Jones Stacie M, Hiller F Charles, Jacobi Sandie E, Foreman Susan K, Pittman Laura M, Cornett Lawrence E
Department of Pediatrics, University of Arkansas for Medical Sciences, Arkansas Children's Hospital, Little Rock, Arkansas 72202, USA.
BMC Pharmacol. 2003 Dec 4;3:15. doi: 10.1186/1471-2210-3-15.
Beta2-adrenergic receptors (beta2AR) 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 beta-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 beta2AR expressed by a recombinant adeno-associated viral (AAV) vector that also included an enhanced green fluorescent protein (EGFP) gene (AAV-beta2AR/EGFP).
By epifluorescence microscopy, approximately 40% of infected HEK 293 cells demonstrated EGFP expression. beta2AR 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 beta2AR. Agonist competition assays with [3H]DHA showed the following rank order of potency: isoproterenol>epinephrine> norepinephrine, consistent with beta2AR 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 beta2AR with vehicle treatment and internalization following isoproterenol treatment.
We conclude that HEK 293 cells infected with AAV-beta2AR/EGFP effectively express beta2AR and that increased expression of these receptors results in enhanced beta2AR signaling. This method of gene transfer may provide an important means to enhance function in in vivo systems.
β2-肾上腺素能受体（β2AR）在多种细胞和器官系统中发挥重要调节作用，是气道和心血管疾病治疗中的重要治疗靶点。长期使用β-激动剂会导致受体下调继发耐受性，从而降低治疗效果。本研究旨在评估重组腺相关病毒（AAV）载体表达的β2AR的信号传导能力，该载体还包含增强型绿色荧光蛋白（EGFP）基因（AAV-β2AR/EGFP）。
通过落射荧光显微镜观察，约40%的感染HEK 293细胞显示出EGFP表达。根据所采用的培养条件，与 mock 感染对照相比，用[3H]二氢阿普洛尔（[3H]DHA）测量的感染细胞中β2AR密度增加了13倍或77倍。[3H]DHA结合至单一受体群体，解离常数为0.42 nM，这与野生型β2AR预期相符。用[3H]DHA进行的激动剂竞争试验显示出以下效力顺序：异丙肾上腺素>肾上腺素>去甲肾上腺素，与β2AR相互作用一致。异丙肾上腺素刺激的环磷酸腺苷（cAMP）水平在感染细胞中比对照高5倍（314±43对63.4±9.6 nmol/培养皿；n = 3）。受体转运显示，用载体处理时β2AR呈表面表达，而异丙肾上腺素处理后会发生内化。
我们得出结论，感染AAV-β2AR/EGFP的HEK 293细胞有效表达β2AR，这些受体表达的增加导致β2AR信号增强。这种基因转移方法可能为增强体内系统功能提供重要手段。
