V-ATPase inhibition prevents recovery from anoxia in Artemia franciscana embryos: quiescence signaling through dissipation of proton gradients.
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.
Division of Cellular, Developmental and Integrative Biology, Department of Biological Science, Louisiana State University, Baton Rouge, LA 70803, USA. jcovi1@lsu.edu
Joseph A
JA
Covi
W Dale
WD
Treleaven
Steven C
SC
Hand
D000136:Acid-Base Equilibrium / Q000502:physiology*; D000818:Animals; D001156:Artemia / Q000378:metabolism* / Q000502:physiology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D000860:Hypoxia / Q000378:metabolism*; D018942:Macrolides / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D010101:Oxygen Consumption / Q000502:physiology; D010759:Phosphorus Isotopes; D011522:Protons; D015398:Signal Transduction / Q000502:physiology*; D014589:Utah; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D018942:Macrolides; D010759:Phosphorus Isotopes; D011522:Protons; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C040929:bafilomycin A1; D025262:Vacuolar Proton-Translocating ATPases