Produção Nacional
Revisado por pares
Alternative Programs of Cell Death in Developing Retinal Tissue
2003; Elsevier BV; Volume: 278; Issue: 43 Linguagem: Inglês
10.1074/jbc.m306547200
ISSN1083-351X
AutoresCinthya A. Guimarães, Marlene Benchimol, Gustavo P. Amarante-Mendes, Rafael Linden,
Tópico(s)Retinal Development and Disorders
ResumoWe examined cell death in developing retinal tissue, following inhibition of protein synthesis, which kills undifferentiated post-mitotic cells. Ultrastructural features were found of both apoptosis and autophagy. Only approximately half of the degenerating cells were either terminal dUTP nick-end labeling (TUNEL)-positive or reacted with antibodies specific for activated caspases-3 or -9. Bongkrekic acid completely inhibited any appearance of cell death, whereas inhibitors of autophagy, caspases-9 or -3, prevented only TUNEL-positive cell death. Interestingly, inhibition of caspase-6 blocked TUNEL-negative cell death. Simultaneous inhibition of caspases-9 and -6 prevented cell death almost completely, but degeneration dependent on autophagy/caspase-9 still occurred under inhibition of both caspases-3 and -6. Thus, inhibition of protein synthesis induces in the developing retina various post-translational, mitochondria-dependent pathways of cell death. Autophagy precedes sequential activation of caspases-9 and -3, and DNA fragmentation, whereas, in parallel, caspase-6 leads to a TUNEL-negative form of cell death. Additional mechanisms of cell death may be engaged upon selective caspase inhibition. We examined cell death in developing retinal tissue, following inhibition of protein synthesis, which kills undifferentiated post-mitotic cells. Ultrastructural features were found of both apoptosis and autophagy. Only approximately half of the degenerating cells were either terminal dUTP nick-end labeling (TUNEL)-positive or reacted with antibodies specific for activated caspases-3 or -9. Bongkrekic acid completely inhibited any appearance of cell death, whereas inhibitors of autophagy, caspases-9 or -3, prevented only TUNEL-positive cell death. Interestingly, inhibition of caspase-6 blocked TUNEL-negative cell death. Simultaneous inhibition of caspases-9 and -6 prevented cell death almost completely, but degeneration dependent on autophagy/caspase-9 still occurred under inhibition of both caspases-3 and -6. Thus, inhibition of protein synthesis induces in the developing retina various post-translational, mitochondria-dependent pathways of cell death. Autophagy precedes sequential activation of caspases-9 and -3, and DNA fragmentation, whereas, in parallel, caspase-6 leads to a TUNEL-negative form of cell death. Additional mechanisms of cell death may be engaged upon selective caspase inhibition. Programmed cell death is a major component of both normal development and disease (1Domen J. Immunol. Res. 2000; 22: 83-94Crossref PubMed Scopus (41) Google Scholar, 2Bijl M. Limburg P.C. Kallenberg C.G. Neth. J. Med. 2001; 59: 66-75Crossref PubMed Scopus (46) Google Scholar, 3Cecconi F. Gruss P. Cell. Mol. Life Sci. 2001; 58: 1688-1697Crossref PubMed Google Scholar, 4Clarke G. Lumsden C.J. McInnes R.R. 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Notwithstanding, autophagy may lead to cell death either per se (29Bursch W. Ellinger A. Kienzl H. Török L. Pandley S. Sikorska M. Walker R. Hermann R.S. Carcinogenesis. 1996; 17: 1595-1607Crossref PubMed Scopus (469) Google Scholar, 30Bursch W. Hochegger K. Torok L. Marian B. Ellinger A. Hermann R.S. J. Cell Sci. 2000; 113: 1189-1198Crossref PubMed Google Scholar, 31Bursch W. Cell Death Differ. 2001; 8: 569-581Crossref PubMed Scopus (568) Google Scholar, 32Paglin S. Hollister T. Delohery T. Hackett N. McMahill M. Sphicas E. Domingo D. Yahalom J. Cancer Res. 2001; 61: 439-444PubMed Google Scholar) or associated with signs of apoptosis (33Jia L. Dourmashkin R.R. Allen P.D. Gray A.B. Newland A.C. Kelsey S.M. Br. J. Haematol. 1997; 98: 673-685Crossref PubMed Scopus (211) Google Scholar, 34Xue L. Fletcher G.C. Tolkovsky A.M. Mol. Cell. Neurosci. 1999; 14: 180-198Crossref PubMed Scopus (392) Google Scholar, 35Uchiyama Y. Arch. Histol. Cytol. 2001; 64: 233-246Crossref PubMed Scopus (218) Google Scholar, 36Bultzingslowen I. Jontell M. Hurst P. Nannmark U. Kardos T. Oral Oncol. 2001; 37: 537-544Crossref PubMed Scopus (18) Google Scholar). Blockade of caspase-mediated apoptosis may allow a complete program of cell death to proceed based on the autophagosomal-lysosomal compartment (31Bursch W. Cell Death Differ. 2001; 8: 569-581Crossref PubMed Scopus (568) Google Scholar, 34Xue L. Fletcher G.C. Tolkovsky A.M. Mol. Cell. Neurosci. 1999; 14: 180-198Crossref PubMed Scopus (392) Google Scholar, 35Uchiyama Y. Arch. Histol. Cytol. 2001; 64: 233-246Crossref PubMed Scopus (218) Google Scholar), thus uncovering alternative pathways of cell death in individual cells. On the other hand, the apoptotic execution machinery is available for action in cells independent of protein synthesis (37Chang S.H. Cvetanovic M. Harvey K.J. Komoriya A. Packard B.Z. Ucker D.S. Exp. Cell Res. 2002; 277: 15-30Crossref PubMed Scopus (18) Google Scholar), and currently identified components of autophagy are also associated with post-translational, rather than transcriptional control mechanisms (27Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (3059) Google Scholar, 28Reme C.E. Wolfrum U. Imsand C. Hafezi F. Williams T.P. Invest. Ophthalmol. Vis. Sci. 1999; 40: 2398-2404PubMed Google Scholar, 29Bursch W. Ellinger A. Kienzl H. Török L. Pandley S. Sikorska M. Walker R. Hermann R.S. Carcinogenesis. 1996; 17: 1595-1607Crossref PubMed Scopus (469) Google Scholar, 30Bursch W. Hochegger K. Torok L. Marian B. Ellinger A. Hermann R.S. J. Cell Sci. 2000; 113: 1189-1198Crossref PubMed Google Scholar, 31Bursch W. Cell Death Differ. 2001; 8: 569-581Crossref PubMed Scopus (568) Google Scholar, 32Paglin S. Hollister T. Delohery T. Hackett N. McMahill M. Sphicas E. Domingo D. Yahalom J. Cancer Res. 2001; 61: 439-444PubMed Google Scholar, 33Jia L. Dourmashkin R.R. Allen P.D. Gray A.B. Newland A.C. Kelsey S.M. Br. J. Haematol. 1997; 98: 673-685Crossref PubMed Scopus (211) Google Scholar, 34Xue L. Fletcher G.C. Tolkovsky A.M. Mol. Cell. Neurosci. 1999; 14: 180-198Crossref PubMed Scopus (392) Google Scholar, 35Uchiyama Y. Arch. Histol. Cytol. 2001; 64: 233-246Crossref PubMed Scopus (218) Google Scholar, 36Bultzingslowen I. Jontell M. Hurst P. Nannmark U. Kardos T. Oral Oncol. 2001; 37: 537-544Crossref PubMed Scopus (18) Google Scholar, 37Chang S.H. Cvetanovic M. Harvey K.J. Komoriya A. Packard B.Z. Ucker D.S. Exp. Cell Res. 2002; 277: 15-30Crossref PubMed Scopus (18) Google Scholar, 38Ohsumi Y. Nat. Rev. Cell. Mol. Biol. 2001; 2: 211-216Crossref PubMed Scopus (1071) Google Scholar). Thus, it appears that, in both forms of cell death, either the nature of the upstream cell death-inducing signals or the metabolic state of each cell determines its mode of triggering an essentially ready-made program of cell demise. Notwithstanding that several programs of cell death have been demonstrated in a variety of cell lines or isolated cultured cells, little is known of the relationship between apoptosis and autophagy within organized tissues, particularly during development. However, an association of autophagy and apoptosis has been shown to underlie the involution of several organs in metamorphosing insects (reviewed in Ref. 12Lockshin R.A. Zakeri Z. Curr. Opin. Cell Biol. 2002; 14: 727-733Crossref PubMed Scopus (207) Google Scholar). Using explants of developing retina, we have previously shown that inhibition of protein synthesis triggers apoptosis restricted to a relatively homogeneous population of undifferentiated, post-mitotic cells located within the neuroblastic (proliferative) layer (39Rehen S.K. Neves D.D. Fragel-Madeira L. Britto L.R. Linden R. Eur. J. Neurosci. 1999; 11: 4349-4356Crossref PubMed Scopus (28) Google Scholar, 40Rehen S.K. Varella M.H. Freitas F.G. Moraes M.O. Linden R. Development. 1996; 122: 1439-1448PubMed Google Scholar). The histotypical retinal explants are a favorable model to study mechanisms of cell death in a complex and highly structured tissue such as the mammalian central nervous system, because they preserve the spatial distribution of various cell types, cell communication including gap junctions, as well as the extracellular matrix (41Linden R. Brain Res. Rev. 2000; 32: 146-158Crossref PubMed Scopus (28) Google Scholar, 42Linden R. Rehen S.K. Chiarini L.B. Prog. Retin. Eye Res. 1999; 18: 133-165Crossref PubMed Scopus (105) Google Scholar), all of which affect the sensitivity to cell death (43Raff M.C. Nature. 1992; 356: 397-400Crossref PubMed Scopus (2506) Google Scholar). In addition, the retinal explant model preserves interactions of cells in various stages of differentiation, a major trait of developing complex tissues. Within this context, programmed cell death induced by inhibition of protein synthesis allows the analysis of the post-translational execution programs, independent of upstream gene expression (37Chang S.H. Cvetanovic M. Harvey K.J. Komoriya A. Packard B.Z. Ucker D.S. Exp. Cell Res. 2002; 277: 15-30Crossref PubMed Scopus (18) Google Scholar). In this study we examined the morphology and mechanisms of cell death induced by inhibition of protein synthesis in retinal tissue, with emphasis on the relationship between apoptosis and autophagy. Materials—Anisomycin (Sigma), an inhibitor of protein synthesis, was used at 1 μg/ml from a stock solution in Me2SO. 3-Methyladenine (3MA 1The abbreviations used are: 3MA, 3-methyladenine; Ac-LEHD-CHO or LEHD, Ac-Leu-Glu-His-Asp-H; Ac-VEID-CHO or VEID, Ac-Val-GluIle-Asp-H; ANI, anisomycin; APAF-1, apoptotic protease activating factor-1; NBL, neuroblastic layer; TUNEL, terminal dUTP nick-end labeling; Z-DEVD-fmk or DEVD, Ac-Asp-Glu-Val-Asp-fluoromethylketone; TBS, Tris-buffered saline; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TNF, tumor necrosis factor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.; Sigma), an inhibitor of autophagy, was used at 1–10 mm from a stock solution in culture medium. Ac-Leu-Glu-His-Asp-H (aldehyde) (Ac-LEHD-CHO), an inhibitor of caspase-9, and Ac-Val-Glu-Ile-Asp-H (aldehyde) (Ac-VEID-CHO), an inhibitor of caspase-6, were purchased from Peptide Institute, while Ac-Asp-Glu-Val-Asp-fluoromethylketone (Ac-DEVD-fmk) was purchased from Calbiochem, and were used at 10–100 μm from stock solutions in Me2SO. Bongkrekic acid (Calbiochem), an inhibitor of adenine nucleotide translocator, was used at 3–100 μm from a stock solution in sterile water. Ac-VEID-p-nitroanilide, a substrate for caspase-6, was used at 200 μm from a stock solution in Me2SO. Tissue Culture—Lister hooded rats were used in this study. Retinal explants were prepared as previously described (44de Araújo E.G. Linden R. Eur. J. Neurosci. 1993; 5: 1181-1188Crossref PubMed Scopus (63) Google Scholar). The animals were killed instantaneously by decapitation at postnatal day 1, their eyes were removed, and the retinas were dissected. Fragments of ∼1 mm2 were cut in culture medium and placed in 25-ml tight-lidded Erlenmeyer flasks with 5 ml of Eagle's basal medium (Invitrogen) with 5% fetal calf serum (WL Immunochemicals) and 20 mm Hepes. The flasks were kept in an orbital shaker at 80–90 revolutions/min and 37 °C for 24 h. Drugs were added at the beginning of the incubation period. Histology—The tissue was fixed with 4% paraformaldehyde in 0.1 m sodium phosphate buffer, pH 7.2, for 1 h and then infiltrated in a solution of 30% sucrose in 0.1 m sodium phosphate buffer, pH 7.2. The explants were oriented under a dissecting microscope, in an aluminum chamber filled with OCT embedding medium, and transverse sections were cut at 10-μm thickness in a cryostat. The sections were either stained with neutral red or processed for immunocytochemistry or TUNEL staining. TUNEL Staining—In situ nick-end labeling of fragmented DNA (TUNEL) was done using a FraGel kit (Oncogene). Immunocytochemistry—Immunocytochemistry for activated caspase-9 was done using cleaved Caspase-9 (Asp-353) antibody (Cell Signaling Technology) as recommended by the manufacturer. Briefly, tissue sections were incubated with Tris-buffered saline (TBS)/Triton, washed with TBS, and then incubated with blocking solution (5.5% normal goat serum in TBS/Triton) for 1 h at room temperature. After blocking, sections were incubated with primary antibody at the suggested dilution (1:100) in 5% bovine serum albumin (BSA) in TBS, overnight at 4 °C. The sections were washed with TBS/Triton and incubated with biotinylated secondary antibody (diluted in TBS plus 3% BSA; 1:200 for secondary antibody from Vectastain HRP-ABC kit, Vector Laboratories) for 1 h at room temperature. After washing with TBS/Triton, sections were incubated for 30 min with 0.6% H2O2 in TBS at room temperature. Sections were washed with TBS/Triton and then incubated for 1 h with ABC reagent at room temperature. After washing with TBS, sections were incubated with diaminobenzidine for 7 min, washed with water, coverslipped in glycerol, and analyzed by light microscopy. Immunocytochemistry for activated caspase-3 was done using the CM1 polyclonal antiserum that recognizes the p20 activated form of human, mouse, and rat caspase-3, without significant cross-reactivity with the zymogen form of 32 kDa (45Namura S. Zhu J. Fink K. Endres M. Srinivasan A. Tomaselli K.J. Yuan J. Moskowitz M.A. J. Neurosci. 1998; 18: 3659-3668Crossref PubMed Google Scholar). This antibody was a gift from Dr. Anu Srinivasan (Idun Pharmaceuticals), and a detailed description was provided in the publication mentioned above. Paraformaldehyde-fixed tissue sections were incubated with 3% H2O2 in phosphate-buffered saline (PBS) for 30 min, followed by a PBS wash. The sections were incubated with a blocking buffer containing 2% normal goat serum, 2% BSA, 0.2% nonfat milk powder, and 0.8% Triton X-100 in PBS for 1 h at room temperature. After this period, the sections were incubated overnight at 4 °C with primary antibody CM1 at 1:5000. The immunocytochemical reaction was developed with a Vectastain HRP-ABC kit, and the sections were coverslipped in glycerol and analyzed by light microscopy. Quantification of Cell Death and Statistics—Dying cells appeared as round condensed profiles, either homogeneously stained with Neutral Red or as outstanding globose profiles under differential interference contrast microscopy, usually found isolated among healthy looking cells. To estimate the rates of cell death, either in Neutral Red-stained material or in immunocytochemistry/TUNEL-stained material, condensed profiles were counted within the neuroblastic layer (NBL) in three random fields of 0.0148 mm2 from each of three distinct explants in each experiment. Data were expressed as means ± S.E. of the replicates from several pooled experiments. The values were normalized with respect to the average rate of cell death within the NBL in anisomycin-treated explants in each experiment, which was taken as 100%. The data were subject to either one-way or two-way analysis of variance, as applicable, followed by Duncan's multiple range test, using an SPSSPC statistical software. p < 0.01 was set as criterion significance. Transmission Electron Microscopy—After 24 h in culture in either control medium or incubated with anisomycin, retinal explants were fixed overnight in a solution containing 2.5% glutaraldehyde, 4% freshly prepared formaldehyde in 0.1 m phosphate buffer, pH 7.3. After fixation the cells were washed in PBS and post-fixed on 1% OsO4 in cacodylate buffer plus 5 mm CaCl2 and 0.8% potassium ferricyanide. Cells were washed, stained with uranyl acetate and lead citrate, dehydrated at 20 °C in an acetone series, and infiltrated at the same temperature in Epon. Polymerization was carried out for 72 h at 37 °C. Thin sections were collected on copper grids, stained with uranyl acetate and lead citrate, and examined in a JEOL 1210 transmission electron microscope. Caspase Activity Assay—Protein samples were prepared as follows. Retinal explants cultured for 24 h in various experimental conditions (indicated in the legend) were incubated with buffer A (150 mm NaCl, 20 mm Tris, pH 7.5, 1% Triton X-100, 10 μg/ml leupeptin, 5 μg/ml aprotinin, 100 μm phenylmethylsulfonyl fluoride) for 10 min on ice. Then buffer B was added (100 mm Hepes, pH 7.4, 150 mm NaCl, 0.2% CHAPS, 4 mm dithiothreitol), the samples were centrifuged, and the pellet was stored at –70 °C. Protein samples (300 μg) were incubated at 37 °C for 4 h with VEID-p-nitroanilide (200 μm in buffer B), and substrate hydrolysis was measured by sample absorbance at 405 nm in a Microplate Reader 3550-UV (Bio-Rad). Inhibition of Protein Synthesis Induces Ultrastructural Features of Both Apoptosis and Autophagy in Retinal Explants—In retinal tissue stained with neutral red, dying cells appear as round and heavily stained profiles, easily detectable among healthy cells. (Fig. 1A). Examples of low and high power views of sections of retinal tissue containing degenerating cells can be found in our previous publications (see, e.g., Refs. 40Rehen S.K. Varella M.H. Freitas F.G. Moraes M.O. Linden R. Development. 1996; 122: 1439-1448PubMed Google Scholar and 46Varella M.H. Correa D.F. Campos C.B. Chiarini L.B. Linden R. Neurochem. Int. 1997; 31: 217-227Crossref PubMed Scopus (20) Google Scholar). These condensed profiles can be also detected by their globose morphology, under differential interference contrast microscopy (Fig. 2, A–C). In explants maintained in control medium, ganglion cells degenerate as a result of axotomy, whereas there is no cell death in the outer stratum of the retina. In retinal tissue treated with the inhibitor of protein synthesis anisomycin (ANI), ganglion cells degeneration is blocked, whereas cell death is induced in the NBL. Although ANI is known to activate mitogen-activated protein kinases at low concentrations, cell death in the NBL is induced only at concentrations of anisomycin that significantly block protein synthesis (46Varella M.H. Correa D.F. Campos C.B. Chiarini L.B. Linden R. Neurochem. Int. 1997; 31: 217-227Crossref PubMed Scopus (20) Google Scholar). 2C. A. Guimarães and R. Linden, unpublished results. The paradoxical effect of anisomycin was described previously by Rehen and co-workers (40Rehen S.K. Varella M.H. Freitas F.G. Moraes M.O. Linden R. Development. 1996; 122: 1439-1448PubMed Google Scholar), who also demonstrated that the cells sensitive to ANI-induced cell death in the developing retina are undifferentiated post-mitotic cells (39Rehen S.K. Neves D.D. Fragel-Madeira L. Britto L.R. Linden R. Eur. J. Neurosci. 1999; 11: 4349-4356Crossref PubMed Scopus (28) Google Scholar). We did not observe massive degenerative areas, and dead cells were always found surrounded by healthy-looking profiles, showing that anisomycin does not induce tissue necrosis.Fig. 2Apoptosis induced in the retina by inhibition of protein synthesis. Sections of retinal explants treated for 24 h with 1 μg/ml anisomycin were stained with the TUNEL procedure (A) or with antibodies to either activated caspase-3 (B) or -9 (C). Condensed profiles of dead cells are visible by differential interference contrast microscopy. D–F, quantification of condensed profiles positive (filled bars) or negative (open bars) for TUNEL (D), activated caspase-3 (E), or caspase-9 (F), in explants maintained either in control medium (CTR), or treated for 24 h with 1 μg/ml anisomycin (ANI). Values in this and in the following figures are means ± S.E. In D–F, each graph is a pool of two independent experiments (n = 6 for each data point), and the data were normalized with reference to the total density of pyknotic profiles in explants treated with anisomycin (100%). *, p < 0.01 versus respective control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Ultrastructural analysis of the NBL in control explants from the retinas of neonatal rats showed the presence mostly of elongated nuclei containing granular and relatively diffuse chromatin, surrounded by very small amounts of cytoplasm, typical of immature cells of the proliferative neuroepithelium (Fig. 1B). No attempt was made to distinguish between the proliferating and post-mitotic cells known to reside within this layer (39Rehen S.K. Neves D.D. Fragel-Madeira L. Britto L.R. Linden R. Eur. J. Neurosci. 1999; 11: 4349-4356Crossref PubMed Scopus (28) Google Scholar). The massive predominance of vertically elongated nuclei within the NBL is probably associated with the ongoing vertical migration of the nuclei of cells within this layer. Proliferating cell nuclei move to and fro as a result of interkinetic nuclear migration, whereas post-mitotic cells usually translocate vertically toward their definitive positions in the emerging retinal layers. Occasional larger, round nuclei are found toward the external margin of the NBL, probably corresponding to a much smaller population of early differentiating horizontal cells (data not shown). In tissue treated with anisomycin, nuclei showing various degrees of chromatin condensation were typical of apoptosis (Fig. 1C). Around the presumptive early apoptotic profiles occasional organelles appeared preserved (data not shown), whereas the whole cell bodies rounded up around the heavily condensed late apoptotic nuclei. Typical autophagic profiles were also frequently found within the cytoplasm of cells in the NBL of retinal tissue treated with anisomycin (Fig. 1, D–F). Either portions of the cytosol or whole organelles were found within double- or multiple-layered vacuoles (Fig. 1D). Occasionally, clusters of mitochondrial profiles were found surrounded by a double-membrane vacuole (Fig. 1, E and F). These ultrastructural features are consistent with the induction by anisomycin of both apoptosis and autophagy in retinal tissue. Anisomycin Induces Cell Death Only in Part through Activation of Caspases-9 and -3 and DNA Fragmentation—Both a DNA ladder and TUNEL staining of degenerating profiles within the NBL have been previously shown in retinal tissue treated with anisomycin (40Rehen S.K. Varella M.H. Freitas F.G. Moraes M.O. Linden R. Development. 1996; 122: 1439-1448PubMed Google Scholar). However, only a fraction of the globose, degenerating profiles observed under differential interference contrast were stained by the TUNEL procedure.2 We systematically quantified TUNEL labeling in the NBL of retinal explants treated with ANI and found that 57 ± 5.6% (mean ± S.E.) of the pyknotic profiles were positive (Fig. 2, A and D). These results suggested that anisomycin-induced cell death may be either TUNEL-positive or -negative. We then examined retinal explants treated with anisomycin using the CM1 antibody raised against the activated form of caspase-3. Only 55 ± 6.3% of the condensed profiles were immunoreactive (Fig. 2, B and E), supporting the hypothesis that anisomycin induces two distinct pathways of cell death, one of which seems to be dependent on caspase-3 activation. Furthermore, only 50 ± 2.2% of condensed cells were immunoreactive with an antibody to activated caspase-9 (Fig. 2, C and F), consistent with the hypothesis that caspase-9 is the protease responsible for the activation of caspase-3 in the retina. Therefore, the quantitative data indicated that a cascade of activation of caspase-9, caspase-3, and DNA fragmentation leading to TUNEL staining appears to be associated with only a fraction of the cell death induced in retinal tissue by inhibition of protein synthesis. It is unlikely that these findings can be explained by a trivial failure of the staining methods to attain all cells, both because of the similar fractions of cells stained with three distinct procedures and because of the consistent effects of selective caspase inhibitors described below. Mitochondria Are Involved in Cell Death Induced by Anisomycin—Activation of caspase-9 is dependent on the assembly of the mitochondrial apoptosome, containing procaspase-9, APAF-1, dATP, and cytochrome c. The release of cytochrome c is often associated with the opening of a permeability transition pore in the outer membrane of the mitochondria. To test whether mitochondria participate in cell death induced by ANI, we used bongkrekic acid, an inhibitor of the adenine nucleotide translocator, which is one of the components of permeability transition pore. Bongkrekic acid completely inhibited cell death induced by ANI (Fig. 3), showing that cell death induced by ANI is dependent on mitochondrial commitment. Inhibitors of Caspases-9, -3, and -6 and Autophagy Block in Part Cell Death Induced by Anisomycin—To test for the role of various caspases in retinal cell death, we treated retinal explants in the presence of ANI together with each one of various peptides that inhibit caspase-9, caspase-3, or caspase-6. LEHD, an inhibitor of caspase-9, partially blocked cell death induced by ANI (fig 4A). LEHD concentrations up to 300 μm had no additional effect, reaching a maximum near 60% inhibition. This is consistent with the immunoreactivity for activated caspase-9, found in roughly half of the degenerating profiles (Fig. 2, C and F). DEVD, an inhibitor of caspase-3, partially blocked cell death induced by ANI (Fig. 4B), also in agreement with immunocytochemical data for activated caspase-
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