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J. Biol. Chem., Vol. 277, Issue 21, 19191-19197, May 24, 2002

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Calcium Oscillation Linked to Pacemaking of Interstitial Cells of Cajal

REQUIREMENT OF CALCIUM INFLUX AND LOCALIZATION OF TRP4 IN CAVEOLAE^*

Shigeko Torihashi^§, Toyoshi Fujimoto, Claudia Trost^¶, and Shinsuke Nakayama

From the Departments of  Anatomy and Cell Biology and  Cell Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan and the ^¶ Institute für Pharmakologie und Toxikologie, Universität des Saarlandes, D-66421 Homburg, Germany

Received for publication, February 20, 2002, and in revised form, March 14, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Interstitial cells of Cajal (ICC) are considered to be pacemaker cells in gastrointestinal tracts. ICC generate electrical rhythmicity (dihydropyridine-insensitive) as slow waves and drive spontaneous contraction of smooth muscles. Although cytosolic Ca²⁺ has been assumed to play a key role in pacemaking, Ca²⁺ movements in ICC have not yet been examined in detail. In the present study, using cultured cell clusters isolated from mouse small intestine, we demonstrated Ca²⁺ oscillations in ICC. Fluo-4 was loaded to the cell cluster, the relative amount of cytosolic Ca²⁺ was recorded, and ICC were identified by c-Kit immunoreactivity. We specifically detected Ca²⁺ oscillation in ICC in the presence of dihydropyridine, which abolishes Ca²⁺ oscillation in smooth muscles. The oscillation was coupled to the electrical activity corresponding to slow waves, and it depended on Ca²⁺ influx through a non-selective cation channel, which was SK&F 96365-sensitive and store-operated. We further demonstrated the presence of transient receptor potential-like channel 4 (TRP4) in caveolae of ICC. Taken together, the results infer that the Ca²⁺ oscillation in ICC is intimately linked to the pacemaker function and depends on Ca²⁺ influx mediated by TRP4.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Interstitial cells of Cajal (ICC)¹ are a distinct and unique cell population distributed in the gastrointestinal (GI) muscle layer of many vertebrates including humans (1-2). They are network-forming cells connected electrically with each other and with smooth muscle cells via gap junctions. GI muscle shows spontaneous rhythmical contractions accompanied by periodic electrical oscillation, i.e. so-called slow waves that are affected by neither tetradotoxin (TTX, blocker of nervous activity) nor dihydropyridine (blocker of L-type calcium channel) (3-5). It has been postulated that ICC are pacemaker cells that generate slow waves and induce spontaneous contractions of the smooth muscles.

In the last decade, ICC were found to express the proto-oncogene c-kit and to develop depending upon activation of c-kit signal pathways (4, 6-8). Expression of c-kit and/or c-Kit receptors has been used commonly as a marker of ICC in the GI muscle layers, and it also enables the isolation of ICC. Recent electrophysiological studies using isolated ICC have demonstrated periodic oscillations of the membrane current (9-11). Thus, ICC have been considered as pacemaker cells in recent years.

The intrinsic properties underlying the pacemaking mechanism in ICC have been emphasized in previous reports (11). Several groups have reported that generation of electrical rhythmicity involves Ca²⁺ release through inositol trisphosphate (IP₃) type 1 receptor in the endoplasmic reticulum (ER) and subsequent Ca²⁺ entry into mitochondria (12-14). From these results, periodic rises in the cytosolic (intracellular) Ca²⁺ concentration ([Ca²⁺]_i) are considered to play a key role in generating slow waves. However, such [Ca²⁺]_i oscillation has not yet been analyzed precisely. Currently, using Ca²⁺ imaging techniques, Yamazawa and Iino (15) have demonstrated Ca²⁺ transients in ICC and longitudinal smooth muscles. Their results suggested that [Ca²⁺]_i plays a crucial role in pacemaking and that Ca²⁺ imaging at the tissue level is a useful technique to investigate slow wave propagation in GI muscle. It is, however, necessary to clearly identify the distribution of ICC and [Ca²⁺]_i in order to analyze the mechanism underlying the generation of electrical rhythmicity in ICC. In the present study we recorded fluorescent Ca²⁺ images in which ICC were identified by c-Kit immunostaining in small cell clusters isolated from GI muscle. The observed [Ca²⁺]_i oscillation was sensitive to neither TTX nor dihydropyridine so that it was isolated from [Ca²⁺]_i in smooth muscles in the presence of nifedipine.

In contrast to the intracellular Ca²⁺ circuit, periodic activation of plasmalemmal channels to generate pacemaker current has not been demonstrated, although several candidates such as non-selective cation channels including TRPs, Cl channels, and/or Ca²⁺-activated K⁺ channels have been reported (5, 8, 16-18). To address this, we investigated the pharmacological properties of [Ca²⁺]_i oscillation and the influx in ICC using cultured cell clusters. We also examined expression of putative channels by immunohistochemistry. Our results indicate that [Ca²⁺]_i oscillation in ICC requires a Ca²⁺ influx and that TRP4 is at best a candidate for the mediator.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Cultured Cell Clusters-- BALB/c mice (10-15 days after birth) of either sex were used. Animals were treated according to the Guide to Animal Use and Care of the Nagoya University School of Medicine. Smooth muscle layers of small intestines were separated from the mucosa, cut into small pieces, and incubated in Ca²⁺-free Hanks' solution containing collagenase (1.3 mg/ml, Wako Chemical), trypsin inhibitors (2 mg/ml), ATP (0.27 mg/ml), and bovine serum albumin (2 mg/ml) for 40-45 min at 37 °C. After rinsing with an enzyme-free solution (without collagenase and trypsin inhibitors), the muscle pieces were triturated with fire-blunted glass pipettes. The resultant small cell clusters were placed onto murine collagen-coated coverslips in 35-mm culture dishes and incubated in a culture medium (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum, streptomycin (100 units/ml), and penicillin (100 µg/ml) at 37 °C. After 2-4 days of incubation, the cultured cell clusters were used for Ca²⁺ imaging.

Ca²⁺ Imaging-- The cultured cell clusters were incubated for 2 h (at room temperature) in a modified Krebs solution containing 10 µM fluo-4 acetoxymethyl ester (Dojindo) and detergents (0.02% Pluronic F-127, Dojindo; or 0.02% cremophor EL, Sigma). A CCD camera system (Argus HiSCA, Hamamatsu Photonics) combined with an inverted microscope was used to monitor oscillation of the intracellular Ca²⁺ concentration ([Ca²⁺]_i). [Ca²⁺]_i in ICC was measured in the presence of 1 µM nifedipine (Sigma). The cell clusters were illuminated at 488 nm, and fluorescent emissions of 515-565 nm were recorded at an intensity of fluo-4. Digital Ca²⁺ images (328 × 247 pixels) were normally collected at 100-400-ms intervals. Because fluo-4 is a single wavelength indicator, it was not possible to apply the ratiometric method for quantitative determination of [Ca²⁺]_i. Therefore, the intensity of fluo-4 fluorescence was normalized in the temporal analysis. The temporal fluorescence intensity of the dyes (Ft) was divided by the fluorescence intensity at the start (F0). These relative values represent integrated [Ca²⁺]_i. After recording the fluorescence intensity with nifedipine, localization of ICC were examined by c-Kit immunohistochemistry, and small c-Kit-positive points (2 µm in diameter) were analyzed as [Ca²⁺]_i in ICC. c-Kit-negative points of the same size were recorded as non-ICC regions. During Ca²⁺ imaging, the temperature of the recording chamber was kept at 35 °C using a modified micro-warm plate system (DC-MP10DM, Kitazato Supply), and the bath solution was circulated at 0.5 ml/s.

Simultaneous Recording of Electrical Activity and [Ca²⁺]_i-- In some cell clusters, electrical activity was also measured using a differential amplifier (DP-301, Warner Instruments) and a recticorder (RJG-4022, Nihon Koden). The amplifier was operated in an AC mode (high pass = 0.1 Hz; gain = 1,000). A cut-off frequency of 100 Hz was applied to reduce the noise. Glass pipettes with tips smaller than 10 µm in diameter were filled with modified Krebs solution (see below) and put on the middle of cell clusters during Ca²⁺ imaging.

Immunohistochemistry and Western Blotting-- The cell clusters used for Ca²⁺ imaging were treated for 5 min with rat anti-c-Kit antibody (ACK2, 10 µg/ml) (7) conjugated with Alexa Fluoro 594 (Molecular Probes). After washing with modified Krebs solution, the cell clusters were fixed with ice-cold acetone for 2 min and observed using a confocal laser microscope (LSM5 PASCAL, Zeiss). These samples were incubated with mouse anti--enteric actin antibody (1:200; ICN Pharmaceuticals) again and subsequently treated with goat anti-mouse IgG conjugated with FITC (1:200; Vector Laboratories) followed by re-examination of the same clusters using confocal microscopy. Instead of the procedure described above, some cell clusters were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4 at 4 °C) for 30 min. They were then incubated with rabbit anti-PGP9.5 antibody (1:8,000; UltraClone) and treated with goat anti-rabbit IgG conjugated with Texas Red (1:200, Vector Laboratories) for confocal microscopy. To examine TRP4 immunoreactivity on ICC, muscle layers fixed with ice-cold acetone for 5 min were treated as whole mount preparations for double labeling with rabbit anti-TRP4 antibody (1:100, either from Alomon Laboratories corresponding to residues 943-958 of mouse TRP4 or Ab236 corresponding to residues 969-981 of bovine TRP4) (20) and ACK2. They were detected by goat anti-rabbit IgG with FITC and goat anti-rat IgG with Texas Red (Vector Laboratories). Rabbit anti-TRP6 antibody (1:100; Alomon Laboratories, corresponding to residues 24-38 of mouse TRP6) was also used in the same manner. Another double staining with anti-TRP4 and mouse anti-caveolin 1 antibodies (1:20; BD Transduction Laboratories, Lexington, KY) was tried to cultured cell clusters after Ca²⁺ imaging and demonstrated by goat anti-rabbit IgG with Texas Red and goat anti-mouse IgG with FITC, respectively. The specificity of immunoreactivity was checked by controls in which primary antibodies were omitted from the initial incubation. For an examination of the expression of TRP4 and TRP6 in the muscle layer, Western blotting was carried out. The muscle layer was homogenized, separated by SDS-PAGE, blotted onto nitrocellulose membrane, and probed by anti-TRP4 and anti-TRP6 antibodies. Antibodies preincubated with excess antigens were also used to check the specificity and were applied to both immunohistochemistry and Western blotting. The membranes were further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Amersham Biosciences), and reactions were visualized using Supersignal West Dura extended duration substrate (Pierce).

Immunoelectron Microscopy-- Muscle layers were fixed with 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, and cut into small pieces. After fixation for 30 min at 4 °C, samples were infused with a mixture of sucrose and polyvinylpyrrolidone (Sigma) (21) and frozen rapidly in liquid nitrogen. Ultrathin cryo-sections (50-70 nm) were incubated with rabbit anti-TRP4 antibody (1:100, Ab236) and processed for indirect immunostaining with 5-nm colloidal gold-conjugated goat anti-rabbit IgG (Amersham Biosciences). The sections were embedded in a mixture of 2% methylcellulose (Nacalai Tesque) and 0.5% uranyl acetate (22). They were then examined using an electron microscope (H-7000, Hitachi).

Solutions-- The modified Krebs solution was used as a normal solution in Ca²⁺ measurements and had the following composition: NaCl, 125 mM; KCl, 5.9 mM; CaCl₂, 2.5 mM; MgCl₂, 1.2 mM; glucose, 11.8 mM, and Hepes, 11.8 mM. pH was adjusted to 7.4-7.5 with Tris base. For a Ca²⁺-free solution, CaCl₂ was replaced with NaCl. Several salts such as MnCl₂, NiCl₂, LaCl₃, and CdCl₂ were added to the bath solution. SK&F 96365 and thapsigargin were purchased from Biomol.

Statistics-- Numerical data are expressed as mean ± standard deviation.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Structure of Cell Clusters and c-Kit Immunopositive Cells-- Small pieces of tissue developed into round or ovoid clusters after 2-4 days in culture and were attached to coverslips. The clusters were mainly composed of smooth muscle cells positively stained with anti--enteric actin antibody (Fig. 1, A and B), and most of them included c-Kit-positive ICC. The distribution pattern of ICC, however, was disturbed (Fig. 1C). In large cell clusters (more than 100 µm in diameter), enteric neurons were also included (Fig. 1D).

View larger version (82K): [in this window] [in a new window]   Fig. 1.   Phase-contrast microscopy and immunohistochemistry of a cell cluster isolated from the mouse small intestine and cultured for 2 days. A, phase-contrast image shows a round cell cluster approximately 100 µm in diameter. Bar, 50 µm. B, immunohistochemistry for -enteric actin on the same cell cluster shown in A indicates that most cells are smooth muscle cells. C, immunohistochemistry for c-Kit identifies ICC in the same cluster in A and B. ICC are located randomly in the cluster. D, immunohistochemistry for PGP9.5 shows enteric neurons. Large clusters (i.e. more than 100 µm in diameter) sometimes contain enteric neurons. Bar, 50 µm.

Calcium Oscillation in Cultured Cell Clusters-- Under superfusion with a modified Krebs solution at 35 °C, the cultured cell clusters showed periodic contractions at a rate of 19 ± 3.8 cycles/min (n = 27). The contractions were always associated with periodic depolarization in the cell membrane, i.e. slow waves (measured using patch clamp techniques).² Emission light of fluo-4 in these clusters was monitored using a CCD camera system. Fig. 2 shows an example of such experiments, and the movement of [Ca²⁺]_i is indicated by pseudocolor ratio images (Fig. 2, A and B). Although the amount of fluorescent dye loaded to each cell varied, many cells clearly showed a synchronized [Ca²⁺]_i oscillation followed by contractions of the cluster. In Fig. 2C changes in the fluorescence intensity were measured at the three points indicated in Fig. 2, A and B. [Ca²⁺]_i oscillation was also monitored in the presence of 250 nM TTX, which should have completely suppressed nerve activities. Neither periodic changes in the [Ca²⁺]_i concentration nor contractility were affected by this treatment (data not shown).

View larger version (58K): [in this window] [in a new window]   Fig. 2.   [Ca2+]i oscillations in a cultured cell cluster. The cluster was loaded with 10 µM fluo-4 acetoxymethyl ester, and the temporal change of the fluorescence intensity was measured. A, the minimal level of [Ca2+]i in control solution (at 35 °C) is shown in a pseudocolor ratio image. B, the maximal level of [Ca2+]i is shown in pseudocolor ratio image. Note that the size of the cluster in A is smaller than that in B, indicating contraction of the cluster. Bar, 50 µm, C, corresponding time courses of [Ca2+]i in the cluster. Green, red, and black lines show oscillations observed at three points indicated by respective colors in A and B. All points demonstrate clear and synchronized [Ca2+]i oscillations. The frequency was 20.4 cycles/min. Ft/F0, temporal fluorescence intensity of the dyes/fluorescence intensity at start.

Dihydropyridine derivatives (nifedipine), which selectively block voltage-sensitive L-type Ca²⁺ channels and consequently prevent smooth muscle contraction, are known to scarcely affect slow waves in the mouse small intestine. In the presence of nifedipine, we further examined properties of [Ca²⁺]_i oscillation in cultured cell clusters. There were regional dihydropyridine-resistant [Ca²⁺]_i oscillations in the cluster although the contractile activity of the cluster was suppressed (Fig. 3, A-C). Points 1 and 2 in Fig. 3, A and B indicate regions inside a cluster showing such [Ca²⁺]_i oscillation during exposure to 1 µM nifedipine. On the other hand, point 3 in Fig. 3, A and B did not show such movement. The maximal and minimal levels of [Ca²⁺]_i indicated by pseudocolor ratio images during [Ca²⁺]_i oscillation are shown in Fig. 3, A and B. The time course analysis of [Ca²⁺]_i oscillation is presented in Fig. 3E (dihydropyridine-resistant at points 1 and 2 and dihydropyridine-sensitive at point 3). The frequency of dihydropyridine-resistant [Ca²⁺]_i oscillation was 21 ± 4.1 cycles/min (n = 30), and results similar to that were seen in the solution without nifedipine described above. The phase-contrast microscopy (Fig. 3C) and c-Kit immunohistochemistry (Fig. 3D) obtained from the same cell cluster used in Fig. 3, A-D indicate localization of c-Kit-positive cells in the cluster. Points 1-3 in Fig. 3D respectively correspond to those indicated in Fig. 3, A and B. Comparison of Fig. 3, A and B with Fig. 3D clearly demonstrates that points 1 and 2 correspond to c-Kit immunopositive regions and generate [Ca²⁺]_i oscillation. On the other hand, point 3 originated from a c-Kit-negative region and did not show [Ca²⁺]_i oscillation. We obtained similar results in 30 of 35 cell clusters examined. This indicates that ICC identified by c-Kit immunostaining show dihydropyridine-resistant [Ca²⁺]_i oscillation and that they play an important role in generating spontaneous rhythmicity in the cluster.

View larger version (50K): [in this window] [in a new window]   Fig. 3.   [Ca2+]i oscillation in a cell cluster in the presence of 1 µM nifedipine. A, pseudocolor ratio image showing the minimal level of [Ca2+]i. B, ratio image showing the maximal level of [Ca2+]i. [Ca2+]i rises only in limited areas, not as a whole. C, phase-contrast micrograph of the same cell cluster shown in A and B. D, immunohistochemistry for c-Kit shows localization of ICC in the same cluster. Points 1-3 are respective to those in A and B. Note that points 1 and 2 correspond to c-Kit-positive ICC, whereas point 3 is in a c-Kit-negative area. Points 1 and 2 showed a significant increase of [Ca2+]i, but point 3 did not. Bar, 20 µm. E, corresponding time courses of [Ca2+]i in the cluster. Green, red, and black lines show the movement obtained from the three points indicated by respective colors in A and B. Point 1 (in black) and point 2 (in red) clearly show [Ca2+]i oscillations at 19.2 cycles/min in frequency. Point 3 (in green) does not show such an oscillation. Ft/F0, temporal fluorescence intensity of the dyes/fluorescence intensity at start.

[Ca²⁺]_i Oscillation in ICC and Electrical Activity-- Electrical activity corresponding to slow waves was measured using an extracellular recording technique. Simultaneous recordings of the electrical activity and [Ca²⁺]_i oscillations in ICC with 1 µM nifedipine are shown in Fig. 4. The frequency of both slow waves and [Ca²⁺]_i oscillation in ICC (thick line) was scarcely affected by nifedipine, although [Ca²⁺]_i in a c-Kit-negative region (thin line) did not show oscillation. We obtained the same results in 4 of 5 cell clusters. The frequency of slow waves and [Ca²⁺]_i oscillation in ICC were the same, and their movements coincided with each other, reflecting a close temporal relationship. The frequency was 17 ± 3.3 cycle/min (n = 4), and the amplitude of the electrical activity averaged 0.25 ± 0.02 mV (n = 4).

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Simultaneous recordings of the electrical activity and [Ca2+]i in ICC. Upper panel shows electrical activity and the lower panel indicates [Ca2+]i movements in ICC (thick line) and non-ICC region (thin line) in the presence of 1 µM nifedipine. Electrical activities corresponding to slow waves were synchronized with [Ca2+]i oscillations in ICC. The frequency was 18.3 cycles/min. Depolarization (0.16 mV) is temporally associated with increases of [Ca2+]i in ICC, and these two show tight temporal relationship. Ft/F0, temporal fluorescence intensity of the dyes/fluorescence intensity at start.

Characteristic Properties of [Ca²⁺]_i Oscillation in ICC-- When Ca²⁺-free solution was applied in the presence of 1 µM nifedipine, the amplitude of [Ca²⁺]_i oscillation in ICC decreased and subsequently disappeared (n = 5; Fig. 5A). Modified manganese quenching (200 µM MnCl₂ and 1 µM nifedipine in a Ca²⁺-free bath solution) also abolished [Ca²⁺]_i oscillation in ICC (n = 4; Fig. 5B). These results indicate that [Ca²⁺]_i oscillation in ICC required extracellular Ca²⁺. Because nifedipine does not block [Ca²⁺]_i oscillation in ICC, the L-type Ca²⁺ channel is not likely to be involved in the Ca²⁺ influx of ICC. We then examined the effects of several cations with 1 µM nifedipine. La³⁺ (50 µM) markedly reduced the amplitude of [Ca²⁺]_i in the cluster and blocked [Ca²⁺]_i oscillation in ICC (n = 7; Fig. 5C). Cd²⁺ (100 µM) increased the intensity of fluo-4 fluorescence in the cluster and disturbed [Ca²⁺]_i oscillation in ICC (n = 6; Fig. 5D). On the other hand, Ni²⁺ (50 µM), known to be a blocker of T-type calcium channel, did not affect [Ca²⁺]_i oscillation rhythms in ICC (n = 5; Fig. 5E). When a small amount of SK&F 96365 (4 µM), which blocks TRP, was added to the bath solution with nifedipine it significantly reduced the amplitude and prevented [Ca²⁺]_i oscillation in ICC (n = 5; Fig. 6A). Some TRPs have been suggested to be involved in Ca²⁺ entry mediated by store depletion (store-operated channels). When thapsigargin (a Ca²⁺ pump blocker) was added at 5 µM, the Ca²⁺ level rose once and then subsided along with a diminution of [Ca²⁺]_i oscillation in ICC (n = 4; Fig. 6B). Thapsigargin combined with nifedipine in the Ca²⁺-free solution quickly blocked [Ca²⁺]_i oscillation in ICC. However, no transient increase in the Ca²⁺ level was recorded (n = 3; data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effects of Ca2+-free condition and various cations on [Ca2+]i oscillations in ICC (thick lines) and non-ICC cells (thin lines) in the presence of 1 µM nifedipine. A, in Ca2+-free solution, [Ca2+]i oscillations in ICC were abolished within 1 min. B, quenching by Mn2+ quickly diminished [Ca2+]i oscillations in ICC. C, La3+ blocked [Ca2+]i oscillations in ICC. D, Cd2+ increased the intensity of fluorescence and disturbed the oscillation. E, Ni2+ (50 µM), a T-type channel blocker, did not affect the oscillation in ICC. Ft/F0, temporal fluorescence intensity of the dyes/fluorescence intensity at start.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effects of SK&F96365 and thapsigargin on [Ca2+]1 oscillation in ICC in the presence of 1 µM nifedipine. A, SK&F96365 abolished [Ca2+]i oscillations in ICC. B, thapsigargin, a blocker of Ca2+ pump in ER, hampered [Ca2+]i oscillations in ICC. It increased and then decreased Ca2+ level and inhibited the oscillation eventually. Ft/F0, temporal fluorescence intensity of the dyes/fluorescence intensity at start.

Immunohistochemistry and Cytochemistry for TRP Proteins-- Expressions of TRP proteins were investigated by immunohistochemistry on a whole mount preparation of the smooth muscle layer. Double staining with anti-c-Kit and anti-TRP4 antibodies revealed the TRP4 immunoreactivity in c-Kit immunopositive ICC at the myenteric plexus level (Fig. 7, A-D). Not only ICC but also other supposedly smooth muscle cells expressed TRP4. TRP4 expression was stronger in ICC than in the putative smooth muscle cells (Fig. 7B). The two antibodies used for TRP4 immunohistochemistry showed similar results. Although TRP6 was also positive in the smooth muscle layer as confirmed by Western blotting (Fig. 7E), double immunostaining for TRP6 and c-Kit did not detect TRP6 in ICC (data not shown). The fine localization of TRP4 in ICC was studied using cell clusters by double labeling immunohistochemistry for TRP4 and caveolin-1 in ICC where [Ca²⁺]_i oscillation was recorded in the presence of nifedipine. This double staining demonstrated that localization of TRP4 largely coincided with caveolin-1 (n = 10; Fig. 8, A-D). Immunoelectron microscopy further confirmed that TRP4 was mostly distributed in caveolae in ICC, which were located between the circular and longitudinal muscle layer and identified as ICC morphologically (n = 5; Fig. 9).

View larger version (46K): [in this window] [in a new window]   Fig. 7.   Immunohistochemistry and Western blotting of TRP. A-D, whole mount preparation of the muscle layer. Bar, 10 µm. A, anti-c-Kit antibody shows distribution of ICC at the level of the myenteric plexus. These are multipolar cells connected with each other to form a network. B, immunolabeling of TRP4. C, merged image of A and B shows colocalization of c-Kit and TRP4 in ICC. Some non-ICC cells express TRP4, but the expression of TRP4 is stronger in ICC than in non-ICC cells. D, a control processed without primary antibodies does not show any immunoreactivity. E, Western blotting analysis proved that both TRP4 and TRP6 are expressed in the muscle layer. Lanes 1, 3, and 5 are probed by two different anti-TRP4 antibodies (a product of Alomon Laboratories, Ab236) and anti-TRP6 antibody, respectively. Lanes 2, 4, and 6 were treated with absorbed antibodies (preincubated with 10-fold excess of the antigens) used in lanes 1, 3, and 5, respectively. Ab236 recognized an additional band below 66,000, which was also absorbed in the control.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Distribution of TRP4 in ICC. A-C, double immunolabeling of TRP4 and caveolin-1 in ICC. Bar, 10 µm. TRP4 (A) and caveolin-1 (B) were both densely distributed along the cell edge (arrowheads). C, merged image of A and B. D, a control processed without primary antibodies does not show any immunoreactivity.

View larger version (148K): [in this window] [in a new window]   Fig. 9.   Immunoelectron microscopy of TRP4 in ICC, which is a caveolae-rich cell located between the circular and longitudinal muscle layers where ICC are densely distributed. The cell has neither myofilaments nor dense bodies. TRP4 immunoreactivity marked by colloidal gold particles are located in caveolae. Asterisks indicate the lumen of caveolae, which were cross-sectioned and thus appeared as vesicles. Control sections processed without primary antibody did not show any specific immunoreactivity (data not shown). Bar, 100 nm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study, we observed slow periodic contractions accompanied by [Ca²⁺]_i oscillations in cell clusters after a 2-4 day culture. Immunohistochemistry revealed that smooth muscle cells, c-kit-positive ICC, and sometimes enteric neurons were included in these clusters (Fig. 1). A blockade of the nervous activities with TTX, however, affected neither contractility nor [Ca²⁺]_i oscillation, suggesting that enteric neurons were not indispensable for the generation of the basic spontaneous activity. We assume that the neurons might modulate the basic rhythmicity and coordinate the activity of neighboring contractile units (23). It is also well known that dihydropyridine (nifedipine) blocks L-type Ca²⁺ channels in smooth muscle cells and inhibits their contractions. Our Ca²⁺ imaging in the presence of nifedipine, therefore, excludes [Ca²⁺]_i oscillation in smooth muscle. In fact, the contractile activity and [Ca²⁺]_i oscillation of the entire cell cluster were blocked with nifedipine.

Recent studies have shown that ICC play the role of pacemaker cells. They generate electrical activity, i.e. slow waves, which is propagated to smooth muscle cells and produces spontaneous contractions of the muscle layer. One of the characteristic features of the slow wave is dihydropyridine (nifedipine)-resistant activity (5, 11). Nifedipine does not prevent slow wave generation. This indicates that besides smooth muscle cells special pacemaker cells are present and that ion channels that are different from L-type Ca²⁺ channels play a critical role in the pacemaking. Ca²⁺ imaging with nifedipine combined with subsequent immunohistochemistry showed that [Ca²⁺]_i oscillation occurs in c-Kit-positive ICC in the cultured cell cluster (Fig. 3). Furthermore, simultaneous recording of [Ca²⁺]_i and electrical activity in ICC revealed that [Ca²⁺]_i oscillation in ICC is synchronized with slow waves and that the two are likely to be causally related (Fig. 4). The slow waves seen in the isolated cell cluster are also similar to those observed in intact tissues in their frequency of oscillation (11, 13, 14) and temperature-dependence.² Therefore, the present preparation is a suitable model system for analysis of the pacemaker mechanism as a minimal unit of the GI muscle layer.

Studies on electrical rhythmicity in ICC have suggested that IP₃ receptor-dependent calcium release from ER is crucial for the generation of slow waves. Circular smooth muscle isolated from mutant mice lacking IP₃ type 1 receptor failed to generate slow waves even though the action potential of the smooth muscle was not affected (12, 24). Another group reported that inhibitors of IP₃ receptor blocked pacemaker activity; a membrane-permeable blocker of IP₃ receptor (xestospongin C) and injection of heparin inhibited pacemaker current and slow waves (13). Thus, Ca²⁺ release from ER through IP₃ receptor is necessary for [Ca²⁺]_i oscillation in ICC. It is also well known that ICC have many mitochondria and that mitochondrial Ca²⁺ uptake is thought to be required for electrical pacemaking in ICC. Pacemaker currents were closely related to mitochondrial Ca²⁺ transient; mitochondrial uncouplers (carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide p-chlorophenylhydrazone (CCCP)) and respiratory chain inhibitors (antimycin) blocked pacemaker current (13). This is consistent with a previous report that pacemaking in ICC is dependent upon metabolic activity (26, 27). All together, these data suggest that an intracellular calcium event depending upon the periodic release of Ca²⁺ from ER through IP₃ receptor and subsequent uptake of Ca²⁺ by mitochondria is necessary for [Ca²⁺]_i oscillation in ICC. Although these two events appear to be primary factors in [Ca²⁺]_i oscillation in ICC, the mechanisms integrating Ca²⁺ handling of ER and mitochondria in ICC and the generation of pacemaker current in the plasma membrane remain to be clarified.

In addition to the intracellular calcium events, the present study of [Ca²⁺]_i oscillation in ICC demonstrated that the pacemaking mechanism in ICC requires extracellular Ca²⁺ because Ca²⁺-free bath solution and Mn²⁺ inhibited [Ca²⁺]_i oscillation in ICC (Fig. 5). The insensitivity to nifedipine clearly shows that Ca²⁺ influx is not mediated by an L-type calcium channel, which is ordinarily required for the contractions of smooth muscle cells. Instead, the channel is thought to be a non-selective cation channel because La³⁺ and Cd²⁺ quickly reduced the amplitude of [Ca²⁺]_i oscillation and eventually blocked it. On the other hand, the T-type calcium channel is not likely to contribute to the influx because a considerable amount of Ni²⁺ did not affect [Ca²⁺]_i oscillation (Fig. 5) (28).

A gene family of TRP channels was cloned recently, and their products are considered to be non-selective cation channels (29-32). Some of them (including TRP4) were reported to be store-operated channels (20, 33). The expression of trp genes in the murine and canine GI muscle layers was recently reported. Only full-length trp4, trp6, and some splice variants were detected in the muscle layer of the mouse GI tract (18). Epperson et al. (34) reported that freshly isolated or cultured ICC also express only trp4 and trp6. These reports suggest that TRP4 and/or TRP6 may be involved in [Ca²⁺]_i oscillation in ICC. In the present study we showed that [Ca²⁺]_i oscillation in ICC was blocked by SK&F 96365, which was used as an inhibitor of TRP4 expressed on Xenopus oocytes (35). Moreover, thapsigargin that blocks ER Ca²⁺-ATPases increased and then decreased [Ca²⁺]_i in ICC and eventually blocked the oscillations. This indicates that the channel involved in oscillation was store-operated and activated by the depletion of Ca²⁺ in ER. Sensitivity to store depletion is higher in TRP4 than in TRP6 (29). These data strongly suggest that the non-selective cation channel involved in [Ca²⁺]_i oscillation in ICC was TRP4. Although it was confirmed that both TRP4 and TRP6 were expressed in the muscle layer of the mouse small intestine by Western blotting (Fig. 7), TRP6 was demonstrated intensely in the smooth muscle but not in ICC by immunohistochemistry (data not shown). We thus think that TRP4 is predominant in ICC, whereas TRP6 is a principal type in smooth muscle. The periodic Ca²⁺ release through IP₃ receptor may cause depletion of the Ca²⁺ store, which then activates TRP4 spontaneously. This Ca²⁺ influx (mediated by TRP4) might provide pacemaker current in ICC.

By immunohistochemistry TRP4 was located mostly in caveolae shown by colocalization with caveolin-1 labeling. The caveolar distribution of TRP4 was corroborated with immunoelectron microscopy. Caveolae are enriched with many signaling molecules and are abundant in ICC (36, 37). G protein-coupled receptors and receptor tyrosine kinases concentrated in caveolae are known to regulate TRP activity (38, 19). Therefore, it is strongly suggested that neurotransmitters and hormones work on caveolae to modulate the pacemaking function in ICC. Moreover, a recent paper reported the possibility that TRP3 in caveolae formed direct physical interaction with IP₃ receptors in ER and mediated Ca²⁺ influx in HEK-293 cells (25). Further studies are necessary to determine the functional relationship between TRP4, caveolae, and IP₃ receptor and to understand the mechanism by which pacemaker currents are generated.

In summary, the [Ca²⁺]_i oscillation we demonstrated in ICC using isolated cell clusters from the mouse small intestine was closely linked to pacemaker activity. Ca²⁺ influx was necessary for [Ca²⁺]_i oscillation, and the non-selective cation channel TRP4 (located mainly in caveolae) was inferred to mediate Ca²⁺ entry in ICC.

	ACKNOWLEDGEMENTS

We thank Dr. Nishi (University of Kumamoto) for antibody ACK2 and Dr. Satoh (Iwate Medical University) for technical advice on calcium imaging. We also thank Drs. Sanders, Horowitz, Ward, and Koh for valuable discussions (University of Nevada in Reno).

	FOOTNOTES

^* This work was supported by the Ministry of Education, Science, Sports, and Culture of Japan and by the Japan Clinical Study Group for the Esophago-cardiac Region. A preliminary account was presented at the 18^th International Symposium on Gastrointestinal Motility, November 15-19, 2001, Madison, WI (Torihashi, S., Fujimoto, T., and Nakayama, S. (2001) Neurogastroenterol. Motil. 13, 437a).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.

^§ To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan. Tel.: 81-52-744-2001; Fax: 81-52-744-2012; E-mail: storiha@med.nagoya-u.ac.jp.

Published, JBC Papers in Press, March 15, 2002, DOI 10.1074/jbc.M201728200

² S. Nakayama and S. Torihashi, unpublished data.

	ABBREVIATIONS

The abbreviations used are: ICC, interstitial cells of Cajal; FITC, fluorescein isothiocyanate; TTX, tetradotoxin; TRP, transient receptor potential; ER, endoplasmic reticulum; IP₃, inositol trisphosphate; GI, gastrointestinal.

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