Angiotensin AT1a shRNA Demonstrates Interactions between Brainstem Angiotensin AT1a Receptors and Angiotensin Converting Enzyme 2

Abstract

Angiotensin (Ang) AT1a receptors and Ang converting enzyme 2(ACE2) are expressed in the brainstem dorsal vagal complex (DVC). Studies were conducted using adenovirus mediated inference small hairpin RNA for AT1a (Ad-AT1a-shRNA) to evaluate the interactions between AT1a and ACE2. We used a system in which Ad-AT1a-shRNA or its Ad-LacZ control was injected bilaterally into the DVC. mRNA levels were measured using in situ hybridization (ISH). Using a double cannulae inserted in the DVC, Ad-AT1a-shRNA or Ad-LacZ (both at 1x 109 cpu/ml, 200nl) was injected. This injection method allows for a side by side comparison of mRNA expression. Brainstems were collected at day 10 after treatment, a time at which receptors are known to be decreased. ISH was performed for Ang AT1a, Ang AT1b, ACE1, and ACE2, monitored using phosphor imaging and autoradiography. Results showed: Ad-AT1a-shRNA decreased Ang AT1a mRNA by 61.2 ± 6.8% compared with the control side (p<0.001). Ad-AT1a-shRNA had no effect on AT1b receptor mRNA expression. ACE2 mRNA expression on the Ad-AT1a-shRNA treated side was reduced by 29.0±14.5 % (P<0.01). Ad-AT1a-shRNA had no effect on ACE1. These results demonstrate that Ad-AT1a-shRNA can site- and subtype- specifically silence AT1a receptor mRNA. AT1a may also modulate brainstem ACE2 expression, perhaps reflecting important interactions between the systems in control of blood pressure and autonomic function.