TISSUE SPECIFIC INHIBITORS OF PHOSPHODIESTERASE

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Irreversible inhibitors of cyclic nucleotide phosphodiesterases will be prepared. The design of these agents will be based on the extensive SAR of inhibitors of cyclic AMP and cyclic GMP phosphodiesterases established in our laboratory over the past seven years. Irreversible inhibitors that can be showed to be affinity labels will be used to study the active site of calmodulin-dependent phosphodiesterase. Proteolytic fragments and/or individual amino acids that are covalently modified by the affinity label will be identified. Since some of the irreversible inhibitors will be relatively specific for either the calmodulin-dependent or the "low Km" form of phosphodiesterase, we will attempt to discern if these agents irreversibly inhibit these phosphodiesterases in the intact cell and determine the relationship between inhibition in the intact tissue and the intracellular levels of cAMP and cGMP. These studies in the intact tissue should help delineate which of the two forms of phosphodiesterase is most important in the in vivo regulation of cAMP and cGMP concentrations. In addition we will prepare a series of adenosine antogonists. Agents that are potent adenosine antagonists but weak phosphodiesterase inhibitors would be of great value as tools to study the physiological roles of adenosine. We will therefore design and prepare agents that inhibit adenosine-receptor interactions that are coupled to adenylate cyclase. The design of these agents will be based on the structure of theophylline which is considerably more potent as an inhibitor of adenosine-receptor interactions than it is as an inhibitor of cyclic nucleotide phosphodiesterase activities.