The beta adrenergic receptor - adenylyl cyclase (betaAR-AC) system expressed by human airway cells is homeostatically essential in the control of airway caliber, reactivity, and the pathogenesis of airway inflammation. However, the effect(s) of inflammatory cytokines like interleukin-1beta (IL1beta) present in the airway in diseases like asthma on the integrated function an regulation of expression of the components of the betaAR-AC (e.g. betaAR, GTP binding proteins, adenylyl cyclase) is poorly understood. Our preliminary data indicate that functional coupling of beta-adrenergic agonists to cAMP production is markedly impaired in human airway epithelial cells treated in vitro with IL1beta despite a 2 - 4 fold increase in beta AR density and beta2 AR mRNA. This extensively revised and refocused project will test the following hypotheses: that 1) IL1beta alters the function of the betaAR-AC system of human airway epithelial cells by affecting the expression of the Galphai2 and beta2AR genes; 2) that Galpha i 2, a recently recognized oncogene, drives beta2AR gene expression; and 3) endobronchial allergen instillation changes human airway epithelial cell betaAR-AC system function. Accordingly, the specific aims of this project are to examine in human airway epithelial cells; 1) the effects of Il1beta on the function of the beta AR-AC system as assessed by betaAR density, binding affinity, Galpha i and Galphas content, cAMP responses to beta AR agonists, and adenylyl cyclase activity; 2) the mechanism(s) underlying the Il1beta effects on betaAR-AC system function; and 3) the effect(s) of local antigen bronchial challenge in well characterized groups of subjects (i.e. atopic asthmatics, atopic non-asthmatics, and non-atopic normal subjects) on the function of the baiter-AC system.
Aims 1 and 2 will be performed primarily on a transformed human airway epithelial cell line (i.e. BEAS-2B) whose betaAR-AC system closely resembles that of normal human cells.
Aim 2 will be performed on airway epithelial cells harvested at bronchoscopy by bronchial brushing. The function of the betaAR-AC will be assessed at the level of the betaAR, G proteins, and adenylyl-cyclase. Control of expression of the betaAR and G proteins will be assessed at the transcriptional and post-transcriptional levels. The following techniques will be applied: ligand binding, pertussis and cholera toxin 32P-ADP ribosylation, immunoblotting, reverse-transcriptase polymerase chain reaction, cell co-transfection with appropriate human beta2AR and Galphai2 gene expression vectors and promoter-reporter constructs, nuclear runoff, and DNA footprinting analysis. Data generated in this proposal are likely to increase our understanding of the regulation of expression of the major protein components comprising the betaAR-AC and the effects of airway inflammation on the function of this system.
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| Aksoy, M O; Bin, W; Yang, Y et al. (2001) Nuclear factor-kappa B augments beta(2)-adrenergic receptor expression in human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 281:L1271-8 |
| Kelsen, S G; Aksoy, M O; Brennan, K et al. (2000) Chronic effects of inhaled albuterol on beta-adrenoceptor system function in human respiratory cells. J Asthma 37:361-70 |
| Kelsen, S G; Anakwe, O; Aksoy, M O et al. (1997) IL-1 beta alters beta-adrenergic receptor adenylyl cyclase system function in human airway epithelial cells. Am J Physiol 273:L694-700 |
