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Calcium-induced Calpain Mediates Apoptosis via Caspase-3 in a Mouse Photoreceptor Cell Line

2004; Elsevier BV; Volume: 279; Issue: 34 Linguagem: Inglês

10.1074/jbc.m401037200

1083-351X

Ashish K. Sharma, Bäerbel Rohrer,

Connexins and lens biology

The rd mouse, an accepted animal model for photoreceptor degeneration in retinitis pigmentosa, has a recessive mutation for the gene encoding the β-subunit of the cGMP phosphodiesterase. This mutation results in high levels of cGMP, which leaves an increased number of the cGMP-gated channels in the open state, thus allowing intracellular calcium (Ca2+) to rise to toxic levels, and rapid photoreceptor degeneration follows. To delineate the events in rd photoreceptor degeneration, we demonstrated an increase in calpain and caspase-3 activity, hypothesizing that Ca2+-mediated apoptosis in photoreceptors is mediated by calpain, involving mitochondrial depolarization and caspase-3 activation. To examine this hypothesis further, a murine photoreceptor-derived cell line (661W) was treated with the Ca2+ ionophore A23187, cGMP-gated channel agonist 8-bromo-cGMP, or phosphodiesterase inhibitor isobutylmethylxanthine to mimic the increased Ca2+ influx seen in the rd photoreceptors. Ca2+-induced cell death in 661W cells was found to be mediated by calpain and caspase-3 and could be completely inhibited by the calpain inhibitor SJA6017, implicating both calpain and caspases in the apoptotic process. The apoptotic events correlated in an SJA6017-inhibitable manner with bid cleavage, mitochondrial depolarization, cytochrome c release, and caspase-3 and -9 activation. We concluded that Ca2+ influx in the rd model of photoreceptor degeneration leads to the activation of the cysteine protease calpain, which executes apoptosis via modulation of caspase-3 activity. The rd mouse, an accepted animal model for photoreceptor degeneration in retinitis pigmentosa, has a recessive mutation for the gene encoding the β-subunit of the cGMP phosphodiesterase. This mutation results in high levels of cGMP, which leaves an increased number of the cGMP-gated channels in the open state, thus allowing intracellular calcium (Ca2+) to rise to toxic levels, and rapid photoreceptor degeneration follows. To delineate the events in rd photoreceptor degeneration, we demonstrated an increase in calpain and caspase-3 activity, hypothesizing that Ca2+-mediated apoptosis in photoreceptors is mediated by calpain, involving mitochondrial depolarization and caspase-3 activation. To examine this hypothesis further, a murine photoreceptor-derived cell line (661W) was treated with the Ca2+ ionophore A23187, cGMP-gated channel agonist 8-bromo-cGMP, or phosphodiesterase inhibitor isobutylmethylxanthine to mimic the increased Ca2+ influx seen in the rd photoreceptors. Ca2+-induced cell death in 661W cells was found to be mediated by calpain and caspase-3 and could be completely inhibited by the calpain inhibitor SJA6017, implicating both calpain and caspases in the apoptotic process. The apoptotic events correlated in an SJA6017-inhibitable manner with bid cleavage, mitochondrial depolarization, cytochrome c release, and caspase-3 and -9 activation. We concluded that Ca2+ influx in the rd model of photoreceptor degeneration leads to the activation of the cysteine protease calpain, which executes apoptosis via modulation of caspase-3 activity. Inherited retinal degeneration diseases such as retinitis pigmentosa result in photoreceptor degeneration in which night blindness and loss of peripheral vision are the initial symptoms finally culminating in a progressive loss of vision (1Berson E.L. Investig. Ophthalmol. Vis. Sci. 1993; 34: 1659-1676PubMed Google Scholar). At least 30 genes have been implicated in the genetics of retinitis pigmentosa, many of which encode photoreceptor-specific proteins such as the structural protein peripherin (2Farrar G.J. Kenna P. Jordan S.A. Kumar-Singh R. Humphries M.M. Sharp E.M. Sheils D. Humphries P. Genomics. 1992; 14: 805-807Crossref PubMed Scopus (49) Google Scholar), rod outer segment membrane protein-1 (3Bascom R.A. Manara S. Collins L. Molday R.S. Kalnins V.I. McInnes R.R. Neuron. 1992; 8: 1171-1184Abstract Full Text PDF PubMed Scopus (204) Google Scholar), rod cGMP phosphodiesterase (4McLaughlin M.E. Sandberg M.A. Berson E.L. Dryja T.P. Nat. Genet. 1993; 4: 130-134Crossref PubMed Scopus (499) Google Scholar), and rhodopsin (5Farrar G.J. McWilliam P. Bradley D.G. Kenna P. Lawler M. Sharp E.M. Humphries M.M. Eiberg H. Conneally P.M. Trofatter J.A. Genomics. 1990; 8: 35-40Crossref PubMed Scopus (78) Google Scholar). Extensive studies of retinal degeneration in animal models such as the rd (6Farber D.B. Park S. Yamashita C. Exp. Eye Res. 1988; 46: 363-374Crossref PubMed Scopus (39) Google Scholar) and the rds (7Blanks J.C. Adinolfi A.M. Lolley R.N. J. Comp. Neurol. 1974; 156: 95-106Crossref PubMed Scopus (114) Google Scholar) mice, as well as several other knock-out and transgenic mice, suggest that apoptosis is the common feature of photoreceptor cell death in all the models (8Chang G.Q. Hao Y. Wong F. Neuron. 1993; 11: 595-605Abstract Full Text PDF PubMed Scopus (569) Google Scholar). Apoptosis is an active mode of cell death that is induced by a variety of physiological and pathological stimuli. Photoreceptor apoptosis resulting in visual deficits occurs in humans and animals secondary to inherited, chemical-, disease-, and injury-induced retinal degeneration. Calcium (Ca2+) overload is suggested to play a fundamental role in the process of photoreceptor apoptosis in chemical-induced and inherited retinal degenerations. Sustained increases in intracellular Ca2+ trigger apoptosis in a diverse array of in vivo and in vitro systems (9Susin S.A. Zamzami N. Kroemer G. Biochim. Biophys. Acta. 1998; 1366: 151-165Crossref PubMed Scopus (757) Google Scholar, 10Nicotera P. Orrenius S. Cell Calcium. 1998; 23: 173-180Crossref PubMed Scopus (400) Google Scholar). Various studies have indicated that elevated rod photoreceptor Ca2+ plays a key role in the process of apoptotic cell death in humans and animal models during inherited retinal degeneration, retinal diseases and injuries, and chemical exposure. These include patients with retinitis pigmentosa and cancer-associated retinopathy (11Thirkill C.E. Roth A.M. Keltner J.L. Arch. Ophthalmol. 1987; 105: 372-375Crossref PubMed Scopus (212) Google Scholar), retinal degeneration mice (12Fox D.A. Poblenz A.T. He L. Ann. N. Y. Acad. Sci. 1999; 893: 282-285Crossref PubMed Scopus (87) Google Scholar), and rats with light-induced damage (13Edward D.P. Lam T.T. Shahinfar S. Li J. Tso M.O. Arch. Ophthalmol. 1991; 109: 554-562Crossref PubMed Scopus (53) Google Scholar) and hypoxicischemic injury (14Crosson C.E. Willis J.A. Potter D.E. J. Ocul. Pharmacol. 1990; 6: 293-299Crossref PubMed Scopus (53) Google Scholar). The genetic defect in the rd mouse is a mutation of the β-subunit of the cGMP phosphodiesterase, which is the same gene affected in human recessive retinitis pigmentosa. The recessive mutation in rd mice leads to rapid photoreceptor degeneration, which completely eliminates photoreceptors by postnatal day 21 (P21) (15Carter-Dawson L.D. LaVail M.M. Sidman R.L. Investig. Ophthalmol. Vis. Sci. 1978; 17: 489-498PubMed Google Scholar). The absence of phosphodiesterase activity leads to an increased accumulation of cGMP in the photoreceptors compared with those in wild type mice (6Farber D.B. Park S. Yamashita C. Exp. Eye Res. 1988; 46: 363-374Crossref PubMed Scopus (39) Google Scholar). In turn, this leads to an increase in Na+ and Ca2+ influx through the cGMP-gated cation channels, which subsequently causes a metabolic overload and toxicity to the photoreceptors. Photoreceptor Ca2+ levels in the rd mouse are elevated, starting at P5 and have been shown to be increased to ∼190% by P15 compared with photoreceptors from wild type mice (12Fox D.A. Poblenz A.T. He L. Ann. N. Y. Acad. Sci. 1999; 893: 282-285Crossref PubMed Scopus (87) Google Scholar). This increase in free intracellular Ca2+ may directly activate apoptosis in photoreceptor degeneration, which is postulated to be the cause of cell death in the rd mouse (8Chang G.Q. Hao Y. Wong F. Neuron. 1993; 11: 595-605Abstract Full Text PDF PubMed Scopus (569) Google Scholar). Although the molecular mechanisms underlying the apoptotic pathway in the rd model remain unknown, there are two possible (but not mutually independent) triggering mechanisms: one is Ca2+ overload, and the other is the generation of reactive oxygen species. There is converging evidence to suggest that mitochondria and caspases play a fundamental role in the execution of Ca2+-induced rod apoptosis (16He L. Poblenz A.T. Medrano C.J. Fox D.A. J. Biol. Chem. 2000; 275: 12175-12184Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). A crucial link is understanding how increased Ca2+ leads to mitochondrial depolarization and/or caspase-3 activation; however, this remains unclear. The two events could be independent (caspase-3 is a Ca2+-activated protease) or dependent (events downstream of mitochondrial dysfunction are know to lead to caspase-3 activation). Interestingly, in unpublished gene expression array experiments comparing retinas from rd and wild type mice, we have observed a significant increase in the levels of mRNA for m-calpain in the rd mouse. 1K. Hulse and B. Rohrer, unpublished results.1K. Hulse and B. Rohrer, unpublished results. Based on that and other observations in various systems, we propose the following pathway of apoptosis in photoreceptor degeneration (Fig. 1): an increase in intracellular Ca2+ activates calpain, a Ca2+-dependent cysteine protease (17Molinari M. Anagli J. Carafoli E. J. Biol. Chem. 1994; 269: 27992-27995Abstract Full Text PDF PubMed Google Scholar), which can cleave the proapoptotic Bcl-2 family protein bid. Interaction of truncated bid (t-bid) with the mitochondrial permeability transition pore (PTP) 2The abbreviations used are: PTP, permeability transition pore; 8-Br-cGMP, 8-bromo-cGMP; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Δψm, mitochondrial membrane potential loss; FLIPR, Fluorometric Imaging Plate Reader System; IBMX, isobutylmethylxanthine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PIPES, 1,4-piperazinediethanesulfonic acid; t-bid, truncated bid.2The abbreviations used are: PTP, permeability transition pore; 8-Br-cGMP, 8-bromo-cGMP; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Δψm, mitochondrial membrane potential loss; FLIPR, Fluorometric Imaging Plate Reader System; IBMX, isobutylmethylxanthine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PIPES, 1,4-piperazinediethanesulfonic acid; t-bid, truncated bid. causes mitochondrial membrane potential loss (Δψm), leading to the release of cytochrome c. The increase in cytoplasmic cytochrome c causes the assembly of the apoptosome (18Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6235) Google Scholar), leading to the activation of caspases, which cleave downstream death substrates and activate endonucleases that cleave genomic DNA into fragments resulting in the apoptotic nuclear morphology (19Sun X.M. MacFarlane M. Zhuang J. Wolf B.B. Green D.R. Cohen G.M. J. Biol. Chem. 1999; 274: 5053-5060Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar). In this study, we first establish that both calpain and caspase-3 are active in the rd mouse retina. To investigate further the Ca2+-activated apoptotic pathways in photoreceptors, we used 661W photoreceptor cells exposed to the Ca2+ ionophore A23187, the cGMP-gated channel agonist 8-Br-cGMP, or the phosphodiesterase inhibitor 3-isobutylmethylxanthine (IBMX). The 661W cells, which express several markers of photoreceptor cells such as cone opsins, transducin, and cone arrestin (20Tan E. Ding X.-Q. Saadi A. Agarwal N. Nash M.I. Al-Ubaidi M.R. Investig. Ophthalmol. Vis. Sci. 2004; 45: 764-768Crossref PubMed Scopus (249) Google Scholar), are sensitive to photooxidative stress similar to normal retinal photoreceptor cells (21Krishnamoorthy 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 (137) Google Scholar) and showed an increase in intracellular Ca2+ after treatment with A23187, 8-Br-cGMP, or IBMX, making them an excellent in vitro model to study the potential pathways of photoreceptor degeneration of the rd mouse retina. Using appropriate enzyme inhibitors for calpain (SJA6017) and caspase-3 (z-devd-fmk), we were able to confirm the molecular mechanisms involved in the Ca2+ influx-activated apoptotic pathway outlined in the hypothesis. Animals—The rd mouse line was generously provided by Dr. Debra Farber (Jules Stein, UCLA), and wild type animals with the same genetic background (C57BL/6) were purchased from Taconic Farms (Germantown, NY) and maintained in the Animal Facility at the Medical University of South Carolina with food and water ad libitum. Animals were handled in accordance with institutional guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Cell Culture—The 661W cell line used for this study was generously provided by Dr. Muayyad Al-Ubaidi (University of Oklahoma). These cells grow readily with a doubling time of ∼24 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. In most assays, we used 100-mm culture dishes with cells seeded at a concentration of 1 × 106 in 5 ml of growth medium and allowed them to expand to ∼85% to 90% confluence. Cell treatments were performed with the following compounds: Ca2+ ionophore A23187 (Molecular Probes), cGMP-gated channel agonist, 8-Br-cGMP (Sigma), phosphodiesterase inhibitor IBMX (Sigma), calpain inhibitor SJA6017 (Senju Pharmaceuticals, Kobe, Japan), caspase-3 inhibitor z-devd-fmk (Alexis Biochemicals), caspase-8 inhibitor z-ietd-fmk (Calbiochem) caspase-9 inhibitor z-lehd-fmk (Calbiochem), and cyclosporin A (Sigma). Enzymatic Assay for Calpain Activity—Calpain activity was measured using Ac-LLY-AFC as the substrate provided in the calpain activity assay kit (Calbiochem). 661W cells (1 × 106) were resuspended in 100 μl of extraction buffer and centrifuged at 10,000 × g for 1 min. The cell lysate was transferred to a 96-well plate to which 10 μl of 10× reaction buffer and 5 μl of calpain substrate were added. After incubation at 37 °C for 1 h, the samples were read in a fluorometer equipped with a 400 nm excitation filter and a 505 nm emission filter. In the case of retinas dissected from rd and wild type mice, the tissue was resuspended in 100 μl of lysis buffer (50 mmol/liter B-glycerophosphate, 20 mmol/liter EDTA, 15 mmol/liter MgCl2, 1 mmol/liter dithiothreitol), kept on ice at 4 °C for 15 min, and then homogenized by tissue grinding and sonication on ice. The supernatant was collected after centrifugation at 20,000 × g for 15 min. Protein content was measured using Bradford Folin's reagent method (Bio-Rad Laboratories), and enzyme activities were expressed as relative fluorescence units/mg of protein of each sample. The arbitrary values were presented as the mean ± S.E. of three to five experiments or conditions. Enzymatic Assay for Caspase Activity—The activities of caspase-3, -8, and -9 were measured using the following fluorogenic enzyme substrates: z-DEVD-AFC (Molecular Probes), IETD-AFC (Clontech), and LEHD-AFC (Alexis Biochemicals), respectively. After the desired duration of treatments, the cells were collected as a pellet and resuspended in 50 μl of cell lysis buffer and incubated on ice for 10 min. Subsequently, the cells were centrifuged at 3,000 × g for 5 min, and the supernatant was transferred to a 96-well plate to which 50 μl of reaction buffer (50 mmol/liter PIPES, pH 7.4, 10 mmol/liter EDTA, 0.5% CHAPS) containing 10 mmol/liter dithiothreitol and 5 μl of the respective substrate was added. The plate was incubated at room temperature for 1 h, and the samples were read in a fluorometer equipped with a 400 nm excitation filter and 505 nm emission filter. In the case of retinas dissected from rd and wild type mice, the tissue supernatant was prepared and collected as described above. The protein content was measured using Bradford Folin's reagent method (Bio-Rad Laboratories), and enzyme activity was expressed as relative fluorescence units/mg of protein. The arbitrary values were presented as the mean ± S.E. of three to five experiments or conditions. Measurements of Intracellular Calcium—The intracellular levels of Ca2+ in 661W cells were measured using the Fluorometric Imaging Plate Reader System (FLIPR) as described in the assay protocol (Molecular Devices). The cells were seeded in a black wall 96-well plate with an optimal cell seeding density to have a uniform, confluent monolayer after an overnight incubation. The next day, cells were loaded with a Ca2+-sensitive fluorescent dye Fluo-3 (4 μmol/liter) in a dye loading buffer (Hanks' buffer without phenol red + 20 mmol/liter HEPES and 1% bovine serum albumin) and incubated at 37 °C for 1 h. After incubation, the cells were washed three to four times with the wash buffer (Hanks' buffer and 20 mmol/liter HEPES), loaded with the ionophore A23187, 8-Br-cGMP, or IBMX with varying concentrations in different wells, and intracellular Ca2+ levels were assayed in the FLIPR system equipped with an 488 nm excitation filter and 570 nm emission filter. MTT Assay—Cells were washed twice with cold phosphate-buffered saline (PBS) after appropriate treatment periods and incubated in culture medium with 0.5 mg/ml MTT dye (Sigma) for 2 h. After aspiration of the medium, the dark blue crystals formed were dissolved with 0.1 n HCl in isopropyl alcohol, and absorbance was measured at 570 nm (background wavelength 630 nm) using a spectrophotometer. Results are presented as percentage of survival, taking control as 100%. Detection and Quantification of Apoptosis—To detect and quantify the number of cells undergoing apoptosis, cells were harvested after appropriate treatments and washed in cold PBS, recentrifuged, and resuspended in annexin binding buffer (50 mmol/liter HEPES, 700 mmol/liter NaCl, 12.5 mmol/liter CaCl2, pH 7.4) and stained with Alexa Fluor 488 annexin V and propidium iodide dyes (Apoptosis Assay Kit, Molecular Probes). Cells were then followed by flow cytometric analysis on a FACScan instrument maintained by the Medical University of South Carolina core facility, measuring the fluorescent emission at 530 nm and 575 nm to detect and quantify the population of live and apoptotic cells. Immunocytochemistry Analysis—Cells were fixed in 4% paraformaldehyde in PBS, pH 7.4, on chamber slides after the appropriate treatment periods. Primary antibodies were added in PBS-TX (0.4% Triton X-100 (TX)) for 2 h at room temperature. The primary antibodies were: rabbit polyclonal anti-activated caspase-3 (1:200, BD Biosciences), goat polyclonal anti-calpain (200 μg/ml, Santa Cruz Biotechnology), rabbit polyclonal t-bid (1:100, BIOSOURCE International), and a sheep polyclonal anti-cytochrome c (1:100, Oncogene Research Products). The primary antibodies were visualized as appropriate using goat anti-rabbit IgG (Jackson Immunoresearch) or rabbit anti-sheep/goat IgG conjugated to fluorescein (Vector Laboratories) at concentrations in accordance with the manufacturer's recommendation. Cells were photographed using a Nikon microscope equipped for fluorescence and a digital camera driven by Axioscope software. Western Blot Analysis—To detect the presence of cleaved bid, equal amounts (40 μg) of protein/cell lysate in sample buffer (62.5 mmol/liter Tris-HCl, pH 6.8, 10% glycerol, 1% SDS, 1% β-mercaptoethanol, 0.1% bromphenol blue) were loaded on a 12% SDS-polyacrylamide gel. Proteins were blotted on a nitrocellulose membrane (Bio-Rad; 0.45-μm pore size). Nonspecific labeling was blocked in 10% blotto (10% nonfat dry milk, 150 mmol/liter Tris-HCl, pH 7.4, 50 mmol/liter NaCl, 0.05% Tween 20) for 1 h, and membranes were incubated overnight at 4 °C with the primary antibody (1 μg/ml rabbit polyclonal t-bid antibody, BIOSOURCE International). After three washes (150 mmol/liter Tris-HCl, pH 7.4, 50 mmol/liter NaCl, 0.05% Tween 20), the membranes were incubated for 2 h at room temperature with anti-rabbit IgG-horseradish peroxidase antibody (1:5,000; Vector Laboratories). The blots were developed with enhanced chemiluminescence reagents (PerkinElmer Life Sciences) and analyzed using the spectrophotometer (BMG Labtechnologies). As a loading control, blots were stripped and reprobed with antibody against β-actin (Sigma). Determination of Δψm—The Δψm was determined by using JC1 dye (Molecular Probes), which accumulates in a potential-dependent manner in the mitochondria indicated by a fluorescence emission shift from the J aggregate (hyperpolarized mitochondria) exhibiting red fluorescence (525 nm), to J monomer (depolarized mitochondria) showing green fluorescence (590 nm). The population of cells undergoing the shift from J aggregate to monomer signifying mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. Cells were grown on chamber slides, and after treatment with ionophore for 6 h with and without the presence of the appropriate inhibitors, cells were washed with cold PBS and analyzed immediately using confocal microscopy (Leica laser scanning confocal imaging system). Detection of Cytochrome c Release—To determine the release of cytochrome c from the mitochondrial to cytosolic fraction, a quantitative determination of cytochrome c concentrations in cell lysates and subcellular fractions was performed. To isolate the mitochondrial and cytosolic fractions, cells were fractionated as described in the Apoalert cell fractionation protocol (Clontech). The pelleted cells were resuspended in fractionation buffer mix (including 500× protease inhibitor mixture and 1 mol/liter dithiothreitol), incubated on ice for 10 min, homogenized, and centrifuged at 10,000 × g for 25 min at 4 °C. The supernatant was collected, and the protein concentration was determined using Bradford reagents. The total amount of cytochrome c, as well as that in the different subcellular compartments, was then assessed using a cytochrome c immunoassay (R&D Systems). Equal amounts of protein from the cytosolic and enriched mitochondrial fractions were plated in microplate wells containing 100 μl of substrate solution. The plate was incubated for 30 min at room temperature, 100 μl of stop solution was added, and the plate was read at A450 nm (correction wavelength at 540 nm). Results are expressed as a measure of cytochrome c concentration (ng/ml) as arbitrary units compared with the untreated controls, in the mitochondrial and cytoplasmic subcellular fractions of the cells. Statistical Analysis—All experiments described were performed at least in triplicate. Statistical significance was determined using Student's t test, with a significance level of p < 0.05. Calpain and Caspase-3 Activity in the rd Mouse Retina—To establish the cornerstones of our hypothesis of the apoptotic pathway in the rd mouse photoreceptors, calpain and caspase-3 activities were measured in rd and wild type mouse retinas. As shown in Fig. 2A, calpain activity in the rd mouse retina was elevated at P7 compared with wild type retinas; activity levels peaked by P10; and after completion of degeneration of photoreceptors (P21), the activity levels were comparable with those found in the wild type mouse retina. Similarly, caspase-3 levels (Fig. 2B) were elevated in the rd mouse retina starting at P7, peaked at P15, and dropped to wild type levels by P21. The temporal pattern of these two enzymes is indicative of a sequential activation. In addition, these results correlate with an earlier report that mouse Ca2+ levels in the rd retinas were increased significantly compared with wild type retinas beginning at P5 and stayed elevated until P17 (12Fox D.A. Poblenz A.T. He L. Ann. N. Y. Acad. Sci. 1999; 893: 282-285Crossref PubMed Scopus (87) Google Scholar). The markedly increased activity levels of calpain and caspase-3 in the rd mice, in parallel with the massive Ca2+ influx, suggest a correlation of these two events. Calcium-induced Apoptosis in 661W Cells—After having established in vivo that both calpain and caspase-3 are activated in the rd retina, we sought to establish a photoreceptor cell culture model to investigate the mechanisms of Ca2+-induced cell death. For this purpose, 661W cells were treated with A23187, 8-Br-cGMP, or IBMX, all of which were used to mimic the massive Ca2+ influx observed in the rd mouse model. Intracellular Ca2+ levels were measured by loading the 661W cells with the Ca2+ indicator Fluo-3 and measuring the fluorescence changes after treatment with the compounds by the FLIPR method. 661W cells showed a multifold increase in the intracellular Ca2+ after treatment with A23187, 8-Br-cGMP, or IBMX compared with the untreated controls (Fig. 3A). Cell viability in response to the prolonged rise in intracellular Ca2+ was measured using the colorimetric MTT assay. 661W were found to undergo cell death in a time- and dose-dependent manner after treatment with A23187 (Fig. 3B). The 5 μmol/liter dose of ionophore A23187, which caused 40 ± 5.8% cell death after 24 h, was chosen for all further experiments. We will refer to the 5 μmol/liter calcium ionophore treatment as A23187 or ionophore treatment. Likewise, when 661W cells were subjected to 1 mmol/liter 8-Br-cGMP or 1 mmol/liter IBMX for different time periods, a pattern of cell death was observed similar to that seen for the Ca2+ ionophore (57.7 ± 2.3% and 66.7 ± 4.8% cell death, respectively) (Fig. 3C). To confirm that ionophore-induced cell death in 661W cells was associated with apoptosis, flow cytometry analysis of fluorescently labeled annexin V binding (plotted on each x axis of Fig. 4) and propidium iodide uptake (plotted on each y axis of Fig. 4) was performed. The untreated cells remained viable as observed in the lower left panel (R1) after 18 h (Fig. 4A) and 24 h (Fig. 4B). Ionophore-treated cells underwent apoptosis as seen by a shift toward the lower and upper right panels (R2 and R4) of Fig. 4, C and D, with a considerable increase in the population of annexin-positive cells showing that 25 ± 2.4% of the cells underwent apoptotic cell death after 18 h, and 40 ± 4.6% underwent apoptotic cell death after 24 h, respectively.Fig. 4Ionophore-induced apoptosis in 661W cells as measured by flow cytometry analysis. 661W cells were treated with A23187 alone and after pretreatment with SJA6017 or z-devd-fmk for 18 and 24 h. Cells were subjected to flow cytometry analysis after being stained with annexin V-fluorescein (y axis) and propidium iodide dyes (x axis) following the basic culture protocol. The population of cells undergoing apoptosis is localized in R2 and R4, representing the annexin-positive cells for phosphatidylserine externalization, whereas the viable population of cells is observed in R1. The untreated, viable cells (A and B) were present predominantly in R1. Cells treated with the A23187 ionophore for 18 h (C) showed a shift of 25 ± 2.4% cells toward R4, indicating the uptake of annexin V and signifying apoptosis. The shift was enhanced further by a 24-h treatment with ionophore (D), at which time 40 ± 4.6% of the cells were annexin-positive. Pretreatment with SJA6017 offered complete protection from the ionophore-induced apoptosis with only 6.8 ± 1.8% (E) and 8 ± 2.2% (F) annexin-positive cells, whereas z-devd-fmk offered only partial protection with 17.7 ± 2.3% (G) and 24 ± 4% (H) cells undergoing apoptosis. FITC, fluorescein isothiocyanate.View Large Image Figure ViewerDownload (PPT) Involvement of Calpain and Caspases in Ca2+-induced Apoptosis—To confirm that the same proteases that are activated in the rd mouse retina (i.e. calpain and caspase-3) were also activated by Ca2+ influx in 661W cells, pretreatment with both the enzyme inhibitors was used for the cell survival assays, enzymatic activity assays, and immunocytochemistry. To examine the effects of the calpain inhibitor SJA6017 and the caspase-3 inhibitor z-devd-fmk on the Ca2+ influx-induced cell death, cells were pretreated with these inhibitors, and the cell viability was measured by MTT assay. Inhibiting calpain by SJA6017 significantly increased the cell survival (Fig. 3D) compared with z-devd-fmk pretreatment (92.2 ± 3.4% and 72 ± 5.8% cell viability, respectively) after A23187 treatment for 24 h. The cell death triggered by 5 μmol/liter A23187 could be attenuated by SJA6017 in a dose-dependent manner (data not shown), with a maximal effect at 100 μmol/liter, but only a partial rescue could be achieved with z-devd-fmk pretreatments (Fig. 3D), with a maximal effect at 2 μmol/liter. These two doses for both inhibitors were subsequently used for all experiments and will therefore only be referred to by name, excluding the dose. Likewise, cell viability was significantly higher after SJA6017 pretreatment (88.8 ± 4.5% and 90.4 ± 5.6%) than z-devd-fmk (72.3 ± 3.4% and 73 ± 4.6%), when cells were exposed to 8-Br-cGMP or IBMX for 24 h. Similarly, using annexin V binding and propidium iodide uptake, pretreatment with SJA6017 (Fig. 4, E and F) was found to offer complete protection (6.8 ± 1.8% and 8 ± 2.2% apoptotic cells, respectively) from the ionophore-induced apoptotic cell death, whereas z-devd-fmk pretreatment (Fig. 4, G and H) offered only a partial rescue at both the time points (17.7 ± 2.3% and 24 ± 4.0% apoptotic cells, respectively). Because caspase-3 activation is a relatively late event in the apoptotic cascade, a significant number of cells may already be in the late stage of apoptosis, detectable by annexin V staining (Fig. 4, G and H). On the other hand, SJA6017 presumably blocks the events upstream of mitochondr