Polyol formation and NADPH-dependent reductases in dog retinal capillary pericytes and endothelial cells.

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Abstract

PURPOSE: Dogs fed a diet containing 30% galactose experience retinal vascular changes similar to those in human diabetic retinopathy, with selective pericyte loss as an initial lesion. In the present study the relationship among reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductases, polyol formation, and flux through the polyol pathway in cultured dog retinal capillary cells were investigated. METHODS: Pericytes and endothelial cells were cultured from retina of beagle dogs. NADPH-dependent reductases were characterized by chromatofocusing after gel filtration. Sugars in cultured cells were analyzed by gas chromatography, and flux through the polyol pathway was investigated by 19F nuclear magnetic resonance (NMR) with 3-fluoro-3-deoxy-D-glucose (3FG) as a substrate. The presence of aldose reductase and sorbitol dehydrogenase in these cells was examined by northern blot analysis. RESULTS: Two distinct peaks corresponding to aldose reductase and aldehyde reductase, the latter being dominant, were observed in pericytes by chromatofocusing. Culture in medium containing either 10 mM D-galactose or 30 mM D-glucose resulted in the accumulation of sugar alcohol in pericytes that was markedly reduced by aldose reductase inhibitors. 19F NMR spectra obtained from pericytes cultured for 5 days in medium containing 2 mM 3FG displayed the marked accumulation of 3-fluoro-deoxysorbitol but not 3-fluoro-deoxyfructose. No 3FG metabolism was observed in similarly cultured endothelial cells. With northern blot analysis, aldose reductase was detected in pericytes but not in endothelial cells. Sorbitol dehydrogenase was below the detectable limit in pericytes and endothelial cells. CONCLUSIONS: Aldose, aldehyde, and glyceraldehyde reductases are present in dog retinal capillary pericytes, with aldehyde reductase being the major reductase present. Polyol accumulation easily occurs in pericytes but not in endothelial cells.