Expression of Ca2+-activated K+ channels, SK3, in the interstitial cells of Cajal in the gastrointestinal tract

Akikazu Fujita1,2, Tadayoshi Takeuchi1,2, Noriko Saitoh1, Jun Hanai3, and Fumiaki Hata1,2

1 Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Science and 2 Department of Molecular Physiology and Biochemistry, Research Institute for Advanced Science and Technology, Osaka Prefecture University, Sakai, Osaka 599-8531; and 3 Department of Pathology and Research, Sakai Municipal Hospital, Sakai, Osaka 590-0064, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A role for small-conductance Ca2+-activated K+ (SK) channels on spontaneous motility of the gastrointestinal tract has been suggested. Although four subtypes of SK channels were identified in mammalian tissues, the subtypes of SK channel expressed in the gastrointestinal tract are still unknown. In this study, we investigated the expression and localization of SK channels in the gastrointestinal tract. RT-PCR analysis shows expression of SK3 and SK4 mRNA, but not SK1 or SK2 mRNA, in the rat intestine. SK3 immunoreactivity was detected in the myenteric plexus and muscular layers of the stomach, ileum, and colon. SK3-immunoreactive cells were stained with antibody for c-kit, a marker for the interstitial cells of Cajal (ICC), but not with that for glial fibrillary acidic protein in the ileum and stomach. Immunoelectron microscopic analysis indicates that SK3 channels are localized on processes of ICC that are located close to the myenteric plexus between the longitudinal and circular muscle layers and within the muscular layers. Because ICC have been identified as pacemaker cells and are known to play a major role in generating the regular motility of the gastrointestinal tract, these results suggest that SK3 channels, which are expressed specifically in ICC, play an important role in generating a rhythmic pacemaker current in the gastrointestinal tract.

small-conductance calcium-activated potassium channel; immunohistochemistry; immunoelectron microscopy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE MOTILITY OF THE gastrointestinal (GI) tract consists of nonpropulsive mixing and propulsive movements (3, 4, 24, 38). It is generally considered that mixing movements are produced by intrinsic pacemaker cells that generate rhythmic contractions (10, 38), and that peristalsis is produced by intrinsic excitatory and inhibitory neural reflex pathways (3, 4, 12, 24, 30, 38). Apamin, which is a selective blocker of small-conductance Ca2+-activated K+ (SK) channels, has been reported to affect the mechanical and electrical activity of the GI tract. The amplitude of a slow wave and spontaneous contractions were enhanced by apamin in the stomach of guinea pig (41), and inhibitory junction potential and relaxations induced by the electrical stimulation of nonadrenergic and noncholinergic inhibitory motor nerves were inhibited by apamin in the stomach or taenia coli of guinea pig (1, 23, 29, 47) and in the stomach (16), proximal duodenum (33), distal colon (19), and jejunum (35) of rat. These indicate that apamin-sensitive SK channels may be expressed and participate in the regulation of the motility and electrical excitability of the GI tract.

The subtypes of SK channels were first isolated from rat brain (22). So far, four subtypes of SK channels have been identified. In the GI tract, the expression of only SK4 channels has been detected in human stomach, small intestine, and colon (15), and in rat colon (52), by using molecular biological techniques. SK2 and SK3, but not SK1 or SK4 channels, expressed in Xenopus oocytes were blocked by apamin (22, 46). However, a recent study showed that the SK1 channel expressed in mammalian cell lines was apamin sensitive (40). It has not been investigated which subtype of apamin-sensitive SK channels is expressed and which cell within the GI tract expresses the channels.

In this study, the results obtained by RT-PCR analyses show that SK3 channels are expressed in rat intestine. The results by immunohistochemical studies also show that SK3 channels are localized in the myenteric plexus and muscular layers of the stomach, ileum, and colon and are colocalized with c-kit, which is a specific marker of the interstitial cells of Cajal (ICC). Furthermore, the results by the immunoelectron microscopic studies show that SK3 channels are specifically localized on the processes of ICC. These results suggest that SK3 channels play an important role in generating a rhythmic pacemaker current in the GI tract.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The animal experiments were performed properly following the guidelines of the Animal Usage Committee of Osaka University Medical School. Male Wistar rats weighing 200-250 g and male ICR mice (8 wk old) were purchased from Nippon Doubutsu (Kyoto, Japan) and Japan SLC (Shizuoka, Japan), respectively.

PCR amplification of SK2 and SK3 channel cDNAs and RT-PCR analysis of expression of SK channels in rat intestine. Total RNAs from adult rat brain, ileum, and colon were isolated with TRIZOL reagent (Life Technologies, Grand Island, NY) and reverse-transcribed with oligo(dT)12-18 primer using SuperScript II RT (Life Technologies), according to the manufacturer's instructions. The cDNAs of coding regions of SK2 and SK3 were amplified by PCR with LA-Taq polymerase (Takara, Tokyo, Japan) using rat brain cDNA and primer pairs specific for rat SK2 [5'-ATGAGCAGCTGCAGGTAC (1-18) and 5'-CTAGCTACTCTCAGATGA-3' (1726-1743; accession number U69882)] and for rat SK3 [5'-ATGGACACTTCTGGGCAC-3' (298) and 5'-TTAGCAACTGCTTGAACT-3' (2479-2496) (accession number AF292389)].

For analysis of expression of SK channel subtypes in rat brain, ileum, and colon, we designed two sets of specific primers for four SK channel subtypes (SK1 to SK4) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer sequences are indicated as follows (first and second sets of primers of each subtype are shown): for rat SK1, 5'-GGCCAAGGCACAGAGCA-3' (1125-1141) and 5'-TCACCCACAGTCTGATGC-3' (1360-1377), 5'-AGTGTCCCTGATCTCGCT-3' (309) and 5'-ACCCGGCTTTGGTCTGGC-3' (996-1013) (U69885); for rat SK2, 5'-ATGAGCAGCTGCAGGTAC-3' (1-18) and 5'-CTTTTTGCTGGACT-3' (326), 5'-TTCCACAACCACTGCAGA-3' (744) and 5'-CTGCGTTTTTTACTC-3' (1256-1270) (U69882); for rat SK3, 5'-ATGGACACTTCTGGGCAC-3' (298) and 5'-CCGCTTGTTGGCTT-3' (1071-1083), 5'-CCTCTACATCAGCCTGGA-3' (1389-1406) and 5'-ACTTTGGCGTGGTCAATC-3' (2073-2090) (AF292389); for rat SK4, 5'- GAACAAGTGAATTCCATGGT-3' (1223-1242) and 5'-CTATGTGGCCTCCTGGATG-3' (1399-1417), 5'-CTGTACCTGCTCTTGGTTAAGTGTTTAA-3' (308) and 5'-CCTTCGAGTGTGTTTGTAGT-3' (1110-1129) (AF156554); and for rat GAPDH, 5'-GGCTGCCTTCTCTTGTGACAA-3' (84) and 5'-CGCTCCTGGAGGATGGTGAT-3' (263) (AF106860). PCR amplification was performed for 30 cycles at 94°C for 1 min, at 55°C for 1 min, and then 72°C for 1 min followed by 72°C for 8 min. Amplified DNA fragments were separated in a 2% agarose gel. Subsequently, the fragments were sequenced with an automatic sequencer (A-381; Perkin-Elmer, Foster City, CA) after TA cloning (Invitrogen, San Diego, CA).

Transfection of SK2 and SK3 channels into HEK293T cells. Rat SK2 and SK3 cDNA were subcloned into the expression vector, pcDNA3.1/His A (Invitrogen), resulting in the SK3 protein having a molecular mass of 81 kDa, because of addition of 34 amino acids. They were transfected with Lipofectamine Plus Reagent (Life Technologies) into HEK293T cells as described previously (18). Two days after transfection, the cells were used for immunoblotting or immunocytochemistry.

Antibody. Rabbit polyclonal and rat monoclonal antisera against the SK3 channel, glial fibrillary acidic protein (GFAP; clone 6F-2), and c-kit (clone ACK-2) were purchased from Alomone Labs (Jerusalem, Israel), DAKO JAPAN (Kyoto, Japan), and Life Technologies, respectively.

Immunohistochemical studies. HEK293T cells transfected with cDNA of SK3 or SK2 channels, or the freshly isolated stomach, ileum, and colon were fixed with 4% (wt/vol) paraformaldehyde in 0.1 M phosphate buffer (PB solution), pH 7.4. Male Wistar rats and mice were deeply anesthetized with pentobarbital sodium (50 mg/kg ip), and the stomach, ileum, and colon were fixed by transcardiac perfusion as described previously (11, 34). The stomach, ileum, and colon were dissected, postfixed with 4% (wt/vol) paraformaldehyde in 0.1 M PB solution for 24 h, dehydrated with 30% (wt/vol) sucrose solution, and then frozen with OCT compound (Tissue-Tek, Sakura Finetechnology, Tokyo, Japan). Light microscopic immunocytochemistry was performed using a method of indirect fluorescence. Tissue sections (12 µm thickness) were made on a cryostat. After they were washed three times with 0.01 M PBS solution (containing 50 mM NaCl in 0.01 M PB, pH 7.4) for 10 min each, the tissue sections were treated with preincubation solution [10% normal goat serum (NGS), 1% BSA, 0.5% Triton X-100 in 0.01 M PBS, pH 7.4] at room temperature for 1 h, and then were incubated with anti-SK3 channel (0.3 µg/ml), anti-GFAP, or anti-c-kit antibodies in primary antibody solution (3% NGS, 1% BSA, 0.5% Triton X-100, 0.05% sodium azide in 0.01 M PBS, pH 7.4) at room temperature overnight. The sections were washed again three times with PBS at room temperature for 10 min each and visualized with FITC-labeled anti-rabbit IgG (EY Laboratories, San Mateo, CA) or Alexa Fluor 568-labeled anti-rat IgG (Molecular Probes, Eugene, OR) in secondary incubation solution (3% NGS, 1% BSA, 0.5% Triton X-100 in 0.01 M PBS, pH 7.4). Immunoreactivity of the sections was observed by a confocal microscope (MRC-1024; Bio-Rad, Hertfordshire, UK).

For electron microscopic immunocytochemistry (53), ileal tissues were fixed with 4% (wt/vol) paraformaldehyde containing 0.1% glutaraldehyde in 0.1 M PB and postfixed in the same solution at 4°C overnight. Tissue sections (50 µm in thickness) were made by a vibratome. After sections were pretreated with solution A [3% (wt/vol) NGS, 3% (wt/vol) BSA, 0.1% (wt/vol) Triton-X-100, and 0.05% (wt/vol) sodium azide in 0.01 M PBS], they were incubated with anti-SK3 channel antibody diluted in solution A at 4°C for 48 h. After removal from anti-SK3 channel antibody, sections were washed with 0.01 M PBS five times for 30 min each and placed in 0.01 M PBS solution containing biotinylated goat anti-rabbit IgG for 24 h at 4°C. The immunoreactivity was visualized by a reaction to 3,3'-diaminobenzidene tetrahydrochloride and platinous potassium chloride. Sections were washed with PBS at 4°C overnight and postfixed in reduced osmium for 1 h at 4°C. After dehydration in ethanol and infiltration in epoxy resin, sections were flat-embedded on the siliconized slide glasses. Small blocks, selected by light microscopical inspection, were cut out, glued to blank epoxy, and sectioned with an ultramicrotome. The ribbons of thin sections (80 nm in thickness) were collected on grids and counterstained with uranyl acetate and Reynold's lead citrate. These were examined with a Hitachi 7400-alpha electron microscope (Hitachi, Tokyo, Japan).

Western blotting of the SK3 channel. The membrane fractions of ileum and brain of adult rat and that of HEK293T cells were prepared as described previously (25). The membrane proteins were separated with SDS-PAGE (9%) and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were overlaid with anti-SK3 antibody at a concentration of 0.3 µg/ml in buffer B, which contained 5% (wt/vol) skimmed milk and 0.2% (wt/vol) Lubrol PX in 50 mM Tris · HCl (pH 8.0) and 80 mM NaCl. After they were washed three times with buffer B for 10 min each, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (EnVision Plus; Dako, Carpinteria, CA) diluted to 1:1,000 (vol/vol) in buffer B, followed by washing three times with buffer B. Immunoreactive bands were developed with ECL Plus chemiluminescence immunostaining kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturer's protocol.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RT-PCR analyses and immunohistochemistry of SK channels in rat gastrointestinal tract. Four members of the mammalian SK channel family have been identified. To identify which subtypes of SK channel are expressed in the intestine, RT-PCR analysis of total RNA prepared from the rat ileum and colon was performed using specific sets of primers (first sets of primers, see METHODS) for four SK channel subtypes: SK1, SK2, SK3, and SK4 (Fig. 1). Each set of primers amplified its specific DNA fragment when cDNA of each subtype was used as a template (Fig. 1). Two subtypes of SK channels, i.e., SK3 and SK4, seemed to be abundantly expressed in the rat ileum and colon (Fig. 1). Only faint bands of SK1 and SK2 were detected. As a control experiment for RT-PCR analysis, expression of SK channel subtypes in the rat brain was reconfirmed. Expression of SK1, SK2, and SK3 was detected in the brain as previously reported (22, 39). No fragments were amplified with the template without RT treatment. The nucleotide sequences of these amplified DNA fragments were confirmed by sequencing. We also obtained the same results as above by using another (second) set of primers for each of four SK channels in the rat ileum and colon (data not shown).


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 1.   RT-PCR analyses of small-conductance Ca2+-activated K+ (SK) channels in rat (r) ileum and colon. PCR products were resolved by 2% agarose gel electrophoresis, and members of the SK channel family were detected by staining with ethidium bromide. Although both the ileum and colon expressed mRNAs of SK3 and SK4, only faint bands of SK1 and SK2 were detected in both. Brain expressed mRNA of SK1, SK2, and SK3, but not SK4. As a positive control, expression of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA was detected in ileum, colon, and brain. No DNA fragment was amplified with the template without RT treatment. Numbers on the right indicate the size makers in base pairs (bp). C, positive control cDNA of each SK subtype used as a template.

Next, we investigated distribution of the SK3 channel in the stomach and ileum by immunohistochemistry using a polyclonal rabbit IgG antibody for the rat SK3 channel (Fig. 2). Specificity of the anti-SK3 channel antibody was confirmed by immunoblotting and immunocytochemical methods using HEK293T cells transfected with cDNA of the SK3 channel. In the immunoblot analysis, the antibody reacted on only one band with molecular mass estimated at ~81 kDa (Fig. 2A, lane 1). However, the antibody did not react on any proteins in the membrane of HEK293T cells transfected with cDNA of the SK2 channel (Fig. 2A, lane 2). Preincubation of the antibody with antigenic peptide prevented immunoreaction (Fig. 2A, lane 3). In the immunocytochemical analysis, immunoreactivity to the antibody was clearly detected in HEK293T cells transfected with SK3 cDNA (Fig. 2B) but not in those without transfection (not shown). These results indicate that polyclonal rabbit IgG antibody specifically reacts to the SK3 channel.


View larger version (67K):
[in this window]
[in a new window]
 
Fig. 2.   Expression of SK3 channel in rat ileum. A: immunoblot analysis of SK3 channel with a polyclonal rabbit antibody against the amino-terminal region of rat SK3 channel. Membrane fractions obtained from HEK293T cells transfected with cDNA of SK3 channel (lanes 1 and 3) or SK2 channel (lanes 2 and 4) were examined on SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and then immunoblotted with anti-SK3 channel antibody. Lanes 3 and 4: immunolabeling was blocked by a preabsorption with antigenic peptides. Numbers noted on the left indicate the positions of molecular mass markers in kDa. B: immunofluorescent images of localization of SK3 channel in HEK293T cells transfected with cDNA of SK3 channel by a confocal microscopy. After the cells were treated with the anti-SK3 channel antibody, the cells were stained with FITC-conjugated anti-rabbit IgG (green). Right, immunoreactivity was not detected when the antibody was preincubated with the antigenic peptides. Scale bar, 10 µm. C: immunoblot analysis of SK3 channel on membrane fractions obtained from rat ileum and brain, and HEK293T cells transfected with cDNA of SK3 channel. Lanes 4-6, antibody was preabsorbed with antigenic peptides.

Figure 2C shows the immunoblot analysis of membrane fractions prepared from ileum and brain of rat with antibody. A single band of 79 or 81 kDa was detected in ileum and brain, and HEK293T cells transfected with SK3 cDNA, respectively (Fig. 2C, lanes 1-3, see METHODS). Preincubation of the antibody with antigenic peptide prevented the immunoreaction (Fig. 2C, lanes 4-6).

Immunoreactivity of anti-SK3 channel antibody was observed in myenteric plexus of the stomach and ileum. It was also observed within both circular and longitudinal muscle layers of the stomach and ileum (Fig. 3A). However, any immunoreactivity of the SK3 channel was not observed in smooth muscle cells (Fig. 3A). The colon showed the same results as stomach and ileum (data not shown). These immunoreactivities were not detected when antibody was preincubated with antigenic peptide (data not shown).


View larger version (114K):
[in this window]
[in a new window]
 
Fig. 3.   Immunohistochemical examination of SK3 channel in stomach and ileum. Immunofluorescence of SK3 channel was detected by a confocal microscopy in stomach and ileum of rat and mouse. Tissue sections were treated with anti-SK3 channel antibody (A, left panels of Ba and Bb, and Ca) or anti-glial fibrillary acidic protein (GFAP) antibody (right panels in Ba and Bb) and stained with FITC-conjugated anti-rabbit IgG antibody (green) or anti-c-kit antibody (Cb) and stained with Alexa Fluor 568-conjugated anti-rat IgG antibody (red). A: in both rat stomach and ileum, immunoreactivity of SK3 channel was detected in the myenteric plexus between longitudinal and circular muscle layers, and within the muscular layers. M, mucosa; SM, submucosa; CML, circular muscular layer; MPL, myenteric plexus layer; LML, longitudinal muscular layer. Scale bar, 125 µm. B: immunofluorescence of SK3 channel and GFAP at higher magnification. Note that SK3 channel-immunopositive cells are localized outside of the myenteric plexus in rat stomach (a) and ileum (b), surrounding or contacting with the myenteric plexus, although the glial cells that were stained with anti-GFAP antibody are present together with unstained neuronal cells in the myenteric plexus. Scale bars: 50 µm (a) and 25 µm (b). C: double staining of mouse ileum with anti-SK3 and anti-c-kit antibodies. Immunoreactivity of SK3 (green, a) was colocalized with that of c-kit (red, b); c, double exposure of both images. Abbreviations are as in A. Scale bar, 25 µm.

Observation at higher magnification revealed that immunoreactive cells were present outside of the myenteric plexus, surrounding or contacting with the myenteric plexus (Fig. 3B). Glial and neuronal cells, the main components within the myenteric plexus, were able to be distinguished, i.e., glial cells were immunostained with antibody for GFAP, a marker of glial cells, whereas neuronal cells lying together with the stained glial cells were not stained with antibody (Fig. 3B). Therefore, it seems likely that the cells immunoreactive to anti-SK3 channel antibody are different from the two cells, glial and neuronal.

ICC have been known to surround the myenteric plexus and localize within circular and longitudinal muscle layers of the stomach and intestine (7, 50). Therefore, we next studied the localization of ICC with the antibody for c-kit protein, which is a specific marker of ICC, and compared with the localization of the SK3 channel. We stained mouse tissues with anti-c-kit antibody because antisera (ACK-2) against c-kit used in the present study was a monoclonal rat IgG antibody. Immunoreactivity of anti-c-kit antibody was localized in the myenteric plexus and muscular layers of mouse ileum (Fig. 3Cb) and stomach (data not shown) as previously reported (7, 50). Double staining of the anti-SK3 channel (green, Fig. 3Ca) and anti-c-kit (red, Fig. 3Cb) produced a prominent yellow signal in the myenteric plexus and muscular layers of mouse ileum (Fig. 3Cc) and stomach (not shown). Thus SK3 channel immunoreactivity seems to be localized in ICC. Weak immunoreactivity of the SK3 channel was also detected in c-kit-immunonegative cells present in muscular and mucosal layers in both the ileum (Fig. 3Cc) and stomach (data not shown).

Immunoelectron microscopic examination of SK3 channel immunoreactivity in ileum. Exact localization of SK3 channels was further studied by immunoelectron microscopic examination with anti-SK3 channel antibody in rat ileum. Immunolabeling of the SK3 channel was observed in cells (Fig. 4) that were identified as ICC on the basis of the following features. Immunolabeling cells are present near the myenteric plexus (Fig. 4A) and within the muscular layer (Fig. 4C). The basement membrane (arrowheads in Fig. 4A) surrounds the myenteric plexus and smooth muscle cells, but not the immunolabeling cells. The processes of the cells often closely contact each other, forming a gap junction (thick arrows in Fig. 4B). Immunolabeled cells have electron dense nuclei with heterochromatin localized in the periphery of the nuclear envelope (Fig. 4B). The cells also have a dense granular cytoplasm and many mitochondria (Fig. 4B) and lipid vesicles in the cytoplasm (Fig. 4, A, C, and D). The plasma membrane of the cells forms many caveoles. All these features of the immunolabeled cells are consistent with those for ICC (6, 7, 14, 17, 26-28, 32, 42, 50). Immunolabeling of the SK3 channel was predominantly localized on the membranes of the processes of ICC (Fig. 4, A-D) and occasionally on the intracellular vesicles in them (open arrowheads, Fig. 4, C and D). However, any immunolabeling of the SK3 channel was not observed in smooth muscle cells, or neuronal or glial cells in the myenteric and muscular layers (Fig. 4, A and C). Observation at higher magnification indicates that the SK3 channels are localized on the plasma membrane and the membrane of intracellular vesicles of ICC in both myenteric plexus and the muscular layer.


View larger version (162K):
[in this window]
[in a new window]
 
Fig. 4.   Immunoelectron microscopy of SK3 channel in rat ileum. A: immunolabeling of SK3 channel was detected in interstitial cells of Cajal (ICC) near the myenteric plexus (MP) and smooth muscle cells (SM). Immunolabeling of SK3 channel is localized predominantly on the membrane of processes of the cell (thin arrows). The basement membrane that surrounds smooth muscle cells and myenteric plexus (solid arrowheads) is not present around the ICC. Note that smooth muscle cells and neuronal and glial cells in the myenteric plexus are not labeled. B: ICC has numerous mitochondria (m), and an electron-dense nucleus (N) with heterochromatin, which is distributed toward the periphery of the nuclear envelope. Processes of ICC made contact with each other through a gap junction (thick arrows). C and D: membrane of vesicles (open arrowheads) in processes of ICC in the circular muscular layer was also labeled with anti-SK3 channel antibody. Scale bars, 0.5 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The important findings in this study are 1) RT-PCR analyses indicate that, among apamin-sensitive Ca2+-activated K+ channels, SK3 channels are predominantly expressed in the rat intestine, and 2) immunohistochemical studies indicate that SK3 channels are specifically present in ICC.

The existence of apamin-sensitive SK channels in smooth muscle cells of the GI tract has been suggested from the following findings. In smooth muscle strips of guinea pig ileum, apamin caused depolarization of the membranes of smooth muscle cells and increased their excitability (2, 8). In segments of guinea pig ileum (8), canine pylorus (49), rat stomach (16), and rat colon (19) and jejunum (35), apamin selectively abolished the fast-hyperpolarizing component of the inhibitory junction potential (37) evoked by electrical stimulation of nonadrenergic and noncholinergic inhibitory motor nerves (5). With a patch-clamp technique, SK channels were detected in smooth muscle cells of mouse ileum and colon at a single channel level (20, 48). SK channels in smooth muscle cells are intracellular and Ca2+ sensitive but voltage independent and have unitary conductances of 5-20 pS. These channels are activated by purinergic stimulation and inhibited by apamin. However, subtypes of these channels in the GI tract have not been identified. In this study, RT-PCR analyses showed that only faint bands of SK1 and SK2 channels were seen in the preparations from rat ileum and colon, indicating that expression of SK1 and SK2 channels was little in these tissues, if any. Immunohistochemical studies showed that SK3 channels were not expressed in smooth muscle cells. These results suggest that smooth muscle cells of the GI tract express apamin-sensitive subtypes of SK channels that were not identified so far.

Spontaneous electrical rhythmicity is a fundamental property of smooth muscle cells of the GI tract and is essential for pacemaking the motility of GI muscles. The rhythmicity forms a slow wave in the membrane potential of smooth muscle cells. The slow wave consists of a rapid upstroke depolarization, partial repolarization, and then a sustained, plateau potential that lasts for several seconds. The slow wave is suggested to be generated in ICC and propagate to electrically coupled smooth muscle cells in the GI tract (9, 13, 14, 26, 27, 31, 36, 43, 45, 51). Recent studies (14, 21, 42) have shown that the cultured ICC exhibited rhythmic inward currents that were probably responsible for the slow wave activity. The amplitude and frequency of the slow wave of cultured ICC were reduced in a low Na+ or Ca2+ concentration in extracellular solution (21), and the rhythmic inward current of cultured ICC was blocked in Ca2+-free solution (44), although slow wave activity of cultured ICC was insensitive to L-type Ca2+ channel blockers (21). These results indicate that pacemaker currents generated by ICC resulted primarily from the activation of a nonspecific cation conductance. The inhibitory effect of reduction in extracellular Ca2+ on the slow wave in ICC seems likely to be due to reduction in an intracellular Ca2+ concentration or loss of Ca2+ from stores. Ward et al. (51) showed that slow wave activity of ICC was blocked by heparin and xestopongin C, inhibitors of inositol 1,4,5-trisphosphate-dependent release of Ca2+ from the endoplasmic reticulum, and by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ reuptake. Thus intracellular Ca2+ or Ca2+ release from stores may regulate the mechanism that initiates slow waves, and the mechanisms responsible for either inward or outward currents in the slow wave of ICC may be Ca2+ dependent.

Outward current responsible for repolarization of the slow wave is also an important factor in autorhythmicity. In this study, we showed that SK3 channels were specifically expressed in ICC. This result and Ca2+ dependency of slow wave activity in ICC (see above) can lead us to the hypothesis that outward currents through the SK3 channel participate in repolarization of the slow wave in ICC. In tissue preparations, apamin has been reported to increase the amplitude of slow waves in stomach smooth muscle cells (41). However, so far there have been no studies reported that examine the effect of apamin on pacemaker currents in isolated ICC. Further electrophysiological studies in isolated ICC are needed to directly certify this hypothesis. Other K+ channels, such as inward rectifier K+ channels, delayed rectifier K+ channels, or ATP-sensitive K+ channels, could also contribute to the repolarization phase of the slow wave activity in ICC. Koh et al. (21) showed that Ba2+, a blocker of inward rectifier K+ channels, depolarized the membranes of ICC in mouse small intestine, although the amplitude and the frequencies of the slow wave were not affected by Ba2+. On the basis of these data, Koh et al. suggested that inward rectifier K+ channels provide a portion of the current necessary for repolarization of the slow wave. Further studies are needed to identify in detail the K+ channels that contribute to the slow wave in ICC.


    ACKNOWLEDGEMENTS

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and by a scholarship from Ono Pharmaceutical Company.


    FOOTNOTES

Address for reprint requests and other correspondence: A. Fujita, Dept. of Veterinary Pharmacology, Graduate School of Agriculture and Life Science, Osaka Prefecture Univ., 1-1 Gakuencho, Sakai, Osaka 599-8531, Japan (E-mail: afujita{at}vet.osakafu-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2 March 2001; accepted in final form 2 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Banks, BE, Brown C, Burgess GM, Burnstock G, Claret M, Cocks TM, and Jenkinson DH. Apamin blocks certain neurotransmitter-induced increases in potassium permeability. Nature 282: 415-417, 1979[ISI][Medline].

2.   Bauer, V, and Kuriyama H. The nature of non-cholinergic, non-adrenergic transmission in longitudinal and circular muscles of the guinea-pig ileum. J Physiol (Lond) 322: 375-391, 1982.

3.   Bayliss, W, and Starling EH. Movements and innervation of the small intestine. J Physiol (Lond) 24: 99-143, 1899.

4.   Bayliss, W, and Starling EH. The movements and innervation of the large intestine. J Physiol (Lond) 26: 107-118, 1900.

5.   Bennett, M, Burnstock G, and Holman ME. Transmission from perivascular inhibitory nerves to the smooth muscle of the guinea-pig taenia coli. J Physiol (Lond) 182: 527-540, 1966[ISI][Medline].

6.   Berezin, I, Huizinga JD, and Daniel EE. Interstitial cells of Cajal in the canine colon: a special communication network at the inner border of the circular muscle. J Comp Neurol 273: 42-51, 1988[ISI][Medline].

7.   Burns, AJ, Lomax AJ, Torihashi S, Sanders KM, and Ward SM. Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach. Proc Natl Acad Sci USA 93: 12008-12013, 1996[Abstract/Free Full Text].

8.   Bywater, RA, and Taylor GS. Non-cholinergic excitatory and inhibitory junction potentials in the circular smooth muscle of the guinea-pig ileum. J Physiol (Lond) 374: 153-164, 1986[Abstract].

9.   Christensen, J. A commentary on the morphological identification of interstitial cells of Cajal in the gut. J Auton Nerv Syst 37: 75-78, 1992[ISI][Medline].

10.   Christensen, J. Physiology of the Gastrointestinal Tract. New York: Raven, 1994, p. 991-976.

11.   Fujita, A, Horio Y, Nielsen S, Nagelhus EA, Hata F, Ottersen OP, and Kurachi Y. High-resolution immunogold cytochemistry indicates that AQP4 is concentrated along the basal membrane of parietal cell in rat stomach. FEBS Lett 459: 305-309, 1999[ISI][Medline].

12.   Hirst, GD, Holman ME, and McKirdy HC. Two descending nerve pathways activated by distension of guinea-pig small intestine. J Physiol (Lond) 244: 113-127, 1975[Abstract].

13.   Horowitz, B, Ward SM, and Sanders KM. Cellular and molecular basis for electrical rhythmicity in gastrointestinal muscles. Annu Rev Physiol 61: 19-43, 1999[ISI][Medline].

14.   Huizinga, JD, Thuneberg L, Kluppel M, Malysz J, Mikkelsen HB, and Bernstein A. W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity. Nature 373: 347-349, 1995[ISI][Medline].

15.   Ishii, TM, Silvia C, Hirschberg B, Bond CT, Adelman JP, and Maylie J. A human intermediate conductance calcium-activated potassium channel. Proc Natl Acad Sci USA 94: 11651-11656, 1997[Abstract/Free Full Text].

16.   Ito, S, Kurokawa A, Ohga A, Ohta T, and Sawabe K. Mechanical, electrical and cyclic nucleotide responses to peptide VIP and inhibitory nerve stimulation in rat stomach. J Physiol (Lond) 430: 337-353, 1990[Abstract].

17.   Jiménez, M, Borderies JR, Vergara P, Wang Y-F, and Daniel EE. Slow waves in circular muscle of porcine ileum: structural and electrophysiological studies. Am J Physiol Gastrointest Liver Physiol 276: G393-G406, 1999[Abstract/Free Full Text].

18.   Kim, D, Fujita A, Horio Y, and Kurachi Y. Cloning and functional expression of a novel cardiac two-pore background K+ channel (cTBAK-1). Circ Res 82: 513-518, 1998[Abstract/Free Full Text].

19.   Kishi, M, Takeuchi T, Suthamnatpong N, Ishii T, Nishio H, Hata F, and Takewaki T. VIP- and PACAP-mediated nonadrenergic, noncholinergic inhibition in longitudinal muscle of rat distal colon: involvement of activation of charybdotoxin- and apamin-sensitive K+ channels. Br J Pharmacol 119: 623-630, 1996[Abstract].

20.   Koh, SD, Dick GM, and Sanders KM. Small-conductance Ca2+-dependent K+ channels activated by ATP in murine colonic smooth muscle. Am J Physiol Cell Physiol 273: C2010-C2021, 1997[Abstract/Free Full Text].

21.   Koh, SD, Sanders KM, and Ward SM. Spontaneous electrical rhythmicity in cultured interstitial cells of Cajal from the murine small intestine. J Physiol (Lond) 513: 203-213, 1998[Abstract/Free Full Text].

22.   Köhler, M, Hirschberg B, Bond CT, Kinzie JM, Marrion NV, Maylie J, and Adelman JP. Small-conductance, calcium-activated potassium channels from mammalian brain. Science 273: 1709-1714, 1996[Abstract/Free Full Text].

23.   Komori, K, and Suzuki H. Distribution and properties of excitatory and inhibitory junction potentials in circular muscle of the guinea-pig stomach. J Physiol (Lond) 370: 339-355, 1986[Abstract].

24.   Kosterlitz, HW, and Robinson JA. Reflex contraction of the longitudinal muscle coat of the isolated guinea-pig ileum. J Physiol (Lond) 146: 369-379, 1959[ISI].

25.   Kusaka, S, Inanobe A, Fujita A, Makino Y, Tanemoto M, Matsushita K, Tano Y, and Kurachi Y. Functional Kir7.1-channels localized at the root of apical processes in rat retinal pigment epithelium. J Physiol (Lond) 531: 27-36, 2001[Abstract/Free Full Text].

26.   Langton, P, Ward SM, Carl A, Norell MA, and Sanders KM. Spontaneous electrical activity of interstitial cells of Cajal isolated from canine proximal colon. Proc Natl Acad Sci USA 86: 7280-7284, 1989[Abstract].

27.   Lee, JCF, Thuneberg L, Berezin I, and Huizinga JD. Generation of slow waves in membrane potential is an intrinsic property of interstitial cells of Cajal. Am J Physiol Gastrointest Liver Physiol 277: G409-G423, 1999[Abstract/Free Full Text].

28.   Liu, LWC, Thuneberg L, and Huizinga JD. Development of pacemaker activity and interstitial cells of Cajal in the neonatal mouse small intestine. Dev Dyn 213: 271-282, 1998[ISI][Medline].

29.   Maas, AJJ, and Den Hertog A. The effect of apamin on the smooth muscle cells of the guinea-pig taenia coli. Eur J Pharmacol 58: 151-156, 1979[ISI][Medline].

30.   Mackenna, BR, and McKirdy HC. Peristalsis in the rabbit distal colon. J Physiol (Lond) 220: 33-54, 1972[Medline].

31.   Maeda, H, Yamagata A, Nishikawa S, Yoshinaga K, Kobayashi S, Nishi K, and Nishikawa S. Requirement of c-kit for development of intestinal pacemaker system. Development 116: 369-375, 1992[Abstract/Free Full Text].

32.   Malysz, J, Thuneberg L, Mikkelsen HB, and Huizinga JD. Action potential generation in the small intestine of W mutant mice that lack interstitial cells of Cajal. Am J Physiol Gastrointest Liver Physiol 271: G387-G399, 1996[Abstract/Free Full Text].

33.   Martins, SL, De Oliveira RB, and Ballejo G. Rat duodenum nitrergic-induced relaxations are cGMP-independent and apamin-sensitive. Eur J Pharmacol 284: 265-270, 1995[ISI][Medline].

34.   Nagelhus, EA, Veruki ML, Torp R, Haug F-M, Laake JH, Nielsen S, Agre P, and Ottersen OP. Aquaporin-4 water channel protein in the rat retina and optic nerve: polarized expression in Müller cells and fibrous astrocytes. J Neurosci 18: 2506-2519, 1998[Abstract/Free Full Text].

35.   Niioka, S, Takeuchi T, Kishi M, Ishii T, Nishio H, Takewaki T, and Hata F. Nonadrenergic, noncholinergic relaxation in longitudinal muscle of rat jejunum. Jpn J Pharmacol 73: 155-161, 1997[ISI][Medline].

36.   Sanders, KM. Ionic mechanisms of electrical rhythmicity in gastrointestinal smooth muscles. Annu Rev Physiol 54: 439-453, 1992[ISI][Medline].

37.   Shuba, MF, and Vladimirova IA. Effect of apamin on the electrical responses of smooth muscle to adenosine 5'-triphosphate and to non-adrenergic, non-cholinergic nerve stimulation. Neuroscience 5: 853-859, 1980[ISI][Medline].

38.   Smith, T, and Sanders K. Textbook of Gastroenterology. Philadelphia, PA: Lippincott, 1995, p. 234-260.

39.   Stocker, M, and Pedarzani P. Differential distribution of three Ca2+-activated K+ channel subunits, SK1, SK2, and SK3, in the adult rat central nervous system. Mol Cell Neurosci 15: 476-493, 2000[ISI][Medline].

40.   Strøbæk, D, Jørgensen TD, Christophersen P, Ahring PK, and Olesen S-P. Pharmacological characterization of small-conductance Ca2+-activated K+ channels stably expressed in HEK 293 cells. Br J Pharmacol 129: 991-999, 2000[Abstract/Free Full Text].

41.   Suzuki, K, Ito KM, Minayoshi Y, Suzuki H, Asano M, and Ito K. Modification by charybdotoxin and apamin of spontaneous electrical and mechanical activity of the circular smooth muscle of the guinea-pig stomach. Br J Pharmacol 109: 661-666, 1993[Abstract].

42.   Thomsen, L, Robinson TL, Lee JCF, Farraway LA, Hughes MJG, Andrews DW, and Huizinga JD. Interstitial cells of Cajal generate a rhythmic pacemaker current. Nat Med 4: 848-851, 1998[ISI][Medline].

43.   Thuneberg, L. Interstitial cells of Cajal: intestinal pacemaker cells? Adv Anat Embryol Cell Biol 71: 1-130, 1982[ISI][Medline].

44.   Tokutomi, N, Maeda H, Tokutomi Y, Sato D, Sugita M, Nishikawa S, Nishikawa S, Nakao J, Imamura T, and Nishi K. Rhythmic Cl-current and physiological roles of the intestinal c-kit-positive cells. Pflügers Arch 431: 169-177, 1995[ISI][Medline].

45.   Torihashi, S, Ward SM, Nishikawa S-I, Nishi K, Kobayashi S, and Sanders KM. c-kit-Dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract. Cell Tissue Res 280: 97-111, 1995[ISI][Medline].

46.   Vergara, C, Latorre R, Marrion NV, and Adelman JP. Calcium-activated potassium channels. Curr Opin Neurobiol 8: 321-329, 1998[ISI][Medline].

47.   Vladimirova, IA, and Shuba MF. Effect of strychnine, hydrastine, and apamin on synaptic transmission in smooth muscle cells. Neurofiziologia 10: 295-299, 1978.

48.   Vogalis, F, and Goyal RK. Activation of small conductance Ca2+-dependent K+ channels by purinergic agonists in smooth muscle cells of the mouse ileum. J Physiol (Lond) 502: 497-508, 1997[Abstract].

49.   Vogalis, F, and Sanders KM. Excitatory and inhibitory neural regulation of canine pyloric smooth muscle. Am J Physiol Gastrointest Liver Physiol 259: G125-G133, 1990[Abstract/Free Full Text].

50.   Ward, SM, Burns AJ, Torihashi S, and Sanders KM. Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine. J Physiol (Lond) 480: 91-97, 1994[Abstract].

51.   Ward, SM, Ordog T, Baker SA, Jun JY, Amberg G, Monaghan K, and Sanders KM. Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria. J Physiol (Lond) 525: 355-361, 2000[Abstract/Free Full Text].

52.   Warth, R, Hamm K, Bleich M, Kunzelmann K, von Hahn T, Schreiber R, Ullrich E, Mengel M, Trautmann N, Kindle P, Schwab A, and Greger R. Molecular and functional characterization of the small Ca2+-regulated K+ channel (rSK4) of colonic crypts. Pflügers Arch 438: 437-444, 1999[ISI][Medline].

53.   Yakushigawa, H, Tokunaga Y, Inanobe A, Kani K, Kurachi Y, and Maeda T. A novel junction-like membrane complex in the optic nerve astrocyte of the Japanese macaque with a possible relation to a potassium ion channel. Anat Rec 250: 465-474, 1998[ISI][Medline].


Am J Physiol Cell Physiol 281(5):C1727-C1733
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society