Boivin Benoit, Lavoie Catherine, Vaniotis George, Baragli Alessandra, Villeneuve Louis-Robert, Ethier Nathalie, Trieu Phan, Allen Bruce G, Hébert Terence E
Centre de recherche, Institut de Cardiologie de Montréal, Canada.
Cardiovasc Res. 2006 Jul 1;71(1):69-78. doi: 10.1016/j.cardiores.2006.03.015. Epub 2006 Mar 24.
We sought to determine if different beta-adrenergic receptor (betaAR) subtypes, and their associated signalling machinery, are functionally localized to nuclear membranes.
Employing enriched nuclear preparations, we assayed the specific presence of betaAR by measuring 125I-cyanopindolol (CYP) binding, Western blotting, confocal microscopy and functional assays.
Western blots of rat heart nuclear fractions and confocal immunofluorescent analysis of adult rat and mouse ventricular cardiomyocytes displayed the presence of beta 1AR and beta 3AR but, surprisingly, not the beta 2AR on nuclear membranes. Nuclear localization of downstream signalling partners Gs, Gi and adenylyl cyclases II and V/VI was also demonstrated. The functional relevance of nuclear betaAR was shown by receptor-mediated stimulation of adenylyl cyclase activity by isoproterenol but not the beta 3AR-selective agonist CL 316243 in enriched nuclear preparations. We also examined the effect of subtype-selective ligands on the initiation of RNA synthesis in isolated nuclei. Both isoproterenol and another beta 3AR-selective agonist, BRL 37344, increased RNA synthesis which was inhibited by pertussis toxin (PTX). Neither a beta 1AR-selective agonist, xamoterol, nor a beta 2AR-selective agonist, procaterol, was able to stimulate transcription. However, both CGP 20712A and ICI 118,551 blocked isoproterenol-mediated effects to varying extents. PTX treatment also revealed that nuclear betaAR may be coupled to other signalling pathways in addition to Gi, as stimulation under these conditions reduced initiation of transcription below basal levels.
These results highlight differential subcellular localization for betaAR subtypes and indicate that betaAR may have specific roles in regulating nuclear function in cardiomyocytes.
我们试图确定不同的β-肾上腺素能受体（βAR）亚型及其相关信号传导机制是否在功能上定位于核膜。
利用富集的细胞核制剂，我们通过测量125I-氰吲哚洛尔（CYP）结合、蛋白质印迹法、共聚焦显微镜和功能测定来检测βAR的特异性存在。
大鼠心脏细胞核组分的蛋白质印迹以及成年大鼠和小鼠心室心肌细胞的共聚焦免疫荧光分析显示核膜上存在β1AR和β3AR，但令人惊讶的是，不存在β2AR。还证实了下游信号传导伙伴Gs、Gi以及腺苷酸环化酶II和V/VI的核定位。在富集的细胞核制剂中，异丙肾上腺素可介导受体刺激腺苷酸环化酶活性，而β3AR选择性激动剂CL 316243则不能，这表明了核βAR的功能相关性。我们还研究了亚型选择性配体对分离细胞核中RNA合成起始的影响。异丙肾上腺素和另一种β3AR选择性激动剂BRL 37344均增加了RNA合成，而百日咳毒素（PTX）可抑制这种增加。β1AR选择性激动剂扎莫特罗和β2AR选择性激动剂丙卡特罗均不能刺激转录。然而，CGP 20712A和ICI 118,551均不同程度地阻断了异丙肾上腺素介导的效应。PTX处理还表明，核βAR除了与Gi偶联外，可能还与其他信号传导途径偶联，因为在这些条件下的刺激会使转录起始低于基础水平。
这些结果突出了βAR亚型在亚细胞定位上的差异，并表明βAR可能在调节心肌细胞核功能中具有特定作用。
