Difference between revisions of "Covi 2005 Thesis"
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|abstract=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. | |abstract=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. | ||
|keywords=anoxia; V-ATPase; pH; proton gradients | |||
|mipnetlab=US LA Baton Rouge Hand SC | |mipnetlab=US LA Baton Rouge Hand SC | ||
}} | }} | ||
Revision as of 14:09, 11 March 2012
| Covi JA (2005) V-ATPase expression and function in the brine shrimp, Artemia Franciscana: Role of proton gradients in anoxia-induced quiescence. Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College, Department of Biological Sciences, PhD Thesis: 101pp. |
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Abstract: 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. • Keywords: anoxia; V-ATPase; pH; proton gradients
• O2k-Network Lab: US LA Baton Rouge Hand SC
Labels:
Stress:z edited"z edited" is not in the list (Cell death, Cryopreservation, Ischemia-reperfusion, Permeability transition, Oxidative stress;RONS, Temperature, Hypoxia, Mitochondrial disease) of allowed values for the "Stress" property., Hypoxia Organism: z edited"z edited" is not in the list (Human, Pig, Mouse, Rat, Guinea pig, Bovines, Horse, Dog, Rabbit, Cat, ...) of allowed values for the "Mammal and model" property., Fish"Fish" is not in the list (Human, Pig, Mouse, Rat, Guinea pig, Bovines, Horse, Dog, Rabbit, Cat, ...) of allowed values for the "Mammal and model" property. Tissue;cell: z edited"z edited" is not in the list (Heart, Skeletal muscle, Nervous system, Liver, Kidney, Lung;gill, Islet cell;pancreas;thymus, Endothelial;epithelial;mesothelial cell, Blood cells, Fat, ...) of allowed values for the "Tissue and cell" property. Preparation: z edited"z edited" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property., Intact Organism"Intact Organism" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property. Enzyme: z edited"z edited" is not in the list (Adenine nucleotide translocase, Complex I, Complex II;succinate dehydrogenase, Complex III, Complex IV;cytochrome c oxidase, Complex V;ATP synthase, Inner mt-membrane transporter, Marker enzyme, Supercomplex, TCA cycle and matrix dehydrogenases, ...) of allowed values for the "Enzyme" property., Complex V; ATP Synthase"Complex V; ATP Synthase" is not in the list (Adenine nucleotide translocase, Complex I, Complex II;succinate dehydrogenase, Complex III, Complex IV;cytochrome c oxidase, Complex V;ATP synthase, Inner mt-membrane transporter, Marker enzyme, Supercomplex, TCA cycle and matrix dehydrogenases, ...) of allowed values for the "Enzyme" property. Regulation: z edited"z edited" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., ATP; ADP; AMP; PCr"ATP; ADP; AMP; PCr" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property.
HRR: Oxygraph-2k
