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通过实时聚合酶链反应在大鼠视交叉上核中鉴定PAC1受体亚型mRNA

Identification of PAC1 receptor isoform mRNAs by real-time PCR in rat suprachiasmatic nucleus.

作者信息

Ajpru Supaporn, McArthur Angela J, Piggins Hugh D, Sugden David

机构信息

Endocrinology and Reproduction Research Group, GKT School of Biomedical Sciences, Kings College London, London SE1 1UL, UK.

出版信息

Brain Res Mol Brain Res. 2002 Sep 30;105(1-2):29-37. doi: 10.1016/s0169-328x(02)00387-x.

Abstract

The pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in the photic resetting of the rodent circadian clock in the suprachiasmatic nucleus (SCN). PACAP can exert its effects via VPAC1, VPAC2 and PAC1 G-protein coupled receptors. PAC1 and VPAC2, but not VPAC1, mRNA is expressed in rat SCN. A variety of PAC1 receptor splice variants have been described showing differences in ligand binding affinity and selectivity, G-protein coupling and ability to activate signal transduction pathways. The present experiments used PCR with isoform specific primers to determine which PAC1 variants are expressed in rat SCN. The PAC1(null) isoform and a variant containing a single 28-amino acid insert in the third intracellular (IC3) loop (hop1/2) were detected. No other IC3 variants (hip, hip-hop), N-terminal variants (PAC1(short), PAC1(very short) and PAC1(3a)) or the variant differing in transmembrane II and IV (PAC1TM4) were detected in SCN obtained at any time of day. A quantitative real-time PCR assay was established which measured combined expression of the PAC1(null/hop) variants in rat SCN during a 12:12-h light:dark (L:D) cycle. There was no significant variation of PAC1 mRNA expression (PAC1(null)+PAC1(hop)) with time of day. Nor was there a significant difference in the proportion of these two variants with time of day. These results indicate that the phase-dependency of the actions of PACAP on SCN firing and circadian behaviour are not mediated by changes in the level of expression of PAC1 receptor mRNA, nor by phase-dependent expression of PAC1 receptor variants with altered ligand binding, G-protein coupling or signalling characteristics.

摘要

垂体腺苷酸环化酶激活多肽(PACAP)与啮齿动物视交叉上核(SCN)生物钟的光重置有关。PACAP可通过VPAC1、VPAC2和PAC1 G蛋白偶联受体发挥作用。PAC1和VPAC2的mRNA在大鼠SCN中表达,但VPAC1不表达。已描述了多种PAC1受体剪接变体,它们在配体结合亲和力和选择性、G蛋白偶联以及激活信号转导途径的能力方面存在差异。本实验使用针对异构体特异性引物的PCR来确定哪些PAC1变体在大鼠SCN中表达。检测到PAC1(null)异构体和在第三个细胞内环(IC3)中含有单个28个氨基酸插入片段的变体(hop1/2)。在一天中任何时间获得的SCN中均未检测到其他IC3变体(hip、hip-hop)、N端变体(PAC1(short)、PAC1(very short)和PAC1(3a))或在跨膜II和IV中不同的变体(PAC1TM4)。建立了一种定量实时PCR测定法,用于测量大鼠SCN在12:12小时光照:黑暗(L:D)周期中PAC1(null/hop)变体的联合表达。PAC1 mRNA表达(PAC1(null)+PAC1(hop))在一天中没有显著变化。这两种变体的比例在一天中也没有显著差异。这些结果表明,PACAP对SCN放电和昼夜节律行为作用的相位依赖性不是由PAC1受体mRNA表达水平的变化介导的,也不是由具有改变的配体结合、G蛋白偶联或信号特征的PAC1受体变体的相位依赖性表达介导的。

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