Lin Zhanyi, Chen Yanfang, Zhang Wenfeng, Chen Alex F, Lin Shuguang, Morris Mariana
Department of Pharmacology & Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45450, USA.
Exp Physiol. 2008 May;93(5):676-84. doi: 10.1113/expphysiol.2007.041657. Epub 2008 Feb 29.
Angiotensin (Ang) AT1 receptors and Ang-converting enzymes (ACE and ACE2) are expressed in the dorsal vagal complex (DVC) of the brainstem. The aim of this study was to examine in vivo interactions between brainstem Ang AT1 receptors, ACE and ACE2 using small, hairpin RNA (shRNA) gene-silencing methods. The study takes advantage of the bilateral brainstem expression of renin-angiotensin system (RAS) markers. Adenovirus vectors (Ad, 2.0 x 10(9) c.f.u. ml(-1), 200 nl) carrying interference small hairpin RNA (shRNA) for either AngAT1a (Ad-AT1a-shRNA) or AngAT1b (Ad-AT1b-shRNA) were microinjected into the right side of the brainstem DVC. The Ad-LacZ control was injected into the left side. Brainstems were processed with in situ hybridization and immunochemistry. Results showed that: (1) Ad-AT1a-shRNA downregulated Ang AT1a mRNA by 61.2 +/- 6.8% (P < 0.01) and Ad-AT1b-shRNA downregulated Ang AT1b mRNA by 51.6 +/- 5.2% (P < 0.01); (2) downregulation of Ang AT1a mRNA was associated with decreased ACE2 mRNA expression (decrease of 29.0 +/- 14.5%, P < 0.01), while reduction in Ang Ad-AT1b mRNA had no effect; (3) ACE mRNA expression was not altered by either RNA interference (RNAi) treatment; and (4) immunochemical staining for Ang AT1 receptors, ACE and ACE2 were in agreement with the mRNA changes observed. These results demonstrate the utility of in vivo gene silencing to examine functional specificity. Both Ad-AT1a-shRNA and Ad-AT1b-shRNA induced site- and subtype-specific downregulation of receptor expression. Gene silencing showed that there were interactions between brainstem Ang AT1a receptors and the RAS regulatory enzyme, ACE2.
血管紧张素（Ang）AT1受体和血管紧张素转换酶（ACE和ACE2）在脑干的迷走神经背核复合体（DVC）中表达。本研究的目的是使用小发夹RNA（shRNA）基因沉默方法，在体内研究脑干Ang AT1受体、ACE和ACE2之间的相互作用。该研究利用了肾素-血管紧张素系统（RAS）标志物在脑干两侧的表达。将携带针对AngAT1a（Ad-AT1a-shRNA）或AngAT1b（Ad-AT1b-shRNA）的干扰小发夹RNA（shRNA）的腺病毒载体（Ad，2.0×10⁹ c.f.u. ml⁻¹，200 nl）微量注射到脑干DVC的右侧。将Ad-LacZ对照注射到左侧。对脑干进行原位杂交和免疫化学处理。结果显示：（1）Ad-AT1a-shRNA使Ang AT1a mRNA下调61.2±6.8%（P<0.01），Ad-AT1b-shRNA使Ang AT1b mRNA下调51.6±5.2%（P<0.01）；（2）Ang AT1a mRNA的下调与ACE2 mRNA表达降低相关（降低29.0±14.5%，P<0.01），而Ang Ad-AT1b mRNA的减少则没有影响；（3）RNA干扰（RNAi）处理均未改变ACE mRNA表达；（4）Ang AT1受体、ACE和ACE2的免疫化学染色与观察到的mRNA变化一致。这些结果证明了体内基因沉默在研究功能特异性方面的实用性。Ad-AT1a-shRNA和Ad-AT1b-shRNA均诱导了受体表达的位点和亚型特异性下调。基因沉默表明脑干Ang AT1a受体与RAS调节酶ACE2之间存在相互作用。
