Adenovirus-Mediated Small-Interference RNA for In Vivo Silencing of Angiotensin AT _1a Receptors in Mouse Brain

Abstract

Abstract-Because of the lack of pharmacological approaches, molecular genetic methods have been required to differentiate between angiotensin type 1(AT 1 ) receptor subtypes AT 1a and AT 1b . RNA interference is a new tool for the study of gene function, producing specific downregulation of protein expression. In this study, we used the small hairpin RNA (shRNA) cassette method to screen target sites for selectively silencing AT 1a or AT 1b receptor subtypes in cultured Neuro-2a cells using real-time RT-PCR. For in vivo functional studies, we used C57BL mice with arterial telemetric probes and computerized licking monitors to test the effect of adenovirus carrying the DNA sequence coding AT 1a shRNA (Ad-AT 1a -shRNA). Ad-AT 1a -shRNA was injected into the lateral ventricle (intracerebroventricular) or the brain stem nucleus tractus solitaries/dorsal vagal nucleus (NTS/DVN) with measurement of water intake, blood pressure (BP), and heart rate (HR) for up to 20 days after injection. Tissue culture studies verified the specificity and the efficiency of the constructs. In animal studies, ␤-galactosidase staining and Ang receptor binding assays showed expression of shRNA and downregulation of Ang AT 1 receptors in the subfornical organ and NTS/DVN by Ͼ70%. Intracerebroventricular injection of Ad-AT 1a -shRNA increased water intake with no effect on BP or HR. In contrast, microinjection of Ad-AT 1a -shRNA into NTS/DVN caused a decrease in BP with no effect on HR or water intake. Results demonstrate the use of the RNA interference method in site-directed silencing of gene expression and provide a method for the in vivo study of Ang AT 1 receptor function. Key Words: blood pressure Ⅲ brain Ⅲ gene regulation Ⅲ renin-angiotensin system B rain angiotensin (Ang) has been implicated in the control of blood pressure (BP), water balance, and hormone secretion. 1-3 Ang type 1 (AT 1 ) receptors are richly expressed in several brain regions, including the subfornical organ (SFO), paraventricular nucleus (PVN), dorsal nucleus vagus (DVN), nucleus tractus solitarii (NTS), and locus coeruleus. 4,5 In rodents, 6 and perhaps in humans, 7,8 there are 2 related AT 1 receptors subtypes, AT 1a and AT 1b . Recent studies by us 4,5 and others 9 demonstrate that AT 1 receptor subtypes AT 1a and AT 1b have similar distribution patterns in the mouse brain. In addition, several lines of reports showed that AT 1a and AT 1b receptor subtypes exhibit individual regulatory functions. 10 -12 Our previous results demonstrated that AT 1a and AT 1b receptors are differentially expressed in response to increased dietary salt and fluid deprivation within mouse brain areas, such as the hypothalamus, anterior pituitary, and brain stem. 4,5,13 However, the precise functional roles of brain AT 1a and AT 1b receptors in specific brain regions remain to be determined.AT 1a and AT 1b receptors share 95% homology in mRNA and amino acid sequences and have similar Ang II binding characteristics. 14 It is difficult to differentiate the subtypes, because there are ...

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