Apoptotic death in erythrocytes of lamprey Lampetra fluviatilis induced by ionomycin and tert-butyl hydroperoxide.

The work examined the effects of Ca overload and oxidative damage on erythrocytes of river lamprey Lampetra fluvialtilis. The cells were incubated for 3h with 0.1-5μM Ca ionophore ionomycin in combination with 2.5mM Ca and 10-100μM pro-oxidant agent tert-butyl hydroperoxide (tBHP). The sensitivity of lamprey RBCs to studied compounds was evaluated by the kinetics of their death. Both toxicants induced dose- and time dependent phosphatidylserine (PS) externalization (annexin V-FITC labeling) and loss of membrane integrity (propidium iodide uptake). Highest doses of ionomycin (1-2μM) increased the number of PS-exposed erythrocytes to 7-9% within 3h, while 100μM tBHP produced up to 50% of annexin V-FITC-positive cells. Caspase inhibitor Boc-D-FMK (50μM), calpain inhibitor PD150606 (10μM) and broad protease inhibitor leupeptin (200μM) did not prevent ionomycin-induced PS externalization, whereas tBHP-triggered apoptosis was blunted by Boc-D-FMK. tBHP-dependent death of lamprey erythrocytes was accompanied by the decrease in relative cell size, loss of cell viability, activation of caspases 9 and 3/7, and loss of mitochondrial membrane potential, but all these processes were partially attenuated by Boc-D-FMK. None of examined death-associated events were observed in ionomycin-treated erythrocytes except activation of caspase-9. Incubation with ionomycin did not alter intracellular K and Na content, while exposure to tBHP resulted in 80% loss of K and 2.8-fold accumulation of Na. Thus, lamprey erythrocytes appear to be more susceptible to oxidative damage. Ca overload does not activate the cytosolic death pathways in these cells.