Apoptotic death in erythrocytes of lamprey Lampetra fluviatilis induced by ionomycin and tert-butyl hydroperoxide.
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.
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, M. Thorez av. 44, Sankt-Petersburg, 194223, Russia. Electronic address: nagalak@mail.ru.
Natalia I
NI
Agalakova
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, M. Thorez av. 44, Sankt-Petersburg, 194223, Russia.
Tatiana I
TI
Ivanova
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, M. Thorez av. 44, Sankt-Petersburg, 194223, Russia.
Gennadii P
GP
Gusev
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, M. Thorez av. 44, Sankt-Petersburg, 194223, Russia.
Anna V
AV
Nazarenkova
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, M. Thorez av. 44, Sankt-Petersburg, 194223, Russia.
Dina A
DA
Sufiyeva
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015415:Biomarkers / Q000097:blood / Q000378:metabolism; D061207:Calcium Ionophores / Q000633:toxicity*; D048429:Cell Size; D002470:Cell Survival / Q000187:drug effects; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D015759:Ionomycin / Q000633:toxicity*; D007700:Kinetics; D007798:Lampreys; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D016877:Oxidants / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects; D045483:Rivers; D012426:Russia; D014874:Water Pollutants, Chemical / Q000633:toxicity*; D020122:tert-Butylhydroperoxide / Q000633:toxicity*
D015415:Biomarkers; D061207:Calcium Ionophores; D016877:Oxidants; D014874:Water Pollutants, Chemical; D015759:Ionomycin; D020122:tert-Butylhydroperoxide
Apoptosis; Cellular K(+) and Na(+) content; Ionomycin; Lamprey erythrocytes; Mitochondrial pathway; Tert-butyl hydroperoxide