MENZE, M.A.*; HAND, S.C.; Louisiana State University; Louisiana State University: Lessons in Apoptosis from an Invertebrate Extremophile, Embryos of Artemia franciscana
Embryos of the brine shrimp A. franciscana are exceptional in their ability to tolerate anoxia and desiccation at room temperature for years and to maintain viability under conditions that are known in mammalian species to open the mitochondrial permeability transition pore (mPTP), which leads to apoptotic and necrotic cell death. We demonstrated previously that brine shrimp mitochondria lack a calcium-induced permeability transition, cytochrome c (cyt-c) is not released via the mPTP, and profound calcium storage capacity likely contributes to the prolonged anoxia tolerance in this species. However, translocation of cyt-c by pro-apoptotic Bcl-2 family members such as Bax and Bak remains a possibility, although involvement of cyt-c in apoptotic signaling of invertebrates is controversial. Low levels of caspase 9- and caspase 3-like activities can be detected in cell free extracts of A. franciscana embryos when measured by cleavage of the fluorogenic substrates Z-DEVD-R110 and Z-LEHD-R110. However, in contrast to our experiments with human hepatoma cells (C3A), adding cyt-c to extracts from A. franciscana embryos fails to elevate caspase-3 like activity. Furthermore, caspase-9 like activity is highly depressed by KCl. A comparison of kinetic models shows that the best fit is achieved by assuming competitive inhibition between KCl and Z-LEHD-R110. By applying this model, an apparent inhibition constant of about Ki = 6 mM is obtained. Thus at a physiological KCl concentration of about 145 mM, a strong inhibition of caspase-9 would occur. Multiple mechanisms are in place that apparently reduce the probability of apoptosis under conditions of energy limitation in this invertebrate. [NIH grant 1-RO1-GM071345-01].
