Abstract
Abstract: :
Purpose: Inhibition of rod cGMP phosphodiesterase activity leads to sustained elevation of intracellular calcium, which may trigger apoptosis in retinal photoreceptors (Fox et al., 1999). We set out to investigate the mechanisms behind calcium-induced apoptosis in photoreceptor degeneration mediated by the combined actions of caspases and calpain in murine photoreceptor derived cell line 661w (Al-Ubaidi, et al., 1992). Methods: 661w cells were assessed for cell survival (MTT assay) and upregulation of caspase-3 activity in response to calcium influx mediated by the calcium ionophore A23187 and after subsequent inhibition of cell death by synthetic peptide inhibitors of caspase-3 (Z-DEVD-FMK) and calpain (SJA6017; Senju Pharmaceutical Co., Ltd.; Kobe, Japan). Caspase-3 antibody was used for immunocytochemical studies. Results: (A) MTT assays on 661w cells revealed a dose- and time-dependent induction of cell death by 5 µM calcium ionophore A23187 (40 ± 5.8% by 24 hrs and 60 ± 4.1% by 30 hrs), which can be inhibited by 2 µM Z-DEVD-FMK (p=0.001) and 100 µM SJA6017 (p=0.01). (B) Immunocytochemical analysis on A23187 exposed 661w cells demonstrated a uniform increase in staining for caspase-3. (C) Spectrophotometric analysis of caspase-3 activity showed an increase after induction by A23187, which could be completely inhibited by the caspase-3 inhibitor. Conclusion: Our preliminary results suggest that calcium-mediated apoptosis in photoreceptors involves calpain- and caspase- 3 dependent pathways. Further experiments examining the interaction between these two pathways will be performed. The presence of these apoptotic pathways in the rd mouse retina will be investigated, using an organ culture system.
Keywords: photoreceptors • apoptosis/cell death • calcium