Endothelium-derived nitric oxide (NO) is a critical modulator of vasomotor tone in developing lung, having a key role in the normal transition to extrauterine life and in the pathophysiology of persistent pulmonary hypertension of the newborn (PPHN). The OVERALL OBJECTIVE of this proposal is to determine how pulmonary endothelial NO production is modified by changes in O2 and how it is regulated developmentally.
Specific Aim No. 1 is to determine the direct effect of ACUTE alterations in O2 on NO production and the role of cytosolic free calcium (Cai 2+) homeostasis in this process. NO production in ovine fetal pulmonary endothelial cells will be assessed during acute changes in O2 by measuring cGMP- in detector smooth muscle cells. Studies with fura-2 will reveal O2-related alterations in endothelial Cai 2+, and pharmacologic methods will reveal the processes regulating Cai 2+ which mediate the changes in NO production.
Aim No. 2 is to determine the effect of PROLONGED hypoxia on NO production and the role of changes in NO synthase activity and expression in this process. Chronic hypobaria in rats will be used to examine prolonged hypoxia in vivo, and ovine fetal pulmonary endothelial cells will be subjected to prolonged alterations in O2 in vitro. NO synthase activity will be determined by measuring 1-arginine conversion to 1-citrulline, the enzyme protein will be quantitated in immunoblots and by immunohistochemistry, and NO synthase mRNA expression will be assessed by ribonuclease protection assays and in situ hybridization.
Aim No. 3 is to delineate the ontogeny of pulmonary endothelial NO production, correlating it with NO synthase activity, protein expression, and steady state MRNA levels. Whole lung, arterial segments, and endothelial cells from early and late third trimester fetal lambs and 1 week and 1 month old newborn lambs will be studied.
Aim No. 4 is to determine the amount of constitutive versus inducible NO synthase in developing pulmonary endothelium and the putative factors regulating their expression. The dependence of NO synthase activity on calcium/calmodulin will be tested and inhibition profiles will be performed with 1-arginine analogues. The modulation of NO production by corticosteroids and cytokines will be examined and the basis of modulation will be determined in studies of NO synthase enzyme activity, protein quantification, and MRNA levels. By revealing for the first time the cellular and molecular processes regulating endothelial NO production in the developing pulmonary circulation, these studies will increase our fundamental knowledge of the mechanisms underlying successful cardiopulmonary transition and the pathophysiology of PPHN.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD030276-08
Application #
6182225
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Program Officer
Spong, Catherine
Project Start
1993-04-01
Project End
2001-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
8
Fiscal Year
2000
Total Cost
$241,557
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Pediatrics
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
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