The objective of this research is to achieve an understanding of the cellular, molecular and functional properties of endothelial cell (EC) cyclic nucleotide phosphodiesterases (CN PDE), the regulatory role(s) of CN PDE in EC cyclic nucleotide metabolism and EC function assessed by relaxing factor (EDRF/NO) production. Our working hypothesis is that a cGMP-activatable CN PDE (PDE II) serves as a novel cellular receptor for cGMP in the EC. CN PDE, cyclic nucleotide accumulation and decay, and the production of EDRF/NO will be studied in cultured bovine coronary EC and isolated primary bovine aortic ECs. Endothelial cells were chosen for study because: 1) coronary artery EC cells have been developed that maintain differentiated functions through many passages in culture, 2) adequate quantities of cultured and primary EC can be obtained for comparative studies, and 3) regulation of EC functions is modified in a variety of vascular pathologies, immune reactions, hypertension, and thrombotic and atherosclerotic diseases. A comprehensive research plan is proposed involving: a) CN PDE isozyme characterization and mRNA analysis, b) examination of the effects of cGMP and PDE II activation on cAMP accumulation and decay in intact EC, c) studies on the possible participation of cyclic nucleotides in the regulation of EDRF/NO production, and d) cloning of PDE II to obtain EC cDNA for future deletion and site-directed mutagenesis to define the structural features of cGMP receptor binding. New isozyme selective inhibitors of CN PDE will be used as probes of cell-free isozyme fractions, intact cell CN PDE isozyme functions and modified EDRF/NO production. Special methods used in these studies include 3-adenine prelabeling to measure cAMP turnover, EC media column transfer bioassays to measure EDRF/NO production, and alanine-scanning mutagenesis. Thorough analysis of endogenous factors (ie. ANF), pharmacological probes and novel transfection experiments using cDNA coding for ANF receptors lacking external membrane domains are planned as methods to modify cGMP and cAMP content and possibly EDRF/NO production EC. We anticipate that an understanding of the fundamental molecular and regulatory mechanisms we propose a study in EC will have broad implications for cardiovascular cellular physiology and pharmacology.
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