Enhanced β(2)-adrenergic receptor (β(2)AR) signaling by adeno-associated viral (AAV)-mediated gene transfer

BACKGROUND: β(2)-Adrenergic receptors (β(2)AR) 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 β(2)AR expressed by a recombinant adeno-associated viral (AAV) vector that also included an enhanced green fluorescent protein (EGFP) gene (AAV-β(2)AR/EGFP). RESULTS: By epifluorescence microscopy, ~40% of infected HEK 293 cells demonstrated EGFP expression. β(2)AR density measured with [(3)H]dihydroalprenolol ([(3)H]DHA) increased either 13- or 77-fold in infected cells compared to mock infected controls depending on the culture conditions used. The [(3)H]DHA binding was to a single receptor population with a dissociation constant of 0.42 nM, as would be expected for wild-type β(2)AR. Agonist competition assays with [(3)H]DHA showed the following rank order of potency: isoproterenol>epinephrine> norepinephrine, consistent with β(2)AR 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 β(2)AR with vehicle treatment and internalization following isoproterenol treatment. CONCLUSIONS: We conclude that HEK 293 cells infected with AAV-β(2)AR/EGFP effectively express β(2)AR and that increased expression of these receptors results in enhanced β(2)AR signaling. This method of gene transfer may provide an important means to enhance function in in vivo systems.