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Hydroxy-α sanshool induces colonic motor activity in rat proximalcolon: a possible involvement of KCNK9.
Kubota K
,
Ohtake N
,
Ohbuchi K
,
Mase A
,
Imamura S
,
Sudo Y
,
Miyano K
,
Yamamoto M
,
Kono T
,
Uezono Y
.
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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.
Fig. 1. Induction of colonic motor activity and intraluminal high-amplitude pressure by hydroxy-α sanshool (HAS) in rat proximal colons. A: an entire recording of intraluminal pressure in a representative experiment is shown. A colonic specimen was placed in the bath and video images and intraluminal pressure were taken from time 0 to 4 h. At 30 min, 10 μM of HAS was added, and at 0.75 h, HAS was washed (WO) out by several exchanges with fresh buffer. At 3 h, 10 μM of HAS was added again, and at 3.25 h, HAS was washed out. Moderate amplitude of pressure peaks continued until â¼2 h and thereafter only very small peaks were detected. HAS added at 30 min and 3 h gave essentially the same motor pattern with similar amplitudes and intervals. B: serial photographs of motility of nontreated (left) and HAS-treated (right) colons. Movie data are shown in Supplementary Video S3. C: relationship between the intraluminal pressure chart and spatiotemporal map. Top: no-treatment control (at 3 h) showing rhythmic propulsive motor complex (RPMC) with low amplitude. Pre means the motility before HAS addition. Bottom: HAS treatment (at 3 h). A representative example of long-distance contraction (LDC). Propulsive contractions are displayed as diagonal streaks of dark color. After the addition of HAS, a brief temporal relaxation is frequently observed in the pressure chart (see 10-fold magnified view), but this is always observed by video imaging. The relaxation response is indicated in the figure (labeled âTemporal relaxationâ) with the change in minimum pressure levels (labeled by âBasal level downâ) before and after HAS application. Nonpropulsive contractions are displayed as short, fragmented streaks of dark color. Pink dots in the spatiotemporal map correspond to those in the intraluminal pressure chart.
Fig. 2. Dose-dependent induction of high-amplitude pressure peaks by HAS. A: typical patterns of intraluminal pressure induced by HAS. B: quantitation of HAS-induced contractility. Ratio of peak frequency (PF), peak pressure amplitude (PPA), and area under the curve (AUC) calculated as the ratio to the PF, PPA, and AUC before drug treatment (%-PF, %-PPA, and %-AUC, respectively) are shown (n = 4â6). *P < 0.05, **P < 0.01, ***P < 0.001 vs. Vehicle control.
Fig. 3. Motor activity by HAS is not mediated by TRPV1 nor TRPA1. Typical changes of intraluminal pressure induced by a TRPV1 agonist capsaicin (A) and a TRPA1 agonist allyl isothiocyanate (AITC, B). C: effects of desensitization (pretreatment with 3 μM capsaicin plus 10 μM AITC), a TRPV1 antagonist BCTC (1 μM), or a TRPA1 antagonist HC-030031 (5 μM) on HAS-induced motility (n = 5â6). Pre means the motility before HAS addition. *P < 0.05. NS, not significant.
Fig. 4. Distribution of mRNA of KCNK channels in rat colon. A: RT-PCR analysis of mRNA expression in rat colon. PCR products were electrophoresed through a 2% agarose gel. RT, reverse transcriptase, SC, spinal cord. B: in situ hybridization of rat colon for KCNK3, KCNK9, and PGP9.5. Top: single staining for KCNK3 or KCNK9. Red dots indicate the target mRNA. Location of the myenteric plexus is indicated by a dashed white circle. Bottom: double staining for PGP9.5 and KCNK9. Red and violet dots indicate PGP9.5 and KCNK9 mRNAs, respectively. KCNK9 mRNA is indicated by arrows. Nuclei were stained with hematoxylin.
Fig. 5. Distribution of KCNK9 and KCNK3 proteins in rat colons A: immunohistochemistry of whole-mount muscle layer of rat colon for KCNK9, phalloidin, and PGP9.5. DAPI, 4â²,6-diamidino-2-phenylindole; LM, longitudinal muscle; MP, myenteric plexus; CM, circular muscle. B: immunohistochemistry of whole-mount muscle layer of rat colon for KCNK3, phalloidin, and PGP9.5. C: double immunostaining of KCNK9 and PGP9.5. in MP. D: double immunostaining of KCNK9 and c-Kit in MP. Scale bar = 30 μm.
Fig. 6. HAS induces depolarization by blocking KCNK3 and KCNK9. A: 2-electrode voltage-clamp analysis with Xenopus oocytes expressing rat KCNK3 or KCNK9. Percent suppression of leak current was determined with a holding potential of +60 mV. Data are expressed as means ± SE for n = 4–8. *P < 0.05, **P < 0.01 vs. DMSO control. #P < 0.05, ###P < 0.001 vs. PBS control. B: kinetics of the changes of membrane potential responses induced by HAS, hydroxy-β sanshool (HBS), and lidocaine (Lid) in rat KCNK3- or KCNK9-expressing CHO-K1 cells. Application of compounds is indicated by “Apply.†Kinetics of the changes in membrane potential responses induced by 90 mM KCl is included as a positive control (n = 4). C: summary of normalized response to KCNK3, KCNK9, and mock cell by HAS, HBS, and Lid. Values represent the ΔF/F0 normalized to that of DMSO or PBS controls (mean ± SE for n = 4). *P < 0.05, **P < 0.01, ##P < 0.01.
Fig. 7. Motor activity induced by HBS and Lid. The typical patterns of HBS (A) and Lid (B) on a spatiotemporal map and intraluminal pressure chart are shown.
Fig. 8. Effect of TTX on HAS- and HBS-induced contraction. A and B: top charts show typical changes in the HAS (left) and HBS (right) motility patterns induced by TTX. Middle and bottom graphs show changes of %-PF, %-PPA, and %-AUC induced by TTX, respectively. Experiment used 10 μM HAS (n = 6) and 30 μM HBS (n = 5 and 6). Pre means the motility before addition of TTX or its vehicle. *P < 0.05, **P < 0.01 vs. Control (vehicle).
Fig. 9. Acceleration of defecation by HAS and TU-100 in normal rats or postoperative ileus (POI) model rats. A: increase of defecation frequency at 5 h after HAS (50 mg/kg po) administration in normal rats. *P < 0.05, **P < 0.01 vs. Vehicle (olive oil 0.5 ml/kg po) (n = 8). B: decrease of defecation frequency by laparotomy. *P < 0.05, ***P < 0.001 vs. Normal (anesthetized only). (n = 8 â¼21, at 7 h after operation). C: increase of defecation frequency after HAS (15 and 50 mg/kg po) dosing at 4 h after administration (i.e., 6 h after operation) in POI rats. *P < 0.05 vs. Vehicle (n = 23â25). D: acceleration of defecation frequency induced TU-100 (1 and 3 g/kg po) dosing 3 h after administration (i.e., 6 h after operation) in POI rats. *P < 0.05, **P < 0.01 vs. Vehicle (water 15 ml/kg po) (n = 8 or 9).
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