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RNA interference shows interactions between mouse brainstem angiotensin AT 1 receptors and angiotensin‐converting enzyme 2
Author(s) -
Lin Zhanyi,
Chen Yanfang,
Zhang Wenfeng,
Chen Alex F.,
Lin Shuguang,
Morris Mariana
Publication year - 2008
Publication title -
experimental physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.925
H-Index - 101
eISSN - 1469-445X
pISSN - 0958-0670
DOI - 10.1113/expphysiol.2007.041657
Subject(s) - small hairpin rna , rna interference , gene silencing , messenger rna , renin–angiotensin system , receptor , brainstem , angiotensin ii , microbiology and biotechnology , in situ hybridization , biology , downregulation and upregulation , endocrinology , gene expression , medicine , chemistry , rna , gene , biochemistry , blood pressure
Angiotensin (Ang) AT 1 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 AT 1 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 × 10 9 c.f.u. ml −1 , 200 nl) carrying interference small hairpin RNA (shRNA) for either AngAT 1a (Ad‐AT 1a ‐shRNA) or AngAT 1b (Ad‐AT 1b ‐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‐AT 1a ‐shRNA downregulated Ang AT 1a mRNA by 61.2 ± 6.8% ( P < 0.01) and Ad‐AT 1b ‐shRNA downregulated Ang AT 1b mRNA by 51.6 ± 5.2% ( P < 0.01); (2) downregulation of Ang AT 1a mRNA was associated with decreased ACE2 mRNA expression (decrease of 29.0 ± 14.5%, P < 0.01), while reduction in Ang Ad‐AT 1b mRNA had no effect; (3) ACE mRNA expression was not altered by either RNA interference (RNAi) treatment; and (4) immunochemical staining for Ang AT 1 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‐AT 1a ‐shRNA and Ad‐AT 1b ‐shRNA induced site‐ and subtype‐specific downregulation of receptor expression. Gene silencing showed that there were interactions between brainstem Ang AT 1a receptors and the RAS regulatory enzyme, ACE2.