Normal functioning of the heart involves several G protein-coupled receptors, including beta-adrenergic, muscarinic, alpha1-adrenergic, endothelin, and angiotensin. Since cardiac performance is profoundly affected by age, it is not surprising that age-related decreases in the responsiveness of cardiac G protein-coupled receptors have been documented. The molecular bases of these decreases, however, remain unknown. Over the past decade, a considerable amount of molecular information on signaling through G protein-coupled receptors has been amassed. Specifically, receptor phosphorylation has emerged as a major factor in GPCR signaling. Extensive studies with beta-adrenergic and muscarinic receptors indicate that these receptors undergo phosphorylation by specific kinases, and this event disrupts receptor/G protein coupling. Catalyzed by receptor-specific kinases (G protein receptor kinase [GRK] isozymes) and second messenger-activated kinases (protein kinase A [PKA] and protein kinase C [PKC] isozymes), receptor phosphorylation appears to be the underlying mechanism for both homologous (agonist-dependent) and heterologous (agonist-independent) desensitization. The proposed studies will delineate the mechanism by which aging produces a decrease in the responsiveness of cardiac G protein-coupled receptors. These studies will be performed on atrial appendages obtained from relatively young (less than or equal to 55 years) and old (greater than or equal to 70 years) patients during cardiac surgery. Our strategy is to first identify the molecular species affected by age and to then determine the underlying mechanism. The first specific aim will test the hypothesis that aging changes the expression of the three main components (receptors/G proteins/effector enzymes) involved in signal transduction through cardiac G protein- coupled receptors. The second specific aim will test the hypothesis that aging diminishes receptor/G protein coupling; here we will examine agonist-stimulated incorporation of 32P-labeled photoaffinity probe azidoanilido-GTP into receptor-associated Galpha-subunits. This approach has not previously been used to assess receptor/G protein coupling in aged heart. The third specific aim will test the hypothesis that aging causes an increase in the activity of protein kinases known to phosphorylate G protein-coupled receptors. The PI's extensive experience in the biochemistry of G protein-coupled receptors and the availability of various reagents in the PI's laboratory (GRK isozymes/G proteins/antibodies) place the PI in a unique position to successfully complete these studies.
