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Aldose Reductase Induced by Hyperosmotic Stress Mediates Cardiomyocyte Apoptosis

2003; Elsevier BV; Volume: 278; Issue: 40 Linguagem: Inglês

10.1074/jbc.m211824200

1083-351X

Anita Gálvez, Juan Alberto Ulloa, Mario Chiong, Alfredo Criollo, Verónica Eisner, L. Felipe Barros, Sergio Lavandero,

Biochemical effects in animals

Cells adapt to hyperosmotic conditions by several mechanisms, including accumulation of sorbitol via induction of the polyol pathway. Failure to adapt to osmotic stress can result in apoptotic cell death. In the present study, we assessed the role of aldose reductase, the key enzyme of the polyol pathway, in cardiac myocyte apoptosis. Hyperosmotic stress, elicited by exposure of cultured rat cardiac myocytes to the nonpermeant solutes sorbitol and mannitol, caused identical cell shrinkage and adaptive hexose uptake stimulation. In contrast, only sorbitol induced the polyol pathway and triggered stress pathways as well as apoptosis-related signaling events. Sorbitol resulted in activation of the extracellular signal-regulated kinase (ERK), p54 c-Jun N-terminal kinase (JNK), and protein kinase B. Furthermore, sorbitol treatment resulting in induction and activation of aldose reductase, decreased expression of the antiapoptotic protein Bcl-xL, increased DNA fragmentation, and glutathione depletion. Apoptosis was attenuated by aldose reductase inhibition with zopolrestat and also by glutathione replenishment with N-acetylcysteine. In conclusion, our data show that hypertonic shrinkage of cardiac myocytes alone is not sufficient to induce cardiac myocyte apoptosis. Hyperosmolarity-induced cell death is sensitive to the nature of the osmolyte and requires induction of aldose reductase as well as a decrease in intracellular glutathione levels. Cells adapt to hyperosmotic conditions by several mechanisms, including accumulation of sorbitol via induction of the polyol pathway. Failure to adapt to osmotic stress can result in apoptotic cell death. In the present study, we assessed the role of aldose reductase, the key enzyme of the polyol pathway, in cardiac myocyte apoptosis. Hyperosmotic stress, elicited by exposure of cultured rat cardiac myocytes to the nonpermeant solutes sorbitol and mannitol, caused identical cell shrinkage and adaptive hexose uptake stimulation. In contrast, only sorbitol induced the polyol pathway and triggered stress pathways as well as apoptosis-related signaling events. Sorbitol resulted in activation of the extracellular signal-regulated kinase (ERK), p54 c-Jun N-terminal kinase (JNK), and protein kinase B. Furthermore, sorbitol treatment resulting in induction and activation of aldose reductase, decreased expression of the antiapoptotic protein Bcl-xL, increased DNA fragmentation, and glutathione depletion. Apoptosis was attenuated by aldose reductase inhibition with zopolrestat and also by glutathione replenishment with N-acetylcysteine. In conclusion, our data show that hypertonic shrinkage of cardiac myocytes alone is not sufficient to induce cardiac myocyte apoptosis. Hyperosmolarity-induced cell death is sensitive to the nature of the osmolyte and requires induction of aldose reductase as well as a decrease in intracellular glutathione levels. Programmed cell death culminating in apoptosis is responsible for normal tissue homeostasis and has increasingly been implicated in mediating pathological cell loss (1Kerr J.R.F. Wyllie A.H. Curie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12927) Google Scholar). Apoptosis is accompanied by characteristic morphological changes, including cell shrinkage, nuclear condensation, plasma membrane blebbing, chromatin condensation, and the formation of apoptotic bodies (2Saraste A. Pulkki K. Cardiovasc. Res. 2000; 45: 528-537Crossref PubMed Scopus (1042) Google Scholar). 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In contrast to these beneficial applications, little is known about the effects of mannitol on cardiomyocyte apoptosis. The primary mechanism of action of hyperosmotic stress appears to be mechanical and related to shrinkage of these cells. However, the underlying mechanisms by which hyperosmotic stress triggers apoptosis in the cardiomyocyte remain unknown. Maeno et al. (21Maeno E. Ishizaki Y. Kanaseki T. Hazama A. Okada Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9487-9492Crossref PubMed Scopus (654) Google Scholar) have shown that apoptotic volume decrease, which is caused by disordered cell volume regulation, is an early prerequisite for events leading to apoptotic cell death. To avoid excessive alterations in volume, cells have developed regulatory mechanisms including ion transport across the membrane and changes in metabolism. The ability of cells to resist osmotic shrinkage by cell volume regulation parallels their resistance to apoptosis after osmotic shock (22Bortner C.D. Cidlowski J.A. Am. J. Physiol. 1996; 271: C950-C961Crossref PubMed Google Scholar). Cells adapt to hyperosmotic stress by a variety of mechanisms that restore cell volume by restoring intracellular salt and osmolyte concentrations (23Burg M.B. Am. J. Physiol. 1995; 268: F983-F996Crossref PubMed Google Scholar). Aldose reductase (AR 1The abbreviations used are: AR, aldose reductase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; PKB, protein kinase B; NAC, N-acetylcysteine; SDH, sorbitol dehydrogenase; BSO, dl-buthionine-(S,R)-sulfoximine; Sor, sorbitol; Man, mannitol; LY, LY-294002; PD, PD-98059; SB, SB-203580; SP, SP-600125; KRPH, Krebs Ringer phosphate HEPES buffer; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; RVI, regulatory volume increase.; EC 1.1.1.21) is the first enzyme in the polyol pathway, which helps to promote resistance of cells to anisotonic perturbations. AR catalyzes the formation of sorbitol from glucose using NADPH as a cofactor (24Garcia-Perez A. Martin B. Murphy H.R. Uchida S. Murer H. Cowley Jr., B.D. Handler J.S. Burg M.B. J. Biol. Chem. 1989; 264: 16815-16821Abstract Full Text PDF PubMed Google Scholar, 25Das B. Srivastava S. Diabetes. 1985; 34: 1145-1151Crossref PubMed Scopus (59) Google Scholar, 26Srivastava S.K. Golblum R.M. Hair G.A. Das B. Biochim. Biophys. Acta. 1984; 800: 220-227Crossref PubMed Scopus (56) Google Scholar). Sorbitol accumulation is considered an adaptive response to hypertonicity that is observed in many cells (27Burg M.B. Kwon E.D. Kültz D. Annu. Rev. Physiol. 1997; 59: 437-455Crossref PubMed Scopus (331) Google Scholar, 28Yancey P.H. Clark M.E. Hand S.C. Bowlus R.D. Somero G.N. Science. 1982; 217: 1214-1222Crossref PubMed Scopus (3031) Google Scholar). Although the induction of AR has been associated with compensatory responses to hyperosmotic stress, it also plays an important role in the development of complications in diabetes (29Cohen M.P. Metabolism. 1986; 35: 55-59Abstract Full Text PDF PubMed Scopus (59) Google Scholar, 30Gabbay K.H. New Engl. J. Med. 1973; 288: 831-836Crossref PubMed Scopus (619) Google Scholar, 31Kador P.F. Kinoshita J.H. Med. Res. Rev. 1985; 8: 325-352Crossref Scopus (293) Google Scholar) and myocardial ischemia-reperfusion injury (32Ramasamy R. Oates P.J. Schaefer S. Diabetes. 1997; 46: 292-300Crossref PubMed Scopus (126) Google Scholar, 33Ramasamy R. Trueblood N.A. Schaefer S. Am. J. Physiol. 1988; 275: H195-H203Google Scholar, 34Ruef J. Rao G.N. Li F. Bode C. Patterson C. Bhatnagar A. Runge M.S. Circulation. 1998; 97: 1071-1078Crossref PubMed Scopus (135) Google Scholar, 35Ruef J. Liu S.Q. Bode C. Tocchi M. Srivastava S. Runge M.S. Bhatnagar A. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1745-1752Crossref PubMed Scopus (93) Google Scholar). The relationship between AR and apoptosis has not been previously examined in the cardiomyocyte. However, in lens epithelial cells and pancreatic β-cells, AR activation induced apoptosis, possibly causing an imbalance in redox status (36Hamaoka R. Fujii J. Miyagawa J. Takahashi M. Kishimoto M. Moriwaki M. Yamamoto K. Kajimoto Y. Yamasaki Y. Hanafusa T. Matsuzawa Y. Taniguchi N. J. Biochem. (Tokyo). 1999; 126: 41-47Crossref PubMed Scopus (30) Google Scholar, 37Murata M. Ohta N. Sakurai S. Alam S. Tsai J. Kador P.F. Sato S. Chem. Biol. Interact. 2001; 130: 617-625Crossref PubMed Scopus (60) Google Scholar). In cultured retinal pericytes, glucose-induced apoptosis is mediated through the AR pathway, involving increased oxidative stress characterized by reduced GSH contents (38Mika K. Nakamura J. Hamada Y. Naruse K. Nakashima E. Kato K. Kasuya Y. Yasuda Y. Kamiya H. Hotta N. Diabetes Res. Clin. Pract. 2003; 60: 1-9Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Whether osmotically active nonionic substances such as sorbitol or mannitol induce AR and whether this enzyme mediates apoptosis as a consequence of hyperosmotic stress in cardiac myocytes has not been investigated. In this study, we addressed such issues in cardiomyocytes and asked whether sorbitol- and mannitol-mediated hyperosmotic stress activate different signaling pathways. We also investigated whether induction of AR and increased intracellular sorbitol levels mediates hyperosmotic stress-induced apoptosis. We demonstrate here in cultured rat cardiomyocytes that mannitol or sorbitol induced hyperosmotic stress, reduced cell volume, and stimulated 2-deoxyglucose uptake to a similar degree. Treatment with sorbitol, but not mannitol, induced and activated AR, decreased sorbitol dehydrogenase levels, stimulated intracellular sorbitol accumulation, increased DNA fragmentation, and decreased the levels of the antiapoptotic Bcl-xL protein. Phosphorylation of the mitogen-activated protein kinase ERK was enhanced in the presence of sorbitol but not with mannitol. Both JNK isoforms (p46 and p54) were differentially activated by sorbitol and mannitol. p38-MAPK and protein kinase B (PKB) were activated to a lesser extent by both sorbitol and mannitol at hyperosmotic concentrations. Increases in AR protein levels and activity by sorbitol-induced hyperosmotic stress were differentially regulated by ERK, p38-MAPK, and phosphatidylinositol 3-kinase (PI3K)/PKB pathways. GSH intracellular levels were decreased by hyperosmotic sorbitol but not by mannitol. Hyperosmotic sorbitol-induced apoptosis was attenuated by zopolrestat, a specific AR inhibitor. DNA fragmentation stimulated by sorbitol was also prevented by N-acetylcysteine (NAC; a GSH precursor). Our data demonstrate that AR contributes to apoptosis triggered by hyperosmotic stress in cultured cardiac myocytes. These data also indicated that our current understanding of the protective mechanisms of mannitol in myocardial reperfusion injury needs to be reevaluated. All studies were performed following the guidelines approved by the bioethics committee of the Faculty of Chemical and Pharmaceutical Sciences at University of Chile, Santiago. The experiments were performed as stipulated in Ref. 88National Institutes of HealthGuide for the Care and Use of Laboratory Animals. National Institutes of Health, Bethesda, MD1985Google Scholar. Materials—Polyclonal antibodies against AR and sorbitol dehydrogenase (SDH; NAD+ oxidoreductase; EC 1.1.1.14) were kindly provided by Dr. N. Taniguchi (Osaka University Medical School, Osaka, Japan) (39Takahashi M. Fujii J. Miyoshi E. Hoshi A. Taniguchi N. Int. J. Cancer. 1995; 62: 749-754Crossref PubMed Scopus (77) Google Scholar). Dulbecco's modified Eagle's medium, medium 199, SDH, agarose, β-NAD, NADPH, GSH, glutathione reductase, NAC, gdl-buthionine-(S,R)-sulfoximine (BSO), sorbitol (Sor), mannitol (Man), and other biochemicals were purchased from Sigma unless stated otherwise. ECL, autoradiographic films, and 2-deoxy-d-[3H]glucose (8.1 Ci/mmol) were from PerkinElmer Life Sciences. Prestained molecular mass standard proteins were purchased from Invitrogen. Protein assay reagents were from Bio-Rad. LY-294002 (LY), PD-98059 (PD) and SB-203580 (SB) were from Calbiochem. SP-600125 (SP) was from Tocris (Ellisville, MO). Polyclonal antibodies against phosphorylated and total ERK, p38-MAPK, JNK, and PKB used in Western blot analysis were purchased from Cell Signaling Technology Inc. (Beverly, MA). Polyclonal antibody against Bcl-xL was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Zopolrestat was kindly provided by Pfizer (Groton, CT). Calcein-acetoxymethylester was from Molecular Probes, Inc. (Eu-gene, OR). Culture and Treatment of Cardiomyocytes—Cardiac myocytes were prepared from hearts of 3-day-old Sprague-Dawley rats as described previously (40Foncea R. Andersson M. Ketterman A. Blakesley V. Sapag-Hagar M. Sugden P.H. LeRoith D. Lavandero S. J. Biol. Chem. 1997; 272: 19115-19124Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Rats were bred in the Animal Breeding Facility of the Faculty of Chemical and Pharmaceutical Sciences, University of Chile (Santiago, Chile). Cardiomyocytes were plated at a final density of 1.4 × 103/mm2 on gelatin-coated 35-, 60-, or 100-mm Petri dishes. For fluorescence measurements, cells were plated on gelatin-coated 25-mm glass coverslips in 35-mm Petri dishes. Cardiac myocytes were cultured in the absence or presence of different concentrations of sorbitol or mannitol dissolved in serum-free Dulbecco's modified Eagle's medium/medium 199. The final osmolarities of culture media containing different amounts of sorbitol or mannitol were determined using a freezing point osmometer (Osmet). Cultured cardiomyocytes were identified using an anti-β-myosin heavy chain antibody. Cell cultures were at least 95% pure. Preparation of Cell Extracts—Cardiomyocytes were scraped into 100 μl of cold lysis buffer: 20 mm Tris-HCl (pH 7.5), 1 mm EDTA, 1 mm EGTA, 20 mm NaF, 1 mm sodium pyrophosphate, 1 mm sodium vanadate, 140 mm NaCl, and 1 mm phenylmethylsulfonyl fluoride containing 10% (v/v) glycerol, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1% (v/v) Triton X-100. Samples were centrifuged at 12,000 × g for 10 min at 4 °C, and the protein content of supernatants was determined by a Bio-Rad Bradford assay using bovine serum albumin as a standard (41Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Soluble fractions were heated at 95 °C with 0.33 volumes of 4× SDS-PAGE sample buffer for Western blot analysis. Western Blot Analysis for Aldose Reductase and Sorbitol Dehydrogenase—Cell lysates were matched for protein (15 μg), separated by SDS-PAGE on 12% (w/v) polyacrylamide gels, and electrotransferred to nitrocellulose (0.45 μm) using a Trans-blot unit (Bio-Rad) for 1.5 h at 100 V. Membranes were blocked with 3% (w/v) bovine serum albumin in PBS (pH 7.4) containing 0.1% (v/v) Tween 20 (PBST) overnight at 4 °C. Anti-AR or -SDH primary antibodies were diluted in blocking solution (1:5,000). Nitrocellulose membranes were incubated with primary antibody for 1 h at 25 °C. After washing in PBST (4 × 15 min each), blots were incubated for 1 h at room temperature with horseradish peroxidase-linked secondary antibody (1:5,000 in 3% (w/v) bovine serum albumin in PBST). Blots were washed again in PBST (4 × 15 min each), and specific binding was detected using ECL with exposure to Kodak film for 2–10 s. Each blot was quantified by scanning densitometry. Aldose Reductase Activity—Cultured cardiomyocytes were exposed to culture media containing different concentrations of sorbitol or mannitol at the indicated times. Cells were then rinsed three times with PBS and lysed with 200 μl of lysis buffer (10 mm potassium phosphate, pH 7.0, containing 5 mm β-mercaptoethanol). Cell extracts were centrifuged at 10,000 × g for1hat4 °C, and the resulting supernatants were dialyzed in 2 liters of lysis buffer overnight. The dialyzed samples were incubated with 1 ml of DEAE-cellulose pre-equilibrated with lysis buffer. AR was eluted with increasing NaCl concentrations (0–300 mm). The fraction with the highest AR activity (50 mm NaCl) was used throughout the study. Enzyme activity was determined spectrophotometrically at 37 °C by monitoring the decrease in absorbance of NADPH at 340 nm for 3 min in the absence or presence of 5 mm d-glucose as a substrate (25Das B. Srivastava S. Diabetes. 1985; 34: 1145-1151Crossref PubMed Scopus (59) Google Scholar). Briefly, the assay, in a total volume of 1 ml, contained 50 mm potassium phosphate, pH 6.0, 5 mm β-mercaptoethanol, 0.4 m Li2SO4, 5 mm d-glucose, enzyme (1–3 μg of protein); the reaction was started by the addition of 100 μm NADPH. One unit of the enzyme was defined as 1 μmol of NADPH oxidized per min at 37 °C. Determination of Intracellular Sorbitol Levels—Intracellular sorbitol level was determined essentially as described by Malone (42Malone J. Diabetes. 1980; 29: 861-864Crossref PubMed Google Scholar). Briefly, cultured cardiac myocytes (4 × 106 cells/60-mm Petri dish) were lysed with 200 μl of 6% (w/v) HClO4 and then centrifuged at 10,000 × g for 10 min at 4 °C. Supernatants were neutralized with 62 mm glycine (pH 9.4). Sorbitol was determined using a 40-μl sample in glycine buffer 50 mm (pH 9.4), 0.64 units of SDH, and 0.2 mm β-NAD, in a final volume of 0.5 ml. After incubation for 30 min at 37 °C, samples were analyzed in a spectrofluorometer (emission, 366 nm; excitation, 452 nm). The acid pellets were resuspended in 150 μl of 1 m NaOH and incubated for 30 min at 50 °C. Protein content was determined by the Lowry method (43Lowry O.H. Rosenbrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Results were expressed as mg of sorbitol per mg of protein. Assessment of Cell Volume—Cardiomyocyte relative volume was determined by calcein measurements using a modified protocol (44Barros L.F. Barnes K. Ingram J.C. Castro J. Porras O.H. Baldwin S.A. Pflugers Arch. 2001; 442: 614-621Crossref PubMed Scopus (49) Google Scholar). Briefly, cardiomyocytes grown on glass coverslips were loaded at room temperature with 5 μm calcein-acetoxymethyl ester for 5–10 min in Krebs Ringer phosphate HEPES buffer (KRPH; 136 mm NaCl, 4.7 mm KCl, 1.25 mm MgSO4, 1.25 mm CaCl2,5mm Na2PO4,20mm HEPES, pH 7.4) supplemented with 25 mm glucose (KRPH-glc). Cells were imaged at room temperature every 30 s at 488-nm excitation/510–545-nm emission using a Zeiss LSM 410 confocal microscope. Under these conditions, dye bleaching was negligible. In order to compare values from different cells, data were standardized by assigning base-line fluorescence (F 0) the value of 1. Glucose Transport Assay—Cardiomyocytes were placed in Dulbecco's modified Eagle's medium/medium 199 containing 25 mm glucose for 2 h at 37 °C. Cells were then washed with KRPH buffer and either untreated or stimulated as described in the legend to Fig. 4. Uptake of 0.2 mm 2-deoxy-d-[3H]glucose was estimated over a period of 2 min in KRPH buffer at 4 °C by a protocol modified from a previous communication (45Barros L.F. Pflugers Arch. 1999; 437: 763-770Crossref PubMed Scopus (20) Google Scholar). Control experiments showed that at these temperatures intracellular hexose concentrations were less then 20% of the extracellular values. Carrier-mediated uptake rates were obtained by subtracting nonspecific uptake. The latter was measured in the presence of 20 μm cytochalasin B and was less than 10% of the basal uptake. Transport was stopped with three washes of 50 μm phloretin in ice-cold PBS. Radioactivity was released with 1% Triton X-100 and measured by liquid scintillation counting (45Barros L.F. Pflugers Arch. 1999; 437: 763-770Crossref PubMed Scopus (20) Google Scholar). Samples were normalized for protein content using the Bradford protein assay. ERK, p38-MAPK, JNK, and PI3K/PKB Signaling Pathway Activation—Western blots were performed as described above. The membranes were subjected to immunoblot analysis with anti-phospho-ERK antibody, anti-phospho-p38-MAPK antibody, anti-phospho-JNK antibody, or anti-phospho-PKB antibody. Membranes were stripped and reprobed with anti-ERK antibody, anti-p38-MAPK antibody, anti-JNK antibody, or anti-PKB antibody. Blots were quantified by laser-scanning densitometry. Results were expressed as the ratio of phosphorylated protein kinase to total protein kinase levels. All blots were controlled for equal loading. Bcl-xL Levels—Bcl-xL levels were determined by Western blot analysis for Bcl-xL. Proteins (20–50 μg) were separated by SDS-PAGE on a 15% polyacrylamide gel and were transferred electrophoretically to nitrocellulose. Nonspecific binding sites were blocked with 3% (w/v) nonfat milk powder in PBS (pH 7.5) containing 0.1% (v/v) Tween 20 (PBST) for 60 min at room temperature. Primary antibody was diluted 1:1,000 in blocking solution. Nitrocellulose was incubated with primary antibody overnight at 4 °C. After washing in PBST (three times for 10 min each), nitrocellulose was incubated for 1.5 h at room temperature with horseradish peroxidase-linked secondary antibody (1:5,000 in 1% (w/v) nonfat milk powder in PBST). After repeating the washing procedure described above, bound antibody was detected by ECL with exposure to Hyperfilm for 0.5–30 min. Blots were quantified by scanning densitometry. DNA Fragmentation—For the detection of DNA fragmentation, cells were washed with cold PBS. DNA was prepared by scraping the cells into 1 ml of lysis buffer consisting of 0.8 mm EDTA (pH 8.0), 8 mm Tris-HCl (TE; pH 8.0) and 4% SDS. The DNA was extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) followed by centrifugation at 12,000 × g for 15 min at 4 °C. The resulting DNA was incubated with proteinase K (50 μg/ml, Sigma) for 1 h at 50 °C to facilitate protein disruption. DNA was re-extracted from supernatants with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1). DNA, precipitated from the upper aqueous phase with 0.1 volume of 3 m sodium acetate (pH 5.2) and 2 volumes of ice-cold ethanol, was left at –20 °C overnight before centrifugation. Pellets were resuspended in 200 μl of TE buffer, followed by a 60-min incubation with DNase-free RNase A (2 mg/ml; Sigma) at 37 °C. Samples were re-extracted, and DNA was precipitated as described above. Pellets were resuspended in TE buffer, and DNA concentrations were quantified from the absorbance at 260 nm. DNA samples were analyzed by electrophoresis on 2% agarose and visualized by staining with a solution containing 0.2 μg/ml ethidium bromide (46Morales M.P. Gálvez A. Eltit J.M. Ocaranza P. Díaz-Araya G. Lavandero S. Biochem. Biophys. Res. Commun. 2000; 270: 1029-1035Crossref PubMed Scopus (35) Google Scholar). Measurement of Intracellular Glutathione—GSH levels were determined using an enzymatic assay according to Anderson and Meister (47Anderson M.E. Meister A. J. Biol. Chem. 1980; 255: 9530-9533Abstract Full Text PDF PubMed Google Scholar). Expression of Results and Statistical Analysis—Data shown are the means ± S.E. of the number of independent experiment indicated (n)or representative experiments performed on at least three separate occasions with similar outcome. Data were analyzed by analysis of variance, and comparisons between groups were performed using a protected Tukey's test. A value of p < 0.05 was set as the limit of statistical significance. Effect of Hyperosmotic Solutions of Sorbitol and Mannitol on Polyol Pathway in Cultured Cardiomyocytes—AR is the first enzyme in the polyol pathway, and different reports have shown that a hyperosmotic environment stimulates the expression and activity of AR in many cell types (48Kaneko M. Carper D. Nishimura C. Millen J. Bock M. Hohman T.C. Exp. Cell Res. 1990; 188: 135-140Crossref PubMed Scopus (57) Google Scholar, 49Mizisin A.P. Li L. Perello M. Freshwater J.D. Kalichman M.W. Roux L. Calcutt N.A. Am. J. Physiol. 1996; 270: F90-F97PubMed Google Scholar). The time course of hyperosmotic AR induction by sorbitol is depicted in Fig. 1A. Induction of AR was slow and only detectable after 8 h of hyperosmotic exposure. Maximum AR levels were reached after 24 h; however, after 32 h, AR levels decreased again. To assess whether hyperosmolarity affects the levels of AR in cardiac myocytes, we compared the amount of AR protein in lysates obtained from isosmotically or hyperosmotically treated cardiomyocytes with sorbitol and mannitol for 24 h. AR was detected as a single band with M r 36,000 in isosmotically and hyperosmotically treated cells. Hyperosmotic challenge with sorbitol (400 and 600 mosmol/kg water) caused an increase (1.4- and 2.5-fold, respectively) in the amount of AR protein after 24 h of treatment (Fig. 1B). Mannitol did not significantly change AR protein levels in hyperosmotically treated cardiomyocytes when compared with control cells maintained under isosmotic conditions (270 mosmol/kg water) (Fig. 1B). Thus, AR induction in cardiac myocyte was dependent on the nature of the osmolyte. Hyperosmolarity is also a known regulator of AR activity in some cell types (23Burg M.B. Am. J. Physiol. 1995; 268: F983-F996Crossref PubMed Google Scholar). Hence, we next determined whether the increases in AR protein levels were paralleled by increases in AR activity levels. Most human tissues have three forms of aldo-keto reductases, namely aldose reductase and aldehyde reductases I and II (26Srivastava S.K. Golblum R.M. Hair G.A. Das B. Biochim. Biophys. Acta. 1984; 800: 220-227Crossref PubMed Scopus (56) Google Scholar). Since there is some overlap in the substrate specificity of various aldo-keto reductases, AR is usually purified by DEAE column chromatography (26Srivastava S.K. Golblum R.M. Hair G.A. Das B. Biochim. Biophys. Acta. 1984; 800: 220-227Crossref PubMed Scopus (56) Google Scholar). Despite the fact that aldehyde reductase is not expressed in cardiac muscle (50Vander Jagt D.L. Robinson B. Taylor K.K. Hunsaker L.A. J. Biol. Chem. 1990; 265: 20982-20987Abstract Full Text PDF PubMed Google Scholar), this partial purification step ensured that measurements in our assay were specific for AR activity. As shown in Fig. 1C, sorbitol-supplemented hyperosmotic medium increased AR activity. In general, AR protein content paralleled the changes in AR activity induced by sorbitol (Fig. 1, B and C). Under all hyperosmotic conditions tested, mannitol failed to increase AR activity in cardiac myocytes (Fig. 1C). Collectively, these results suggest that mannitol neither activated AR nor induced AR expression in rat cardiac myocytes. Several studies in other cell types have suggested that the osmopr