Involvement of calpain in hypoxia-induced damage in rat retina in vitro

Our previous study suggested that calpain isoforms played an important role in retinal ganglion cell death induced by ischemia-reperfusion in rats [Curr. Eye Res. 21 (2000) 571]. The purpose of the present study was to further establish the direct involvement of calpain in hypoxia-induced damage by administering calpain inhibitor SJA6017 to oxygen-starved, cultured retinas. Retinas were incubated in RPMI medium with glucose and 95% O₂/5% CO₂ to supply sufficient oxygen for retinal cell survival. To induce a hypoxic condition, retinas were incubated with 95% N₂/5% CO₂. Leakage of LDH in the medium was measured to assess retinal cell damage. Activation of calpain and proteolysis of calpain substrate α-spectrin were analyzed by casein zymography and immunoblotting. Large amounts of LDH leaked into the medium from retinas under hypoxic conditions for 12 h, and SJA6017 significantly reduced LDH leakage. Caseinolytic activity of μ- and m-calpains decreased with hypoxia for 5 and 12 h, suggesting calpain activation followed by autolytic degradation. SJA6017 partially inhibited decreased calpain activities. Proteolysis of 230 kDa α-spectrin to 150 and 145 kDa breakdown products was observed in retinas with hypoxia. SJA6017 completely inhibited production of the 145 kDa breakdown product and partially inhibited production of the 150 kDa breakdown product. These results confirm the direct involvement of calpains in retinal cell damage induced by hypoxia in vitro.