Doctor of Philosophy (PhD)
Upon exposure to anoxia, encysted embryos of the brine shrimp, Artemia franciscana, enter a severely depressed metabolic state, the transition into which is facilitated in part by one of the largest intracellular acidifications ever reported for a eukaryotic organism. However, the endogenous origin of this pH shift remains largely unexplained. We hypothesize that the unexplained acidification is produced by a net flux of protons into the cytoplasm from compartments acidified by a V-type proton pump (V-ATPase) during aerobic development. Northern blots demonstrate expression of the V-ATPase subunit-B mRNA during early development, while Western blots demonstrate the expression of at least 6 constituent subunits of the V-ATPase in both heavy membranes and microsomal vesicles. Inhibition of embryo hatching with the highly specific V-ATPase inhibitor, bafilomycin A1, confirmed a requirement for V-ATPase activity during the anoxia tolerant stages of development. Given that the V-ATPase is responsible for acidification of intracellular compartments in eukaryotes, these data indicate the presence of compartmentalized proton stores in A. franciscana embryos. 31P-NMR studies using intact embryos demonstrate that V-ATPase inhibition with bafilomycin A1, severely limits intracellular alkalinization during recovery from anoxia without affecting the restoration of cellular nucleotide triphosphates. Based on these data, it appears that oxidative phosphorylation and ATP resynthesis can only account for the first 0.3 pH unit alkalinization observed during aerobic recovery from 1 h of anoxia. The additional 0.7 pH unit increase requires proton pumping by the V-ATPase. Furthermore, aerobic incubation with the protonophore, carbonyl cyanide 3-chlorophenylhydrazone, produces an intracellular acidification similar to that observed after 1 h of anoxia, and subsequent anoxic exposure yields little additional acidification. When combined with protons generated from net ATP hydrolysis, these data show that the dissipation of proton chemical gradients is sufficient to account for the reversible acidification associated with quiescence in these embryos. Whole embryo respirometry demonstrates that V-ATPase activity and processes immediately dependent on that activity constitute approximately 31% of the aerobic energy budget of the preemergent embryo. Downregulation of such a costly transporter would be essential in order to attain the level of metabolic depression observed in the whole embryo under anoxia.
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Covi, Joseph Antonio, "V-ATPase expression and function in the brine shrimp, Artemia franciscana: role of proton gradients in anoxia-induced quiescence" (2005). LSU Doctoral Dissertations. 2331.
Steven C. Hand