• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제14권2호
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• pp.171-180
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• 2011

목 적: 고지방식이를 섭취한 흰쥐에서 식이 유발인자인 NPY와 장내 운동 조절인자인 Cajal 간질세포의 변화를 관찰하고자 하였다. 대상 및 방법: 동일 연령의 Sprague-Dawley계 수컷 흰쥐를 임의로 대조군(7마리)과 고지방식이군(7마리)으로 분리한 후, 대조군에는 일반 흰쥐 사료를 충분히 섭취시켰으며, 고지방식이군에게는 60% kcal 지방이 포함된 사료(중앙실험동물센터)를 6주 동안 물과 함께 충분히 섭취하도록 하였다. 6주 후 시상하부 조직 절편에 rabbit polyclonal NPY (1 : 1000, Abcam)항체를 반응시키고, 소장의 조직 절편에 rabbit polyclonal c-kit (1 : 400, Neuromics)항체를 반응시켜서 면역조직화학 검사를 시행하였다. 결 과: 고지방식이군의 시상하부에서 NPY의 면역 반응성이 강하게 나타났으며, 특히 PVN에서 NPY의 면역 반응 세포수의 증가를 관찰할 수 있었다. 고지방식이군에서 소장의 Auerbach's plexus 부위의 Cajal 간질 세포의 면역 반응성이 떨어졌으며, 환상근과 종주근 내 Cajal 간질 세포 수도 현저히 감소하였다. 결 론: 고지방식이를 섭취한 흰쥐에서 시상하부 NPY 면역반응 신경세포수와 발현의 증가는 NPY의 저항성과 관련되며, 소장에서 Cajal 간질 세포의 감소는 장운동의 이상을 초래할 수 있음을 나타낸다.

• The Korean Journal of Physiology and Pharmacology
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• 제12권6호
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• pp.323-330
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• 2008

The properties of voltage dependent $Ca^{2+}$ current (VDCC) were investigated in interstitial cells of Cajal (ICC) distributed in the myenteric layer (ICC-MY) of guinea-pig antrum. In tissue, ICC-MY showed c-Kit positive reactions and produced driving potentials with the amplitude and frequency of about 62 mV and 2 times $min^{-1}$ respectively, in the presence of $1{\mu}M$ nifedipine. Single ICC-MY isolated by enzyme treatment also showed c-Kit immunohistochemical reactivity. These cells were also identified by generation of spontaneous inward current under $K^+$ -rich pipette solution. The voltage clamp experiments revealed the amplitude of - 329 pA inward current at irregular frequency. With $Cs^+$-rich pipette solution at $V_h=-80\;mV$, ICC-MY produced voltage-dependent inward currents (VDIC), and nifedipine ($1{\mu}M$) blocked VDIC. Therefore, we successfully isolated c-Kit positive single ICC from guinea-pig stomach, and found that ICC-MY potently produced dihydropiridine sensitive L-type voltage-dependent $Ca^{2+}$ currents ($VDCC_L$).

• 대한한의학방제학회지
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• 제32권1호
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• pp.29-37
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• 2024

Objectives : The purpose of this study was to examine the effects of insurance herbal medicines on colonic interstitial Cells of Cajal (ICC) in mice. Methods : Among the insurance herbal medicines, we chose Gamisoyo-san (GSS), Banhasasim-tang (BHSST) and Bojungikki-tang (BGIKT). We made the ICC culture in large intestine in mice and used the electrophysiological method to record pacemaker potentials. Also we used MTT assay to check cell viability and examined the ICC protein expression by western blot. Results : 1. GSS (1-10 mg/ml) induced the pacemaker potential depolarization and decreased frequency with concentration-dependent manners in colonic ICC. EC50 is 2.99 mg/ml. BHSST (1-10 mg/ml) induced the pacemaker potential depolarization and decreased frequency with concentration-dependent manners in colonic ICC. EC50 is 2.76 mg/ml. BGIKT (1-10 mg/ml) induced the pacemaker potential depolarization and decreased frequency with concentration-dependent manners in colonic ICC. EC50 is 4.49 mg/ml. 2. GSS, BHSST and BGIKT had no effects on cell viability in colonic ICC. 3. GSS and BGIKT increased the Anoctamin-1 (ANO1) protein expression and BHSST increased the transient receptor potential melastatin-subfamily member 7 (TRPM7) protein expression in colonic ICC. Conclusions : These results suggest that GSS, BHSST, and BGIKT have shown the potential to regulate gastrointestinal (GI) motility by regulating colonic ICC and may show the potential to treat colon-derived GI diseases such as irritable bowel syndrome (IBS).

• 대한한의학방제학회지
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• 제27권4호
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• pp.237-244
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• 2019

Objectives : The purpose of this study was to investigate the effects of Carthami flos on pacemaker potentials of small intestinal and colonic Interstitial Cells of Cajal (ICC). Methods : To dissociate the ICC, we used enzymatic digestions from the small intestine and colon in mice. In the ICC, the electrophysiological whole-cell patch-clamp configuration was used to record pacemaker potentials in the cultured ICC. Results : 1. The ICC generated pacemaker potentials in the murine small intestine and colon. 2. Pretreatment with a Ca²⁺ free solution and thapsigargin, a Ca²⁺-ATPase inhibitor in the endoplasmic reticulum, stopped the pacemaker potentials. In the case of Ca²⁺-free solutions, Carthami flos did not induce membrane depolarizations in the murine small intestine and colon. However, when thapsigargin in a bath solution was applied, Carthami flos induced membrane depolarizations only in the murine colon. 3. Pretreatment with 2-APB (transient receptor potential melastatin (TRPM) channel inhibitor) abolished the pacemaker potentials and suppressed Carthami flos-induced effects in the murine small intestine and colon. 4. However, pretreatment with T16Ainh-AO1 (Ca²⁺ activated Cl- channel; anoctamin 1 (ANO1) inhibitor) did not affect the pacemaker potentials and induced Carthami flos-induced effects only in the murine small intestine. Conclusions : These results suggest that Carthami flos can modulate the pacemaker activity of ICC and the mechanisms underlying pacemaking in ICC might be different in the small intestine and the colon.

• The Korean Journal of Physiology and Pharmacology
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• 제12권1호
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• pp.25-30
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• 2008

Although many studies show that thromboxane $A_2\;(TXA_2)$ has the action of gastrointestinal (GI) motility using GI muscle cells and tissue, there are no reports on the effects of $TXA_2$ on interstitial cells of Cajal (ICC) that function as pacemaker cells in GI tract. So, we studied the modulation of pacemaker activities by $TXA_2$ in ICC with whole cell patch-clamp technique. Externally applied $TXA_2\;(5{\mu}M)$ produced membrane depolarization in current-clamp mode and increased tonic inward pacemaker currents in voltage-clamp mode. The tonic inward currents by $TXA_2$ were inhibited by intracellular application of GDP-${\beta}$-S. The pretreatment of ICC with $Ca^{2+}$ free solution and thapsigargin, a $Ca^{2+}$-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the $TXA_2$-induced tonic inward currents. However, chelerythrine or calphostin C, protein kinase C inhibitors, did not block the $TXA_2$-induced effects on pacemaker currents. These results suggest that $TXA_2$ can regulate intestinal motility through the modulation of ICC pacemaker activities. This modulation of pacemaker activities by $TXA_2$ may occur by the activation of G protein and PKC independent pathway via extra and intracellular $Ca^{2+}$ modulation.

• The Korean Journal of Physiology and Pharmacology
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• 제12권3호
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• pp.111-115
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• 2008

The effects of (-)-epigallocatechin gallate (EGCG) on pacemaker activities of cultured interstitial cells of Cajal (ICC) from murine small intestine were investigated using whole-cell patch-clamp technique at $30^{\circ}C$ and $Ca^{2+}$ image analysis. ICC generated spontaneous pacemaker currents at a holding potential of -70 mV. The treatment of ICC with EGCG resulted in a dose-dependent decrease in the frequency and amplitude of pacemaker currents. SQ-22536, an adenylate cyclase inhibitor, and ODQ, a guanylate cyclase inhibitor, did not inhibit the effects of EGCG. EGCG-induced effects on pacemaker currents were not inhibited by glibenclamide, an ATP-sensitive $K^+$ channel blocker and TEA, a $Ca^{2+}$-activated $K^+$ channel blocker. Also, we found that EGCG inhibited the spontaneous $[Ca^{2+}]_i$ oscillations in cultured ICC. In conclusion, EGCG inhibited the pacemaker activity of ICC and reduced $[Ca^{2+}]_i$ oscillations by cAMP-, cGMP-, ATP-sensitive $K^+$ channel-independent manner.

• The Korean Journal of Physiology and Pharmacology
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• 제11권1호
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• pp.15-20
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• 2007

To investigate whether hydrogen peroxide($H_2O_2$) affects intestinal motility, pacemaker currents and membrane potential were recorded in cultured interstitial cells of Cajal(ICC) from murine small intestine by using a whole-cell patch clamp. In whole cell patch technique at $30^{\circ}C$, ICC generated spontaneous pacemaker potential under current clamp mode(I=0) and inward currents(pacemaker currents) under voltage clamp mode at a holding potential of -70 mV. When ICC were treated with $H_2O_2$ in ICC, $H_2O_2$ hyperpolarized the membrane potential under currents clamp mode and decreased both the frequency and amplitude of pacemaker currents and increased the resting currents in outward direction under voltage clamp mode. Also, $H_2O_2$ inhibited the pacemaker currents in a dose-dependent manner. Because the properties of $H_2O_2$ action on pacemaker currents were same as the effects of pinacidil(ATP-sensitive $K^+$ channels opener), we tested the effects of glibenclamide(ATP-sensitive $K^+$ channels blocker) on $H_2O_2$ action in ICC, and found that the effects of $H_2O_2$ on pacemaker currents were blocked by co- or pre- treatment of glibenclamide. These results suggest that $H_2O_2$ inhibits pacemaker currents of ICC by activating ATP-sensitive $K^+$ channels.

• 대한한의학방제학회지
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• 제28권1호
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• pp.71-80
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• 2020

Obejectives : The purpose of this study was to investigate the effects of Yijin-tang on pacemaker potentials of small intestinal interstitial Cells of Cajal (ICC). Methods : To dissociate the ICC, we used enzymatic digestions from the small intestine in mice. The electrophysiological whole-cell patch-clamp configuration was used to record pacemaker potentials in the cultured ICC and the in vivo effects of Yijin-tang on GI motility were investigated by calculating percent intestinal transit rates (ITR). Results : 1. The ICC generated pacemaker potentials in the murine small intestine. Yijin-tang produced membrane depolarization with concentration-dependent manners in the current clamp mode. 2. Pretreatment with a Ca²⁺ free solution and thapsigargin, a Ca²⁺-ATPase inhibitor in the endoplasmic reticulum, stopped the pacemaker potentials. In the case of Ca²⁺-free solutions and thapsigargin, Yijin-tang did not induce membrane potential depolarizations. 3. U73122, a phospholipase C (PLC) inhibitors, blocked the Yijin-tang-induced membrane potential depolarizations. However, U73343, an inactive PLC inhibitors, did not block. 4. In the presence of protein kinase C (PKC) inhibitors, staurosporine or Rottlerin, Yijin-tang depolarized the pacemaker potentials. However, in the presence of Go6976, Yijin-tang did not depolarize the pacemaker potentials. 5. In mice, intestinal transit rate (ITR) values were significantly and dose-dependently increased by the intragastric administration of Yijin-tang. Conclusions : These results suggest that Yijin-tang can modulate the pacemaker activity of ICC through an internal/external Ca²⁺ and PLC/PKC-dependent pathway in ICC. In addition, Yijin-tang is a good candidate for the development of a prokinetic agent.

• 동의생리병리학회지
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• 제27권1호
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• pp.112-117
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• 2013

We studied the modulation of pacemaker activities by Samchulkunbi-tang (SCKB) in cultured interstitial cells of Cajal (ICC) from murine small intestine with the whole-cell patch-clamp technique. Externally applied SCKB produced membrane depolarization in the current-clamp mode. The pretreatment with $Ca^{2+}$-free solution and thapsigargin, a $Ca^{2+}$-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker potentials and suppressed the SCKB-induced action. The application of flufenamic acid (a nonselective cation channel blocker) abolished the generation of pacemaker potentials by SCKB. However, the application of niflumic acid (a chloride channel blocker) did not inhibit the generation of pacemaker potentials by SCKB. In addition, the membrane depolarizations were inhibited by not only GDP-${\beta}$-S, which permanently binds G-binding proteins, but also U-73122, an active phospholipase C inhibitor. These results suggest that SCKB modulates the pacemaker activities by nonselective cation channels and external $Ca^{2+}$ influx and internal $Ca^{2+}$ release via G-protein and phospholipase C-dependent mechanism. Therefore, the ICC are targets for SCKB and their interaction can affect intestinal motility.

• The Korean Journal of Physiology and Pharmacology
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• 제14권5호
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• pp.317-324
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• 2010

We elucidated the distribution of interstitial cells of Cajal (ICC) in human stomach, using cryosection and $c-Kit$ immunohistochemistry to identify $c-Kit$ positive ICC. Before $c-Kit$ staining, we routinely used hematoxylin and eosin (HE) staining to identify every structure of human stomach, from mucosa to longitudinal muscle. HE staining revealed that the fundus greater curvature (GC) had prominent oblique muscle layer, and $c-Kit$ immunostaining $c-Kit$ positive ICC cells were found to have typical morphology of dense fusiform cell body with multiple processes protruding from the central cell body. In particular, we could observe dense processes and ramifications of ICC in myenteric area and longitudinal muscle layer of corpus GC. Interestingly, $c-Kit$ positive ICC-like cells which had morphology very similar to ICC were found in gastric mucosa. We could not find any significant difference in the distribution of ICC between fundus and corpus, except for submucosa where the density of ICC was much higher in gastric fundus than corpus. Furthermore, there was no significant difference in the density of ICC between each area of fundus and corpus, except for muscularis mucosa. Finally, we also found similar distribution of ICC in normal and cancerous tissue obtained from a patient who underwent pancreotomy and gastrectomy. In conclusion, ICC was found ubiquitously in human stomach and the density of ICC was significantly lower in the muscularis mucosa of both fundus/corpus and higher in the submucosa of gastric fundus than corpus.