Research article

Enhanced β₂-adrenergic receptor (β₂AR) signaling by adeno-associated viral (AAV)-mediated gene transfer

Stacie M Jones¹ , F Charles Hiller² , Sandie E Jacobi³ , Susan K Foreman⁴ , Laura M Pittman⁴ and Lawrence E Cornett⁵

¹  Departments of Pediatrics and Physiology and Biophysics University of Arkansas for Medical Sciences Arkansas Children's Hospital Little Rock, Arkansas, USA 72202

²  Department of Internal Medicine University of Arkansas for Medical Sciences John L. McClellan Veteran's Administration Hospital Little Rock, Arkansas, USA 72205

³  Department of Internal Medicine University of Arkansas for Medical Sciences Little Rock, Arkansas, USA 72205

⁴  Department of Pediatrics University of Arkansas for Medical Sciences Arkansas Children's Hospital Little Rock, Arkansas, USA 72202

⁵  Departments of Physiology and Biophysics and Internal Medicine University of Arkansas for Medical Sciences Little Rock, Arkansas, USA 72205

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BMC Pharmacology 2003, 3:15doi:10.1186/1471-2210-3-15

Published:	4 December 2003

Abstract

Background

β₂-Adrenergic receptors (β₂AR) play important regulatory roles in a variety of cells and organ systems and are important therapeutic targets in the treatment of airway and cardiovascular disease. Prolonged use of β-agonists results in tolerance secondary to receptor down-regulation resulting in reduced therapeutic efficiency. The purpose of this work is to evaluate the signaling capabilities of the β₂AR expressed by a recombinant adeno-associated viral (AAV) vector that also included an enhanced green fluorescent protein (EGFP) gene (AAV-β₂AR/EGFP).

Results

By epifluorescence microscopy, ~40% of infected HEK 293 cells demonstrated EGFP expression. β₂AR density measured with [³H]dihydroalprenolol ([³H]DHA) increased either 13- or 77-fold in infected cells compared to mock infected controls depending on the culture conditions used. The [³H]DHA binding was to a single receptor population with a dissociation constant of 0.42 nM, as would be expected for wild-type β₂AR. Agonist competition assays with [³H]DHA showed the following rank order of potency: isoproterenol>epinephrine> norepinephrine, consistent with β₂AR interaction. Isoproterenol-stimulated cyclic AMP levels were 5-fold higher in infected cells compared to controls (314 ± 43 vs. 63.4 ± 9.6 nmol/dish; n = 3). Receptor trafficking demonstrated surface expression of β₂AR with vehicle treatment and internalization following isoproterenol treatment.

Conclusions

We conclude that HEK 293 cells infected with AAV-β₂AR/EGFP effectively express β₂AR and that increased expression of these receptors results in enhanced β₂AR signaling. This method of gene transfer may provide an important means to enhance function in in vivo systems.