|Molecular Vision 2001;
Received 23 May 2001 | Accepted 16 July 2001 | Published 18 July 2001
Synthesis and evaluation of novel aldose reductase inhibitors: Effects on lens protein kinase Cg
S. Lewis,1 J. Karrer,1 S. Saleh,1 Y.
Chen,2 Z. Tan,2 D. Hua,2 J.
McGill,2 Y.-P. Pang,5 B. Fenwick,3 A.
Brightman,4 D. Takemoto1
Departments of 1Biochemistry, 2Chemistry, 3Pathology, and 4Clinical Sciences, Kansas State University, Manhattan, KS; 5Department of Pharmacology, Mayo Clinic, Rochester, MN
Correspondence to: Dr. D. Takemoto, Department of Biochemistry, Willard Hall, Kansas State University, Manhattan, KS, 66506; Phone: (785) 532-7009; FAX: (785) 532-7278; email: email@example.com
Purpose: To synthesize novel aldose reductase inhibitors (ARI) that will normalize losses in protein kinase Cg (PKCg) observed during diabetes and galactosemia.
Methods: ARI were synthesized as tricyclic pyrones 1-6 (HAR-1 through HAR-6) from 3-methyl-1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b]benzopyran and (5aS,7S)-7-isopropenyl-3-methyl-1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b]benzopyran and were tested by inhibition of aldose reductase enzyme activity in vitro and by inhibition of polyol formation in lens epithelial cells in culture. Identified compounds were further tested in galactosemic rat lens in vivo for (a) normalized PKCg levels by Western blot, (b) reduction of phosphorylation of the gap junction protein Cx46 by analyses of co-immunoprecipitated proteins, and (c) by normalization of gap junction activity as measured by dye transfer.
Results: HAR-1 (1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b]benzopyran-3-acetic acid) was identified as an ARI with IC50 for aldose reductase inhibition at 2 nM. Polyol accumulation in lens epithelial cells was reduced by 80% at 10 mM. Rats fed 40% galactose for 9 days had an 80% reduction in PKCg levels which were normalized by HAR-1 at 100 mg/kg/day, fed orally. Phosphorylation of Cx46 was increased by 50% and this was normalized in HAR-1 treated rats (6 day treatment). Gap junction activity of galactosemic rats was reduced by 55% and this was normalized by HAR-1 in six day-treated rats.
Conclusions: HAR-1 is a novel ARI which normalized losses of PKCg, changes in Cx46 phosphorylation, and gap junction activity.
Diabetes affects an estimated 13 million Americans and over 200 million people worldwide. The high levels of glucose in diabetic tissues results in the increased generation of sorbitol by an enzyme called aldose reductase (AR). AR, the first enzyme in the polyol pathway, reduces glucose and other aldoses through an NADPH dependent pathway to sorbitol . In the second step of the polyol pathway sorbitol is oxidized by sorbitol dehydrogenase to fructose. Sorbitol dehydrogenase has a lower rate constant than aldose reductase and, thus, sorbitol is not readily metabolized and accumulates within the cell [2,3]. This increase in polyol causes cell edema and an increase in cell permeability. This results in a loss of potassium, free amino acids, and myo-inositol, and in an accumulation of sodium and chloride within the cell. This, in turn, causes the cells to swell and form vacuoles, which ultimately results in a loss of integrity . This process has been implicated in many diabetic complications including retinopathy, neuropathy, nephropathy and cataract formation [5,6]. Galactose, which is also a substrate for AR, is not a substrate for sorbitol dehydrogenase and thus cannot proceed through the second step of the polyol pathway. Galactitol formed by AR is not readily metabolized. This makes galactosemia a convenient model system.
Many investigators have used galactosemic models because deterioration of cells proceeds more quickly . Galactosemic cataracts are similar to diabetic cataracts both in progressive stages of deterioration biochemically and structurally [8-11]. It has been shown that, following the induction of diabetes or during galactosemia, there is an up-regulation of diacylglycerol in some tissues. This, in turn, leads to an increase in protein kinase Cb (PKC)b isoform . PKCb is not a major isoform in the lens. The predominant isoforms found in the lens are PKCg and PKCa. Primary lens epithelial cells grown in high galactose media show a decrease in PKCg. This difference is normalized with the administration of tolrestat . PKCg is the isoform which phosphorylates and inhibits gap junctions .
Because of its role in diabetic complications, AR inhibition has been investigated as a possible preventive treatment. There are currently two main classes of AR inhibitors, cyclic imides such as sorbinil and carboxcylic acid derivatives such as tolrestat and epalrestat . Figure 1 shows the structures of these inhibitors and the newly synthesized class investigated in this paper. Current inhibitors are not very water soluble except the corresponding sodium salts of tolrestat and epalrestat. The new class of inhibitors described in this report are various tetrahydro-pyranopyrone derivatives that are water-soluble and will, most likely, be transported to the eye.
Several derivatives of the tricyclic pyrones such as HAR-1 through HAR-6 were synthesized (Figure 1). Computer-aided enzyme docking studies were performed on HAR-1 and sorbinil with aldose reductase . Solvation energies of HAR-1, HAR-5, HAR-6, and sorbinil were also calculated using the AMSOL program to estimate the relative hydrophobicities of the synthesized compounds. Computer generated enzyme docking studies were performed on HAR-1, HAR-5, and HAR-6 with aldose reductase. It was found that the binding energies for HAR-1, HAR-5, and HAR-6 were -77.2727, -80.4147, and -79.9923 kcal/mol, respectively . These new ARI's have been tested for AR enzyme inhibition, normalization of polyol levels, and for effects on PKCg levels and gap junction activity.
Synthesis of novel inhibitors
Tricyclic pyrone carboxylic acids 1 and 5 (HAR-1 and HAR-5, Figure 1) were synthesized separately from 3-methyl-1H,7H-5a,6,8,9-tetahydro-1-oxopyrano[4,3-b]benzopyran (substrate A) and (5aS,7S)-7-isopropenyl-3-methyl-1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b]benzopyran (substrate B) by the treatment with lithium diisopropylamide (LDA) in tetrahydrofuran (THF) at -78 °C followed by carbon dioxide in 94% and 90% yield, respectively. Preparations of the aforementioned compounds have been previously described . Similarly, compound 2 (HAR-2, Figure 1) was prepared from the lithiation of substrate A with LDA followed by n-butyl glyoxylate (87% yield) and basic hydrolysis with 1% NaOH in THF and water (72% yield). Compound 3 (HAR-3, Figure 1) was obtained from substrate A by the following sequence: (i) Lithiation with LDA followed by n-butyl glyoxylate; (ii) hydroxylation at C10 of the resulting adduct with the BH3THF followed by 30% H2O2 and 0.1% NaOH; and (iii) hydrolysis with 1% NaOH (73% yield). Compound 4 (HAR-4, Figure 1) was synthesized from substrate A by the following sequence: (i) Lithiation with LDA followed by n-butyl glyoxylate; (ii) mesylation with methanesulfonyl chloride and triethylamine followed by dehydromesylation with 1,8-diazabicyclo[5.4.0]undec-7-ene in toluene (69% yield); (iii) Michael addition of the resulting ene ester with methylamine in THF; and (iv) hydrolysis with 1% NaOH in THF (35% yield in 2 steps). Compound 6 (HAR-6, Figure 1) was obtained from substrate B by the following sequence: (i) Lithiation with LDA followed by benzyl chloroformate (83% yield); (ii) selective hydroxylation of C-7 isopropenyl group with 1 equivalent of BH3THF followed by 30% H2O2 and 0.5% NaOH (69% yield); and (iii) hydrolysis with 1% NaOH in THF (89% yield). HAR-1 (1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b]benzopyran-3-acetic acid) has the highest AR inhibitory activity.
Toxicity of inhibitors
The N/N 1003a rabbit lens epithelial cells were grown in six well plates with Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (Atlanta Biological, Norcross, GA) and 50 mg/ml gentamycin. The cells were grown to 40% confluency, then the media was changed and supplemented with the following concentrations of inhibitor, 1 nM, 10 nM, 100 nM, 1 mM, or 10 mM. The cells were observed under light microscopy for morphological changes and then harvested for cell viability using hemocytometer counting and trypan blue dye exclusion.
Isolation of aldose reductase
E. coli cells that overexpress recombinant rat aldose reductase were a gift from Sanai Sato (NEI). The cells were grown overnight at 37 °C in 5 ml of LB media (5 g Bactotryptone, 2.5 g yeast extract, 5 g NaCl, 1.5 g Agar, 500 mL H20 final volume, and 40 mg/ml Ampicillin). A 5 ml aliquot of log phase bacteria was then transferred to 500 ml of LB media and allowed to grow for an additional 6 h at 37 °C. The cells were then pelleted by centrifugation at 5,000x g for 15 min. The supernatant was removed and the pellet was taken up in 10 ml of phosphate buffered saline supplemented with 10 mM mercaptoethanol. The cell membranes were then disrupted by sonication (Sonic Disemembrator 50, Fisher Scientific, Pittsburgh, PA) and the mixture was centrifuged at 500x g for 40 min. The supernatant was used for the enzyme assay.
AR enzyme assay
Using a double beam spectrophotometer, 25 mM xylose, 0.15 mM NADPH, 0.2 M Na2SO4, various inhibitor concentrations, and enough 0.1 M phosphate buffer, pH 6.1, to make a final volume of 1 ml was added to a control cuvette. Enzyme (200 mg/ml) and control mixture was added to the test cuvette, and the oxidation of NADPH was monitored as a decrease in absorbance at 340 nm for 1 min .
Inhibition of polyol accumulation
The N/N 1003a rabbit lens epithelial cells were grown in tissue culture flasks with control media (5 mM glucose), normal media with 40 mM galactose, or 40 mM galactose and 1 mM to 100 mM HAR-1 inhibitor, for 48 h. The media was then removed and the cells were washed three times with PBS. The cells were then pelleted by centrifugation and the pellet was taken up in 50 ml of PBS. Cell membranes were disrupted by sonication and 0.1 mg of 3-O-methoxyglucose was added as an internal standard. The mixture was centrifuged for 20 min at 20,000x g. The supernatant was reserved and 50 ml of 0.3 M ZnSO4 and 50 ml of 0.3 M BaSO4 were added and the solution was vortexed and then centrifuged at 10,000x g for 10 min. The supernatant was removed and lyophilized. Deriva-Sil (Regis Technology, Morton Grove, IL, 50 ml) and 50 ml of pyridine were added to the lyophilized cell extract. The solution was then incubated at 70 °C for 1 h, mixing every 15 min. The relative galactose and galactitol content of each extract was then measured using GC/MS (Hewlett Packard GC/MS, Palo Alto, CA) with a Rtx-1 30 mm x 0.25 mm capillary column (Restek, Bellefonte, PA). Peaks were integrated and compared to standards of glucose, galactose or galactitol.
In vivo galactosemic study
Six week old Sprague Dawley Rats (250-300 g) were fed normal chow (Bioserve, Laurel, MD; rodent grain base diet, 40% fiber F3975), high galactose chow (Bioserve Rodent grain base diet, 40% galactose F1624), normal chow with HAR-1, or high galactose chow with either HAR-1 (100 mg/kg body weight per day) or tolrestat (100 mg/kg body weight per day). The rats were given food supplemented with HAR-1 or tolrestat in the morning and then given food and water ad libitum the rest of the day. They were kept on a 12 h on and 12 h off light cycle. After 6 or 9 days the rats were sacrificed with an overdose of CO2 and then eyes were taken and immediately frozen on dry ice for later polyol and PKC analyses. Experiments on all rats conformed to the ARVO Resolution on the Use of Animals in Research.
Polyol content of galactosemic rat lens
The lenses were removed, weighed and placed in 500 ml of PBS supplemented with 15 ml of 3-O-methoxyglucose as internal standard. The tubes containing the lenses were then sealed and boiled for 20 min in a water bath, and 100 ml of 0.3 M ZnSO4 and 100 ml of BaSO4 were added, and the mixture was centrifuged for 15 min at 10,000x g. The supernatant was removed and lyophilized for GC/MS analysis as described above. Peaks were integrated and expressed as percent galactose or galactitol of total area of both peaks.
PKC analyses of diabetic galactosemic rat lens
The lenses were removed from the enucleated eyes, weighed, and homogenized in 200 ml of lysis buffer (50 mM Tris, 100 mM NaCl, M-Per Pierce [Pierce, Rockford, IL]) Zwitterionic detergent (60% v/v), 5 mM NaF, 1 mM Na3VO4, 40 mM b-glycerophosphate, 6 mg/ml chymotrypsin, 10 mM 3,4-dichlorocoumarin, 10 mM E-64, 1 mg/ml leupeptin, 1 mg/ml pepstatin A, 1 mg/ml aprotinin, 1 mM PMSF, and 5 mM EDTA. The homogenate was centrifuged for 20 min at 0 °C at 1000x g. The supernatant was then analyzed for protein content. Equal amounts of protein were loaded and separated on a 10% SDS polyacrylamide gel (100 mg/lane). The western blot was blocked with 3% milk in PBS, and then mouse anti-PKCg (1:5000) or PKCa (1:1000) antisera (Transduction Laboratories, San Diego, CA) were applied in a 3% milk solution overnight. The membrane was then washed 3 times in TDN (0.05 M NaCl, 2 mM EDTA, 0.01 M Tris, pH 7.0) and goat anti-mouse IgG (1:5000, Promega, Madison, WI) was applied. The blot was developed using supersignal chemoluminescent substrate from Pierce.
Immunoprecipitation of PKCg and Cx46 from normal or galactosemic rat lens
Cortical fiber cells of three lenses per data point were dissected and washed in ice cold PBS, then centrifuged at 2,000x g for 10 min. The PBS was removed and cortical fiber cells were left on ice and mixed with 0.5 ml lysis buffer (20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 25 mg/ml aprotinin, 25 mg/ml leupeptin), then the cells were sonicated, while in lysis buffer, on power 9 for 3-4 times, each time for 5 s, and kept on ice for 30 min. The lysed cells were centrifuged at 20,000x g for 20 min, then the supernatant was collected. To reduce non-specific adsorption, the supernatant was pre-cleared by incubation with 20 ml (1:1,v/v, in PBS) protein G agarose beads and preimmune sera at 1:1,000 in 2.5% bovine serum albumin (BSA) at 4 °C for 15 min with shaking. The samples were then centrifuged at 12,000x g for 25 s and the pellet was discarded. The supernatants were then mixed with anti-Cx46 specific antibodies in a total concentration of 5 mg/ml and incubated overnight at 4 °C with constant mixing. Twenty ml of protein G agarose beads were added to the samples and they were incubated for another 2 h at 4 °C. The samples were washed three times with lysis buffer and centrifuged at 12,000x g for 25 s. The immunoprecipitates were mixed with 20 ml of sample buffer, incubated for 5 min in boiling water and analyzed by 12.5% SDS-PAGE and western blotting. The transferred proteins on the nitrocellulose membrane were probed with anti-PKCg (1:1,000), anti-phosphoserine (Transduction Labs, 1:1000), or anti-Cx46 antibodies (a gift of Dr. L. Takemoto, Kansas State University, Manhattan, KS). Protein load was adjusted so that levels of Cx46 and of PKCg were equal in each lane.
Gap junction activity of normal and galactosemic rat lens
Rat eyes were removed immediately after the death of the animal and washed in PBS, pH 7.2. The lenses were dissected using a dissection scissors and forceps, then washed in PBS and mounted in 3% agar in a small tissue culture plate so that the anterior of the lens was at the top [19,20]. For dye injection, 2.5 mg/ml lucifer yellow in PBS was microinjected (10 ml) into the superficial cortical fibers. The lenses were incubated at room temperature for 30 min to allow dye transfer. They were examined by laser scanning confocal microscopy to evaluate the extent of dye transfer. The excitation filter on the confocal microscope was BP 485/20 and the emission filter was BP 515-565. The extent of dye transfer was determined as the distance, in mm, that lucifer yellow transferred from the site of injection to the interior fiber cells in 30 min. For each experimental group three lenses were used from the three rats/group and the distance of dye transfer presented in the figure represent the mean of dye transfer in the lenses. Time from animal sacrifice to dye injection was approximately 10 min.
Cell morphology and in vivo toxicity
The N/N 1003a rabbit lens epithelial cells incubated with and without inhibitor were observed for up to a week. There were no differences in appearance or growth patterns between the control and experimental cells in culture. Animals treated solely with novel inhibitor at 100 mg/kg body weight per day were analyzed by the Kansas State Veterinary Medical School Pathology Department. There were no significant toxic effects observed.
Aldose reductase activity
The solvation energies of compounds HAR-1, HAR-5, HAR-6, and sorbinil (Figure 1) calculated using the SM5.4PDA AM1 method available in the AMSOL program are -140.3, -67.0, -67.8, and -35.6 kcal/mol, respectively . Computer-aided enzyme docking studies revealed that the binding energies of aldose reductase for HAR-1 and sorbinil are -80.1 and -35.2 kcal/mol, respectively . These data suggest that HAR-1 is more soluble in water and more potent in inhibiting aldose reductase than sorbinil and HAR-5 and HAR-6. The recombinant rat aldose reductase extracted from the E. coli cells had good enzymatic activity. The results of the inhibitor studies are summarized in Table 1. Inhibitor HAR-1 showed the greatest inhibitor effect with an IC50 of 2 nM, followed by inhibitor HAR-3 with IC50 of 20 nM while sorbinil had an IC50 of 1 mM (not shown).
Inhibition of polyol accumulation by ARI's
The N/N 1003a rabbit lens epithelial cells were incubated in 40 mM galactose with or without HAR-1 inhibitor. The cells were then harvested and analyzed by GC/MS for relative galactose and galactitol content. The results are summarized in Table 2 and Table 3. Tolrestat was the most effective inhibitor with an IC50 of about 2 mM. HAR-1 was the most effective of the new inhibitors with an IC50 of about 3 mM and with the polyol level diminishing to 0 at an inhibitor concentration of about 10 mM. In whole rat lens from nine-day galactose-fed animals, polyol levels were approximately 48% normalized in rats fed 100 mg/kg body wt/day HAR-1. In galactose fed rats without inhibitor, 43% and 56% of the combined GC/MS peaks were galactose and galactitol, respectively. In galactose fed rats with HAR-1, 72% and 27% of the combined GC/MS peaks were galactose and galactitol, respectively.
Changes in PKCg in vivo during galactosemia
Six week old Sprague Dawley rats were fed a high galactose diet (40%) supplemented with 100 mg/kg body weight/day of HAR-1 for 6 or 9 days. After sacrifice, their eyes were removed and frozen. Four lenses from each group of rats were homogenized and analyzed for PKCg content. As shown in Figure 2, PKCg decreased in galactose-treated animals at 9 days, and this decrease was normalized to control levels in the animals treated with HAR-1. This decrease was specific to PKCg as connexin46 levels did not change (data not shown). No change in PKCg levels were observed at 6 days (data not shown).
Serine phosphorylation on Cx46 in the lens cortex of normal and galactosemic rats: PKCg is activated
In rat lens cortical regions Cx46 antisera co-immunoprecipitated PKCg. When equal PKCg and equal Cx46 per lane are compared, the amount of serine phosphorylation of Cx46 was greater in 6 day galactosemic versus normal lens (Figure 3A,B). This was reduced in rats treated with galactose and HAR-1 inhibitor at 100 mg/kg body wt/day for six days (40% galactose diet). PKCg has been found to phosphorylate Cx43 and Cx46 on serine . Therefore, although total PKCg levels were not changed at 6 days, the PKCg activity, as measured by Cx46 phosphorylation, was increased. When equal loads of Cx46 were compared, the Cx46 from galactosemic lens samples had higher serine phosphorylation. This was normalized to control levels by HAR-1.
Gap junction activity of normal and galactosemic rat lens
Increased PKCg activity is associated with decreased gap junction activity . When gap junction activity was measured in fresh whole rat lens the extent of dye transfer was reduced in 6 day galactosemic lens (34.7 mM) when compared to normal lens (77 mM). This was returned to normal by use of the ARI, HAR-1 at 100 mg/kg body wt/day (79.2 mM, Figure 4 and Table 4).
Aldose reductase inhibition has been investigated as a possible means of preventing some diabetic complications such as cataract formation, retinopathy, neuropathy and nephropathy. In this study a novel class of water-soluble inhibitors (HAR-1 through HAR-6) were synthesized. Computer generated enzyme docking studies were performed on HAR-1, HAR-5, and HAR-6 with aldose reductase. It was found that the binding energies for HAR-1, HAR-5, and HAR-6 were -77.2727, -80.4147, and -79.9923 kcal/mol, respectively . These computational data suggested that HAR-1, HAR-5, and HAR-6 have similar strong binding energies with aldose reductase.
HAR-1 is the smallest of the compounds tested. A short carboxylic acid side chain off of the C3 atom seems to be all that is required for high inhibition (IC50 0.002 mM). When the chain was extended and a secondary alcohol added to the C3 the inhibitory ability decreased (HAR-2, IC50 >500 mM). However, it was partially recovered by the addition of a hydroxyl group at C10 (HAR-3, IC50 0.02 mM). When the model compound was made larger and bulkier by the addition of carbon chains at the bottom of the ring system, the AR inhibitor ability decreased (HAR-5 and HAR-6, no inhibition).
These inhibitors were tested in vitro by an enzyme assay. Only four of the six inhibitors tested showed AR inhibitor activity (Table 1). HAR-1 showed the greatest effect with an IC50 of 2 nM, followed by HAR-3 that had an IC50 of 0.02 mM. Inhibitors HAR-1 through HAR-4 were then tested for their ability to penetrate lens epithelial cell membranes and inhibit polyol formation. Lens epithelial cells were grown in high galactose media supplemented with 10 mM of inhibitor for 48 h. The cells were harvested and the carbohydrate fraction was analyzed for relative galactose and galactitol content. As seen in Table 2 there was a marked decrease in galactitol accumulation with the administration of 10 mM of HAR-1. At 10 mM concentration, tolrestat inhibited 95% of accumulation of galactitol within the cell compared to 80% inhibition by HAR-1 and 40% by HAR-4. The IC50 for the HAR-1, however, was 2 mM while the IC50 for polyol accumulation in intact cells on animals was significantly higher. This is, most likely, due to the differences in uptake of the compounds across the cell and/or to the differences in the in vivo stability. Thus, while the in vitro aldose reductase inhibitor may closely parallel the binding energies in the model system, the in vivo protocol must also reflect uptake and stability. It should be mentioned, however, that the HAR-1 is within the same order of magnitude as tolrestat for intact cells (Table 2 and Table 3). Whole lens polyol levels were also normalized by 48% (Table 3). This demonstrated efficacy of this new class of aldose reductase inhibitors in vitro, in cell cultures, and in animals.
HAR-1 was then tested in a galactosemic rat model. As seen in Figure 2 lens epithelial PKCg was decreased in animals fed a 40% galactose diet for 9 days. This is in agreement with previous work done showing that PKCg decreased in primary lens epithelial cells treated with 40 mM galactose supplemented media . When the animals were also supplemented with our AR inhibitor, HAR-1, the PKCg levels were normalized. tolrestat did not have this effect Figure 2. We are uncertain as to why tolrestat does not normalize PKCg levels but does restore polyol levels (Table 2, Table 3, and Figure 2). Since diabetes and galactosemia both cause increased diacylglycerol, perhaps this is not as well controlled by tolrestat. Future studies would need to be done to determine diacylglycerol levels using these two inhibitors, in vivo, in a galactosemic and/or diabetic animal.
PKCg is the isoform of PKC which inhibits gap junctions . Overexpression of PKCg, but not of PKCa, will reduce gap junction activity of lens epithelial cells in culture . Gap junctions are important for cell-cell communication. They are especially important in the lens because of the lack of vascularization. Gap junctions are a major means of cell-cell communication within the interior fiber cells. The major fiber cell gap junction proteins are Cx46 and Cx50 [19,20]. The role of PKCg in control of lens cortical Cx46 or Cx50 has not been determined.
When rats were fed a 40% galactose diet for only 6 days no change in PKCg levels were observed . However, PKCg and Cx46 could be co-immunoprecipitated and lens cortical samples from 6 day galactosemic rats have increased serine phosphorylation levels on the immunoprecipitated and separated Cx46. This was normalized by HAR-1 (Figure 3). This increased phosphorylation of Cx46 parallels the inhibition of the gap junction activity (Figure 4 and Table 4).
The decrease in PKCg protein levels that is observed at 9 days may be due to down regulation of PKC. When cells are exposed to high glucose or galactose there is an observed increase in the flux of glucose through the pentose phosphate pathway, and, an increase in de novo synthesis of diacylglycerol [23-25]. This would result in early activation of PKC's. Since Cx46 is a substrate for PKCg  this may result in decreased gap junction activity due to the PKCg phosphorylation of Cx46.
It is well known that galactosemia causes increased diacylglycerol production . If this causes translocation of PKCg to membranes, then, increased phosphorylation of Cx46 may result. This would then cause the observed decrease in gap junction activity. However, the overall decrease in PKCg levels would result from persistent activation of this diacylglycerol-dependent PKC isoform, resulting in down-regulation of PKCg after longer exposures to galactose (i.e., 9 days). This could be normalized by an aldose reductase inhibitor (ARI). Diacylglycerol is also normalized in the lens cortical region by other ARI's [23-25]. The normalized PKCg levels would not, however, result in increased PKCg activity since enzyme activity is low under low diacylglycerol levels. It is only in the galactosemic, or perhaps, the diabetic state that PKCg activity for gap junctions would be increased at early time periods as a result of de novo diacylglycerol synthesis. Our recent work with HAR-1 in dogs supports a down-regulation mechanism as mRNA levels for PKCg were not decreased after galactosemia but PKCg protein levels were decreased .
At later times this may reverse, once PKCg levels are totally down-regulated. In the enucleated fiber cells of the cortical region the PKCg could not be resynthesized. Thus, we would predict that, where PKCg was totally depleted, the phosphorylation of Cx46 should decrease and gap junction activity should, in fact, increase after longer galactosemia. Such a situation has been observed in late diabetes .
These results suggest that some ARI's can be useful to normalize changes in gap junction activity, perhaps in part, through correction of PKCg activity and levels, by normalization of the activation/down regulation mechanism. It is of interest that one of the early changes observed in the lens, after two weeks of diabetes in rats is a very localized swelling in a discrete cortical band of outer fiber cells . This is despite the fact that the lens epithelial cells would be the first to experience the diabetic-induced glucose fluctuation. If activation and subsequent down regulation of PKCg occurred in these cortical regions, the pathology would be observed here, first, in regions which had become enucleated and, therefore, could not resynthesize the lost PKCg.
The authors thank John Reddan for providing the N/N 1003a cells, Dr. Sanai Sato for the E. coli expressing recombinant rat aldose reductase, Dr. Brad Fenwick for the pathology of rats and, Dr. Marjorie Lou, and Steve Zatechka for their technical advice. Financial Support from Kansas State University Special Group Incentive Research Award and Great Plains Diabetes Research, Inc is greatly appreciated. HAR-1 is under patent application S/N 08/902.053, filed 7/29/97, US Patent Office.
1. Kinoshita JH. Mechanism initiating cataract formation. Invest Ophthalmol Vis Sci 1974; 13:713.
2. Van Heningen R. Formation of polyol by the lens of the rat with "sugar" cataract. Nature 1959; 184:194-5.
3. Hers H. Mechanism of formation of seminal fructose and foetal fructose. Biochim Biophys Acta 1960; 37:127-38.
4. Robison WG Jr, Houlder N, Kinoshita JH. The role of lens epithelium in sugar cataract formation. Exp Eye Res 1990; 50:641-6.
5. Newfield RS, Polak M, Marchase R, Czernichow P. Epidemiology and genetics of diabetic complications. Diabetologia 1997; 40:B62-4.
6. Caprio S, Wong S, Alberti KG, King G. Cardiovascular complications of diabetes. Diabetologia 1997; 40:B78-82.
7. Crabbe MJ, Goode D. Aldose reductase: a window to the treatment of diabetic complications? Prog Retin Eye Res 1998; 17:313-83.
8. Kinoshita JH, Fukushi S, Kador P, Merola LO. Aldose reductase in diabetic complications of the eye. Metabolism 1979; 28:S462-9.
9. Kinoshita JH, Kador P, Datiles M. Aldose reductase in diabetic cataracts. JAMA 1981; 246:257-61.
10. Friedenwald R. Contributions to the histopathology of cataract. Arch Ophthalmol 1955; 53:825-31.
11. Kuwabara T, Kinoshita JH, Cogan DG. Electron microscopic study of galactose-induced cataract. Invest Ophthalmol Vis Sci 1969; 8:133-49.
12. Xia P, Inoguchi T, Kern TS, Engerman RL, Oates PJ, King GL. Characterization of the mechanism for the chronic activation of diacylglycerol-protein kinase C pathway in diabetes and hypergalactosemia. Diabetes 1994; 43:1122-9.
13. Gonzalez K, Udovichenko I, Cunnick J, Takemoto DJ. Protein kinase C in galactosemic and tolrestat-treated lens epithelial cells. Curr Eye Res 1993; 12:373-7.
14. Saleh SM, Takemoto DJ. Overexpression of protein kinase Cgamma inhibits gap junctional intercellular communication in the lens epithelial cells. Exp Eye Res 2000; 71:99-102.
15. Constantino L, Rastelli G, Vianello P, Cignarella G, Barlocco D. Diabetes complications and their potential prevention: aldose reductase inhibition and other approaches. Med Res Rev 1999; 19:3-23.
16. Pang YP, Kozikowski AP. Prediction of the binding site of 1-benzyl-4-[(5,6-dimethoxy-1-indanon-2-yl)methyl]piperidine in acetylcholinesterase by docking studies with the SYSDOC program. J Comput Aided Mol Des 1994; 8:683-93.
17. Hua DH, Chen Y, Sin H, Maroto MJ, Robinson PD, Newell S, Perchellet SM, Ladesich JB, Freeman JA, Perchellet JP, Chiang P. A one-pot condensation of enals and pyrones. Synthesis of novel 1H,7H-5a,6,8,9-Tetrahydro-1-oxopyrano[4,3-b]benzopyrans. J Org Chem 1997; 62:6888-96.
18. Ellingboe J, Alessi T, Millen J, Sredy J, King A, Prusiewicz C, Guzzo F, VanEngen D, Bagli J. (Pyrimidinyloxy)acetic acids and pyrimidineacetic acids as a novel class of aldose reductase inhibitors. J Med Chem 1990; 33:2892-9.
19. Shestopalov VI, Bassnett S. Exogenous gene expression and protein targeting in lens fiber cells. Invest Ophthalmol Vis Sci 1999; 40:1435-43.
20. Eckert R, Adams B, Kistler J, Donaldson P. Quantitative determination of gap juctional permeability in the lens cortex. J Membr Biol 1999; 169:91-102.
21. Takemoto DJ, Takemoto LJ, Zoukhri D, Saleh S. The association between PKCg and connexin 46 in the lens cortex. Invest Ophthalmol Vis Sci 2001; 42:S541.
22. Karrer JE, Saleh S, Boyle D, Takemoto DJ. Effect of galactosemia on gap junctions in the lens. Invest Ophthalmol Vis Sci 2001; 42:S541.
23. Keogh RJ, Dunlop ME, Larkins RG. Effect of inhibition of aldose reductase on glucose flux, diacylgycerol formation, protein kinase C, and phopholipase A2 activation. Metabolism 1997; 46:41-7.
24. Kapor-Drezgic J, Zhou X, Babazono T, Dlugosz JA, Hohman T, Whiteside C. Effect of high glucose on mesangial cell protein kinase C-delta and -epsilon is polyol pathway-dependent. J Am Soc Nephrol 1999; 10:1193-203.
25. Derylo B, Babazono T, Glogowski E, Kapor-Drezgic J, Hohman T, Whiteside C. High glucose-induced mesangial cell altered contractility: role of the polyol pathway. Diabetologia 1998; 41:507-15.
26. Harris R, Browning M, Karrer J, Brightman A, Hua D, Wagner L, Takemoto D. Normalization of PKCg in galactosemic dogs by a novel aldose reductase inhibitor. Invest Ophthalmol Vis Sci 2001; 42:S541.
27. Abdullah K, Luthra G, Bilski JJ, Abdullah SA, Reynolds LP, Redmer DA, Grazul-Bilska AT. Cell-to-cell communication and expression of gap junctional proteins in human diabetic and nondiabetic skin fibroblasts: effects of basic fibroblast growth factor. Endocrine 1999; 10:35-41.
28. Bond J, Green C, Donaldson P, Kistler J. Liquefaction of cortical tissue in diabetic and galactosemic rat lenses defined by confocal laser scanning microscopy. Invest Ophthalmol Vis Sci 1996; 37:1557-65.