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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL046494-06
Application #
2028613
Study Section
Lung Biology and Pathology Study Section (LBPA)
Project Start
1992-02-01
Project End
2001-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
6
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of South Alabama
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
Mobile
State
AL
Country
United States
Zip Code
36688
Zhu, Bing; Kelly, John; Vemavarapu, Lakshmi et al. (2004) Activation and induction of cyclic AMP phosphodiesterase (PDE4) in rat pulmonary microvascular endothelial cells. Biochem Pharmacol 68:479-91
Tarpey, Siobhan B; Sawmiller, Darrell R; Kelly, Claire et al. (2003) Phosphodiesterase 3 activity is reduced in dog lung following pacing-induced heart failure. Am J Physiol Lung Cell Mol Physiol 284:L766-73
Liu, Li; Underwood, Tashandra; Li, Han et al. (2002) Specific cGMP binding by the cGMP binding domains of cGMP-binding cGMP specific phosphodiesterase. Cell Signal 14:45-51
Thompson, W Joseph; Ashikaga, Takashi; Kelly, John J et al. (2002) Regulation of cyclic AMP in rat pulmonary microvascular endothelial cells by rolipram-sensitive cyclic AMP phosphodiesterase (PDE4). Biochem Pharmacol 63:797-807
Francis, Sharron H; Bessay, Emmanuel P; Kotera, Jun et al. (2002) Phosphorylation of isolated human phosphodiesterase-5 regulatory domain induces an apparent conformational change and increases cGMP binding affinity. J Biol Chem 277:47581-7
Liu, L; Li, H; Underwood, T et al. (2001) Cyclic GMP-dependent protein kinase activation and induction by exisulind and CP461 in colon tumor cells. J Pharmacol Exp Ther 299:583-92
Zhou, L; Thompson, W J; Potter, D E (1999) Multiple cyclic nucleotide phosphodiesterases in human trabecular meshwork cells. Invest Ophthalmol Vis Sci 40:1745-52
Stevens, T; Creighton, J; Thompson, W J (1999) Control of cAMP in lung endothelial cell phenotypes. Implications for control of barrier function. Am J Physiol 277:L119-26
Moore, T M; Chetham, P M; Kelly, J J et al. (1998) Signal transduction and regulation of lung endothelial cell permeability. Interaction between calcium and cAMP. Am J Physiol 275:L203-22
Kelly, J J; Moore, T M; Babal, P et al. (1998) Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+ and permeability. Am J Physiol 274:L810-9

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