Caspase activity during cell stasis: avoidance of apoptosis in an invertebrate extremophile,Artemia franciscana

Abstract

Evaluation of apoptotic processes downstream of the mitochondrion reveals caspase-9- and low levels of caspase-3-like activities in partly purified extracts of Artemia franciscana embryos. However, in contrast to experiments with extracts of human hepatoma cells, cytochrome c fails to activate caspase-3 or -9 in extracts from A. franciscana. Furthermore, caspase-9 activity is sensitive to exogenous calcium. The addition of 5 mM calcium leads to a 4.86 ± 0.19 fold (SD) ( n = 3) increase in activity, which is fully prevented with 150 mM KCl. As with mammalian systems, high ATP (>1.25 mM) suppresses caspase activity in A. franciscana extracts. A strong inhibition of caspase-9 activity was also found by GTP. Comparison of GTP-induced inhibition of caspase-9 at 0 and 2.5 mM MgCl2indicates that free (nonchelated) GTP is likely to be the inhibitory form. The strongest inhibition among all nucleotides tested was with ADP. Inhibition by ADP in the presence of Mg2+is 60-fold greater in diapause embryos than in postdiapause embryos. Because ADP does not change appreciably in concentration between the two physiological states, it is likely that this differential sensitivity to Mg2+-ADP is important in avoiding caspase activation during diapause. Finally, mixtures of nucleotides that mimic physiological concentrations in postdiapause and diapause states underscore the depressive action of these regulators on caspase-9 during diapause. Our biochemical characterization of caspase-like activity in A. franciscana extracts reveals that multiple mechanisms are in place to reduce the probability of apoptosis under conditions of energy limitation in this embryo.