Stimulation of amylase release by Orexin is mediated by Orexin 2 receptor in AR42J cells
Stimulation of amylase release by Orexin is mediated by Orexin 2 receptor in AR42J cells
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DOI：
10.1097/00006676-200211000-00014
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发表时间：
2002-11-01
期刊：
影响因子：
2.9
通讯作者：
Wu, SV
中科院分区：
文献类型：
--
作者：
Harris, DM;Go, VLW;Wu, SV
介绍和目标：食欲素已被证明主要具有中枢生理功能，包括调节食物和水的摄入、睡眠和唤醒。然而，很少有人知道他们的直接周边影响，如果有的话。作为理解食欲素在非神经元组织或细胞中的作用的第一步，我们开始研究检查食欲素受体（OXR）在已建立的胰腺肿瘤细胞系AR 42 J中的表达。其次，我们想确定各种分子形式的食欲素是否有活性刺激AR 42 J细胞中任何已知的胰腺细胞功能。方法：进行逆转录-PCR分析以鉴定特异性食欲素受体亚型的存在。在用食欲素A和B肽、它们各自的C-末端十肽片段和下丘脑泌素-2-gly（甘氨酸延伸的食欲素B）刺激后测量细胞内钙动员和cAMP水平。在用该组食欲素肽急性刺激30分钟后，在条件培养基中测量α-淀粉酶的释放。在无血清条件下用食欲素处理24小时后，通过H-3-胸苷掺入来测定细胞增殖。结果：RT-PCR和测序结果显示，AR 42 J细胞主要表达Orexin受体亚型2（OX 2 R）。食欲素刺激细胞内钙动员的剂量依赖性增加，食欲素A的EC 50为0.05 nM，食欲素B为0.1 nM，但即使在微摩尔浓度下也不能刺激任何显著的cAMP积累或DNA合成。食欲素-A和-B，但不是下丘脑泌素-2-甘氨酸，也刺激剂量依赖性增加淀粉酶释放的AR 42 J细胞。食欲素-A和-B羧基末端十肽引起了显着的，但低得多的钙和淀粉酶的反应。结论：我们的数据表明，OX 2 R介导的Ca 2+依赖性淀粉酶的释放在AR 42 J细胞，这表明Orexins可能在胰腺肿瘤细胞中具有分泌功能。
Introduction and Aims: Orexins have been demonstrated to have mainly central physiological functions, including regulation of food and water intake, sleep, and arousal. However, little is known about their direct peripheral effects, if any. As a first step toward understanding the role of Orexin in non-neuronal tissues or cells, we initiated studies to examine expression of Orexin receptors (OXR) in an established pancreatic tumor cell line AR42J. Secondly, we wanted to determine whether Orexins, in various molecular forms, are active to stimulate any known pancreatic cell functions in AR42J cells. Methodology: Reverse transcription-PCR analysis was performed to identify the presence of specific Orexin receptor subtypes. Intracellular calcium mobilization and cAMP levels were measured following stimulation by Orexin A and B peptides, their respective C-terminal decapeptide fragments, and hypocretin-2-gly (glycine-extended Orexin B). Release of alpha-amylase was measured in conditioned media after acute stimulation with the set of Orexin peptides for 30 minutes. Cell proliferation was determined by H-3-thymidine incorporation after 24 hours following treatment with Orexins under serum-free condition. Results: RT-PCR and sequencing results showed that Orexin receptor subtype 2 (OX2R) was the main form expressed in AR42J cells. Orexins stimulated dose-dependent increases in intracellular calcium mobilization with EC50 0.05 nM for Orexin A and 0.1 nM for Orexin B but were unable to stimulate any significant cAMP accumulation or DNA synthesis even at micromolar concentrations. Both Orexin-A and -B, but not hypocretin-2-gly, also stimulated dose-dependent increases in amylase release in the AR42J cells. Orexin-A and -B carboxyl-terminal decapeptides elicited significant but much lower calcium and amylase responses. Conclusion: Our data demonstrate that OX2R mediates Ca2+-dependent amylase release in AR42J cells, suggesting that Orexins may have secretory functions in pancreatic tumor cells.