The metabolic downregulation critical for long-term survival of Artemia franciscana embryos under
anoxia is mediated, in part, by a progressive intracellular acidification. However, very little is known about the mechanisms responsible for the pH transitions associated with exposure to, and recovery from,
oxygen deprivation. In the present study, we demonstrate with 31P-NMR that incubation of intact embryos with the V-
ATPase inhibitor
bafilomycin A1 severely limits intracellular alkalinization during recovery from
anoxia without affecting the restoration of cellular
nucleotide triphosphate levels. 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 the 1 pH unit acidification produced during 1 h of
anoxia. The additional 0.7 pH unit increase requires
proton pumping by the V-
ATPase. Aerobic incubation with bafilomycin also suggests that V-
ATPase inhibition alone is not enough to induce an acute dissipation of
proton gradients under
anoxia. In intact embryos, the dissipation of
proton gradients and uncoupling of oxidative phosphorylation with
carbonyl cyanide 3-chlorophenylhydrazone (
CCCP) leads to an intracellular acidification similar to that seen after 1 h of
anoxia. Subsequent exposure to
anoxia, in the continued presence of
CCCP, yields little additional acidification, suggesting that
proton gradients are normally dissipated under
anoxia. 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.