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Identification of novel gastrointestinal hormones using orphan G protein-coupled receptors

使用孤儿 G 蛋白偶联受体鉴定新型胃肠激素

基本信息

批准号：
13470113
负责人：
TATEMOTO Kazuhiko
金额：
$ 7.23万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

The principal aim of this proposal is to study the identification and physiological characterization of bombesin receptor subtype-3 (BRS-3) ligand and to study physiological and biochemical characterization of apelin, a novel gastrointestinal hormone isolated from the stomach.STUDY 1: Bombesin receptor subtype-3 (BRS-3) is an orphan G protein-coupled receptor that is activated by a receptor agonist, [D-Phe^6, β-Ala^<11>, Phe^<13>, Nle^<14>]Bn(6-14). We found that this agonist stimulated the laminin-mediated adhesion of a human small cell lung cancer cell line, NCI-N417, which expresses native BRS-3. The enhancement of adhesion was accompanied by an increase in vinculin-like immunoreactivity, and diminished in the presence of an anti-Pj integrin antibody, suggesting that the receptor activation stimulates focal adhesion formation. We also suggest that mobilization of intracellular calcium is involved in the mechanism of BRS-3-mediated adhesion of NCI-N417 cells.STUDY 2: Apelin, a novel … More 36-amino acid peptide, has been identified to be the endogenous ligand for the APJ receptor. We have examined the presence of apelin mRNA in isolated mouse adipocytes and mouse 3T3-L1 cells, and investigated the effects of various hormones on apelin expression in 3T3-L1 adipocytes. We found that both apelin mRNA and APJ mRNA were expressed in isolated mouse adipocytes, while apelin mRNA, but not APJ mRNA, was detected in 3T3-L1 adipocytes. Furthermore, we found that the level of apelin mRNA increased to 3.9-fold during the differentiation of 3T3-L1 cells to adipocytes. Administration of insulin (1 nM-100 nM) increased 2-to 3-fold the apelin mRNA levels in 3T3-L1 adipocytes, while dexamethasone, (0.1 nM-100 nM) decreased the apelin mRNA levels in a dose-dependent manner. In 3T3-L1 adipocytes, dexamethasone reversed insulin-induced increase in apelin mRNA expression, and insulin reversed dexamethasone-induced decrease in the expression. Thus, insulin and glucocorticoids may act as positive and negative regulators of apelin gene expression in 3T3-L1 adipocytes.STUDY 3: Apelin, the endogenous peptide ligand of the APJ receptor, is expressed in brain regions implicated in food and water intake. This study investigated for the first time, the effect of apelin-12 oh spontaneous (nocturnal) feeding. Rats received 1, 3, and 10 nmol apelin-12 or saline vehicle in random order by intracerebroventricular injection 10 minutes prior to lights out. Acute effects were observed, with dose-dependent reductions in food intake 2-4 hours after injection. Thus, apelin-12 exerted a delayed inhibitory effect on nocturnal feeding.STUDY 4: Apelin is an endogenous ligand of the human orphan receptor APJ. We detected apelin-like immunoreactivity localized within the endothelia of Small arteries in various organs, adipocytes, gastric mucosa, and Kupffer cells in the liver. We also found that apelin lowers blood pressure. Mean arterial pressure after intravenous administration of apelin-12, apelin-13, and apelin-36 at a dose of 10 nmol/kg in anesthetized rats was reduced by 26 ± 5 mmHg, 11 ± 4 mmHg, and 5 ± 4 mmHg, respectively. In the presence of a nitric oxide (NO) synthase inhibitor, the effect of apelin-12 on blood pressure was abolished. Furthermore, the administration of apelin-12 (10 nmol/kg) in rats produced a transitory elevation of the plasma nitrite/nitrate concentration from a basal level of 21.4 ±1.6 mM to 27.0 ±1.5 mM. Thus, apelin may play an important role in the regulation of blood pressure. Less

研究1：蛙皮素受体亚型-3（BRS-3）是一种孤儿G蛋白偶联受体，可被受体激动剂[D-Phe ^6，β-Ala ^，Phe ^，Nle ^] Bn（6 - 14）激活。<11><13><14>我们发现，这种激动剂刺激层粘连蛋白介导的粘附的人小细胞肺癌细胞系，NCI-N417，表达天然BRS-3。粘附的增强伴随着黏着斑蛋白样免疫反应性的增加，并且在抗Pj整联蛋白抗体的存在下减少，这表明受体活化刺激粘着斑形成。我们还表明，细胞内钙的动员参与了BRS-3介导的NCI-N417细胞粘附的机制。 ...更多信息 36-氨基酸肽，已被鉴定为APJ受体的内源性配体。我们检测了在分离的小鼠脂肪细胞和小鼠3T3-L1细胞中apelin mRNA的存在，并研究了各种激素对3T3-L1脂肪细胞中apelin表达的影响。我们发现apelin mRNA和APJ mRNA在分离的小鼠脂肪细胞中表达，而apelin mRNA在3T3-L1脂肪细胞中检测到，但不检测到APJ mRNA。此外，我们发现在3T3-L1细胞向脂肪细胞分化期间，apelin mRNA的水平增加至3.9倍。胰岛素（1 nM-100 nM）给药使3T3-L1脂肪细胞中的apelin mRNA水平增加2 - 3倍，而地塞米松（0.1 nM-100 nM）以剂量依赖性方式降低apelin mRNA水平。在3T3-L1脂肪细胞中，地塞米松逆转胰岛素诱导的apelin mRNA表达增加，胰岛素逆转地塞米松诱导的表达减少。因此，胰岛素和糖皮质激素可以作为3T3-L1脂肪细胞中apelin基因表达的正和负调节剂。研究3：apelin，APJ受体的内源性肽配体，在涉及食物和水摄入的脑区域中表达。这项研究首次调查了apelin-12 OH自发（夜间）喂养的影响。大鼠在熄灯前10分钟通过脑室内注射以随机顺序接受1、3和10 nmol爱帕琳-12或盐水媒介物。观察到急性效应，注射后2 - 4小时摄食量呈剂量依赖性减少。因此，爱帕琳-12对夜间进食产生延迟抑制作用。研究4：爱帕琳是人孤儿受体APJ的内源性配体。我们检测到apelin样免疫反应性定位于各种器官的小动脉内皮、脂肪细胞、胃粘膜和肝脏的枯否细胞内。我们还发现爱帕琳肽可以降低血压。在麻醉大鼠中以10 nmol/kg的剂量静脉内施用爱帕琳-12、爱帕琳-13和爱帕琳-36后，平均动脉压分别降低26 ± 5 mmHg、11 ± 4 mmHg和5 ± 4 mmHg。在一氧化氮（NO）合酶抑制剂的存在下，apelin-12对血压的影响被取消。此外，给予大鼠apelin-12（10 nmol/kg）可使血浆亚硝酸盐/硝酸盐浓度从基础水平21.4 ± 1.6 mM暂时升高至27.0 ± 1.5 mM。因此，apelin可能在血压调节中发挥重要作用。少