Our goals are 1. to develop and analyze and experimental cell model that mimics the coupling properties of human heart beta1- and beta2- adrenoceptors; and 2. to investigate factors that are important in receptor mediated regulation of intracellular Ca2+ concentrations ([Ca2+]i). For both aims we will express by recombinant DNA techniques, varying densities of cloned human receptors and GTP-binding proteins in a murine cell line (L cells), thus providing a common biochemical background for all, and then analyze, as appropriate, coupling parameters and the regulation of [Ca2+]i. Thus, in heart it has been shown that beta2-adrenoceptors couple better to adenylyl cyclase (AC) than beta1-adrenoceptors, and that chronic treatment of patients with beta1- selective adrenergic receptor blockers (e.g., atenolol) results in a potentiation of the beta2-adrenoceptor effects. beta-Adrenoceptors are coupled to AC by the G protein Gs. Experiments are proposed to define how relative concentrations of receptors and the Gs proteins determine how agonists and partial agonists stimulate AC, in terms of positions and heights of dose-response curves, ligand binding and its regulation by guanine nucleotides, and stimulation of GTPase activity, and to test whether the effect of the blocker seen in human heart is due to cross-sensitization among Gs protein-coupled receptors, and whether this may occur also among receptors that are coupled by a G protein of another kind, such as muscarinic acetylcholine receptors that cause changes in intracellular Ca2+ levels, or among receptors that are each coupled by different types of G proteins. To investigate regulation of [Ca2+]i we will use cell lines with transfected receptors that mobilize Ca2+ (muscarinic, alpha1-adrenergic and serotonin), and compare their effects in terms of oscillatory nature of the changes in [Ca2+]i, and test whether patterns of changes in [Ca2+]i are related to """"""""pacing"""""""" functions encoded in GTP binding proteins of various types and known to hydrolyze the nucleotide at a molecular frequency not unlike that of the Ca2+ oscillations. A better understanding should develop of the mechanisms by which G protein- coupled receptors act, of how one receptor may affect the workings of another and of how non-excitable cells regulate [Ca2+]i. Part of this research has clear physiologic and therapeutic implications.

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National Heart, Lung, and Blood Institute (NHLBI)
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Pharmacology A Study Section (PHRA)
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Baylor College of Medicine
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