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 co ntrast, the fate of GTP during ischemia remains unknown. We used LLC-PK pro ximal 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 incre ased to 29%. We also investigated steady-state models of selective ATP or G TP depletion. Combinations of antimycin A and mycophenolic acid selectively reduced GTP to 51% or 25% of control. Similarly, alanosine selectively red uced ATP to 61% or 26% of control. Selective GTP depletion resulted in sign ificant apoptosis. Selective ATP depletion caused mostly necrosis. These mo dels of ATP or GTP depletion can prove useful in dissecting the relative co ntribution of the two nucleotides to the ischemic phenotype.