CONTROL OF SORBITOL METABOLISM IN RENAL INNER MEDULLA OF DIABETIC RATS - REGULATION BY SUBSTRATE, COSUBSTRATE AND PRODUCTS OF THE ALDOSE REDUCTASE REACTION

Citation
Rw. Grunewald et al., CONTROL OF SORBITOL METABOLISM IN RENAL INNER MEDULLA OF DIABETIC RATS - REGULATION BY SUBSTRATE, COSUBSTRATE AND PRODUCTS OF THE ALDOSE REDUCTASE REACTION, Biochimica et biophysica acta, 1225(1), 1993, pp. 39-47
Citations number
29
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biophysics,Biology
ISSN journal
0006-3002
Volume
1225
Issue
1
Year of publication
1993
Pages
39 - 47
Database
ISI
SICI code
0006-3002(1993)1225:1<39:COSMIR>2.0.ZU;2-H
Abstract
Streptozotocin diabetes induces a 4-fold increase in the maximal veloc ity of inner medullary aldose reductase as determined in vitro but inc reases sorbitol synthesis in intact inner medullary collecting duct (I MCD) cells only 1.3-fold [1]. In order to resolve this discrepancy we investigated the importance of intracellular factors in controlling th e role of cellular sorbitol synthesis. These factors include glucose c oncentration, sorbitol concentration, the activity of the NADPH-regene rating pentose phosphate pathway, intracellular NADP and NADPH content , and intracellular reduced (GSH) and oxidized glutathione (GSSG). It was found that the apparent K-m of cellular sorbitol production for gl ucose was identical in control and diabetic rats (56 +/- 18 vs. 59 +/- 14 mmol/l D-glucose), whereas V-max increased by 31% in diabetes. In inner medullary collecting duct cells of diabetic rats containing 146 +/- 5 mu mol sorbitol/g protein, sorbitol synthesis was slightly lower (-15%), compared to cells which had been sorbitol-depleted prior to t he experiment (87 +/- 4 mu mol sorbitol/g protein). However, no inhibi tory effect of sorbitol (up to 200 mmol/l) was observed on aldose redu ctase activity in vitro. In diabetic rats the content of NADPH was abo ut 32% lower than in the control rats (3.8 +/- 0.3 vs. 5.6 +/- 0.4 mu mol/g protein) and the ratio of NADPH/NADP was decreased from 25.6 +/- 5.1 to 8.6 +/- 1.7. In homogenates of the inner medulla the activity of 6-phospho-gluconate dehydrogenase (EC 1.1.1.43) was identical in bo th experimental groups, so the pentose phosphate shunt seems to be una ltered. GSH content in diabetic rats was also diminished (4.02 +/- 0.6 7 mu mol/g protein vs. 7.41 +/- 0.5 mu mol/g protein) and the GSH/GSSG ratio fell from 92.6 to 57.4. In enzyme tests in vitro an apparent K- m of 7.3 +/- 1.9 mu mol/l of the aldose reductase for NADPH was found; NADP acted as competitive inhibitor with an apparent K-i of 183 +/- 3 1 mu mol/l. Aldose reductase activity was also found to be strongly in hibited by the SH-group reagent p-chloromercurybenzoesulfonate (appare nt K-i = 0.85.10(-6) mol/l). Combining the results obtained on the pro perties of the aldose reductase in vitro and the observation made in t he intact cells, the investigators suggest that the decrease in NADPH/ NADP ratio, as well as changes in the redox state in the cells of diab etic animals, can play a significant role in the control of sorbitol s ynthesis.