In purified ventricular myocytes from adult rat heart, alpha adrenergic stimulation accelerates cAMP degradation. In rabbit cardiocytes, phosphodiesterase (PDE) inhibitors abolish apparent compartmentation of cAMP. We propose that these are two examples of intracellular modulation of cAMP metabolism to achieve specificity within the cell. We will investigate the mechanism of hormonal activation of PDE activity and the role of PDE in maintaining compartmentation of cAMP in adult cardiac myocytes. Using a new HPLC micromethod, isozymes of PDE will be resolved and characterized in terms of substrates, modulators and inhibitors, and subcellular localization. Such information will help identify PDEs involved when inhibitors are applied to whole cells to prevent alpha-adrenergic activation of PDE. The mechanism by which alpha- adrenergic stimuli activate PDE will be investigated (direct coupling to G-protein? cAMP stimulation of cAMP hydrolysis? any effect of electrical stimulation?, etc.). Attempts will be made to isolate stably altered PDE following alpha-adrenergic stimulation, and to reproduce activation in broken cell- reconstituted systems. Using fluorescent protein kinase inhibitor, regions of altered activation of cAMP-protein kinase will be mapped by fluorescent and light microscopy, and effects of hormones, modulators, and specific inhibitors studied to indicate localization of compartments created by PDE activities. Similar techniques will be applied to analysis of compartmentation of PGE1 response in cardiocytes. The cardiac myocyte alpha-1 receptor will be analyzed with respect to subtype and coupling to second messengers, especially cell Ca++ (assessed by FURA-2 fluorescence) and phosphatidylinositol metabolism. These studies will demonstrate cell specific localization, and possibly sub-cellular localization, of PDE isozymes, as well as the means by which hormones can alter these activities. This work will increase our understanding of how cells inegrate their responses to multiple hormonal signals, of how hormonal signals are transduced, and of how specificity and different responses may be achieved in response to elevation of cAMP, a general signal. The studies will also delineate the role played by PDE in maintenance of sub-cellular compartments of cAMP. Knowledge of these matters will be helpful in controlling specific aspects of cardiovascular function independently or coordinately of others using PDE inhibitors.
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