RESUMO
The work examined the effects of Ca2+ overload and oxidative damage on erythrocytes of river lamprey Lampetra fluvialtilis. The cells were incubated for 3h with 0.1-5µM Ca2+ ionophore ionomycin in combination with 2.5mM Ca2+ 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. Ca2+ overload does not activate the cytosolic death pathways in these cells.
