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In sugar cataract formation in rats, aldose reductase (AR) activity is not only linked to lenticular sorbitol (diabetic) or galactitol (galactosemic) formation but also to signal transduction changes, cytotoxic signals and activation of apoptosis. Using both in vitro and in vivo techniques, the interrelationship between AR activity, polyol (sorbitol and galactitol) formation, osmotic stress, growth factor induction, and cell signaling changes have been investigated. For in vitro studies, lenses from Sprague Dawley rats were cultured for up to 48 h in TC-199-bicarbonate media containing either 30 mM fructose (control), or 30 mM glucose or galactose with/without the aldose reductase inhibitors AL1576 or tolrestat, the sorbitol dehydrogenase inhibitor (SDI) CP-470,711, or 15 mM mannitol (osmotic-compensated media). For in vivo studies, lenses were obtained from streptozotocin-induced diabetic Sprague Dawley rats fed diet with/without the ARIs AL1576 or tolrestat for 10 weeks. As expected, lenses cultured in high glucose/galactose media or from untreated diabetic rats all showed a decrease in the GSH pool that was lessened by ARI treatment. Lenses either from diabetic rats or from glucose/galactose culture conditions showed increased expression of basic-FGF, TGF-β, and increased signaling through P-Akt, P-ERK1/2 and P-SAPK/JNK which were also normalized by ARIs to the expression levels observed in non-diabetic controls. Culturing rat lenses in osmotically compensated media containing 30 mM glucose or galactose did not lead to increased growth factor expression or altered signaling. These studies indicate that it is the biophysical response of the lens to osmotic stress that results in an increased intralenticular production of basic-FGF and TGF-β and the altered cytotoxic signaling that is observed during sugar cataract formation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Chronic inflammatory diseases such as autoimmune and bacterial infections are associated with an elevated risk of ocular inflammation. Ciliary epithelial cells that play an important role in maintaining aqueous humor dynamics and homeostasis of anterior segment of eye are continuously exposed to inflammatory markers during infections and injury. Lipopolysacchharide (LPS), a Gram-negative bacterial endotoxin, dysregulates aqueous humor (AqH) homeostasis by inducing inflammatory changes. We have investigated how inhibition of a polyol pathway enzyme, aldose reductase (AR), alters LPS-induced inflammatory changes in human non-pigmented ciliary epithelial cells (hNPECs). The stimulation of hNPECs with LPS (1 microg/ml) caused increased secretion of inflammatory markers such as PGE(2) and NO in the culture medium as well as increased expression of COX-2 and iNOS proteins in cell extracts. LPS also increased phosphorylation of MAPKs (ERK1/2) and SAPK/JNK and activation of redox-sensitive transcription factors NF-kappaB and AP-1 in hNPECs and inhibition of AR by zopolrestat and sorbinil ameliorated these changes. Further, LPS-induced decrease in the expression of Na/K-ATPase in hNPECs was restored by AR inhibitors. Similar results were observed in ciliary bodies of LPS-injected rats. Taken together, our results suggest that AR plays an important role in the LPS-induced inflammatory changes in hNPECs and that inhibition of AR could be a novel therapeutic approach for ocular inflammation., ((c) 2010 Elsevier Ltd. All rights reserved.)

Retinal ischemic injury is common in patients with diabetes, atherosclerosis, hypertension, transient ischemia attack and amaurosis fugax. Previously, signs of ischemic stress, such as pericyte loss, blood-retinal barrier breakdown and neovascularization, which can lead to occlusion of retinal vessels, have been prevented in diabetic db/db mice with aldose reductase (AR) null mutation. To determine the role in retinal ischemic injury of AR and sorbitol dehydrogenase (SDH), the first and second enzymes in the polyol pathway, mice with deletion of AR (AR(-/-)) or SDH-mutation (SDH(-/-)), or C57BL/6N mice treated with AR or SDH inhibitors were subjected to transient retinal artery occlusion (2h of occlusion and 22h of reperfusion) by the intraluminal suture method. Neuronal loss and edema observed in wildtype (AR(+/+)) retinas after transient ischemia were prevented in the retinas of AR(-/-) mice or C57BL/6N mice treated with an AR inhibitor, Fidarestat. Fewer TUNEL-positive cells and smaller accumulations of nitrotyrosine and poly(ADP-ribose) were also observed in the retinas of AR(-/-) mice. However, SDH(-/-) mice and C57BL/6N mice treated with SDH inhibitor, CP-470,711, were not protected against ischemia-induced retinal damage. Taken together, AR contributes to retinal ischemic injury through increased edema and free radical accumulation. Therefore, AR inhibition should be considered for the treatment of retinal ischemic injury often observed in diabetic patients.

Aldehydes are widespread environmental and industrial compounds, which cause cytotoxicity, tissue damage, mutagenicity, and carcinogenicity leading to various disease conditions such as cardiovascular, bronchial, and visual complications. We have shown earlier that aldose reductase (AR) besides reducing glucose to sorbitol, efficiently reduces various toxic lipid-derived aldehydes, generated under oxidative stress, with K(m) in the physiological range. We have identified the role of AR in the prevention of various lipid aldehyde-induced cytotoxic signals leading to apoptosis in human lens epithelial cells (HLEC). HLEC were cultured without or with AR inhibitors followed by addition of various saturated and unsaturated lipid aldehydes with a carbon chain length varying from C3 to C10. The cell viability was assessed by cell counts and MTT assay, and apoptosis was measured by evaluating nucleosomal degradation and caspase-3 activation using specific ELISA kits. Although all the aldehydes caused apoptosis of HLEC, the unsaturated aldehydes were more toxic than saturated aldehydes. Inhibition of AR by sorbinil potentiated while the over-expression of AR prevented the apoptosis induced by various lipid aldehydes. AR over-expression also prevented the lipid aldehyde-induced activation of caspase-3, MAPK, JNK and the expression of Bcl-2 family of proteins in HLEC. The results indicate that the lipid aldehydes generated under oxidative stress are cytotoxic to HLEC leading to apoptosis and that the reduction of lipid aldehydes by AR would prevent it.

Numerous animal studies indicate that aldose reductase inhibitors (ARIs) are beneficial for the prevention or amelioration of diabetic complications such as neuropathy, nephropathy and the ocular complications of cataract, retinopathy and keratopathy. To aid in the identification of novel potent ARIs, we have previously developed a screening method that is based on the formation of a non-covalent ternary tight-binding enzyme-inhibitor-nucleotide (AR-ARI-NADPH) complex that can be isolated using YM-10 filter units. Here, we report a modification of this method that permits us to rapidly identify tight binding ARIs that are isolated by denaturation from AR-ARI-NADPH complexes that are free of possible contamination resulting from the reaction of methanol with the YM-10 filter units. For the development of this procedure, nine structurally diverse ARIs were mixed with purified recombinant rat lens aldose reductase (RLAR) bound with NADPH to form tight-binding RLAR-ARI-NADPH complexes. These complexes were purified by high pressure Sephadex 75 size exclusion chromatography using ammonium acetate buffer and the formation of each complex was confirmed by electrospray ionisation mass spectrometry (ESI-MS). Each of the complexes was then denatured with methanol, rechromatographed on the size exclusion column, and the identity of the bound ARIs was confirmed by ESI-MS. The apparent ARI binding with aldose reductase to form a tight binding ARI complex appeared proportional to their IC50 values. This procedure allows for the rapid identification of tight binding ARIs with apparent IC50s<0.1 microm.

Apoptosis of lens epithelial cells (LECs) is implicated in the pathogenesis of several types of cataract formation. The high intracellular levels of polyol induce histological change in the LECs, which is considered the earliest event in sugar cataractogenesis. This study was designed to investigate whether high galactose exposure induces apoptosis in LECs during the development of sugar cataract. The effect of an aldose reductase inhibitor, SNK-860, was also examined. We induced sugar cataract in Sprague-Dawley rats by feeding them a 50% galactose-containing diet with or without SNK-860. The percentage of LECs undergoing apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL) method, and DNA fragmentation analyses were performed. Galactitol levels in the lens epithelium were quantified by gas chromatography. The number of TUNEL-positive cells gradually increased throughout the period of galactose exposure, up to 5 days. DNA fragmentation analysis in LECs of rats fed a galactose-rich diet demonstrated an apparent ladder pattern. SNK-860 reduced the percentage of TUNEL-positive cells, the amount of intracellular galactitol, and the levels of DNA laddering. To explore the mechanism of the apoptotic process, the expression of p53, a potent mediator of apoptosis, was examined. Based on Western blot and real-time reverse transcription-polymerase chain reaction results, the amount of p53-expression increased at both the protein and mRNA levels after galactose exposure, and the increase in p53-expression was inhibited by SNK-860. Based on these results, we concluded that apoptosis occurs in rat lens epithelial cells following galactose exposure. Furthermore, the reduction of apoptosis by aldose reductase inhibitor suggests that this apoptosis is associated with the accumulation of sugar alcohols. It is probable that the mechanism of apoptosis during sugar cataract formation involves the increased expression of p53.

In order to define the ability of bovine lens aldose reductase (ALR2) to generate polyols from aldoses, the quantitative determination of glycerol in the presence of glyceraldehyde was performed by gas chromatography after derivatization with trifluoroacetic anhydride. The proposed method appears to be useful in quantifying low amounts of glycerol in the presence of relatively high concentrations of glyceraldehyde and in following glycerol formation in enzyme assay conditions. The generation of one equivalent of glycerol in the presence of ALR2, is paralleled by the oxidation of one equivalent of NADPH. A similar result was obtained when S-glutathionyl-modified ALR2 was used, instead of the native enzyme, as a catalyst of glyceraldehyde reduction. Sorbinil, a classical ALR2 inhibitor, present in the enzyme assay mixture, inhibits to the same extent both NADPH oxidation and glycerol formation. The demonstration of the stoichiometric ratio of 1:1 occurring in the presence of bovine lens ALR2 between the synthesis of glycerol from D, L -glyceraldehyde and the oxidation of NADPH, rules out doubts concerning the ability of the enzyme to catalyse the reduction of aldoses to the corresponding polyalcohols. Possible autooxidation processes of glyceraldehyde, in the enzyme assay conditions, appear to be irrelevant with respect to the enzyme-catalysed reduction of the aldose. This would indicate that the spectrophotometric monitoring of NADPH oxidation at 340 nm, in the presence of ALR2, is a reliable method to assay the enzyme activity., (Copyright 2000 Academic Press.)

The pathogenesis of pericyte loss, an initial deficit in the early stage of diabetic retinopathy, remains unclear. Polyol pathway hyperactivity has been implicated in the pathogenesis of diabetic retinopathy, and recent studies have suggested that apoptosis may be involved in pericyte loss. The present study was conducted to investigate whether high glucose induces apoptosis in cultured bovine retinal pericytes. The effect of an aldose reductase inhibitor, SNK-860, was also examined. After a 5 day incubation with various concentrations of glucose (5.5-40 m M) in the presence or absence of SNK-860, the cell viability and the percentages of dead cells were measured, and staining with the TUNEL method and Hoechst 33342, and DNA electrophoresis were performed. High glucose reduced the viability and increased the percentages of dead cells. TUNEL-positive cells were observed in pericytes under high glucose, but not in those under 5.5 m M glucose. In the staining of nuclei with Hoechst 33342, the percentage of apoptotic cells in total cells counted under high glucose was higher than that under 5.5 m M glucose. DNA electrophoresis of pericytes cultured with high glucose demonstrated a 'ladder pattern'. Hyperosmolarity also induced apoptosis in pericytes, but less than that by high glucose. SNK-860 inhibited the glucose-induced apoptosis in pericytes. These observations suggest that the pericyte loss in diabetic retinopathy involves an apoptotic process, and that the polyol pathway hyperactivity plays an important role in inducing apoptosis in pericytes by high glucose., (Copyright 2000 Academic Press.)

Aldose reductase inhibition is one of the therapeutic strategies that has been proposed to prevent or ameliorate long term diabetic complications including retinopathy and sugar cataract. Rats were fed with a galactose rich diet and the aldose reductase inhibitor Tolrestat was topically delivered by ocular instillation. The levels of lens aldose reductase activity, galactitol and the onset of cataract were evaluated during and after treatment with the inhibitor. Topical application of 1-3% Tolrestat (10 microl) four times daily resulted, after 9 days, in a significant decrease in the enzyme activity. Well after interrupting treatment with the drug, the enzyme activity remained impaired and galactose induced cataract was prevented. Our findings may represent the basis for therapeutic plans to prevent sugar cataract by long term cyclic treatments with aldose reductase inhibitors, with reduction in drug doses and side effects., (Copyright 1999 Academic Press.)

The effects of aldose reductase inhibitors on lens protein modifications induced by naphthalene-1,2-dihydrodiol were investigated in vitro to confirm the role of aldose reductase on naphthalene cataract formation. HPLC analysis of naphthalene-1, 2-dihydrodiol incubated with aldose reductase and NAD+indicated the formation of a metabolite peak corresponding to 1,2-naphthoquinone. Soluble proteins from rat lenses prepared by gel filtration of crude lens extracts through Sephadex PD-10, incubated with naphthalene-1, 2-dihydrodiol in the presence of NAD+displayed an absorbance ca 450 nm and their spectra were essentially identical to those of 1, 2-naphthoquinone-protein adducts. Similar spectra were also obtained from proteins isolated from the intact rat lens after in vitro incubation in medium containing naphthalene-1,2-dihydrodiol. The spectra obtained from lens proteins incubated with 1, 2-dihydroxynaphthalene were distinct from those of either naphthalene-1,2-dihydrodiol or 1,2-naphthoquinone. Aldose reductase inhibitors possessing either hydantoin or carboxylic acid groups prevented protein modification induced by naphthalene-1, 2-dihydrodiol but not protein modification induced by 1, 2-dihydroxynaphthalene or 1,2-naphthoquinone. Therefore, the metabolite formed from naphthalene-1,2-dihydrodiol by aldose reductase is 1,2-naphthoquinone. Lens proteins modified by naphthalene-1,2-dihydrodiol appear essentially identical to protein adducts formed with 1,2-naphthoquinone and their formation can be prevented by both hydantoin and carboxylic acid containing aldose reductase inhibitors., (Copyright 1999 Academic Press.)

The relationship between sugar cataract formation and radical production was investigated. The in vivo formation of free radicals in the lenses of rats fed on a diet containing 25% and 50% galactose was studied using the electron spin resonance (ESR) spin trapping method. Effects of treatment with aldose reductase inhibitor (ARI) SNK-860 on free radical formation were determined in 25% and 50% galactose fed rats. Hydroxyl radical (*OH) adduct of the spin trap 5,5'-dimethyl-1-pyrroline- N -oxide (DMPO) was directly detected in the superficial cortical cataract obtained from 25% and 50% galactose-fed rats. *OH production was completely inhibited by ARI SNK-860 in both galactose groups. Polyol accumulated in rat lenses given 50% galactose with a peak within the first 2 weeks, and was significantly inhibited by SNK-860. The increase in *OH production was considered with the polyol accumulation in both galactose groups. The dose of SNK-860 to inhibit *OH by 50% level was estimated at 3 m by the method of kinetic competition in vitro experiment. SNK-860 is not an effective *OH scavenger compared to other *OH scavengers. The results of the present study suggest that *OH is indirectly inhibited by SNK-860 resulting from decreasing polyol and *OH formation is related to sugar cataract formation in early stages, possibly via the Fenton reaction involving H2O2 produced from the activated polyol pathway. We suppose that *OH may accelerate damage to the cell membrane of lens fibers resulting from polyol accumulation *OH may play an important role in the early stage of sugar cataract process., (Copyright 1999 Academic Press.)

Increased iris vessel permeability observed in diabetics has also been reported to occur in diabetic animals and galactose-fed rats. The potential role of aldose reductase in the induction of iris vessel changes has been investigated in rats fed a 50% galactose diet with/without the aldose reductase inhibitors AL 1576, sorbinil or ponalrestat for 7 to 18 months. Compared to normal control rats, long-term galactose-fed rats display a breakdown of the blood-aqueous barrier due to iris vessel changes that include focal straightening, dilation, constriction, increased permeability, ischemia and new vessel proliferation. The onset and progression of these iridal vessel changes were prevented by the aldose reductase inhibitors AL 1576 and sorbinil, and reduced by Ponalrestat. Computerized analyses of lumen areas of iris vessels indicated an 18-fold decrease in the vascular area near the pupillary boarder in untreated galactose-fed rats compared with age-matched controls and galactose-fed rats treated with aldose reductase inhibitors. These observations linking iris vessel changes with galactose-feeding, coupled with the fact that aldose reductase inhibitors also prevent these changes, strongly suggest a link between the sorbitol pathway and the appearance and progression of iris vessel changes., (Copyright 1999 Academic Press.)

Several recent studies with the sorbitol dehydrogenase inhibitors 4-[4-(N,N-dimethylsulfamoyl)-piperazino]-2-methylpyrimidine, SDH-1, and its active metabolite 4-[4-(N, N-dimethylsulfamoyl)piperazino]-2-hydroxymethylpyrimidine , SDH-2, suggest that inhibition of sorbitol dehydrogenase may be beneficial in delaying the onset of diabetic complications due to their ability to ameliorate redox changes associated with polyol metabolism. To compare the relative importance of sorbitol dehydrogenase versus aldose reductase inhibition on sugar cataract formation, cataract formation was monitored in 50% galactose-fed and diabetic rats treated with/without the sorbitol dehydrogenase inhibitors SDH-1 or SDH-2 or the aldose reductase inhibitors AL 1576 or Ponalrestat. For these studies, diabetes was induced in young 50 g rats with streptozotocin while galactosemia was produced by feeding a diet containing 50% galactose. Inhibitors were administered in the diet with the diet containing 0.06% (w/w) of the sorbitol dehydrogenase inhibitors or Ponalrestat, and 0.0125% (w/w) of AL 1576. Cataract formation was monitored by hand-held slit lamp and polyol levels were measured by gas chromatography. Sugar cataract formation was accelerated in diabetic rats treated with sorbitol dehydrogenase inhibitors while no difference in cataract formation was observed in galactose-fed rats treated with/without SDH inhibitors. Cataract formation was inhibited in both diabetic and galactosemic rats by either Ponalrestat or AL 1576. These results support the concept that sugar cataract formation is initiated by the aldose reductase catalysed intracellular accumulation of polyols and that these sugar cataracts can be prevented through inhibition of aldose reductase., (Copyright 1998 Academic Press.)

Structural impairments of optic nerve fibers in the streptozotocin-induced diabetic rat were investigated using morphometric analysis. The effect of aldose reductase inhibitor (ARI) on abnormalities in myelinated nerve fibers was also evaluated. Three months after the induction of diabetes, loss of body weight and significantly elevated levels of serum glucose were observed. Light microscopic examination revealed that the mean size of the optic nerve in the diabetic rat remained unchanged. Electron microscopic morphometry showed the significantly smaller cross-sectional size of axons and myelin but no change of myelinated fiber number. Reductions of myelinated fiber size was especially remarkable in the larger fibers. ARI treatment improved structural abnormalities without any changes in body weight and blood glucose level. Reduction of axon size and myelin/axon ratio was completely inhibited by ARI treatment. These findings suggest that structural impairment may contribute to the abnormalities of psychophysical and electrophysiological measurements detected in diabetes. Moreover, ARI treatment, which can improve the polyol metabolism, may have a beneficial effect on optic nerve impairment in diabetes., (Copyright 1998 Academic Press Limited.)

Sugar cataracts rapidly develop in dogs fed a diet containing 30% galactose. While studies on the formation and progression of these sugar cataracts suggest that they are osmotic in nature and are linked to aldose reductase, sugar cataract formation in the dog to date has not been completely prevented by the administration of aldose reductase inhibitors sorbinil and M79175. To demonstrate that the formation and progression of sugar cataracts in galactose-fed dogs can be dose-dependently inhibited by the administration of aldose reductase inhibitors, 9-month old male beagles were placed on diet containing 30% galactose with/without 10 or 16 mg kg-1 day-1 of M79175 for up to 39 months. Cataract progression in all dogs was followed by periodic slit lamp examination and documented by retroillumination photography. Although large variations in cataract formation and progression were observed, all dogs fed a 30% galactose diet for 39 months developed cataracts. Lens changes were significantly less in galactose-fed dogs treated with either 10 or 16 mg kg-1 M79175 and no cataract formation was observed in 3 of 6 galactose-fed dogs treated with 16 mg kg-1 M79175. These observations confirm that aldose reductase plays a key role in initiating cataract formation in galactose-fed dogs and that cataract formation can be prevented by adequate inhibition of aldose reductase., (Copyright 1998 Academic Press Limited.)

In the past, almost all studies on naphthalene cataract were based on in vivo experiments. Such studies are laborious and time-consuming and are complicated by systemic toxicity arising from the metabolites of naphthalene. In order to study the direct effects of naphthalene metabolites on the lens, we established an in vitro 'naphthalene cataract' model system by exposing rat lens to naphthalene dihydrodiol (2.5 x 10(5) M) containing medium for 48 hr. Under these conditions, we analysed several biochemical parameters including the glutathione level, protein mixed disulfides, protein patterns on SDS-gels, active transport, NA+/K(+)-ATPase activities and the measurement of naphthalene metabolites in the cultured lenses. The results showed that both the morphological and biochemical changes were very similar to those observed in lenses of rats fed naphthalene (1 g kg-1 day-1). Furthermore, ALO1576 completely blocked the in vitro changes as it did in vivo. Therefore, this model system can be used as a new tool to investigate the mechanism of naphthalene cataract formation. Other naphthalene metabolites such as 1-naphthol, 2-naphthol, 1,2-dihydroxynaphthalene and 1,2-naphthoquinone were also studied in vitro and the results showed that the effects of these naphthalene metabolites were very different from those observed in naphthalene cataracts in vivo.

The naphthalene-induced cataract in rats has been studied for many years as a possible model of human aging-related cataract. While the molecular mechanism of this cataract is unclear, it has recently been demonstrated that the aldose reductase inhibitor ALO1576 can prevent lens opacification in this system. The present study was undertaken to investigate the molecular basis for the effects of naphthalene on the lens and the role of pigmentation in the cataractogenic mechanism. Cataracts were induced in five strains of rats (two pigmented, three albino) by oral administration of naphthalene. Initial lens changes were observed after 1 week by slit-lamp; by 3 weeks a distinct shell-like opacity was present in the deep cortex. Little difference in the course of opacification was found between the pigmented and albino strains. Major biochemical effects were a decrease of 20-30% in glutathione (GSH) by 1 week of feeding, disulfide cross-linking of lens proteins present by 3 weeks, and a nearly 20-fold increase in the content of protein-GSH mixed disulfide. No effect was seen in the ability of the affected lenses to accumulate activity [3H]choline or 86Rb from the medium in organ culture nor in the activity of the Na+/K(+)-ATPase. ALO1576 (10 mg kg-1 day-1) completely prevented all morphological and biochemical changes in the lenses of the naphthalene-fed rats in both pigmented and non-pigmented strains. These results indicate that pigmentation is not required for induction of naphthalene cataract in rats. Naphthalene dihydrodiol was found in the aqueous humor and lens of naphthalene-fed rats. It is proposed that naphthalene dihydrodiol produced in the liver reaches the aqueous humor and penetrates the lens where it is further metabolized ultimately to form the toxic species, naphthoquinone.

Sorbitol-3-phosphate (S3P) and fructose-3-phosphate (F3P) are novel phosphorus compounds recently discovered and identified in the crystalline lens as well as other tissues. These phosphates increased with diabetes progression in streptozotocin-diabetic rat lenses. Treatment of these rats with an orally administered aldose reductase inhibitor eliminated S3P and intramuscularly injected insulin obliterated F3P. These results indicate that enzymes catalyzing S3P and F3P formation co-activate with aldose reductase activation and hyperglycemia, respectively.

Our previously reported investigations showed that cataracts could be induced in fetal lenses through the maternal feeding of galactose during pregnancy. We also reported that the lens opacity present at birth reverses completely by 30 days of age if there is no further post-natal exposure to galactose. This investigation was designed to investigate if an aldose reductase inhibitor (ARI) has any cross-placental effect in preventing galactose-induced cataracts in fetuses. We have also evaluated if there are any milk-mediated effects of galactose on cataract induction and of galactose and ARI on the maintenance or reversal of opacities induced in utero. Pregnant Sprague-Dawley rats were fed either 50% galactose or rat chow with or without the ARI, (2R,4S)-6-fluoro-2-methyl spirochroman-4,4'-imidazolidine-2',5'-dione (Eisai compound E-0722). Following parturition, pups of mothers from the different dietary groups were either fed by their own mothers or foster fed by lactating females fed either rat chow or 50% galactose with or without the ARI. Lenses of the pups were examined at desired intervals with light and scanning electron microscopes. We observed that: (a) both galactose and the ARI had a cross-placental effect on the fetal lenses in the development and inhibition of cataracts, respectively; (b) galactose had very little, if any, milk-mediated effect on either the induction of cataracts in newborn pups that were born with transparent lenses or the maintenance of cataracts induced in utero; (c) the ARI appeared to have a milk-mediated effect, which accelerates the reversal of cataract associated alterations in lenses of pups with cataracts induced in utero, leading to further reinstatement of lens transparency; and (d) the presence of ARI in the diet of rats during pregnancy and/or post-parturition provided continued protection to the lenses of pups that were exposed to a galactose diet following birth.

Human lens epithelial (HLE) cells in tissue culture accumulated significant levels of galactitol when they were cultured for 72 hr in medium containing 30 mM D-galactose. Polyol accumulation was accompanied by the appearance of vacuoles as seen by transmission electron microscopy. The number and size of intracellular vacuoles increased when the culture period was extended to 7 days. In addition, polyol accumulation was accompanied by loss of myoinositol. None of these changes occurred in cells exposed to 30 M L-galactose which is not a substrate for aldose reductase. The accumulation of galactitol, intracellular vacuole formation and loss of myoinositol observed in D-galactose-exposed cells were prevented by the inclusion of the aldose reductase inhibitor, sorbinil, in the culture medium. Comparison of the relative efficacies of two aldose reductase inhibitors indicate that AL 1576 is nearly 20 times more potent than sorbinil in inhibiting the human lens enzyme. It is concluded that vacuole formation in HLE cells is due to the osmotic effect of polyol formation brought about by the action of aldose reductase and that the etiology of human diabetic cataract may also involve the polyol pathway.

Crystallin-bound non-tryptophan fluorescence and protein cross-linking were studied in chronic galactosemia. Fluorescence (excitation/emission--370/440 nm) was significantly higher in galactosemic rats compared to controls (P less than 0.001). Accumulation of fluorescence was significantly reduced both in water soluble (P less than 0.001) and insoluble (P less than 0.005) lens fractions in galactosemic rats receiving the aldose reductase inhibitor sorbinil. High performance liquid chromatography of water soluble lens crystallins showed an increase in the content of high molecular weight proteins and the fluorescence associated with it. Sorbinil partly prevented the formation of such fluorescent high molecular weight proteins. Under reducing conditions, sodium dodecyl sulfate polyacrylamide gel electrophoresis of water soluble proteins revealed a distinct high molecular weight protein of 60 kDa in galactosemia. Sorbinil treatment completely abolished the formation of this protein. Western blotting using rabbit antiserum to bovine alpha-, beta- and gamma-crystallins revealed the presence of gamma, but not alpha- and beta-crystallins in the 60-kDa protein. Formation of this protein may result from trimerization of gamma-crystallins or from an association of gamma-crystallin with a non-crystallin cytosolic protein or mixed protein cross-linking of gamma-crystallins with membrane protein(s). The present study shows that polyol pathway in cataractogenesis also extends to protein cross-linking and formation of fluorescent compounds, the nature of which remains to be elucidated.

The onset and progression of diabetic-like retinopathy has been investigated in age-matched male beagle dogs fed a 30% galactose diet. Examination of the intact retinal vessels, isolated by gentle trypsin digestion, reveals that the destruction of pericytes to form pericyte ghosts is the earliest observable retinal vessel change. This results in an irregular distribution of endothelial cell nuclei near pericyte ghosts and the subsequent formation of acellular capillaries containing neither endothelial cells or pericytes. This was followed by the histological appearance of microaneurysms and later, by the funduscopic appearance of intraretinal hemorrhages. Studies in similar galactose-fed dogs treated with aldose reductase inhibitors indicate that all of these changes are linked to the initial aldose reductase associated destruction of pericytes.

The presence of several NADPH-dependent reductases has been observed in the dog lens. Applying the purification procedures of gel filtration, affinity chromatography and chromatofocusing to dog lens homogenates resulted in the purification of aldose reductase. This enzyme appeared similar to dog kidney aldose reductase in molecular weight, isoelectric point, kinetic properties, and susceptibility to inhibition by aldose reductase inhibitors. Evidence for the presence of trace amounts of aldehyde reductase in the dog lens was also observed, although this enzyme is not present in sufficient quantities for isolation and characterization. The presence of a labile third enzyme that is immunologically distinct from either aldose reductase or aldehyde reductase was also detected. This enzyme utilizes only glyceraldehyde as substrate and is not inhibited by aldose reductase inhibitors.

In addition to the results of our clinical trial of epalrestat in diabetic retinopathy (the open study), the current progress in the clinical trials of aldose reductase inhibitors for the 'triopathy' of complications (neuropathy, retinopathy and nephropathy) in Japan is reported. No data from the placebo-controlled double-blind studies in Japan are shown because a detailed analysis of the effects of epalrestat on diabetic neuropathy and retinopathy is now under way. However, it must be stressed that in the phase III placebo-controlled double-blind studies in neuropathy and retinopathy, epalrestat was effective.

Lens hydration was monitored by laser Raman spectroscopy in WBN/Kob rats which form spontaneous diabetes at about 1 yr of age and develop cataracts approximately 6 months after the onset of diabetes. Lens hydration in response to chronic hyperglycemic stress in these rats appeared predominantly in the cortical region while the hydration state in the nuclear region was fairly preserved. The 9-month treatment of similarly diabetic rats with the aldose reductase inhibitor Ponalrestat sufficiently suppressed lens hydration in the cortical and nuclear regions.

Following the identification of potent, orally active inhibitors of aldose reductase, the next challenge comes in demonstrating clinical utility in the treatment of diabetic complications. It is not feasible to conduct pivotal studies in all indications within the same trial and neuropathy evolved as the lead indication. There was little precedent for clinical trials in any diabetic complication and one of the challenges has been the derivation of standardized methodology for quantifying changes. In order to establish the value of any intervention in a chronic disease, it is necessary to demonstrate sustained benefit in long-term trials. This has been complicated by the absence of quantitative prospective data on the natural history of the disease process. In well-controlled clinical trials, patients are likely to improve their diabetic control which will have an additional effect over and above the usual placebo effect seen in any trial. In the absence of any standard therapy, the definition of clinical as opposed to statistical significance of any effect needs to be established. In the case of neuropathy this is further complicated by the heterogeneity of the disease and questions over the relevance of small changes in electrophysiology as an index of improvement in histopathology. The majority of the animal studies have concentrated on prevention of changes rather than reversal. However, in diabetics, complications develop slowly and significant tissue damage has occurred before clinical signs can be detected. To date, all clinical studies have concentrated on treatment of complications. The dose of ARI chosen for such studies has been based on the surrogate of erythrocyte aldose reductase activity confirmed by short-term studies on disease reversal.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent evidence obtained from both galactosemic dogs and rats indicated that aldose reductase inhibitors could prevent several capillary lesions which were similar to those typical of diabetic retinopathy in humans. The present study demonstrates that diabetes-like histopathological changes in the intact retina, which were not visible in the vessel whole mounts used previously, can also be prevented. Sprague-Dawley rats were fed diets containing 50% galactose with or without an aldose reductase inhibitor (tolrestat). After 28 months of galactose feeding, the findings from retinal transections examined by light and electron microscopy were consistent with reports on vessel whole mounts, but showed several additional changes. There was a marked increase in the thickness of the retinal inner limiting membrane, as well as in the capillary basement membranes. There was extensive gliosis and disruption of retinal layers as well as pericyte degeneration, endothelial cell proliferation, accellularity, capillary dilation, and microaneurysm formation. The contents of pericyte compartments in the capillary wall were often replaced with non-pericyte cytoplasm, which appeared glial cell-like. Many of the lumens of acellular capillaries were occluded with debris from degenerating endothelial cells or with cytoplasm possibly originating from glial cell processes. Structures suggestive of degenerated microaneurysms were present mainly in the inner nuclear and outer plexiform layers. The microaneurysms and other major changes were limited to the central and paracentral retina. All these retinal lesions were prevented with orally administered tolrestat. Thus, long-term galactosemia in rats induced histopathologically visible angiopathies, simulating those occurring in background diabetic retinopathy in humans, and these were prevented by treatment with an aldose reductase inhibitor.

Biochemical alterations in the composition of retinal capillary basement membrane components were investigated in galactosemic rats, an animal model that develops basement membrane lesions comparable to those of diabetic retinopathy. Normotensive Wistar-Kyoto rats fed a 30% galactose diet for 9 months developed significant thickening of retinal capillary basement membranes by comparison with animals fed a control test diet (P less than 0.001), or animals on a diet containing 30% galactose and 250 mg kg-1 of the aldose reductase inhibitor sorbinil (P less than 0.001). A quantitative electron microscopic immunogold technique applied on ultrathin sections of the retinas of these animals showed that the labeling densities of collagen type IV and laminin per unit cross-sectional area (which is presumably proportional to the concentrations of these molecules) were significantly increased in the retinal capillary basement membranes of galactose-fed rats, compared with animals on the control test diet. Increases in these two components of basement membranes were prevented by addition of sorbinil to the diet. However, there was no significant change in the labeling density of heparan sulfate proteoglycan (HSPG) core protein in the basement membranes of galactose-fed rats in comparison to animals on either the control diet or galactose-sorbinil diet. Two types of striated fibrillar materials were frequently found in areas of focal thickening of basement membranes of galactose fed rats only. Thinner fibrils reacted strongly with collagen type III antibody, whereas thicker fibrils reacted weakly with collagen type I antibody. Our results indicate that there is an increase in labeling densities of collagen type IV and laminin in thickened basement membranes of retinal capillaries of galactosemic rats along with the expression of interstitial collagens like collagen type III and an abnormal collagen that weakly cross-reacts with antibody to collagen type I, and these effects of galactosemia on the basement membranes are preventable by an aldose reductase inhibitor.

Unlabelled: To investigate the aetiology of complications secondary to experimental diabetes in the rat, the concentrations of glucose, sorbitol, fructose, fructose-1-phosphate, lactate and inositol were measured in the retina and sciatic nerve of streptozotocin-diabetic and normal rats treated for 22 days with ICI 105552 (1-(3,4-dichlorobenzyl)-3-methyl-1,2-dihydro-2-oxoquinol-4-ylacetic acid, sodium salt; 50 mg/kg body wt daily), an inhibitor of aldose reductase (E.C. 1.1.1.21) the first enzyme of the sorbitol pathway. In the diabetic nerve, where accumulation of sorbitol may be pathogenic, treatment with ICI 105552 reduced the accumulations of sorbitol (70%), fructose (47%) and lactate (34%) without affecting glucose, fructose-1-phosphate or inositol. In the nerves of controls, the inhibitor reduced both sorbitol (23%) and fructose (20%) levels without other effects. In the diabetic retina where accumulation of sorbitol or lactate might be pathogenic, treatment with ICI 105552 had no statistically significant effect upon the concentrations of glucose, sorbitol, fructose, fructose-1-phosphate or inositol. However, the inhibitor reduced the concentration of lactate to below the non-diabetic level. In the retinas of controls, dosage with ICI 105552 reduced the concentration of sorbitol by 36% without other effects., The Results: (1) demonstrated that ICI 105552 was a potent inhibitor of aldose reductase in sciatic nerve and suggested that a proportion of nerve lactate in diabetes could result from sorbitol pathway activity; (2) implied that flux through the sorbitol pathway, eventually to form lactate, increases in diabetic retina; and (3) indicated, by the drug's reduction of retinal lactate concentration, that inhibitors of aldose reductase might be of potential use in diabetic retinopathy.

Nuclear magnetic resonance (NMR) spectroscopy has been utilized in the study of the metabolism of intact, functioning rabbit lenses maintained in organ culture. The sorbitol pathway and aldose reductase inhibition have been studied using carbon-13 NMR spectroscopy. Incubation of lenses in high concentration [1-13C] glucose medium with and without added inhibitors allows the sorbitol pathway and glycolysis to be monitored. Various aldose reductase inhibitors have been studied and are ranked based on percentage of inhibition as follows: tolrestat greater than or equal to sorbinil greater than sulindac greater than sulindac sulfide much greater than indomethacin greater than acetylsalicylic acid greater than quercetin greater than tandearil greater than salicylic acid greater than 3,3-Tetramethyleneglutaric acid (TMG). It has been demonstrated that 13C NMR spectroscopy provides an effective method of screening potential inhibitors of aldose reductase. The aspirin substitutes ibuprofen and acetaminophen have been studied and are found to reduce sorbitol accumulation in intact rabbit lenses. The effects of myo-inositol and vitamin E on sorbitol accumulation have also been investigated. Results suggest that the various metabolic pathways within the lens are intricately connected. In a preliminary manner, the effect of diabetes on metabolism in intact lenses has been investigated using 13C NMR spectroscopy. Increased sorbitol production has been observed for diabetic lenses. 31P NMR spectroscopy has also been utilized in the study of lens metabolism and aldose reductase inhibitors. Inclusion of various inhibitors in the high concentration glucose medium results in maintenance of essentially normal phosphorus-containing metabolite levels in the lens. No clear relationship was observed between lens clarity and phosphorus metabolite levels as determined using NMR.

Previous cytochemical and biochemical studies have shown an increase in the activity of acid phosphatase and arylsulfatase during the induction of galactose cataracts in rat lenses. It was postulated that these enzymes may be involved in lens fiber degradation observed during cataractogenesis, however, the role of these enzymes in the repair process was not ruled out. The present investigation has evaluated the level of acid phosphatase activity in lenses in which the induction of opacity is inhibited with the aldose reductase inhibitor sorbinil and during the recovery of galactose induced opacity. Sprague-Dawley rats received 50% galactose diet, or galactose diet with sorbinil, or laboratory chow diet. Following 20 days on this diet all rats received lab chow plus 50 mg kg-1 sorbinil (recovery diet). The lenses were removed at desired intervals following the initiation of the above three diets and following the transfer of animals to the recovery diet. Cytochemical localization and biochemical quantitation of acid phosphatase activity were performed with methods previously reported. Most of the enzyme activity was localized within the epithelial cells and superficial cortical fibers. In the epithelial cell layer, the enzyme activity was primarily localized in lysosomes and at extracellular sites near the epithelial cell membrane which abut each other and cortical fibers. In cortical fibers the enzyme activity was observed at various extracellular sites between the cell membranes of neighboring fibers. The effect of sorbinil, if any, and the possible role of acid hydrolases in the repair process during cataract reversal is discussed.

Polyol accumulation and myo-inositol depletion were accompanied by extensive vacuole formation in cultured canine lens epithelial cells that were incubated for up to 96 hr in growth medium supplemented with 30 mM D-galactose or 30 mM D-glucose. These changes did not occur in cells incubated in a hypergalactosemic or hyperglycemic medium which also contained an aldose reductase inhibitor (20 microM sorbinil). In addition, these changes were not observed in lens cells incubated in growth medium supplemented with either 30 mM mannitol, which is known to enter cells only slowly, or in 30 mM L-galactose, which is not a substrate for aldose reductase. The vacuoles were visible at the ultrastructural level after 6 hr of incubation in 30 mM D-galactose and increased in both number and size with time. These vacuoles had a unique fine structure. They did not result from swelling of mitochondria or other cell organelles. As demonstrated cytochemically, they did not represent either lysosomes or Golgi saccules. The proliferation pattern of cells incubated with 30 mM D-galactose was clearly different from that of control cells, but approached normal when an aldose reductase inhibitor was added to the incubation medium. Together these findings suggest that vacuole formation and altered cell proliferation were caused by polyol accumulation and/or myo-inositol loss, both of which result from aldose reductase activity.

Depletion of lens glutathione (GSH) occurs quickly and drastically following induction of diabetes or galactosemia in rats as well as in lens culture. The explanation for this dramatic loss of GSH has been investigated by many laboratories but the solution has been elusive. There are several possible causes for the change in the reducing power of the lens under hyperglycemia. (a) The enzyme glutathione reductase which reduces oxidized glutathione to GSH is inhibited. (b) The cofactor NADPH which both the aldose reductase of polyol pathway and glutathione reductase require becomes depleted under hyperglycemia to the point that there is an insufficient amount for glutathione reduction. (c) Membrane permeability is increased, due to osmotic-induced lens hydration. We explored all the above possibilities in the mechanism of GSH depletion and studied the effect of aldose reductase inhibitor (ARI) on osmotic change. We found that under hyperglycemic condition, there was no change in the enzyme glutathione reductase activity. There was an initial drop in NADPH level but there was sufficient remaining for glutathione reductase use. Both NADPH and glutathione depletion could be prevented completely by ARI. In addition, ARI could also prevent any hyperglycemic-induced abnormal transport and leakage of amino acids. We have therefore concluded that only the decreased membrane transport of amino acids which are needed for glutathione biosynthesis and the simultaneous loss of GSH through leaky membrane as initiated by the polyol pathway can be responsible for the drastic GSH depletion.

Weanling male Sprague-Dawley rats were given a 50% galactose diet with or without an aldose reductase inhibitor (0.04% tolrestat, Ayerst) for 88 weeks. Quantitative computer planimetry on electron micrographs of perpendicularly transected retinal capillaries showed mean basement membrane thicknesses to be 263 +/- 50 nm in galactose-fed rats, 153 +/- 10 nm in galactose-fed rats given tolrestat, and 114 +/- 16 nm in rats given control feed. The retinal capillary basement membrane thickening in galactose-fed rats was accompanied by other ultrastructural alterations mimicking changes observed in diabetic microangiopathy, such as multi-lamination and the formation of vacuoles and dense inclusions. The results extended previously reported (44-week) findings, showed greater galactose-induced changes in capillary basement membranes, and demonstrated the preventative effect of an aldose reductase inhibitor.

Intact paired rabbit lenses were incubated in media containing 5.5 mM and 35.5 mM glucose (both at 290 +/- 3 mOsm) and examined by phosphorus-31 nuclear magnetic resonance spectroscopy. Lenses in 35.5 mM glucose exhibited an altered metabolic steady-state characterized by elevated alpha-glycerophosphate and depressed adenosine triphosphate concentrations. Time course studies revealed that these metabolic changes occurred chiefly during the initial 48 hr of incubation under high glucose conditions. The inclusion of an aldose reductase inhibitor in the medium prevented these changes in lenticular metabolism.

Rats were maintained on a 50% galactose diet with and without the aldose reductase inhibitor, Sorbinil, or on a normal diet of rat chow, and the retinas and retinal pigment epithelium were examined ultrastructurally at time points ranging from 4 weeks to 20 months. Several ultrastructural changes were observed in the retinal pigment epithelium and retinal capillaries of galactosemic rats that were not seen in rats on a normal diet. Only some of these changes are similar to those that have been previously noted in diabetic (glucosemic) rats. As in diabetics, galactosemic rats demonstrated thickening of the basal laminae of the retinal pigment epithelium and retinal capillaries. They also manifested vacuolization and degenerative foci in the retinal pigment epithelium that were similar, but not identical, to changes seen in diabetic rats. Each of these changes was significantly inhibited by Sorbinil. Unlike diabetics, galactosemic rats did not have dilated basal infoldings, but did have outer retinal folds and a significant increase in large-lipofuscin-like aggregates in the retinal pigment epithelium, both of which were partly prevented by Sorbinil. These data suggest that multiple mechanisms may be involved in retinal and retinal pigment epithelium changes in diabetic and galactosemic rats, and that enhanced polyol metabolism is likely to be involved in some of the changes in both.

Two studies to investigate the penetration of the aldose reductase inhibitor, Sorbinil, were conducted. In the first study, 24 diabetic patients undergoing intracapsular extraction were randomised into three groups on a double masked basis. In the week immediately preceding the operation, all patients were requested to take two capsules daily before breakfast. Each capsule contained 200 mg Sorbinil, 100 mg Sorbinil, or a placebo. On measuring Sorbinil levels in lens, plasma and erythrocytes using HPLC, three clearly defined groups of patients were obtained. In one group no Sorbinil was detected, in the second group there were moderate levels of Sorbinil, while the third group had significantly higher levels of Sorbinil. The ratio of erythrocyte/plasma Sorbinil was 0.225, while the ratio for lens/plasma was 0.7, for both groups where Sorbinil was detected. In a second study, 20 patients were treated topically with a single dose of 0.5 mg ophthalmic Sorbinil at times ranging from 0-14 hr preoperatively. Sorbinil was detected in both lens and aqueous. Transport into the lens was complete within about 2 hr, and although aqueous levels were negligible after 6 hr, Sorbinil persisted up to 14 hr in the lens. Three out of 16 patients taking Sorbinil orally developed a maculopapular rash with pyrexia approximately 8 days after commencing the drug. No side effects were noted in any patients given the topical ophthalmic preparation.

Phosphorylcholine (P-choline) is a precursor of the phospholipids in the lens membrane. A human lens normally contains approx. 1 mM P-choline but this is significantly lowered in some cataractous lenses. A normal rat lens contains a very high concentration (11 mM). We found that rat lens P-choline was depleted drastically when the lenses were exposed to hyperglycemic conditions either in culture, with galactose or xylose, or in vivo by streptozotocin-induced diabetes. The lens P-choline level was measured by fractionating the organic phosphates in the lens homogenate using an ion exchange column, or by quantitating the P-choline 31P NMR intensity in intact lenses. The results of both the chemical method and the noninvasive method agreed remarkably well. Besides the change in P-choline, the choline influx was also drastically reduced both in lenses from diabetic rats and in lenses incubated with 30 mM xylose. In addition, the ATP concentration was greatly diminished under similar conditions. The changes in P-choline, choline, and ATP could all be prevented in the presence of an aldose reductase inhibitor (ARI). It is thus concluded that these changes in phospholipid precursors may result from lenticular membrane defects caused by hyperglycemic stress. The effect of the lowered precursors on lipid biosynthesis was observed, and surprisingly showed a more rapid phospholipid-biosynthesis in the 2-week diabetic rat lens than in the 3-day diabetic rat lens.

In diabetes, glucosylation of the Na,K-ATPase of the lens epithelium makes the pump inefficient. K+ transport and ATP hydrolysis (at near saturating ATP concentrations) are inhibited and the kinetics of ATP hydrolysis become substrate inhibition type. The AR inhibitor (AL1576, Alcon Laboratories) stimulates K+ transport and ATP hydrolysis by glucosylated bovine lens Na,K-ATPase. This inhibitor has a slight stimulatory effect upon the unmodified enzyme function also. The AR inhibitor is not able to prevent glucosylation of the pump in high-glucose-containing medium.

Lens ultrastructure and Na- K-ATPase activity in the lenses of rats fed galactose and a galactose + sorbinil diet (aldose reductase inhibitor) were studied. Lenses of rats on the galactose diet exhibited development of peripheral opacity within 3-4 days. This opacity progressed with the continuation of the galactose feeding, and by 20 days mature cataracts were observed in these animals. The formation of vacuoles, cysts, membrane disruption in the epithelium and fibers, and swelling of fibers accompanied the development of opacity. With the progression of opacity there was a considerable drop in lens Na- K-ATPase activity in the galactose-fed animals. However, the lenses of rats that were treated with sorbinil did not show any of the alterations in the ultrastructure of the epithelium and fibers that accompany galactose cataractogenesis. The level of Na-K-ATPase activity in the sorbinil-treated animals was similar to that found in lenses from the laboratory chow-fed group of rats. These observations further substantiate the role of aldose reductase in sugar-cataract development.

P31 NMR spectroscopy was used to study dynamic changes in rat lens phosphate metabolites under hyperglycemic conditions. NMR control spectra were collected at 37 degrees C with normal media; NMR spectra were also collected under the same conditions except the normal media was substituted for one containing 30 mM xylose. There was a significant increase in intensity of the sugar phosphate NMR signal as well as a moderate decrease in intensity for the three ATP peaks. In separate experiments normal rat lenses were pre-treated with perfusion media containing the ARIs; either Spiro-(2-fluorofluorene-9,4'-imidazolidine)-2',5'-dione (AL theta 1567) or Spiro-(2,7-difluorofluorene-9,4'-imidazolidine)-2',5'-dione (AL theta 1576) for three hours before changing to a xylose plus ARI media. After overnight perfusion, no appreciable change in the rat lens NMR spectra was found. This NMR observation indicates that lenses can be protected from hyperglycemic stress in the presence of the ARIs AL theta 1567 or AL theta 1576.

The absolute rate of collagen synthesis by cultured bovine retinal capillary pericytes, determined using the specific radioactivity of proline in the cellular amino acid pool, was compared in media containing different concentrations of glucose (5, 10 or 40 mM) and Sorbinil (0.0 or 0.1 mM), an inhibitor of aldose reductase. The absolute rate of collagen synthesis, in proline molar terms, by pericytes in medium with 5 mM glucose was 3.3 +/- 1.9 (S.D.) pmol 10(-7) cells 24 hr-1, and increased significantly to 8.8 +/- 3.7 pmol 10(-7) cells 24 hr-1 when the glucose concentration was increased to 40 mM. Sorbinil (0.1 mM) reduced the elevated sorbitol contents of pericytes induced by high concentrations of glucose, but did not significantly change the absolute rate of collagen synthesis per cell.

To test the possible involvement of the sorbitol pathway in the pathogenesis of retinopathy in long-term experimentally-diabetic rats, streptozotocin-diabetic and normal rats were dosed orally (50 mg/kg body weight daily) for up to 373 days with an aldose reductase inhibitor (ICI 105552) or a placebo. Long-term treatment with ICI 105552 (1,(3,4-dichlorobenzyl)-3-methyl-1,2-dihydro-2-oxoquinol-4-ylaceti c acid; sodium salt) markedly reduced the normal accumulations of sorbitol and fructose in the sciatic nerves (86 and 69% reductions) and seminal vesicles with coagulating glands (75 and 49% reductions). Thus, by these criteria, the inhibitor was as effective after several months of dosing as after three weeks. There was no evidence that treatment with this aldose reductase inhibitor had any protective effect against the development of pathological changes in the retina and kidney of these rats.

Both phosphoinositidase (PIase) and individual species of inositol phospholipid (IPL) of bovine retinal capillary pericytes (BRCP) were quantitatively determined. When glucose in growth medium was increased from 5- to 15- or 30 mM, PIase activity was attenuated to 82% or 55%, respectively. In contrast, when glucose (5-, 15-, 30 mM) was added to an enzyme extract from cells grown in the standard growth medium (5 mM glucose, 0.04 mM myo-inositol) the PIase activity was not changed, indicating that the reduced PIase activity was not due to the direct effect of glucose. When IPLs from BRCP were analysed by HPLC and TLC, we observed reduction of the total and newly formed IPLs including the substrate of PIase. Phosphatidylinositol bisphosphate (PIP2). Reduced levels of IPLs were associated with a decrease in myo-inositol and an increase in sorbitol. The changes in IPL metabolism were reversed by adding either free myo-inositol or AL1576, an aldose reductase inhibitor (ARI), to the high-glucose medium. However, the addition of myo-inositol to the growth medium with a standard concentration of glucose only caused a marked increase in phosphatidylinositol, but not in PIP or PIP2, while the supplement of AL1576 in the standard medium did not cause any changes in IPL formation. These findings suggest that the alteration in IPL metabolism in BRCP may be related to insufficient myo-inositol or activated sorbitol pathway under high-glucose conditions. Further explanation of the role of the altered hydrolysis of PIP2 triggered by PIase may provide clues to understanding of the mechanism of decreased pericyte viability in the presence of high glucose concentrations.