Oxidative Stress-induced Apoptosis in Retinal Photoreceptor Cells Is Mediated by Calpains and Caspases and Blocked by the Oxygen Radical Scavenger CR-6

A critical role for reactive oxygen species (ROS) in photoreceptor apoptosis has been established. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. This study delineates the molecular events that occur after treatment of the photoreceptor cell line 661W with the nitric oxide donor sodium nitroprusside (SNP). Cytosolic calcium levels increased during photoreceptor apoptosis, leading to activation of the calcium-dependent proteases calpains. Furthermore, caspase activation also occurred following SNP insult. However, although treatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone inhibited caspase activity per se in SNP-treated 661W cells, it did not prevent apoptosis. On the other hand, CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) acted as a scavenger of ROS and reduced 661W photoreceptor apoptosis induced by SNP by preventing the activation of a pathway in which calpains have a key role. In summary, we report for the first time that both caspases and calpains are involved in 661W photoreceptor apoptosis and that calpain activation can be prevented by the ROS scavenger CR-6. A critical role for reactive oxygen species (ROS) in photoreceptor apoptosis has been established. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. This study delineates the molecular events that occur after treatment of the photoreceptor cell line 661W with the nitric oxide donor sodium nitroprusside (SNP). Cytosolic calcium levels increased during photoreceptor apoptosis, leading to activation of the calcium-dependent proteases calpains. Furthermore, caspase activation also occurred following SNP insult. However, although treatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone inhibited caspase activity per se in SNP-treated 661W cells, it did not prevent apoptosis. On the other hand, CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) acted as a scavenger of ROS and reduced 661W photoreceptor apoptosis induced by SNP by preventing the activation of a pathway in which calpains have a key role. In summary, we report for the first time that both caspases and calpains are involved in 661W photoreceptor apoptosis and that calpain activation can be prevented by the ROS scavenger CR-6. The cell death process of apoptosis is characterized by a series of morphological and biochemical changes, including membrane blebbing, loss of plasma membrane asymmetry, chromatin cleavage, and DNA fragmentation (1Kaufmann S.H. Hengartner M.O. Trends Cell Biol. 2001; 11: 526-534Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar, 2Ferri K.F. Kroemer G. Nat. Cell Biol. 2001; 3: E255-E263Crossref PubMed Scopus (1322) Google Scholar). Apoptosis plays a central role in tissue modeling during development and, together with the cell-generating process of mitosis, is responsible for the maintenance of cell numbers in multicellular organisms. Deregulation of apoptosis has been well documented in several human pathologies, including cancer, neurodegenerative diseases, and AIDS (3Migliore L. Coppede F. Mutat. Res. 2002; 512: 135-153Crossref PubMed Scopus (149) Google Scholar, 4Olinski R. Gackowski D. Foksinski M. Rozalski R. Roszkowski K. Jaruga P. Free Radic. Biol. Med. 2002; 33: 192-200Crossref PubMed Scopus (263) Google Scholar). Apoptosis also appears to be responsible for the cell loss seen in several disorders of the retina, including retinitis pigmentosa (a heterogeneous group of inherited disorders), glaucoma, and macular degeneration (5Chang G.Q. Hao Y. Wong F. Neuron. 1993; 11: 595-605Abstract Full Text PDF PubMed Scopus (578) Google Scholar, 6Li Z. Milan A. Anderson R.M.L. Hollyfield J. Degenerative Diseases of the Retina. Plenum Publishing Corp., New York1995Google Scholar, 7Dunaief J.L. Dentchev T. Ying G.S. Milam A.H. Arch. Ophthalmol. 2002; 120: 1435-1442Crossref PubMed Scopus (450) Google Scholar, 8Carella G. Eur. J. Ophthalmol. 2003; 3: S5-S10Crossref Scopus (13) Google Scholar). Experiments aimed at unraveling the signaling pathways of apoptosis have identified several distinct mechanisms, and it has largely been accepted that caspases play a key role in both the initiation and execution pathways of apoptosis. However, the involvement of caspases does not seem to be clear-cut in some tissue systems. For example, there is still considerable controversy as to whether caspases play a role in retinal cell death (9Liu C. Li Y. Peng M. Laties A.M. Wen R. J. Neurosci. 1999; 19: 4778-4785Crossref PubMed Google Scholar, 10Carmody R.J. Cotter T.G. Cell Death Differ. 2000; 7: 282-291Crossref PubMed Scopus (127) Google Scholar, 11Donovan M. Carmody R.J. Cotter T.G. J. Biol. Chem. 2001; 276: 23000-23008Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 12Donovan M. Cotter T.G. Cell Death Differ. 2002; 9: 1220-1231Crossref PubMed Scopus (126) Google Scholar). There is also some uncertainty about the role of caspases in neurodegenerative conditions (13Okuno S. Shimizu S. Ito T. Nomura M. Hamada E. Tsujimoto Y. Matsuda H. J. Biol. Chem. 1998; 273: 34272-34277Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 14Zhang X. Chen J. Graham S.H. Du L. Kochanek P.M. Draviam R. Guo F. Nathaniel P.D. Szabo C. Watkins S.C. Clark R.S. J. Neurochem. 2002; 82: 181-191Crossref PubMed Scopus (232) Google Scholar, 15Selznick L.A. Zheng T.S. Flavell R.A. Rakic P. Roth K.A. J. Neuropathol. Exp. Neurol. 2000; 59: 271-279Crossref PubMed Scopus (97) Google Scholar). Recent work from our laboratory has indicated that photoreceptor death in animal models of retinitis pigmentosa proceeds in the absence of caspase activity, suggesting a caspase-independent mechanism of cell destruction (11Donovan M. Carmody R.J. Cotter T.G. J. Biol. Chem. 2001; 276: 23000-23008Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 12Donovan M. Cotter T.G. Cell Death Differ. 2002; 9: 1220-1231Crossref PubMed Scopus (126) Google Scholar, 16Doonan F. Donovan M. Cotter T.G. J. Neurosci. 2003; 23: 5723-5731Crossref PubMed Google Scholar). The exact mechanisms operating in photoreceptor death are still unclear but may involve calpains rather than caspases as the executing enzymes. These studies also suggested a key role for reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; RNI, reactive nitrogen intermediates; SNP, sodium nitroprusside; Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone; PARP, poly(ADP-ribose)polymerase; Ac-DEVD-pNA, acetyl-Asp-Glu-Val-Asp p-nitroanilide; PBS, phosphate-buffered saline; RNI, reactive nitrogen intermediate(s); DHE, dihydroethidium; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ER, endoplasmic reticulum. and reactive nitrogen intermediates (RNI) since inhibitors of nitric-oxide synthase blocked the cell death seen. These results are quite interesting since several studies have indicated that the eye is particularly sensitive to oxidative stress and therefore, modifications of the cellular redox state of the eye have been reported to play an important role in retinal degeneration processes (17Liang F.Q. Godley B.F. Exp. Eye Res. 2003; 76: 397-403Crossref PubMed Scopus (491) Google Scholar, 18Carmody R.J. McGowan A.J. Cotter T.G. Exp. Cell Res. 1999; 248: 520-530Crossref PubMed Scopus (97) Google Scholar, 19Crawford M.J. Krishnamoorthy R.R. Rudick V.L. Collier R.J. Kapin M. Aggarwal B.B. Al-Ubaidi M.R. Agarwal N. Biochem. Biophys. Res. Commun. 2001; 281: 1304-1312Crossref PubMed Scopus (63) Google Scholar, 20Krishnamoorthy R.R. Crawford M.J. Chaturvedi M.M. Jain S.K. Aggarwal B.B. Al-Ubaidi M.R. Agarwal N. J. Biol. Chem. 1999; 274: 3734-3743Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Additional support for the involvement of ROS and oxidative stress in photoreceptor apoptosis comes from several studies in which antioxidants appeared to retard or inhibit the degenerative pathology (21Lam S. Tso M.O. Gurne D.H. Arch. Ophthalmol. 1990; 108: 1751-1757Crossref PubMed Scopus (58) Google Scholar, 22Ranchon I. Gorrand J.M. Cluzel J. Droy-Lefaix M.T. Doly M. Investig. Ophthalmol. Vis. Sci. 1999; 40: 1191-1199PubMed Google Scholar, 23Rosner M. Lam T.T. Fu J. Tso M.O. Arch. Ophthalmol. 1992; 110: 857-861Crossref PubMed Scopus (27) Google Scholar). However, the mechanisms of action of these anti-apoptotic molecules are unclear, and further work is necessary to resolve whether oxidative stress acts as a common mediator of retinal degeneration in retinitis pigmentosa. The retina is composed of several different cell types, and this complicates any studies aimed at delineating the underlying mechanism of photoreceptor apoptosis. The production and characterization of the photoreceptor cell line 661W by Al-Ubaidi et al. (24Al-Ubaidi M.R. Font R.L. Quiambao A.B. Keener M.J. Liou G.I. Overbeek P.A. Baehr W. J. Cell Biol. 1992; 119: 1681-1687Crossref PubMed Scopus (188) Google Scholar) have greatly facilitated work in this area. This cell line expresses several markers of photoreceptors and has proved useful for in vitro studies investigating photoreceptor apoptosis (19Crawford M.J. Krishnamoorthy R.R. Rudick V.L. Collier R.J. Kapin M. Aggarwal B.B. Al-Ubaidi M.R. Agarwal N. Biochem. Biophys. Res. Commun. 2001; 281: 1304-1312Crossref PubMed Scopus (63) Google Scholar, 20Krishnamoorthy R.R. Crawford M.J. Chaturvedi M.M. Jain S.K. Aggarwal B.B. Al-Ubaidi M.R. Agarwal N. J. Biol. Chem. 1999; 274: 3734-3743Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 25Roque R.S. Rosales A.A. Jingjing L. Agarwal N. Al-Ubaidi M.R. Brain Res. 1999; 836: 110-119Crossref PubMed Scopus (95) Google Scholar, 26Tuohy G. Millington-Ward S. Kenna P.F. Humphries P. Farrar G.J. Investig. Ophthalmol. Vis. Sci. 2002; 43: 3583-3589PubMed Google Scholar). In the context of this work, we have used this cell line to investigate the role played by oxidative stress in photoreceptor apoptosis induced by the nitric oxide donor sodium nitroprusside (SNP). This constitutes a direct extension of previous work from this laboratory (11Donovan M. Carmody R.J. Cotter T.G. J. Biol. Chem. 2001; 276: 23000-23008Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) in which we showed that nitric oxide and ROS play a key role in driving photoreceptor apoptosis in vivo. In this study, we show that SNP induced ROS production in the mitochondrion and that this in turn triggered apoptosis, with both calpains and caspases playing a role. Treatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD-fmk) inhibited the activation of caspases, but does not appear to be a useful strategy to prevent oxidative stress-induced apoptosis in 661W photoreceptor cells. On the other hand, in the context of this work, we show that CR-6 (a vitamin E analog that has been shown to prevent glutamate neurotoxicity in cultured neurons due to its role as a nitric oxide scavenger (27Montoliu C. Llansola M. Saez R. Yenes S. Messeguer A. Felipo V. Biochem. Pharmacol. 1999; 58: 255-261Crossref PubMed Scopus (19) Google Scholar)), interfered with oxidative stress-induced apoptosis in 661W cells by preventing the activation of the calpain-mediated apoptotic pathway. Drugs, Reagents, and Antibodies—SNP was purchased from Sigma (Poole, United Kingdom). The synthesis of CR-6 has been described elsewhere (28Casas J. Gorchs G. Sanchez-Baeza F. Teixidor P. Messeguer A. J. Agric. Food Chem. 1992; 40: 585-590Crossref Scopus (28) Google Scholar). Cell Signaling Technology (Beverly, MA) provided antipoly(ADP-ribose) polymerase (PARP) (catalog no. 9542), anti-caspase-3 (catalog no. 9662), anti-caspase-9 (catalog no. 9504), and anticaspase-12 (catalog no. 2202) antibodies. Anti-Calpain-1 (catalog no. 208753) and anti-calpain-2 (catalog no. 208755) antibodies were purchased from Calbiochem. Anti-calpastatin (sc-7561) and anti-calpain small regulatory subunit (C-20; sc-7528) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-actin antibody was from Sigma. Peroxidase-conjugated anti-rabbit, anti-goat, and anti-mouse secondary antibodies were obtained from Dako Corp. The broad-spectrum caspase inhibitor Z-VAD-fmk was purchased from Bachem Ltd. (Meyerside, United Kingdom). Alexis Co. (Läufefingen, Switzerland) provided the caspase-3 substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA). Cell Culture—661W cells were kindly provided by Muayyad Al-Ubaidi, Department of Cell Biology, University of Oklahoma (Oklahoma City, OK). 661W cells were routinely grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum (both from Sigma) and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO2 atmosphere as described (20Krishnamoorthy R.R. Crawford M.J. Chaturvedi M.M. Jain S.K. Aggarwal B.B. Al-Ubaidi M.R. Agarwal N. J. Biol. Chem. 1999; 274: 3734-3743Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). SNP was used to induce apoptosis at the doses indicated; SNP stock was prepared at 100 mm in phosphate-buffered saline (PBS) (pH 7.4). Pretreatment of 661W photoreceptors with CR-6 was done for 15 min at 37 °C as described above. A 100 mm CR-6 stock was prepared in Me2SO. Treatments carried out in 6-well plates (Nalge Nunc International, Hereford, United Kingdom) were as follow. Cells (75 × 104/well) were seeded and allowed to attach for 20 h at 37 °C. Following insult, cells were detached with a trypsin/EDTA solution (Sigma) and collected together with their supernatants for flow cytometric analysis. Cells for Western blot analysis were grown in 75-cm2 flasks (Sarstedt AG & Co., Nümbrecht, Germany). Initially, cells (8 × 105/flask) were seeded and allowed to attach before treatment. Samples were collected 24 or 48 h post-insult. Analysis of Generated RNI: Griess Reaction—Determination of the presence of RNI was done by means of the Griess reaction (29Marzinzig M. Nussler A.K. Stadler J. Marzinzig E. Barthlen W. Nussler N.C. Beger H.G. Morris Jr., S.M. Bruckner U.B. Nitric Oxide. 1997; 1 (S. M. J.): 177-189Crossref PubMed Scopus (297) Google Scholar). Briefly, cells (5 × 104) were incubated for 15 min in the dark at room temperature with 40 μl of Griess reagent (Alexis Corp.). Nitrites present in the samples react with sulfanilic acid and N-(1-naphthly)ethylenediamine dihydrochloride in the presence of phosphoric acid, which produces a colored azo dye that can be measured at 548 nm. Analysis of Intracellular ROS Generation—Measurement of superoxide anion levels was carried out as described previously (30Gorman A. McGowan A. Cotter T.G. FEBS Lett. 1997; 404: 27-33Crossref PubMed Scopus (208) Google Scholar). Briefly, cells were loaded with 10 μm dihydroethidium (DHE) (Molecular Probes, Inc., Leiden, The Netherlands), prepared from a 10 mm stock in Me2SO, for 15 min at 37 °C. Superoxide anion oxidizes DHE intracellularly to produce ethidium bromide, which fluoresces upon interaction with DNA. The fluorescence due to ethidium bromide in a BD Biosciences FACScan flow cytometer with excitation and emission settings of 488 and 590 nm, respectively, was monitored to assess superoxide anion levels. CellQuest software was used for data analysis, and 10,000 events/sample were acquired. Cell Death Measurement—Propidium iodide (Sigma) was used to quantify cell death. Treated cells were collected as described above, washed once with ice-cold PBS, and resuspended to a final concentration of 1 × 105 cells/ml. Propidium iodide (50 μg/ml) was added immediately before flow cytometric analysis. Fluorescence was measured at FL2 (590 nm), and 10,000 events/sample were acquired. Measurement of Intracellular Free Ca2+—Intracellular Ca2+ levels were determined using the intracellular Ca2+ probe Fluo-3/acetoxymethyl ester (Molecular Probes, Inc.), which binds Ca2+ with a 1:1 stoichiometry. After trypsinization, cells were washed once with PBS and resuspended in fresh buffer. Cells were incubated in the darkness with 250 nm Fluo-3, prepared from a 500 μm stock, for 30 min at 37 °C. Fluorescence was measured at FL1 (530 nm) in a BD Biosciences FACScan flow cytometer with excitation at 488 nm, and CellQuest software was employed for subsequent data analysis. At least 10,000 events/sample were acquired. Western Blot Analysis—After exposure to drug, whole cell extracts were obtained and resolved by denaturing SDS-PAGE. Briefly, harvested cells were washed twice with ice-cold PBS; resuspended in cell lysis buffer (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1 mm Na3VO4, 1 mm NaF, 1 mm EGTA, 1% Nonidet P-40, and 0.25% sodium deoxycholate) containing antipain (1 μg/ml), aprotinin (1 μg/ml), chymostatin (1 μg/ml), leupeptin (0.1 μg/ml), pepstatin (1 μg/ml), and 0.2 mm 4-(2-aminoethyl)benzenesulfonyl fluoride; and incubated on ice for 20 min. Supernatants were recovered by a 10-min centrifugation (10,000 × g) at 4 °C, and protein concentration was determined with the Bio-Rad protein assay using bovine serum albumin as a standard. Proteins (20–40 μg) were diluted in 2× sample buffer (10% SDS and 100 mm each dithiothreitol, glycerol, bromphenol blue, and Tris-HCl) and resolved on 6–12% SDS-polyacrylamide gels. Then proteins were transferred onto nitrocellulose membranes (Schleicher & Schüll, Dassel, Germany), and the blots were blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline and 0.1% Tween 20 for 1 h at room temperature. Membranes were incubated overnight at 4 °C with the appropriate dilution of primary antibody (1:5000 for anti-m-calpain and anti-μ-calpain antibodies and 1:1000 for all other antibodies). After three 5-min washes with Tris-buffered saline and 0.1% Tween 20, the blots were incubated with the corresponding peroxidase-conjugated secondary antibody (1:1000 dilution) for 1 h at room temperature. They were then washed again three times with Tris-buffered saline and 0.1% Tween 20, rinsed briefly with PBS, and developed with enhanced chemiluminescence reagents (ECL, Amersham Biosciences, Buckinghamshire, United Kingdom). Detection of β-actin (1:5000 antibody dilution) was used as control for equal loading of protein. Determination of Ac-DEVD-pNA Cleavage—661W cells (8 × 105) were grown in 75-cm2 flasks and preincubated at 37 °C with the caspase inhibitor Z-VAD-fmk (50 μm) for 1 h prior to insult with 0.3 mm SNP. Untreated and 0.3 mm SNP-treated 661W cells were used as negative and positive controls, respectively. After a 24-h incubation, cells were collected as described above and centrifuged at 500 × g for 5 min. The pellet was resuspended in 1 ml of ice-cold 1× PBS and transferred to a microcentrifuge tube. Subsequently, the pellet was resuspended in 50 μl of ice-cold lysis buffer (100 mm HEPES (pH 7.4), 1 m NaCl, 1% CHAPS, 1 m dithiothreitol, 10 mm EDTA, and 1% Nonidet P-40) and incubated on ice for 10 min. Following a 20-s sonication, cell lysates were centrifuged for 10 min at 12,500 × g. The protein content of each sample was determined by the Bio-Rad protein assay using bovine serum albumin as a standard. An equal quantity of protein (50 μg) was loaded into each well of a microtiter plate, and the final volume was made up to 90 μl with assay buffer (same as lysis buffer minus 1% Nonidet P-40). Lysates were incubated with 0.2 mm Ac-DEVD-pNA at 37 °C for 20 h. Cleavage of the peptide substrate DEVD-pNA was monitored by liberation of chromogenic pNA in a SpectraMax-340 plate reader (Molecular Devices, Menlo Park, CA) by measuring absorption at 405 nm. Annexin V Assay—A combined staining with fluorescein isothiocyanate-conjugated annexin V and propidium iodide was performed as a measure of apoptosis. Harvested cells were washed once with Ca2+ binding buffer (10 mm HEPES (pH 7.4), 140 mm NaCl, and 2.5 mm CaCl2) and resuspended in 100 μl of the same buffer containing fluorescein isothiocyanate-conjugated annexin V (IQ Products, Groningen, The Netherlands). After a 15-min incubation in the dark at room temperature, cells were diluted with 400 μl of binding buffer, and propidium iodide was added before flow cytometric analysis. Fluorescence was measured as described above. SNP Induces ROS Production and Cell Death in the Photoreceptor Cell Line 661W—Oxidative stress has been reported to play an important role in photoreceptor cell death (18Carmody R.J. McGowan A.J. Cotter T.G. Exp. Cell Res. 1999; 248: 520-530Crossref PubMed Scopus (97) Google Scholar, 20Krishnamoorthy R.R. Crawford M.J. Chaturvedi M.M. Jain S.K. Aggarwal B.B. Al-Ubaidi M.R. Agarwal N. J. Biol. Chem. 1999; 274: 3734-3743Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Previous work carried out in this laboratory demonstrated that nitric oxide mediates retinal degeneration in vivo and that ROS significantly contribute to photoreceptor cell death (11Donovan M. Carmody R.J. Cotter T.G. J. Biol. Chem. 2001; 276: 23000-23008Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Despite these findings, the mechanisms leading to photoreceptor apoptosis are still not fully understood, and little is known about oxidative pathways involved in retinal death. Therefore, to gain a better understanding of the events triggered by RNI and ROS in photoreceptors, the 661W cell line was treated with increasing concentrations of the nitric oxide donor SNP (0.1–0.5 mm) for 20 h. Previous studies have shown that SNP can trigger apoptosis in neurons (31Tamatani M. Ogawa S. Nunez G. Tohyama M. Cell Death Differ. 1998; 5: 911-919Crossref PubMed Scopus (113) Google Scholar). The production of nitric oxide metabolites was detected with the Griess reaction (29Marzinzig M. Nussler A.K. Stadler J. Marzinzig E. Barthlen W. Nussler N.C. Beger H.G. Morris Jr., S.M. Bruckner U.B. Nitric Oxide. 1997; 1 (S. M. J.): 177-189Crossref PubMed Scopus (297) Google Scholar). In this method, nitrite metabolites are detected with a colorimetric reaction. Therefore, the value of the resulting absorbance is proportional to the amount of RNI present in the samples. As illustrated in Fig. 1A, an increase in nitric oxide metabolites was observed in 661W photoreceptor cells upon treatment with up to a concentration of SNP of 0.3 mm. Higher concentrations of SNP did not significantly modify the concentration of RNI in the cells.Fig. 1SNP-induced RNI and ROS production and apoptotic cell death in the photoreceptor cell line 661W. 661W cells were treated with increasing concentrations of SNP (0.1–0.5 mm) for 20 h. A, the presence of nitric oxide metabolites was detected colorimetrically by the Griess reaction. The error bars correspond to the S.D. of three independent experiments done in duplicate. B, superoxide anion levels were quantified by flow cytometry using the probe DHE prior to SNP treatment (0 mm) and after treatment with 0.1–0.5 mm SNP. The percentage of cells displaying increased levels of ROS is shown at each SNP concentration. Results are representative of three independent experiments done in duplicate. C, cell death measurements were done by flow cytometry using propidium iodide. Dot plots show that treatment of 661W photoreceptor cells with the nitric oxide donor SNP induced oxidative stress, leading to cell death. Results are representative of three independent experiments carried out in duplicate. FSC-H, forward-angle light scatter.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In parallel with this study, superoxide anion formation was monitored using the probe DHE (Fig. 1B). Contrary to results observed for RNI, treatment with the lowest concentrations of SNP (0.1–0.2 mm) did not significantly alter the levels of superoxide anions present in 661W cells, and increased levels of ROS were not detected until treatment with 0.3 mm SNP. Incubation with 0.5 mm SNP increased superoxide anion levels up to 73%, indicating a modification of the redox state in 661W cells. Quantification of cell death was performed by flow cytometric analysis. Propidium iodide was used to quantify the population of cells in which membrane integrity was lost. As expected, treatment of 661W cells with SNP (0.1–0.5 mm) induced cell death, with only 29% of cells surviving post-insult with 0.5 mm SNP (Fig. 1C). Nevertheless, an SNP concentration of 0.3 mm, which resulted in ∼50% cell death, was chosen for further studies. Apoptotic cell death was then assessed by detection of DNA fragmentation by DNA gel electrophoresis after treatment with 0.3 mm SNP. The presence of the DNA ladder was detected 48 h post-insult (data not shown). In conclusion, these results confirm and extend previous observations showing the involvement of ROS in photoreceptor cell death (11Donovan M. Carmody R.J. Cotter T.G. J. Biol. Chem. 2001; 276: 23000-23008Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 12Donovan M. Cotter T.G. Cell Death Differ. 2002; 9: 1220-1231Crossref PubMed Scopus (126) Google Scholar, 16Doonan F. Donovan M. Cotter T.G. J. Neurosci. 2003; 23: 5723-5731Crossref PubMed Google Scholar). 661W Photoreceptor Cell Death Induced by SNP Is Mediated by an Increase in Intracellular Ca2+Levels and Calpain Activation—In the cell, oxidative stress induces Ca2+ influx from the extracellular environment and efflux from intracellular stores, leading to an increase in cytoplasmic Ca2+ levels, which has been associated with apoptosis in diverse in vivo and in vitro systems (32Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1807) Google Scholar). It has also been shown that elevated cytosolic Ca2+ levels play a role in rod photoreceptor apoptosis (11Donovan M. Carmody R.J. Cotter T.G. J. Biol. Chem. 2001; 276: 23000-23008Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 33Frasson M. Sahel J.A. Fabre M. Simonutti M. Dreyfus H. Picaud S. Nat. Med. 1999; 5: 1183-1187Crossref PubMed Scopus (207) Google Scholar, 34He L. Poblenz A.T. Medrano C.J. Fox D.A. J. Biol. Chem. 2000; 275: 12175-12184Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). In this study, intracellular Ca2+ levels were determined using the fluorescent probe Fluo-3/acetoxymethyl ester. To ensure that SNP treatment of 661W photoreceptor cells increased [Ca2+]i, calcium levels in untreated 661W cells were compared with those in cells incubated with 0.3 mm SNP for 20 h. As depicted in the histogram in Fig. 2A, treatment of 661W cells with 0.3 mm SNP produced an increase in FL1 fluorescence, indicating an increased concentration of intracellular calcium. Activation of calcium-dependent proteases such as calpains is thought to play an important role in certain models of oxidative stress-induced apoptosis (35Nakagawa T. Yuan J. J. Cell Biol. 2000; 150: 887-894Crossref PubMed Scopus (1057) Google Scholar, 36Ray S.K. Fidan M. Nowak M.W. Wilford G.G. Hogan E.L. Banik N.L. Brain Res. 2000; 852: 326-334Crossref PubMed Scopus (185) Google Scholar, 37O'Donovan C.N. Tobin D. Cotter T.G. J. Biol. Chem. 2001; 276: 43516-43523Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). In addition, several studies have shown that calpains can contribute to neuronal death (38McCollum A.T. Nasr P. Estus S. J. Neurochem. 2002; 82: 1208-1220Crossref PubMed Scopus (59) Google Scholar, 39Neumar R.W. Xu Y.A. Gada H. Guttmann R.P. Siman R. J. Biol. Chem. 2003; 278: 14162-14167Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 40Rami A. Neurobiol. Dis. 2003; 13: 75-88Crossref PubMed Scopus (149) Google Scholar), and calpain inhibitors have been used to block apoptosis (41Jordan J. Galindo M.F. Miller R.J. J. Neurochem. 1997; 68: 1612-1621Crossref PubMed Scopus (171) Google Scholar, 42Rami A. Agarwal R. Botez G. Winckler J. Brain Res. 2000; 866: 299-312Crossref PubMed Scopus (114) Google Scholar, 43McGinnis K.M. Whitton M.M. Gnegy M.E. Wang K.K. J. Biol. Chem. 1998; 273: 19993-20000Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). However, to date, little is known about the involvement of calpains in photoreceptor cell death. Previous work from this laboratory has reported the activation of calpains during light-induced retinal degeneration (12Donovan M. Cotter T.G. Cell Death Differ. 2002; 9: 1220-1231Crossref PubMed Scopus (126) Google Scholar). To verify the activation of calpains in 661W cells following increased levels of intracellular Ca2+, we performed immunoblot analyses using polyclonal antibodies against the calpain isoforms m-calpain and μ-calpain. Moreover, we also analyzed the common small regulatory subunit of calpains, which is dissociated in response to increased levels of cytosolic calcium (44Pal G.P. Elce J.S. Jia Z. J. Biol. Chem. 2001; 276: 47233-47238Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). As illustrated in Fig. 2B, a decrease in the total amount of m-calpain was observed after a 24-h treatment of 661W cells with 0.3 mm SNP (50% cell death). The reduction in calpain levels was even more evident after 48 h (76% cell death). Although calpains require only an increase in [Ca2+]i to become active, the autoproteolytic cleavage further enhanced their activity. Fig. 2B shows that SNP-treated 661W cells contained the autolysed form of μ-calpain (78 kDa), indicating that this isoform is also activated by SNP. Consistent with the results of the immunoblot analysis of the latent forms of calpains, a decrease in the intensity of the band corresponding to the calpain small subunit (28 kDa) was observed after 24 h of treatment, whereas after 48 h, the band was not