal specimen. Similar results were obtained in two additional esophageal specimens.

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).


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Fig. 7.   Demonstration of capacitative Ca2+ entry (CCE) in human LES SMCs. Fura 2 Ca2+ fluorescence was used to measure free intracellular Ca2+ ([Ca2+]i). ACh (10 µmol/l, 10 s) produced a transient rise in [Ca2+]i. With zero Ca2+ in the bathing medium, inhibition of the sarcoplasmic reticulum (SR)-Ca2+-ATPase with 2 µmol/l thapsigargin (TG, applied for 60 s) caused a rise in [Ca2+]i as intracellular Ca2+ stores were depleted. This resulted in activation of CCE, as evidenced by the transient increase in [Ca2+]i elicited on application of 1 mmol/l Ca2+ by pressure ejection from a glass pipette. CCE was not blocked by L-type Ca2+ channel blocker nifedipine (A, Nif, 1 µmol/l) but was substantially inhibited by 10 µmol/l La3+ (B), properties consistent with CCE. Application of Ca2+ without prior exposure to TG did not cause any change of [Ca2+]i, indicating that inhibition of the SR-Ca2+-ATPase was required for activation of CCE (C). Typical sample traces obtained from LES cells after 7 days in culture are shown here. Similar results were obtained in EB cells. D: effects of blockers (La3+, P < 0.001, n = 9 cells from 4 specimens; Nif, not significant, n = 10 cells from 3 specimens).

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).


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Fig. 8.   LES SMCs show enhanced CCE compared with EB SMCs. CCE in EB (A) and LES (B) cells were determined using the protocol described in Fig. 7. The peak rise in [Ca2+]i was compared between EB and LES cells (n = 21 cells, 5 specimens). C: basal [Ca2+]i and the peak transient rise in response to ACh (10 µmol/l, 10 s) were similar in EB and LES. D: rise in [Ca2+]i following application of TG (2 µmol/l) was ~2-fold greater in LES cells than in EB cells. In addition, the peak rise in [Ca2+]i with application of extracellular Ca2+ (CCE) was 2-fold greater in LES than in EB. E: this difference was also apparent when results of individual cells were normalized as the ratio of the CCE and ACh [Ca2+]i transients, suggesting that the differences were not due to cell size. *P < 0.01.

To further investigate the properties of the CCE pathway in LES and EB SMCs, we used extracellular Mn2+ as a surrogate ion for Ca2+. Others have shown that Mn2+ can enter the cell via SOCs and that the rate of entry can be determined by monitoring quenching of fura 2 fluorescence at an excitation wavelength of 360 nm, the isosbestic point (11). As seen in Fig. 9, the rate of quenching by Mn2+ was significantly more rapid in LES cells than in EB SMCs, indicating a greater rate of influx of Mn2+ through SOCs, providing further evidence for CCE being enhanced in LES compared with EB SMCs.


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Fig. 9.   Mn2+ influx in LES and EB SMCs. A: the rate of quenching of fura 2 fluorescence represents the rate of Mn2+ influx following external application of 200 µmol/l Mn2+. TG (2 µmol/l) was added 2 min before Mn2+, resulting in irreversible blockade of the SR-Ca2+-ATPase and activation of store-operated channels. Representative traces from an EB SMC and a LES SMC derived from the same specimen are shown superimposed. B: the half-time of Mn2+ entry was significantly shorter for LES SMCs compared with EB SMCs (n = 19 from 4 specimens, P < 0.005), further supporting the observed enhanced CCE in LES compared with EB.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

5.   Chamley-Campbell, J, Campbell GR, and Ross R. The smooth muscle cell in culture. Physiol Rev 59: 1-61, 1979[Free Full Text].

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 Function---Changes 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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

19.   Putney, JW, Jr. TRP, inositol 1,4,5-trisphosphate receptors, and capacitative calcium entry. Proc Natl Acad Sci USA 96: 14669-14671, 1999[Free Full Text].

20.   Putney, JW, Jr. Muscarinic, alpha -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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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].


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