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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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-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.
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RESULTS |
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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).
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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.
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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