Targeting G protein-coupled receptors (GPCRs) in pancreatic cells is feasible to modulate glucose-induced insulin secretion. Because pancreatic islets consist of several cell types and GPCRs can couple to more than one G-protein family, results obtained in pancreatic cell lines do not always match the response in primary cells or intact islets. Therefore, we set out to establish a protocol to analyze second messenger activation in mouse pancreatic islets. Activation of Gq/11-coupled receptor expressed in primary β cells increased the second messenger IP1 in an accumulation assay. Applying a Gq/11 protein inhibitor completely abolished this signal. Activation of the V1 vasopressin and ghrelin receptors, predominantly expressed in the less abundant alpha and delta cells, was not sufficient to induce a significant IP1 increase in this assay. However, fura-2-based fluorescence imaging showed calcium signals upon application of arginine vasopressin or ghrelin within intact pancreatic islets. Using the here established protocol we were also able to determine changes in intracellular cAMP levels induced by receptors coupling to Gs and Gi/o proteins. Detection of the second messengers IP1, cAMP, and calcium, can be used to reliably analyze GPCR activation in intact islets.

中文翻译：

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探索原发性胰岛中的 G 蛋白偶联受体信号传导。

靶向胰腺细胞中的 G 蛋白偶联受体 (GPCRs) 可以调节葡萄糖诱导的胰岛素分泌。由于胰岛由多种细胞类型组成，并且 GPCR 可以与多个 G 蛋白家族偶联，因此在胰腺细胞系中获得的结果并不总是与原代细胞或完整胰岛中的反应相匹配。因此，我们着手建立一个协议来分析小鼠胰岛中的第二信使激活。在积累测定中，在原代 β 细胞中表达的 Gq/11 偶联受体的激活增加了第二信使 IP1。应用 Gq/11 蛋白抑制剂完全消除了这种信号。V1 加压素和生长素释放肽受体（主要在较少丰富的 alpha 和 delta 细胞中表达）的激活不足以在该测定中诱导 IP1 显着增加。然而，基于 fura-2 的荧光成像显示在完整胰岛内应用精氨酸加压素或生长素释放肽后的钙信号。使用此处建立的协议，我们还能够确定由与 Gs 和 Gi/o 蛋白偶联的受体引起的细胞内 cAMP 水平的变化。第二信使 IP1、cAMP 和钙的检测可用于可靠地分析完整胰岛中的 GPCR 活化。