Defects in the function of microvessels are responsible for much of the retinal injury that occurs in diabetes. Our long term goal is to determine the biochemical mechanisms by which retinal microvessels are damaged in diabetes. When exposed to high concentrations of glucose in tissue culture, retinal pericytes and endothelial cell ssynthesize sorbitol. We believe that intracellular sorbitol may interfere with receptor mediated turnover of inositol phospholipids and subsequent activation of protein kinase C and this may, in part, underlie the microangiopathy. To test this hypothesis we will expose retinal pericytes and endothelial cells in tissue culture to high concentrations of glucose and investigate their metabolism and transport of inositol, turnover of inositol phospholipids, actiatin of protein kinase C, and several aspects of cell function including sodium transport, prostaglandin release, and cell growth. Inositol entry into microvascular cells will be determined by studying the uptake of 3H-inositol. The intracellular concentration of inositol and sorbiotal will be determine as a function of external inositol and glucose concentrations. To measure alterations in the utrhover of inositol phospholipids, the cells will be labeled with 3H-inositol and the release of inositol phosphates as well as changes in the specific activity of inositol phospholipids in the cell membranes will be measured. The activation of protein kinase C by specific agonists will be determined in both the cytosol and membrane fractions of cells growing in normal and high concentrations of glucose. Mitogenesis will be assayed by the incorporation of thymidine into DNA. Sodium transport will be measured as amilioride sensitive Na-H exchange, ouabain sensitive K uptake, binding of ouabain to membrane fractions and sodium dependent uptake of amino acids. Formation of prostaglandins by the microvascular cells will be measured by radioimmunoassay. We will determine the effect of Sorbinil, an inhibitor of aldose reductase upon all functions altered by exposure of the cells to high concentrations of glucose.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
7R01EY003772-10
Application #
3258210
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1988-07-01
Project End
1990-11-30
Budget Start
1988-07-01
Budget End
1988-11-30
Support Year
10
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Kennedy Krieger Research Institute, Inc.
Department
Type
DUNS #
City
Baltimore
State
MD
Country
United States
Zip Code
21205
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Wolff, J E; Belloni-Olivi, L; Bressler, J P et al. (1992) Gamma-glutamyl transpeptidase activity in brain microvessels exhibits regional heterogeneity. J Neurochem 58:909-15
Bressler, J; Goldstein, G W (1992) Effect of elevated ambient glucose upon polyphosphoinositide turnover in bovine retinal endothelial cells and rat astrocytes. Exp Eye Res 54:871-7
Wolff, J E; Laterra, J; Goldstein, G W (1992) Steroid inhibition of neural microvessel morphogenesis in vitro: receptor mediation and astroglial dependence. J Neurochem 58:1023-32
Belloni-Olivi, L; Bressler, J P; Goldstein, G W (1992) Retinal microvessels express less gamma-glutamyl transpeptidase than brain microvessels. Curr Eye Res 11:203-11
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Laterra, J; Guerin, C; Goldstein, G W (1990) Astrocytes induce neural microvascular endothelial cells to form capillary-like structures in vitro. J Cell Physiol 144:204-15
Robertson, P L; Markovac, J; Datta, S C et al. (1988) Transforming growth factor beta stimulates phosphoinositol metabolism and translocation of protein kinase C in cultured astrocytes. Neurosci Lett 93:107-13
Markovac, J; Goldstein, G W (1988) Transforming growth factor beta activates protein kinase C in microvessels isolated from immature rat brain. Biochem Biophys Res Commun 150:575-82

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