The pulmonary endothelial cell and its plasma membrane are critical targets in hyperoxic lung injury. The mechanism of the membrane injury is poorly understood. The over all objective of this proposal is to test the hypothesis that high partial pressures of O2, which can cause peroxidative cleavage of membrane lipids, alter the fluidity of the lipid bilayer of the plasma membrane of pulmonary endothelial cells, and these alterations in fluidity mediate derangements in plasma membrane function, e.g., depression of serotonin (5-hydroxytryptamine;5-HT) uptake, that are prominent manifestations of pulmonary endothelial cell O2 toxicity.
The specific aims are (1) to identify the effects of high O2 tensions on plasma membrane lipid dynamics in endothelial cells, (2) to define the relationship between alterations in plasma membrane fluidity and transmembrane transport of 5-HT by endothelial cells, (3) to determine whether alterations in plasma membrane lipids are directly responsible for hyperoxic depression of 5-HT uptake, and (4) to correlate changes in the lipid composition of the plasma membrane with changes in its fluidity. Fluorescence spectroscopy will be used to evaluate the effects of hyperoxia on fluidity in several distinct lipid domains within the plasma membrane of endothelial cells in monolayer culture. To define the relationship between the alterations in fluidity and 5-HT transport, we will assess the effects of a membrane fluidizer (cis vaccenic acid), a membrane rigidizer (cholesterol), and antioxidants (Alpha-tocopherol, butylated hydroxytoluene) on membrane fluidity and 5-HT transport in control and hyperoxic endothelial cells. In addition, we will compare the time course and dose-response curve for hyperoxic alterations in fluidity with the time course and dose-response curve for hyperoxic depression of 5-HT uptake by cultured endothelial cells. We will measure 5-HT transport and fluidity in a reconstituted membrane system. The 5-HT carrier complex from control and hyperoxic cells will be solubilized and reassembled into liposomes formed from plasma membrane lipids derived from control or hyperoxic endothelial cells. Finally, we will analyze the lipid composition of control and hyperoxic endothelial cell plasma membranes. Elucidation of the mechanisms of hyperoxic membrane injury will allow for the logical development of ways to prevent the manifestations of hyperoxic endothelial cell and lung injury.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL035908-02
Application #
3350327
Study Section
Pathology A Study Section (PTHA)
Project Start
1986-07-01
Project End
1989-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Florida
Department
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Yang, W; Block, E R (1995) Effect of hypoxia and reoxygenation on the formation and release of reactive oxygen species by porcine pulmonary artery endothelial cells. J Cell Physiol 164:414-23
Block, E R; Herrera, H; Couch, M (1995) Hypoxia inhibits L-arginine uptake by pulmonary artery endothelial cells. Am J Physiol 269:L574-80
Rao, U J; Denslow, N D; Block, E R (1994) Hypoxia induces the synthesis of tropomyosin in cultured porcine pulmonary artery endothelial cells. Am J Physiol 267:L271-81
Bhat, G B; Block, E R (1992) Effect of hypoxia on phospholipid metabolism in porcine pulmonary artery endothelial cells. Am J Physiol 262:L606-13
Bhat, G B; Block, E R (1992) Serotonin transport in reconstituted endothelial cell plasma membrane proteoliposomes: effect of hypoxia. Am J Respir Cell Mol Biol 6:633-8
Hart, C M; Tolson, J K; Block, E R (1992) Quantitative fatty acid analyses in cultured porcine pulmonary artery endothelial cells: the combined effects of fatty acid supplementation and oxidant exposure. J Cell Physiol 153:76-87
Block, E R (1991) Hydrogen peroxide alters the physical state and function of the plasma membrane of pulmonary artery endothelial cells. J Cell Physiol 146:362-9
Herskowitz, K; Bode, B P; Block, E R et al. (1991) Characterization of L-glutamine transport by pulmonary artery endothelial cells. Am J Physiol 260:L241-6
Herskowitz, K; Bode, B P; Block, E R et al. (1991) The effects of endotoxin on glutamine transport by pulmonary artery endothelial cells. J Surg Res 50:356-61
Hart, C M; Tolson, J K; Block, E R (1991) Supplemental fatty acids alter lipid peroxidation and oxidant injury in endothelial cells. Am J Physiol 260:L481-8

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