The metabolites of glutamine prevent hydroxyl radical-induced apoptosis through inhibiting mitochondria and calcium ion involved pathways in fish erythrocytes

The present study explored the apoptosis pathways in hydroxyl radicals (∙OH)-induced carp erythrocytes. Carp erythrocytes were treated with the caspase inhibitors in physiological carp saline (PCS) or Ca2+-free PCS in the presence of 40 μM FeSO4/20 μM H2O2. The results showed that the generation of reactive oxygen species (ROS), the release of cytochrome c and DNA fragmentation were caspase-dependent, and Ca2+ was involved in calpain activation and phosphatidylserine (PS) exposure in ∙OH-induced carp erythrocytes. Moreover, the results suggested that caspases were involved in PS exposure, and Ca2+ was involved in DNA fragmentation in ∙OH-induced fish erythrocytes. These results demonstrated that there might be two apoptosis pathways in fish erythrocytes, one is the caspase and cytochrome c-dependent apoptosis that is similar to that in mammal nucleated cells, the other is the Ca2+-involved apoptosis that was similar to that in mammal non-nucleated erythrocytes. So, fish erythrocytes may be used as a model for studying oxidative stress and apoptosis in mammal cells. Furthermore, the present study investigated the effects of glutamine (Gln)’s metabolites [alanine (Ala), citrulline (Cit), proline (Pro) and their combination (Ala10Pro4Cit1)] on the pathways of apoptosis in fish erythrocytes. The results displayed that Ala, Cit, Pro and Ala10Pro4Cit1 effectively suppressed ROS generation, cytochrome c release, activation of caspase-3, caspase-8 and caspase-9 at the physiological concentrations, prevented Ca2+ influx, calpain activation, PS exposure, DNA fragmentation and the degradation of the cytoskeleton and oxidation of membrane and hemoglobin (Hb) and increased activity of anti-hydroxyl radical (AHR) in ∙OH-induced carp erythrocytes. Ala10Pro4Cit1 produced a synergistic effect of inhibited oxidative stress and apoptosis in fish erythrocytes. These results demonstrated that Ala, Cit, Pro and their combination can protect mammal erythrocytes and nucleated cells against oxidative stress and apoptosis. The studies supported the use of Gln, Ala, Cit and Pro as oxidative stress and apoptosis inhibitors in mammal cells and the hypothesis that the inhibited effects of Gln on oxidative stress and apoptosis are at least partly dependent on that of its metabolites in mammalian.