Neurotensin Modulates Pacemaker Activity in Interstitial Cells of Cajal from the Mouse Small Intestine

이준,전제열,김영대,박찬국,김만우,장인엽,Dong Chuan Zuo,Pawan Kumar Shahi,최석,염철호 한국분자세포생물학회 2012 Molecules and cells Vol.33 No.5

Neurotensin, a tridecapeptide localized in the gut to dis-crete enteroendocrine cells of the small bowel mucosa, is a hormone that plays an important role in gastrointestinal secretion, growth, and motility. Neurotensin has inhibitory and excitatory effects on peristaltic activity and produces contractile and relaxant responses in intestinal smooth muscle. Our objective in this study is to investigate the effects of neurotensin in small intestinal interstitial cells of Cajal (ICC) and elucidate the mechanism. To determine the electrophysiological effects of neurotensin on ICC, whole-cell patch clamp recordings were performed in cultured ICC from the small intestine. Exposure to neurotensin depolarized the membrane of pacemaker cells and produced tonic inward pacemaker currents. Only neurotensin receptor1 was identified when RT-PCR and immunocytochemistry were performed with mRNA isolated from small intestinal ICC and c-Kit positive cells. Neurotensin-induced tonic inward pacemaker currents were blocked by external Na+-free solution and in the presence of flufenamic acid, an inhibitor of non-selective cation channels. Furthermore, neurotensin-induced action is blocked either by treatment with U73122, a phospholipase C inhibitor, or thapsigargin, a Ca2+-ATPase inhibitor in ICC. We found that neurotensin increased spontaneous intracellular Ca2+ oscillations as seen with fluo4/AM recording. These results suggest that neurotensin modulates pacemaker currents via the activation of non-selective cation channels by intracellular Ca2+-release through neurotensin receptor1.

Neurotensin Changes Propulsive Activity into a Segmental Motor Pattern in the Rat Colon

( Hongfei Li ),( Ji-hong Chen ),( Zixian Yang ),( Min Huang ),( Yuanjie Yu ),( Shiyun Tan ),( Hesheng Luo ),( Jan D Huizinga ) 대한소화기기능성질환·운동학회(구 대한소화관운동학회) 2016 Journal of Neurogastroenterology and Motility (JNM Vol.22 No.3

Background/Aims Neurotensin is a gut-brain peptide with both inhibitory and excitatory actions on the colonic musculature; our objective was to understand the implications of this for motor patterns occurring in the intact colon of the rat. Methods The effects of neurotensin with concentrations ranging from 0.1-100 nM were studied in the intact rat colon in vitro, by investigating spatio-temporal maps created from video recordings of colonic motility before and after neurotensin. Results Low concentration of neurotensin (0.1-1 nM) inhibited propagating long distance contractions and rhythmic propagating motor complexes; in its place a slow propagating rhythmic segmental motor pattern developed. The neurotensin receptor 1 antagonist SR- 48692 prevented the development of the segmental motor pattern. Higher concentrations of neurotensin (10 nM and 100 nM) were capable of restoring long distance contraction activity and inhibiting the segmental activity. The slow propagating segmental contraction showed a rhythmic contraction-- relaxation cycle at the slow wave frequency originating from the interstitial cells of Cajal associated with the myenteric plexus pacemaker. High concentrations given without prior additions of low concentrations did not evoke the segmental motor pattern. These actions occurred when neurotensin was given in the bath solution or intraluminally. The segmental motor pattern evoked by neurotensin was inhibited by the neural conduction blocker lidocaine. Conclusions Neurotensin (0.1-1 nM) inhibits the dominant propulsive motor patterns of the colon and a distinct motor pattern of rhythmic slow propagating segmental contractions develops. This motor pattern has the hallmarks of haustral boundary contractions. (J Neurogastroenterol Motil 2016;22:517-528)

한국재래산양 중뇌 중심회색질의 neurotensin 분포에 관한 면역조직화학적 연구

이인세,이흥식,이성준,Lee, In-se,Lee, Heungshik S.,Yi, Seong-joon 대한수의학회 1993 大韓獸醫學會誌 Vol.33 No.3

The midbrain periaqueductal gray is a midline structure that encircles the mesencephalic aqueduct of midbrain and plays an important role in anaglgesia and modulation of nociceptive input to the central nervous system. It has been demonstrated that the periaqueductal gray contains several neuropeptides including neurotensin, which has been postulated antinociceptive effect to the periaqueductal gray. The present study was performed to provide immunohistochemical localization of neurotensin of midbrain periaqueductal gray in the Korean native goat by using immunohistochemical method. Neurotensin-like immunireactive neurons were localized throughout the midbrain periaqueductal gray, although more immunoreactive neurons were present in the middle and caudal parts of periaquductal gray than the rostral part. Dense neurotensin-like immunoreactive neurons were much more numerous in the ventral lateral division of the mid- and caudal periaqueductal grays. Neurotensin-like immunoreactive neurons were much larger and more prominent near the external margin of the gray than in the juxta-aqueductal region. Neurotensin-like immunoreactive fibers were observed as short processes extending from immunoreactive cells and some small immunoreactive puncta and varicose-like fibers were also seen.

흰쥐중심편도체핵에서 Neurotensin 면역양성세포의 다리옆핵으로의 투사

박인식,최정화,박봉수,백선용,김봉선,김재봉,김진정 대한해부학회 1996 Anatomy & Cell Biology Vol.29 No.2

Expression of Neurotensin/Neuromedin N Precursor in Murine Mast Cells

Hyun Jong Ahn,Jeong Je Cho 대한생리학회-대한약리학회 2001 The Korean Journal of Physiology & Pharmacology Vol.5 No.6

<P> We have cloned the mouse neurotensin/neuromedin N (NT/N) gene from the murine mast cell line Cl.MC/C57.1 for the first time. The murine NT/N cDNA clone consisted of 765 nucleotides and coded for 169 peptide residues with an N-terminal signal peptide, and the C-terminal region contained of one copy of neurotensin (NT) and one copy of neuromedin N (NN). Total of four Lys-Arg dibasic motifs were present; one each at the middle of the open reading frame, at the N-terminal of NN, at the C-terminal of NT, and between NN and NT. Amino acid sequence analysis of the mouse NT/N revealed 90% homology to that of the rat NT/N gene. NT/N is expressed in murine mast cell lines (Cl.MC/C57.1 and P815), but not in murine bone marrow-derived mast cells (BMMCs), murine macrophage cell line (RAW 264.7), nor in murine T cell line (EL-4). NT/N mRNA in C1.MC/C57.1 is highly inducible by IgE cross-linking, phorbol myristate acetate, neurotensin, and substance P. Following the treatment of demethylating agent, 5-azacytidine (5-azaC), the NT/N gene was induced in BMMCs in response to IgE cross-linking. 5-azaC-treated BMMCs did not express the NT/N gene without additional stimuli. These findings suggested that the regulation of NT/N gene expression was dependent on the effects of not only gene methylation but also enhancer and/or repressor proteins acting on the NT/N promoter.

흰쥐 결장운동에서 Neurotensin의 수축작용과 자율신경계의 영향

지정훈 ( Jeong Hoon Ji ),김성무 ( Sung Moo Kim ),서의근 ( Euikeun Seo ),조영심 ( Young Shim Cho ),유숙희 ( Suk Hee Yoo ),한정호 ( Joung Ho Han ),채희복 ( Hee Bok Chae ),박선미 ( Seon Mee Park ),윤세진 ( Sei Jin Youn ) 대한장연구학회 2010 Intestinal Research Vol.8 No.2

Background/Aims: Although neurotensin (NT) stimulates colon motility and the passage of intestinal contents, the associated mechanism of action remains unclear. The objective of this study was to investigate the effects of NT on colon motility using isolated rat colon. Methods: Intraluminal pressure was measured at both the proximal and distal portions of the isolated colon. An isolated rat colon was perfused with Krebs solution via the superior mesenteric artery. After stabilization, NT was administered in concentrations of 14, 28, 138 and 276 pM. After pretreatment with phentolamine, propranolol, hexamethonium, atropine or tetrodotoxin, NT was administered at a concentration of 276 pM, and then the intraluminal pressure was monitored. Results: NT significantly increased colon motility at concentrations of 14, 28, 138, and 276 in the proximal colon (25.1±6.5%, 175.4±117.0%, 240.8±115.1% and 252.3±110.6%, respectively) and in the distal colon (35.6±11.8%, 97.5±35.1%, 132.7±36.7% and 212.1±75.2%, respectively). The stimulant effect of NT was more potent in the proximal colon, in a concentration-dependent manner (P<0.05). The stimulant effect of NT was significantly inhibited by atropine at both the proximal and distal colon and by tetrodotoxin at the proximal colon, but not by tetrodotoxin at the distal colon and not by propranolol, phentolamine, or hexamethonium at both the proximal and distal colon. Conclusions: NT increased colon motility at both the proximal and distal portions of the rat colon. The effects were more prominent at the proximal portion. The results of this study suggest that the stimulant action of NT may be mediated by local cholinergic muscarinic receptors. (Intest Res 2010;8:162-171)

정신분열병과 Neurotensin 수용체 유전자 다형성의 연합 연구

이유상,김형배,한진희,채영규,이정식,이혜순,주연호,김형섭,최인근,양병환 大韓神經精神醫學會 1999 신경정신의학 Vol.38 No.6

연구목적: Neurotensin(NT)은 NT수용체와 결합하여 그 효과를 나타내는 neuromodulator 혹은 neurotransmitter로서 대뇌에서 도파민의 분비를 조절하는데 중요한 역할을 한다. 근래의 연구에 의하면 NT와 그 수용체는 대뇌에서 항정신병 약물의 효과를 매개하는 것으로 생각되고 있으며 약물치료를 받지 않은 정신분열병 환자의 뇌척수액에서 NT의 양이 적으로 보고되고 있어 이들은 정신분열병과 깊은 관련을 가지고 있을 것으로 추정된다. 최근 NT수용체의 유전자의 3`인접영역에서 정보가치가 높은 4 염기반복 다형성이 발견되어 이를 유전 표지자로 하여 정신분열병과의 연합을 알아보았다. 방 법: 서로 혈연관계에 있지 않은 정신분열병 환자 120명(남자 91명, 여자 29명)과 정상 대조군 106명(남자 84명, 여자 22명)을 대상으로 하였다. PANSS를 사용하여 양성 및 음성을 알아보았다. 말초혈액에서 DNA를 분리한 후에 중합효소연쇄반응을 사용하여 3`인접영역에 있는 4 염기 반복 다형성을 증폭하였고 silver staining한 후에 유전자형을 관찰하였다. chi-square 검증과 Bonferroni`s correction을 사용하여 환자군과 정상 대조군간의 대립유전자 빈도의 차이를 알아보았다. 또한 양성 및 음성 환자군으로 나누어 차이를 알아보았다. 결 과: 총 23개의 대립유전자가 관찰되었으며, 399bp의 대립유전자(A10)의 빈도가 환자군보다 정상대조군에서 통계적으로 유의하게 높았다(χ²=16.49, df=1, p<0.001). 음성 정신분열병 환자군과 정상대조군 사이의 비교에서는 정상대조군의 A10의 빈도가 환자군보다 유의하게 높았다(χ²=21.33, df=1, p<0.001). 성별 비교에서 남자 정신분열병 환자군은 대조군에 비하여 A10의 분포가 유의하게 적었다. (χ²=13.71, df=1, p<0.001) 결 론: NT 수용체 유전자와 정신분열병사이에 음성연합이 관찰되었다. NT 수용체 유전자가 일부 정신분열병의 발병과정에서 확실하지는 않지만 어떤 종류의 보호기능을 할 수도 있다는 것을 암시한다. Objectives: Neurotensin(NT), of which functions are evoked by its interaction with neurotensin receptors(NTR), coexists with mesolimbic dopamine and regulates endogenous dopamine release. Recent studies have shown that NT with NTR exerts neuroleptic-like activity within the central nervous system and may play an important role in the pathogenesis and in the treatment of schizophrenia. We have examined the gentic association between schizophrenia and tetranucleotide repeat polymorphism in the 3-flanking region of the NTR gene to investigate the possible contribution of the NTR gene to the schizophrenia susceptibility. Methods: Among 23 alleles identified, the subjects were 120 patients(male 91, female 29)with schizophrenia and 106 normal healthy controls(male 84, female 22). They were unrelated native Korean. PANSS was used to determine positive or negative subgroup in the schizophrenic patients. Using polymerase chain reaction and polyacrylamide gel electrophoresis, tetranucleotide repeat polymorphism(CCTT and CTT) in the 3`-flanking region of NTR gene was observed. For a comparison of NTR gene`s allelic frequencies between patients with schizophrenia and normal healthy controls, chi-square test and Bonferroni`s correction was performed. Results: The frequency of A10 allele(base pair size=399)was significantly higher in normal healthy controls than schizophrenia(χ²=16.4902, df=1, p<.000). In the comparison between schizophrenic patients with negative symptoms and normal controls, the frequency of A10 allele was significantly higher in normal healthy control subjects than patients with schizophrenia(χ²=21.33, df=1, p<0.001). In the case of male, the frequency of A10 allele of schizophrenia was significantly higher than normal controls(χ²=13.71, df=1, p<0.001). Conclusions: NTR gene was negatively associated with schizophrenia. NTR gene`s tetranucleotide repeat polymorphism may provide some protective function against schizophrenia.

흰쥐 중뇌중심회색질내에서의 substance P, neurotensin 및 vasoactive intestinalpolypeptide 함유신경세포 및 축삭종말의 분포

손은익,조희중,홍해숙,주강 대한해부학회 1995 Anatomy & Cell Biology Vol.28 No.1

Validation of Neurotensin Receptor 1 as a Therapeutic Target for Gastric Cancer

Hafeza Akter,윤정환,유영숙,강민정 한국분자세포생물학회 2018 Molecules and cells Vol.41 No.6

Gastric cancer is the fifth most common type of malignancy worldwide, and the survival rate of patients with advanced-stage gastric cancer is low, even after receiving chemotherapy. Here, we validated neurotensin receptor 1 (NTSR1) as a potential therapeutic target in gastric cancer. We compared NTSR1 expression levels in sixty different gastric cancer-tissue samples and cells, as well as in other cancer cells (lung, breast, pancreatic, and colon), by assessing NTSR1 expression via semi-quantitative real-time reverse transcription polymerase chain reaction, immunocytochemistry and western blot. Following neurotensin (NT) treatment, we analyzed the expression and activity of matrix metalloproteinase-9 (MMP-9) and further determined the effects on cell migra-tion and invasion via wound-healing and transwell assays. Our results revealed that NTSR1 mRNA levels were higher in gastric cancer tissues than non-cancerous tissues. Both of NTSR1 mRNA levels and expression were higher in gastric cancer cell lines relative to levels observed in other cancer-cell lines. Moreover, NT treatment induced MMP-9 expression and activity in all cancer cell lines, which was significantly decreased following treatment with the NTSR1 antagonist SR48692 or small-interfering RNA targeting NTSR1. Furthermore, NT-mediated metastases was confirmed by observing epithelial-mesenchymal transition markers SNAIL and E-cadherin in gastric cancer cells. NT-mediated invasion and migration of gastric cancer cells were reduced by NTSR1 depletion through the Erk signaling. These findings strongly suggested that NTR1 constitutes a potential therapeutic target for the inhibition of gastric cancer inva-sion and metastasis.

Validation of Neurotensin Receptor 1 as a Therapeutic Target for Gastric Cancer

Akter, Hafeza,Yoon, Jung Hwan,Yoo, Young Sook,Kang, Min-Jung Korean Society for Molecular and Cellular Biology 2018 Molecules and cells Vol.41 No.6

Gastric cancer is the fifth most common type of malignancy worldwide, and the survival rate of patients with advanced-stage gastric cancer is low, even after receiving chemotherapy. Here, we validated neurotensin receptor 1 (NTSR1) as a potential therapeutic target in gastric cancer. We compared NTSR1 expression levels in sixty different gastric cancer-tissue samples and cells, as well as in other cancer cells (lung, breast, pancreatic, and colon), by assessing NTSR1 expression via semi-quantitative real-time reverse transcription polymerase chain reaction, immunocytochemistry and western blot. Following neurotensin (NT) treatment, we analyzed the expression and activity of matrix metalloproteinase-9 (MMP-9) and further determined the effects on cell migration and invasion via wound-healing and transwell assays. Our results revealed that NTSR1 mRNA levels were higher in gastric cancer tissues than non-cancerous tissues. Both of NTSR1 mRNA levels and expression were higher in gastric cancer cell lines relative to levels observed in other cancer-cell lines. Moreover, NT treatment induced MMP-9 expression and activity in all cancer cell lines, which was significantly decreased following treatment with the NTSR1 antagonist SR48692 or small-interfering RNA targeting NTSR1. Furthermore, NT-mediated metastases was confirmed by observing epithelial-mesenchymal transition markers SNAIL and E-cadherin in gastric cancer cells. NT-mediated invasion and migration of gastric cancer cells were reduced by NTSR1 depletion through the Erk signaling. These findings strongly suggested that NTR1 constitutes a potential therapeutic target for the inhibition of gastric cancer invasion and metastasis.