Study The Regulation Of β-adrenergic Receptor Conduction In Asthma Lung By A β-adrenergic Receptor Kinase Inhibitor Gene Transfer

ObjectivePhosphorylation of serine and threonine residues at the carboxy-terminus of the agonist-occupied β receptor catalysed by β ARK1 has been delineated as a molecular mechanisms that result inβ 2-adrenoceptor desensitization in asthma lung. Construct a recombination plasmid encoding the carboxyl terminus of β ARK1( β ARKct) which contains the domain responsible for binding to the βγ-subunits of activated G proteins (Gβγ), a process required for the activation of endogenous β ARK1, and study the expression and influence of objective gene on β receptor signal conduction of asthma lung through gene transfer in vivo.Method:1. The cDNA encoding murine βARK1 residues G1P495 to Leu689 amplified using RT-PCR is inserted into a plasmid to construct a recombination expression plasmid pcDNA- β ARKct.2. Establish an asthma animal model and an asthma animal treated with P AR agonist.3. We have delivered lipid-pcDNA-β ARKct complexes in vivo intravenously or intratracheally to the lung of animal.4. Werstern-blot is used to detect the expression of β ARKct in lung, the changes of β AR and cAMP are evaluated by radioimmunology.Result1. The expression of β ARKct in lung was detectable. But, the β ARKct expressed more in lung of mice intratracheal delivery plasmid in vivo than intravenous.2. The down-regulation of β AR and cAMP were seen in asthma mice and the asthmatic mice treated with agonist, but the degrees of down-regulation were not the same.3. β AR and cAMP were increased in lung of asthmatic mice treated with recombination plasmid.Conclusion1. The recombination plasmid encoding β ARKct expressed the objective gene in lung of mice through gene transfer in vivo. But the various DNA doses and gene delivery pathways decided the expression level.2. Gene transfer β ARKct to asthma mice and asthmatic mice treated with agonist could regulate upward the level of β AR and cAMP which were down regulation once. But the roles β ARKct play are different on the β AR up-regulation of this two animal model.