Hydroxy-α sanshool induces colonic motor activity in rat proximal colon: a possible involvement of KCNK9

Various colonic motor activities are thought to mediate propulsion and mixing/absorption of colonic content. The Japanese traditional medicine daikenchuto (TU-100), which is widely used for postoperative ileus in Japan, accelerates colonic emptying in healthy humans. Hydroxy-α sanshool (HAS), a readily absorbable active ingredient of TU-100 and a KCNK3/KCNK9/KCNK18 blocker as well as TRPV1/TRPA1 agonist, has been investigated for its effects on colonic motility. Motility was evaluated by intraluminal pressure and video imaging of rat proximal colons in an organ bath. Distribution of KCNKs was investigated by RT-PCR, in situ hybridization, and immunohistochemistry. Current and membrane potential were evaluated with use of recombinant KCNK3- or KCNK9-expressing Xenopus oocytes and Chinese hamster ovary cells. Defecation frequency in rats was measured. HAS dose dependently induced strong propulsive “squeezing” motility, presumably as long-distance contraction (LDC). TRPV1/TRPA1 agonists induced different motility patterns. The effect of HAS was unaltered by TRPV1/TRPA1 antagonists and desensitization. Lidocaine (a nonselective KCNK blocker) and hydroxy-β sanshool (a geometrical isomer of HAS and KCNK3 blocker) also induced colonic motility as a rhythmic propagating ripple (RPR) and a LDC-like motion, respectively. HAS-induced “LDC,” but not lidocaine-induced “RPR,” was abrogated by a neuroleptic agent tetrodotoxin. KCNK3 and KCNK9 were located mainly in longitudinal smooth muscle cells and in neural cells in the myenteric plexus, respectively. Administration of HAS or TU-100 increased defecation frequency in normal and laparotomy rats. HAS may evoke strong LDC possibly via blockage of the neural KCNK9 channel in the colonic myenteric plexus.


INTRODUCTION
Control of the mechanical activity of the intestines is complex and our understanding of the mechanisms involved is still in its infancy.Elements of the regulation of motor activity and the electrical and mechanical properties of the intestines have been found to be different depending on the species, regions of intestines under investigation, conditions of the specimen (in vivo or in vitro, unstimulated or stimulated, etc.) and methodology used for evaluation.In the rat colon, several distinct motor patterns have been demonstrated by Huizinga and colleagues (3,11).Among them, the patterns termed "rhythmic propulsive motor complex (RPMC)/long distance contraction (LDC)" and "rhythmic propagating ripple (RPR)" have been proposed to be created, at least partly, by two independent networks of interstitial cells of Cajal (ICC).It has been suggested that RPMC/LDC and RPR may be related to the propulsion and mixing/absorption of the luminal contents, respectively.Therefore, agents affecting these motor patterns could lead to the development of new therapeutic options for colonic dismotility diseases such as constipation.
TU-100, a pharmaceutical-grade traditional Japanese (kampo) medicine, has been widely used for the treatment of various gastrointestinal disorders including postoperative ileus and ischemic intestinal disorders (17).The drug has been approved as a prescription drug by the Ministry of Health, Labor and Welfare of Japan and integrated into the modern medical care system in Japan.A double-blind, placebo-controlled study on healthy volunteers in the U.S. has shown that treatment 7 with TU-100 significantly accelerates ascending colon emptying (22).Several double blind placebo-controlled trials on the patients with postoperative ileus (POI), Crohn's disease, functional constipation and irritable bowel syndromes are currently underway in the U.S. and Japan.The drug is an extract powder made from a mixture of Japanese pepper, processed ginger and ginseng radix, with maltose powder as an additive.Hydroxy--sanshool (HAS) contained in Japanese pepper has been elucidated as one of the main active compounds responsible for the efficacy of TU-100 to POI and adhesive intestinal obstruction (31,32).Furthermore, HAS is rapidly absorbed in the gut and reaches high concentrations in the blood when TU-100 is administered orally (12,24,25).
Hydroxy--sanshool (HBS), a geometrical isomer of HAS is also rapidly absorbed into the bloodstream (12,24,25).HAS and HBS, which have been known as agonists to transient receptor potential vanilloid type 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) (18), are now recognized as selective blockers to certain two-pore domain potassium (KCNK) channels: HAS for TASK-1 (KCNK3), TASK-3 (KCNK9) and TRESK (KCNK18) and HBS for KCNK3 (1).TRPA1 expresses abundantly in enterochromaffin cells and TRPA1 stimulation induces serotonin (5-HT) release resulting in enteric nerve activation (26), which may be a trigger for stimulus-induced colonic motility (10).KCNK3 has been reported to express in colonic smooth muscle cells (SMC) and to be involved in the determination of the resting potential (and therefore excitability) of SMC, which may affect the contractility of colonic smooth muscle (28).In the present study, we

Animals
Male SD rats (Japan SLC, Hamamatsu, Japan) at 7-12 weeks old were housed under controlled light environmental conditions and had free access to food and water.All experimental procedures were ethically approved by the Laboratory Animal Committee of Tsumura and Co. and performed according to the institutional guidelines for the care and use of laboratory animals, which is in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Isolated rat proximal colon tract and measurement of intraluminal pressure
Rats were fasted overnight and sacrificed by decapitation before removing their entire colon.A 2-cm to 3-cm segment of the proximal colon was placed into an organ bath (100 mL volume), which was continuously perfused with warm Krebs solution (3.5 mL/min, 34-5 °C).The oral and aboral ends of the proximal colon segment were securely attached with string to an input and output port of the saline, respectively.In order to monitor intraluminal pressure (cmH 2 O), a Mikro-Tip catheter pressure transducer (SPR-524, Millar Instruments, Houston, TX) was set in the lumen of the aboral end.Motility was initiated by loading an intraluminal pressure to approximately 4 cmH 2 O by elevating the drain tube.After an equilibration period of 120-240 min, contraction had reached a consistent pattern, in terms of amplitude and frequency.The intraluminal pressure waves were evaluated by a data acquisition and analysis system (MP100, BIOPAC System, Goleta, CA).The motility was macroscopically observed through video images (PCR-SR87, SONY, Tokyo, Japan).
All drug solutions were added to Krebs solution in the organ bath (serosal side).Peak frequency (PF) was calculated as the mean number of pressure peaks per minute during a defined period.The peak pressure amplitude (PPA) was calculated as the mean pressure of peaks during an allotted time period.The area under the curve (AUC) during the allotted time period was also calculated.
The %-PF, % -PPA and %-AUC of each period were calculated as the ratio to the PF, PPA and AUC before drug treatment, respectively.

Spatiotemporal mapping
A "spatiotemporal map", which is an image representation of motor activity, was generated according to the method described by Huisinga et al. (11).Colon width (coded as image intensity, black to white) is calculated at each point along the length of the colon (image Y-axis), for each 11 video frame (image X-axis) using ImageJ software.As shown in Figure 1C, propagating contractions are represented as the diagonal streaks of dark color.Broad relaxation is represented as white area.

RT-PCR
Muscle layer was carefully detached from the rat ascending colon and the remaining layer was used as the mucosal layer.Total RNA was prepared with RNeasy Universal Tissue Kit (QIAGEN, Hilden, Germany) and cDNA was synthesized with High Capacity cDNA Reverse Transcription Kits (Life Technologies) according to the manufacturer's instructions.Sequences of sense and anti-sense primers for RT-PCR analysis were as follows: rat KCNK3 sense: 5'-TCATCACCACAATCGGCTAT-3', anti-sense: 5'-AGCGCGTAGAACATGCAGAA-3', rat

In situ hybridization
In situ hybridization (ISH) was performed by using QuantiGene ViewRNA.Gene specific probe sets for rat KCNK3, KCNK9, PGP9.5 mRNAs were designed by Affymetrix (Santa Clara, CA).

Immunohistochemistry
Whole mount immunohistochemistry was performed as follows: Intestinal specimens were opened along the mesenteric border.The specimens were stretched taut and pinned out flat to a silicone ring and fixed with ice cold acetone for 30 minutes.After fixation, each preparation was washed three times for 10 minutes each in phosphate-buffered saline (PBS; 0.9% NaCl in 0.1 M sodium phosphate buffer, pH 7.0).The preparations were placed in Superblock (Thermo Fischer Scientific, Rockford, IL) containing 0.3% Triton X-100 overnight at 4°C.The preparations were then placed in primary antibody diluted in antibody diluent (DAKO Japan, Tokyo, Japan) overnight at 4°C.After removal from the primary antibody, the tissues were rinsed for 3 × 10 minutes with PBS and incubated with

Preparation of plasmids and cRNAs
Rat KCNK3 cDNA was amplified from rat whole brain cDNA (Takara, Otsu, Japan) and introduced into pcDNA-DEST40 by Gateway system (Life Technologies, Carlsbad, CA).Rat KCNK9 cDNA was purchased from OriGene (Rockville, MD).cRNAs were prepared with mMessage mMachine Kit (Life Technologies) following the manufacturer's instructions.

Membrane potential assay
Chinese hamster ovary (CHO)-K1 cells (ATCC, Manassas, VA) were seeded into 96well plates (~7,000cells/well).Rat KCNK3 or KCNK9 were then transiently expressed in CHO-K1 with FuGENE HD Transfection Reagent (Promega, Madison, WI) following the manufacturer's instructions.About 48hr after transfection, a membrane potential assay was performed using a FLIPR Membrane Potential Assay Kit (Molecular Devices).The change of membrane potential was monitored in terms of fluorescence intensity, measured using a FlexStation3 (Molecular Devices).

Analysis of defecation frequency
Normal Rats: HAS was orally administered and thereafter the rat's feces were counted cumulatively during a 5 hr period under fasting.Laparotomy rats: Rats (7 -8 week old) were incised about 4 cm in the median line of the abdomen after being anesthetized by intraperitoneal injection of Somnopentyl (Kyoritsu Seiyaku, Tokyo, Japan).The small intestine, cecum and large intestine were exteriorized and covered with sterile gauze dampened with saline for 1 hr before Kubota et al.

15
returning to the abdominal cavity and closing the abdomen.Rat feces were counted cumulatively over a 7 hr period under the fasting state.Rats in the non-operation group were only anesthetized.
HAS and TU-100 were administered orally to rats by the gavage technique 3 or 4 hr after abdominal closure.

Evaluation of colonic motor activity by intraluminal pressure peaks and video imaging and effect of HAS
Motility of isolated segments of the proximal colon was analyzed by the measurement of intraluminal pressure and video image recordings.During a 2 hr period after the beginning of the experiment, moderate amplitude pressure peaks were observed (see Figure 1A).Thereafter the amplitude of these pressure peaks weakened and by the 3 hr time point only very small peaks could be detected (see Figure 1A, from time 3 hr to 4 hr).Nonetheless, low amplitude contractions were still detected by video imaging, which appeared to be propulsive (Figure 1A; details shown in the spatiotemporal map of Figure 1C) until the end of the experiment.These types of contractions do not always produce evident changes in pressure peaks.We performed quantitative analysis of colonic motility mainly by assessing changes in the pressure because they are in good agreement with the motility observed in video-imaging, at least for the high amplitude propagating motor activity.
Relationship between the intraluminal pressure chart and the video images is shown in Supplementary Movie M1 and M2, and the relationship between the intraluminal pressure chart and spatiotemporal map is shown in Figure 1C.It should be noted that for the low amplitude motor activity, not all of the activity produces evident changes in the pressure peaks.Therefore, analyses based on the pressure peaks may tend to underestimate its frequency.

17
As shown in Figure 1A, addition of HAS to the bath solution from the serosal side at 30 min and 3 hr induced high amplitude periodic pressure peaks with similar frequency and potency.The motility is characterized by a strong squeezing over a broad range of the proximal colon (Figures 1B, 1C, movie files are shown in Supplementary Movie M3), after a brief temporal relaxation (Figure 1C).
The dose-dependency of pressure peak pattern, PF, PPA and AUC are shown in Figure 2.

Contraction induced by HAS is not a result of stimulation of TRPV1/TRPA1
Firstly, we investigated whether TRPV1/TRPA1 agonists evoke motility similar to that induced by HAS.A TRPV1 agonist capsaicin (0.1 -10 M) evoked a single contraction peak only, both at 30 min (data not shown) and 3 hr (Figure 3A) though its PPA was potent.A TRPA1 agonist arylisothiocyanate (AITC) induced periodic contractions, although the amplitude was very small even at high concentrations (Figure 3B represents the result of AITC added at 100 M at 3 hr; Supplementary Movie M5).Furthermore, administration of TRPV1 or TRPA1 inhibitor (BCTC and HC-030031, respectively), and desensitization of TRPV1/TRPA1 by bolus application of high doses of capsaicin plus AITC, gave no, or only a modest, suppression of colonic motility evoked by HAS (Figure 3C).

Localization of KCNK9 and KCNK3 proteins
In order to investigate the possible involvement of these KCNK channels in HAS-induced motility, RT-PCR was performed using specific primers for rat KCNK3, KCNK9 and KCNK18.Our results show that KCNK3 and KCNK9 mRNAs are expressed in the muscle layer, whereas KCNK18 mRNA is barely detectable in the rat proximal colon (Figure 4A).The localization of KCNK3 and KCNK9 was evaluated by ISH and immunohistochemistry.As shown in Figure 4B, KCNK3 mRNA was localized in the longitudinal muscle (LM) layer and myenteric plexus (MP) and KCNK9 mRNA in MP (Figure 4B).A neuronal marker PGP9.5 mRNA co-existed with KCNK9 mRNA (Figure 4B).
Immunohistochemistry of whole mount preparation of colonic muscle layers confirmed the predominant localization of KCNK9 in MP and in the enteric nerves in the circular muscle (CM) layer (Figure 5A), and that of KCNK3 in LM SMCs (Figure 5B).Co-immunostaining analysis with anti-KCNK9 and anti-PGP9.5antibodies (Figure 5C) showed that KCNK9 is expressed in 35.8 % of PGP9.5 + MP cells (n = 148).More than a quarter of KCNK9 + cells in MP are PGP9.5 -.
Furthermore, in co-immunostaining experiments with KCNK9 and an ICC marker c-kit (Figure 5D),

HAS induced membrane depolarization via blocking rat KCNK9
It is not known whether HAS and HBS inhibit rat KCNK3 and/or KCNK9.Thus, we conducted two electrode voltage clamp assays using Xenopus oocytes expressing rat KCNK3 or KCNK9.Because both channels were reported to be regulated by extracellular pH, application of pH6.5 solution was used as a positive control.Inhibitory effect of test compounds were determined with a holding potential of +60 mV.LID was used as a non-selective KCNK channel blocker, which strongly inhibited both KCNK3 and KCNK9.As shown in Figure 6A, HAS showed significant and dose dependent inhibition against KCNK3 and KCNK9 while HBS inhibited only KCNK3.These results were comparable to those observed for murine KCNKs (1).
KCNK channels regulate the excitability of cells such as smooth muscle and neurons by adjusting their membrane potential.To address whether HAS inhibition against KCNK3 and KCNK9 led to membrane depolarization, we examined the membrane potential of rat KCNK3-or KCNK9-expressing CHO-K1 cells (Figures 6B, 6C) by using a membrane potential dye.In this assay, the fluorescent signal increases during membrane depolarization and decreases during membrane hyperpolarization.For example, the application of KCl increased the fluorescent signal, which represents a depolarizing membrane potential.The KCl-induced changes in fluorescence intensity for KCNK3 and KCNK9 expressing cells were larger than that observed for the mock cells (Figure 6B).Data are represented by the normalized change in fluorescence (F/F 0 ).Inhibition of KCNK3 or 9 was determined at 100 seconds after compound application.The application of HAS

21
(0.3 M), a neuroleptic agent (Figure 8A).By contrast, the contractions induced by HBS were not abolished but its shape was changed by TTX (Figure 8B).Addition of high doses (up to 3 mM) of TTX did not alter the effect of LID (1 mM, data not shown).

HAS accelerates defecation in normal and POI model rats.
In order to investigate whether HAS accelerates defecation in vivo we examined the amount of feces accumulated during a short period after treatment with the agents.The accumulated number of fecal pellets increased significantly at 5 hr after oral administration of 50 mg/kg HAS to normal rats (Figure 9A).
For rats that had undergone abdominal surgery, the number of pellets decreased significantly 7 hr after suturing (Figure 9B).Oral administration of HAS at 3 hr after the operation significantly increased the number of pellets 4 hr after administration (i.e., 7 hr after the operation) in a dose-dependent manner (Figure 9C).Accordingly, a single oral administration of TU-100 3 hr after the operation also increased the number of pellets 3 hr after drug treatment (i.e., 6 hr after the operation) in a dose-dependent manner.A significant outcome was obtained at a dose of 3 g/kg TU-100 (Figure 9D).

DISCUSSION
HAS was found to induce high amplitude of contractions with significant levels of periodicity.The peak frequency (~ 0.5/min), propagating/propulsive property, sensitivity to TTX and the presence of relaxation phase preceding a very strong contractions are in good agreement with those reported for LDC (11).LID, however, was found to induce low amplitude contractions with high frequency (~6/min), which were non-propulsive and insensitive to TTX (i.e., in good agreement with RPR ( 11)).The increase in amplitude induced by HBS might be periodic, but the interval between consecutive peaks were wider and the incidence of the peaks appeared more irregular compared to those induced by HAS.As shown in Figure 3, "LDC" induced by HAS was not caused by the agonistic activity of TRPV1/TRPA1, which is well documented for HAS and HBS.We reasoned that the target molecules of HAS, HBS and LID may be KCNK channels because these agents, especially HAS and HBS, have a high specificity for KCNK3 and/or KCNK9.Bautista et al (1) have screened various ion channels including KCNK1~6, 9, 10, 13, 16 and 18 and reported specific inhibition of KCNK3 by HBS and of KCNK3/9/18 by HAS.LID blocks a broad range of KCNKs.
Although many of the biological activities of LID can be attributed to its inhibitory activity against multiple voltage-gated sodium channels, the effect on KCNK3 has been suggested to be involved in LID-induced seizure in mice (6).
KCNK ion channels comprise a family of potassium-selective channels that share the unique structural feature of four transmembrane domains and two pore-forming domains (8).The KCNK channel family are open across the physiological voltage range and are therefore believed to underlie many of the background K + currents (also known as resting, baseline, or leak currents) that regulate the resting membrane potential and excitability of many mammalian cells (2,7,21).KCNK channels are found in neuronal and non-neuronal tissues, and they provide, in addition to setting of resting membrane potentials (13,23), a wide variety of important functions, including sensing of oxygen and pH, neuroprotection, and mechanosensitivity (28).These channels are also candidates for the action of volatile anaesthetics on neural excitability (27).Molecular analysis of GI smooth muscle myocytes revealed expression of KCNK genes, including KCNK2, KCNK10 ( 14), KCNK3 and KCNK5 (28), and the properties of the currents produced by these channels have been shown to contribute to the native currents observed in GI smooth muscle (4).These data suggest that certain KCNK family channels may be involved in the regulation of GI motility.
Interestingly, we found that the KCNK9/KCNK3 blocker HAS evokes a unique motor pattern with a high-degree of periodicity while its geometric isomer, HBS, specifically blocked KCNK3 and induced a motor pattern different from that of HAS.This observation suggests that the highly periodic LDC evoked by HAS may be mediated by KCNK9.In the present study, we show that KCNK3 is located in LM SMC and presumably a small portion of neuronal cells in MP, whereas KCNK9 is located mainly in a portion of neuronal cells in MP and CM layer.These findings are in good accordance with previous immunohistochemical studies using mouse (14,28) and human (19) GI tissue.The location of KCNK9 suggests that the channel may be involved in the determination of resting potential and excitability of a substantial portion of enteric neurons innervating the MP and CM layer.The "LDC" induced by HAS was completely abrogated by TTX treatment.If HAS blocks KCNK9 in these motor neurons, their excitability will be augmented.Under such conditions, even if signals with similar intensity are provided from upstream neurons (e.g., interneurons and intrinsic primary afferent neurons) and/or enteroendocrine cells, the strength of the signals to the ICC/SMC will differ substantially.Furthermore, while our data indicates that KCNK9 is not expressed in SMC, it addressed the possibility of its expression in ICC in MP (ICC-MP).ICC-MP network is a pacemaker and has been suggested to actively propagate rhythmic transient depolarizations responsible for RPMC and/or LDC (11,29).If HAS affects ICC-MP to augment its functions, (i.e., to generate more intense and well-regulated electrical periodicity), the agent would evoke potent and regular "LDC" as demonstrated in the present results.
Because KCNK3 exists predominantly in LM SMC, it is thought to be involved in the regulation of excitability of SMC, which may chiefly affect the tone of muscle contractility.The present study indicates that KCNK3 might be expressed in some neurons in MP.This fiding is intriguing because the effect of HBS, the blocker of KCN3, was not abrogated but its potency and frequency were changed by TTX, suggesting a possible involvement of motility control system of both neurogenic and myogenic origin.Chen et al. ( 3) has demonstrated that myogenic mechanisms could be involved in certain LDC-like rhythmic activity.Thus, further detailed examination is necessary to clarify the effect of KCNK3 on colonic motility.
The properties of the LID-induced motility are in good accordance with those of RPR, which is thought to be mediated by the submuscular ICC (ICC-SMP) network (11).LID is primarily a potent inhibitor of several voltage-gated sodium channels and has a wide array of biological activities including the effects on other KCNK family channels (5,20) in which the blocking of KCNK3 is only one item of the array.Accordingly, 1 mM LID significantly increased the membrane potential in KCNK3-, KCNK9-and mock-transfected CHO-K1 cells, which strongly suggests that the depolarization by LID may be due to a mechanism other than its inhibition of KCNK3/9.
HAS is a major ingredient of TU-100, which has been integrated into the modern medical system under the approval of the Ministry of Health, Labor and Welfare of Japan.Basic research has revealed the various beneficial effects of TU-100 on intestinal motility (9,30,32), adhesion (31), vasodilatation (16), and inflammation (15).Clinical trials of TU-100 are currently underway in the U.S. aimed at the development of novel therapeutic treatments for various intestinal disorders.These studies include research on GI and colonic transit by validated scintigraphy, which has indicated TU-100 significantly accelerates ascending colon empting in healthy human volunteers (22).
Pharmacokinetics studies have shown that, when TU-100 is administered orally, HAS is rapidly absorbed in the gut and reaches high concentrations in the blood (approximately 1 M in human and rats) within 15 min (12,24,25).The present experimental settings for the bath application of HAS to the proximal colon is in reasonable agreement with the clinical situation of orally-administered TU-100 in terms of the pharmacological and pharmacokinetic properties of the drug.Furthermore, we have observed that low dosages of HAS, which are too low to induce "LDC" alone, enhance motility triggered by other stimuli such as bethanechol, capsaicin or gingerol (data not shown).This observation is in good accordance with the assumed mechanism of action of HAS, i.e., augmentation of excitability of enteric motor neurons via blocking KCNK9.
In order to investigate whether the induction/enhancement of "LDC" in vitro relates to the colonic transit in vivo, we have examined the effect of HAS or TU-100 on defecation of normal and postoperative rats.A single administration of HAS or TU-100 increased the number of fecal pellets accumulated over a short period of time (3-4 hr after drug treatment and 6-7 hr after surgery).
Although the present study may provide potentially interesting findings, there are several points to be validated and investigated in future studies.Firstly, involvement of KCNK channels in HAS/HBS/LID-mediated motility is still unclear.The use of more specific agonists and/or antagonists, and gene-manipulated mice is necessary to clarify this point.Secondly, further investigation into the biological function of KCNK channels in the generation of slow wave activity, rhythmic transient depolarizations and colonic motility is needed.In the present study, we have 27 demonstrated that KCNK channels strongly affect the membrane potential in the cell line.However, detailed analyses on gastrointestinal cells and tissues, such as the measurement of inhibitory junction potential, have yet to be performed.Thirdly, whether the KCNK9 channel is expressed in ICC-MP should be determined by more conclusive and quantitative methods such as more extensive morphometrical immunohistochemistry of the tissues and analysis of KCNK9 protein and mRNA in purified ICCs.If KCNK9 is expressed in a subpopulation of ICC-MP, it will be reasonable for HAS to modulate the pace-making of colonic motility.Elucidation of these points will undoubtedly contribute to a deeper understanding of the physiology and molecular biology of colonic motor activities.
In conclusion, we have demonstrated that HAS may induce or enhance "LDC" of the proximal colon presumably elevating the excitability of enteric nerves via KCNK9 blocking.The present findings provide not only a clarification of the mechanism of action of a promising new medicine TU-100, but also a way to develop a novel therapeutic strategy for the treatment of intestinal dysmotility.The number of peaks during a set period of time was counted in the pressure peak chart and peak frequency (PF) was calculated as the number of peaks per minute.N = 4 -7.
the relevant secondary antibody conjugated to Alexa fluorochromes (Molecular Probes, Eugene, OR) diluted in antibody diluent (DAKO Japan) overnight at 4°C.After a final set of rinses, the 13 preparations were mounted on microslides and coverslipped with Prolong Gold antifade reagent (Molecular Probes).The slides prepared from whole mount or 7μm-thick sliced specimens were observed using confocal laser microscopy FV-100D (Olympus, Tokyo, Japan).The following antibodies were used: a mouse monoclonal antibody to KCNK9 (Sigma-Aldrich), a guinea pig polyclonal antibody to PGP9.5 (Abcam plc, Cambridge, UK), rabbit polyclonal antibodies to KCNK3 (Santa Cruz Biotechnology Inc. Dallas, TX), CD117 (DAKO Japan), platelet-derived growth factor receptor (PDGFR)- (Santa Cruz Biotechnology) and smooth muscle actin and nuclei were visualized by staining with Alexa568-conjugated phalloidin and 4',6-diamidino-2-phenylindole (DAPI), respectively (Molecular Probes).In double immunostaining, each cell was identified by a combination of DAPI and a respective marker, and double positive-and single positive-cells were counted by visual inspection.

22. 4 %
of c-kit + cells in MP were found to be KCNK9 + (n = 156) though the double positive cells had relatively weaker c-kit immunosignals (i.e., most of the cells with the strongest c-kit immunosignals lacked KCNK9 immunosignal).Nearly half of KCNK9 + cells in MP were c-kit - (often located adjacent to c-kit + cells).KCNK9 signals were not detected in PDGFR--positive fibroblast-like interstitial cells (data not shown), which are thought to participate in inhibitory neurotransmission of enteric nerves.

Figure 1 .
Figure 1.Induction of colonic motor activity and intraluminal high amplitude pressure by HAS in

Figure 2 .
Figure 2. Dose-dependent induction of high amplitude pressure peaks by HAS. A. Typical patterns

Figure 3 .
Figure 3. Motor activity by HAS is not mediated by TRPV1 nor TRPA1.Typical changes of

Figure 4 .
Figure 4. Distribution of mRNA of KCNK channels in rat colon.A. RT-PCR analysis of mRNA

Figure 5 .
Figure 5. Distribution of KCNK9 and KCNK3 proteins in rat colons A. Immunohistochemistry of

Figure 7 .
Figure 7. Motor activity induced by HBS and LID.The typical patterns of HBS (A) and LID (B) on

Figure 8 .Table 1 .
Figure 8.The effect of TTX on HAS-, and HBS-induced contraction.A., B. Upper charts show