Many hormones that stimulate cardiac contraction do so by elevating cardiac cyclic AMP content. Curiously, some agents, such as alpha- adrenergic agonists and endothelin, lower cyclic AMP and also have positive inotropic effects. Others, such as PGE1, elevate cyclic AMP but do not alter contractility. Hence the central hypothesis of this proposal: that modulation of cyclic AMP synthesis (via Gs and Gi) provides an incomplete picture that does not account for the effects of many inotropic agents. Proposed studies will employ isolated adult ventricular myocytes to examine three aspects of this central hypothesis: 1. Functional compartmentation of cyclic AMP exists in single myocytes and is detectable as distinct spatial relationships among components of the cyclic AMP response pathway. Approaches include use of fluorescent PKI and PKA, assessment of protein phosphorylation by PKA in specific subcellular regions, injection of single cells with fluorescent PKA and antibodies to specific components in the cyclic AMP pathway, and use immunocytochemical techniques. 2. Activation of alpha1-receptors lowers cyclic AMP via stimulation of cyclic AMP phosphodiesterases mediated by phospholipid metabolism. Approaches include assessment of DAG/PS effects or HPLC-resolved PDEs, translocation of PDEs, effects of isoform-specific PDE inhibitors and antibodies, and consideration of roles of PKCs and direct alpha-receptor- G-protein-PDE coupling and the application of isoform specific antibodies and immunocytochemistry. 3. Alpha1 agonists exert positive contractile effects via alterations in Na+/H+ exchange. Approaches include assessing changes in cell length, ionic content (ion-sensitive fluorophores), cyclic AMP (fluorescent PKA) and involvement of specific G-proteins and PKCs, using techniques of single cell injection and monitoring of cell fluorescence and motion. These studies will describe signal cross-talk and integration in myocytes, with implications for short term control of contractility and for control of hypertrophic responses that end in heart failure.
Showing the most recent 10 out of 18 publications
