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This Article Full Text Full Text (PDF) All Versions of this Article: 93/5/676    most recent expphysiol.2007.041657v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Google Scholar Articles by Lin, Z. Articles by Morris, M. PubMed PubMed Citation Articles by Lin, Z. Articles by Morris, M. Related Collections Themed Issue papers Cardiovascular control

¹ Department of Pharmacology & Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45450, USA ² Guangdong Cardiovascular Institute, Guangdong Provincial People Hospital, Guangzhou 510080, China ³ Department of Pharmacology & Toxicology, Michigan State University School of Medicine, East Lansing, MI 48824, USA

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 10⁹ 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 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-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.

(Received 3 December 2007; accepted after revision 25 February 2008; first published online 29 February 2008)
Corresponding authors: M. Morris and Y. Chen: Department of Pharmacology & Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45450, USA. Email: mariana.morris{at}wright.edu or yanfang.chen{at}wright.edu

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