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

本课题主要研究bombesin receptor subtype-3 （BRS-3）配体的鉴定和生理特性，以及胃中分离的新型胃肠激素apelin的生理生化特性。研究1:Bombesin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^<11>, Phe^<13>, Nle^<14>]Bn激活（6-14）。我们发现这种激动剂刺激层粘连蛋白介导的人小细胞肺癌细胞系NCI-N417的粘附，该细胞系表达天然BRS-3。黏附的增强伴随着血管素样免疫反应性的增加，并且在抗pj整合素抗体存在时减弱，这表明受体激活刺激了局灶性黏附的形成。我们还认为，细胞内钙的动员参与了brs -3介导的NCI-N417细胞粘附的机制。研究2:Apelin是一种具有36个氨基酸的新型多肽，被认为是APJ受体的内源性配体。我们检测了小鼠离体脂肪细胞和小鼠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。我们发现在小鼠离体脂肪细胞中apelin mRNA和APJ mRNA均有表达，而在3T3-L1脂肪细胞中检测到apelin mRNA，但未检测到APJ mRNA。此外，我们发现在3T3-L1细胞向脂肪细胞分化的过程中，apelin mRNA的水平增加了3.9倍。胰岛素（1 nM-100 nM）使3T3-L1脂肪细胞中apelin mRNA水平升高2- 3倍，而地塞米松（0.1 nM-100 nM）使apelin mRNA水平呈剂量依赖性降低。在3T3-L1脂肪细胞中，地塞米松逆转胰岛素诱导的apelin mRNA表达升高，胰岛素逆转地塞米松诱导的apelin mRNA表达降低。因此，胰岛素和糖皮质激素可能是3T3-L1脂肪细胞中apelin基因表达的正调控因子和负调控因子。研究3:APJ受体的内源性肽配体Apelin在与食物和水摄入有关的大脑区域中表达。本研究首次探讨了apelin-12对自然（夜间）摄食的影响。熄灯前10分钟，大鼠随机接受1、3、10 nmol apelin-12或生理盐水灌胃。观察到急性效应，注射后2-4小时食物摄入量呈剂量依赖性减少。因此，apelin-12对夜间摄食具有延迟抑制作用。研究4:Apelin是人类孤儿受体APJ的内源性配体。我们在不同器官的小动脉内皮、脂肪细胞、胃粘膜和肝脏的库普弗细胞中检测到apelin样免疫反应性。我们还发现apelin可以降低血压。以10 nmol/kg剂量静脉给药apelin-12、apelin-13和apelin-36后，麻醉大鼠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）合成酶抑制剂的存在下，apelin-12对血压的影响被消除。此外，大鼠血浆中apelin-12 （10 nmol/kg）可使亚硝酸盐/硝酸盐浓度从基础水平21.4±1.6 mM短暂升高至27.0±1.5 mM。由此可见，apelin可能在调节血压中起重要作用。少

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