Aldose reductase in human retinal pigment epithelial cells.
Sugar alcohols have been reported to accumulate in retinal pigment epithelium (RPE) of diabetic animals. This finding has raised interest in the role of RPE in diabetes-associated retinal changes such as cystoid macular edema. To confirm the presence of aldose reductase in this tissue, the NADPH-dependent enzyme was purified to an apparent homogeneity from cultured human RPE cells, characterized, and its biochemical properties investigated. The induction of aldose reductase by hypertonic stress was also examined. The purification of aldose reductase was performed by a series of chromatographic steps which include gel filtration, affinity chromatography and chromatofocusing. Final purity achieved was monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The kinetic properties and susceptibility to inhibition of the purified aldose reductase were essentially identical to aldose reductase purified from human placenta and kidney. In addition to aldose reductase, chromatofocusing demonstrated the presence of aldehyde reductase, another NADPH-dependent reductase. However, the amounts of aldehyde reductase present were much smaller than those of aldose reductase and the levels of aldehyde reductase appeared too small to contribute to the polyol production in the RPE cells. Culture of RPE cells in hypertonic medium containing 150 mM sodium chloride (600 mosmol total) increased both reductase activity, monitored with DL-glyceraldehyde as substrate, and immunoblot staining for aldose reductase. Chromatofocusing of RPE cells cultured in hypertonic media resulted in a prominent increase in the peak corresponding to aldose reductase compared to the peak height of cells grown in control medium. No increase in aldehyde reductase from RPE cells cultured in hypertonic medium was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Sato
S
S
Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, MD 20892.
Lin
L R
LR
Reddy
V N
VN
Kador
P F
PF
D000449:Aldehyde Reductase / Q000032:analysis*; D002478:Cells, Cultured; D004790:Enzyme Induction; D005965:Glucuronates / Q000378:metabolism; D006801:Humans; D009997:Osmotic Pressure; D010857:Pigment Epithelium of Eye / Q000201:enzymology*; D013379:Substrate Specificity
D005965:Glucuronates; D000449:Aldehyde Reductase