Modeling ischemia in vitro: selective depletion of adenine and guanine nucleotide pools

Intracellular ATP depletion is a hallmarkevent in ischemic injury. It has been extensively characterized inmodels of chemical anoxia in vitro. In contrast, the fate of GTP duringischemia remains unknown. We used LLC-PK proximal tubular cells tomeasure GTP and ATP changes during anoxia. In 45 min, antimycin Adecreased 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 to65% while ATP increased to 29%. We also investigated steady-statemodels 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 orGTP depletion can prove useful in dissecting the relative contributionof the two nucleotides to the ischemic phenotype.