Physiological role of NO produced by constitutive NO synthase in pancreatic islet of Langerhans

朗格汉斯胰岛组成型NO合酶产生NO的生理作用

• 批准号: 13670094
• 负责人: ISHIKAWA Tomohisa
• 金额: $ 2.24万
• 依托单位: University of Shizuoka
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670094/
• 关键词: islet of Langerhans; β-cells; rat; NO; NO synthase; Ca^<2+>; glucose; DAF-2; α細胞; cGMP

METHODS: Pancreatic islets of Langerhans and β cells were isolated from male Wistar rats (9-12 weeks old) by collagenase digestion technique. (1) Double immunostaining with antiserum to NOS1 or NOS3 and that to glucagon, insulin, somatostatin, or pancreatic polypeptide was performed in the isolated islets, and the fluorescent images were analyzed in a confocal laser scanning microscope. (2) The production of NO was measured with the fluorescent NO indicator DAF-2 DA in a confocal laser scanning microscope. (3) [Ca^<2+>]i was measured in fura-PE3-loaded β cells by Argus-50/CA system. (4) The secretion of insulin from isolated islets was measured by the batch incubation method and EIA. (5) ATP-sensitive K^+ currents were measured by the patch clamp method.RESULTS AND DISCUSSION: (1) Double immunostaining showed that both NOS1- and NOS3-immunoreactivity are present in rat β celles. (2) Glucose produced NO in the β cells in a concentration-dependent manner. (3) In the presence of L-NNA, the NO donor NOC7 at 0.5 and 1.0 μM increased the amplitude of [Ca^<2+>]i oscillations induced by 11.1 mM glucose, and at 10 μM terminated them. A soluble guanylyl cyclase inhibitor suppressed the stimulatory action of NOC7 at low concentrations, whereas it did not affect the inhibitory one at high concentrations, suggesting that the former is mediated by cGMP but the latter is not. (4) Insulin secretion induced by 11.1 mM glucose was facilitated by 0.5 μM NOC7, whereas it was suppressed by 10 μM NOC7.CONCLUSION: NO is an endogenous regulator of insulin secretion, which facilitates and inhibits glucose-induced insulin secretion at low and high concentrations, respectively

方法：采用胶原酶消化法分离9-12周龄雄性Wistar大鼠胰岛和胰岛β细胞。(1)用抗NOS 1或NOS 3抗体和抗胰高血糖素、胰岛素、生长抑素或胰多肽抗体对分离的胰岛进行双重免疫染色，并在共聚焦激光扫描显微镜下分析荧光图像。(2)在激光共聚焦扫描显微镜下，用NO荧光指示剂<$A-2 DA测定NO的产生。(3)用Argus-50/CA系统测定fura-PE 3负载的β细胞中的[Ca^<2+>]i。(4)用分批培养法和EIA法测定胰岛分泌胰岛素的能力。(5)结果与讨论：（1）免疫双标染色显示大鼠β细胞中存在NOS 1-和NOS 3-免疫反应性。(2)葡萄糖以浓度依赖性方式在β细胞中产生NO。(3)在L-NNA存在下，0.5和1.0 μM的NO供体NOC 7增加11.1 mM葡萄糖诱导的[Ca^<2+>]i振荡幅度，10 μM时终止该振荡。可溶性鸟苷酸环化酶抑制剂在低浓度下抑制NOC 7的刺激作用，而在高浓度下不影响抑制作用，这表明前者是由cGMP介导的，而后者不是。(4)0.5 μM的NOC 7促进11.1mM葡萄糖诱导的胰岛素分泌，而10 μM的NOC 7则抑制胰岛素分泌。结论：NO是胰岛素分泌的内源性调节剂，在低浓度和高浓度时分别促进和抑制葡萄糖诱导的胰岛素分泌

项目成果

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Nakayama 等人：“酪氨酸激酶、蛋白激酶 C 和 Rho/Rho 激酶系统在血管平滑肌机械转导中的相互作用”生物流变学。

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Ishikawa T., Nejishima H., Imamura T., Nakayama K.: "Non-contribution of renin-angiotensin system to pressor response to N^G-nitro-L-arginine in dogs"Fundam Clin Pharmacol. 16. 15-21 (2002)

Ishikawa T.、Nejishima H.、Imamura T.、Nakayama K.：“肾素-血管紧张素系统对狗 N^G-硝基-L-精氨酸的升压反应无贡献”Fundam Clin Pharmacol。