Modeling ischemia in vitro: selective depletion of adenine and guanine nucleotide pools.
Intracellular ATP depletion is a hallmark event in ischemic injury. It has been extensively characterized in models of chemical anoxia in vitro. In contrast, the fate of GTP during ischemia remains unknown. We used LLC-PK proximal tubular cells to measure GTP and ATP changes during anoxia. In 45 min, antimycin A decreased ATP and GTP to 8% and 2% of controls, respectively. Ischemia in vivo resulted in comparable reductions in GTP and ATP. After 2 h of recovery, GTP levels in LLC-PK cells increased to 65% while ATP increased to 29%. We also investigated steady-state models of selective ATP or GTP depletion. Combinations of antimycin A and mycophenolic acid selectively reduced GTP to 51% or 25% of control. Similarly, alanosine selectively reduced ATP to 61% or 26% of control. Selective GTP depletion resulted in significant apoptosis. Selective ATP depletion caused mostly necrosis. These models of ATP or GTP depletion can prove useful in dissecting the relative contribution of the two nucleotides to the ischemic phenotype.
Dagher
P C
PC
Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA. pdaghe2@iupui.edu
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000409:Alanine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000968:Antimycin A / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006151:Guanosine / Q000378:metabolism / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007511:Ischemia / Q000378:metabolism*; D007672:Kidney Cortex / Q000098:blood supply* / Q000737:chemistry; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008297:Male; D008954:Models, Biological; D009173:Mycophenolic Acid / Q000494:pharmacology; D009336:Necrosis; D010085:Oxidative Phosphorylation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D013552:Swine
D000903:Antibiotics, Antineoplastic; D004791:Enzyme Inhibitors; D006151:Guanosine; C000082:alanosine; D000968:Antimycin A; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D003847:Deoxyglucose; D009173:Mycophenolic Acid; D000409:Alanine