Ca(2+) and caspases are involved in hydroxyl radical-induced apoptosis in erythrocytes of Jian carp (Cyprinus carpio var. Jian)

There are young erythrocytes and mature erythrocytes in the peripheral blood of fish. The present study explored the apoptosis in hydroxyl radical ((·)OH)-induced young and mature erythrocytes of Jian carp (Cyprinus carpio var. Jian). Carp erythrocytes from the peripheral blood were separated into the young fraction, the intermediate fraction and the mature fraction using fixed-angle centrifugation. The erythrocytes in three age fractions were treated with the caspase inhibitors (zVAD-fmk) in physiological carp saline (PCS) or Ca(2+)-free PCS in the presence of 40 μM FeSO4/20 μM H2O2. The results showed that the (·)OH-induced reactive oxygen species (ROS) generation, phosphatidylserine (PS) exposure and DNA fragmentation are caspase dependent in carp erythrocytes. Furthermore, the ROS generation, PS exposure and DNA fragmentation in the more young fraction are more dependent on the caspase activity. This suggested that the caspases are involved in the (·)OH-induced apoptosis in the young erythrocytes of fish. Results also indicated that Ca(2+) is involved in (·)OH-induced calpain activation, PS exposure and DNA fragmentation in carp erythrocytes. Moreover, the calpain activation, DNA fragmentation and PS exposure in the more mature fraction are more dependent on the levels of Ca(2+). This revealed that (·)OH-induced apoptosis is Ca(2+) dependent in the mature erythrocytes of fish. Taken together, there might be two apoptosis pathways in fish erythrocytes: one is the caspase-dependent apoptosis in the young erythrocytes and the other is the Ca(2+)-involved apoptosis in the mature erythrocytes.