Beta-adrenergic responsiveness is dependent upon the expression of beta-adrenoreceptors. This expression will partly be dependent upon the rate of receptor synthesis, cellular processing, insertion into the plasma membrane and coupling with other components of the system and the rate of degradation. Thus any alterations in these processes may alter responsiveness. Currently, little is known about the turnover and processing of the beta- adrenoreceptor. The long-term objective of the proposed research is to characterize, with the use of irreversible receptor ligands, the relationship between the regeneration of beta- adrenoreceptors and receptor-mediated responses. Using cultured cells, the endogenous receptors are irreversibly blocked and the time course of receptor recovery is determined with respect to: antagonist binding sites (both total and cell surface), agonist high (receptor-guanine nucleotide binding protein coupling) and low affinity states, cellular localization and the ability of the receptor to mediate adenylate cyclase stimulation. The ability of various processing inhibitors and other parameters on the receptor-response recovery period will be tested. These include temperature, membrane potential, ions, ATP levels and inhibitors of protein synthesis, microtubules and glycosylation. Comparisons will be made to a rapid receptor synthesis phase in human A431 cells. After irreversible blockade in vivo, receptor recovery in atria will be determined with respect to: agonist affinity states, the ability of the receptor to mediate cyclase stimulation and atrial tension development and the beta-1 and beta-2 subtypes (several tissues). The atrial recovery studies will then be extended to conditions where it is known that receptor expression and/or responsiveness is altered. These will initially include thyroid status and denervation with 6-hydroxydopamine. Finally, it is proposed to extend some characterization studies on some novel carbostyril based beta-agonists that show either noncovalent, nondissociating or covalent binding to the receptor. Both types of compounds produce irreversible cyclase activation in vitro. The noncovalent agonists will be radiolabeled for direct agonist binding and to study receptor processign during agonist- induced internalization with the agonist attached. The alkylating irreversible agonist will be used in several receptor regeneration studies for comparison to the irreversible antagonist. The proposed studies will provide basic information on the regeneration and cellular processing of beta-adrenoreceptors and its relationship to the ability of the receptor to mediate a response. This information may point to areas where lesions could occur during disease or therapeutic interventions may be useful to alter receptor-effector coupling and receptor mediated responses.
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