This proposal seeks to provide the opportunity for the Principal Investigator (PI) to gain knowledge and skills in molecular biology and genetic epidemiology, under the direct supervision of a highly qualified sponsor, in order to enhance his potential to develop into an independent investigator. The PI is trained in Pediatric Pulmonary Medicine and has recently completed three years research in the cell biology of airway smooth muscle. The first two years of this proposal will incorporate the necessary course-work, seminars, and hands-on experience in new molecular biology and genetic techniques to enable to PI to complete and intensive research experience over the full five years of the proposal. The overall scientific goal of this proposal is to establish the associations between beta2-adrenergic receptor (beta2-AR) polymorphisms and desensitization and to understand the mechanism by which they occur. Because airway smooth muscle is the ultimate and primary target of beta-agonist therapy, cultured human airway smooth muscle (HASM) cell will be used. The hypothesis is that beta2-AR polymorphisms influence acute and long-term desensitization by altering basal cell-surface beta2-AR density, and this will be evaluated by 4 specific aims: First, beta2-AR density, cell stiffness, and cAMP formation will be studied at baseline and in response to Isoproterenol (Iso) in HASM cells exposed to Iso short-and long-term, and the data will be stratified by beta2-AR genotype. Second, heterologous desensitization will be evaluated in a similar manner by pretreating HASM cells with PGE2, IL-1beta, or dibutyryl cAMP (db-cAMP). Third, given the proposed mechanisms by which beta2-AR polymorphisms increase desensitization, dexamethasone (Dex) will be expected to increase beta2-AR density and inhibit desensitization only in those polymorphisms where baseline beta2-AR density is low. Fourth, to study the effects of polymorphisms in the 5'-flanking region on transcription of the beta2-AR gene in HASM cells, transient transfection analysis using luciferase-promoter reporter constructs, which include this 5'-flanking region, will be performed at baseline and in cells pretreated with db-cAMP or Dex. Defining the beta2-AR genotypes that alter beta-agonist responses may thus have significant therapeutic consequences on the use of beta-agonists.
