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
alpha-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 mu- 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
alpha-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.