The effect of hypertonicity on aldose reductase, alpha B-crystallin, and organic osmolytes in the retinal pigment epithelium.
OBJECTIVE
Aldose reductase (AR), an enzyme implicated in diabetic complications of ocular tissues, has been suggested to play a physiologic role in kidney and, possibly, other tissues by elevating the organic osmolytes in conditions of heightened extracellular tonicity. Hypertonicity has been shown to induce AR and alpha B-crystallin in some cells. To examine if similar mechanisms are operating in the retinal pigment epithelium (RPE), another target tissue of diabetic complications, we studied the effect of hypertonic media on the induction of AR, alpha B-crystallin, myoinositol, taurine, and other free amino acids.


METHODS
Human RPE cells were cultured in normal and hypertonic media containing 150 mmol/l NaCl or 200 mmol/l cellobiose in combination with 30 mmol/l galactose from 0-8 days. Western blot analysis with antibodies were used to measure the expression of AR and alpha B-crystallin. Hybridization of northern blots using AR and alpha B-crystallin complementary DNA probes were employed for the measurement of the respective messenger RNA for these proteins. Changes in the levels of myoinositol, galactitol, taurine, and other free amino acids were determined biochemically.


RESULTS
AR and alpha B-crystallin messenger RNA levels rose 16-fold and 4-fold, respectively, when human RPE cells were cultured for 3 days in media supplemented with either 150 mmol/l NaCl or 200 mmol/l cellobiose. AR and alpha B-crystallin protein levels also increased significantly, as seen by western blots. Consistent with the increased AR, galactitol accumulated to a greater extent when human RPE cells were grown in media containing 30 mmol/l galactose plus 150 mmol/l NaCl compared with cells grown in 30 mmol/l galactose alone. An 11-fold increase in cellular myoinositol and a 1.4-fold increase in taurine was observed in cells exposed to hypertonic media.


CONCLUSIONS
These findings suggest that human RPE cells are responsive to hypertonic stress by elevating AR activity and use intracellular organic solutes in an interactive manner to help regulate intracellular tonicity.
Eye Research Institute, Oakland University, Rochester, Michigan.
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Lin
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Carper
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Yokoyama
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Reddy
D000328:Adult; D000449:Aldehyde Reductase / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D002849:Chromatography, Gas; D003459:Crystallins / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006982:Hypertonic Solutions; D007294:Inositol / Q000378:metabolism; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013654:Taurine / Q000378:metabolism
D000596:Amino Acids; D003459:Crystallins; D003470:Culture Media; D006982:Hypertonic Solutions; D012333:RNA, Messenger; D013654:Taurine; D007294:Inositol; D000449:Aldehyde Reductase; D005978:Glutathione