The objective of this research is to understand the cellular, molecular and regulatory properties of endothelial cell (EC) cyclic nucleotide phosphodiesterase (PDE) and the actions of inhibitors of these enzymes. The role of the PDE4 gene family of PDE in the integrated regulation of EC cyclic nucleotide metabolism and calcium transients is sought. Second messenger regulation of EC permeability will also be studied. Rat pulmonary microvascular EC will be studied in culture and monolayer. Pulmonary endothelial cells were chosen for study because control of bood vessel permeability is part of the etiology of vascular pathologies, thrombotic and atherosclerotic disease, and respiratory distress syndromes. The working hypothesis of the project is that PDE4 selective inhibitors act on pulmonary microvascular EC to reverse enhanced permeability injury through cAMP-dependent activation of PKA amd subsequent inhibition of sodium-hydrogen exchange (NHE) activity. The functional coupling of inhibited NHE to sodium-calcium exchange (NCX) will cause efflux of calcium in the presence of PDE inhibition. The phosphoprotein NHE regulatory factor (NHERF) is proposed to mediate cAMP regulation of the NHE. A comprehensive research plan is proposed including aims to: a) define the site of action of PDE4 inhibitors in RPMVEC known to express splice variants possibly with different subcellular loci and covalent regulation, b) study the effects of selected PDE inhibitors on monolayer permeability with newer transport techniques to verify PDE4 participation, c) determine the effects of selected PDE inhibitors on reversal of ischemia-reperfusion injury measured with capillary filtration coefficients in the isolated, perfused rat lung, and d) use high resolution single cell imaging of calcium along with intact cell cAMP and CGMP turnover techniques to study the reciprocal regulation of cAMP accumulation and calcium transients in microvascular Ecs. The research plan is comprehensive and multidimensional. This research offers the potential for rationale design of new therapeutic interventions in EC functions based on PDE isoform expression. We anticipate that understanding the mechanisms and actions of cylic nucleotide PDE inhibitors on microvascular EC will have broad implications for cardiovascular and pulmonary physiology and pharmacology.
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