RESUMO
BACKGROUND: The beta2-adrenergic receptor (beta2AR) is a primary target for medications used to treat asthma. Due to the low abundance of beta2AR, very few studies have reported its localization in tissues. However, the intracellular location of beta2AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to beta-agonist medications. Thus, a method for visualizing beta2AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant beta2AR in primary cell cultures. METHODS: A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat beta2AR expressed in HEK 293 cells. Antibodies capable of recognizing rat beta2AR were identified and used to localize native beta2AR in primary cultures of rat airway smooth muscle and epithelial cells. beta2AR expression was confirmed by performing ligand binding assays using the beta-adrenergic antagonist [3H] dihydroalprenolol ([3H]DHA). RESULTS: Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human beta2AR (Ab-Bethyl) specifically recognized human but not rat beta2AR. An antibody developed against the C-terminal domain of the mouse beta2AR (Ab-sc570) specifically recognized rat but not human beta2AR. An antibody developed against 78 amino acids of the C-terminus of the human beta2AR (Ab-13989) was capable of recognizing both rat and human beta2ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat beta2AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface. CONCLUSION: Antibodies have been identified that recognize human beta2AR, rat beta2AR or both rat and human beta2AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of beta2AR in tissues.