Enhanced β2-adrenergic receptor (β2AR) signaling by adeno-associated viral (AAV)-mediated gene transfer
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
BMC Pharmacology 2003, 3:15 doi:10.1186/1471-2210-3-15Published: 4 December 2003
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).
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.
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.