Energy depletion in culture. Adenine nucleotides are altered as in vivo.
Modeling of ischemic phenomena in vitro has been hindered by the inability to create specific alterations in the variables of interest over a defined time-frame. In particular, changes in the adenine nucleotide pool have been quite difficult to mimic because of the putative low metabolic rate in culture and the long times necessary to achieve even partial chemical energy depletion. Here we present evidence for a rapid method of producing a profound chemical energy depletion with the combination of a NADH dehydrogenase inhibitor (amytal) and a mitochondrial proton ionophore (CCCP). Treatment with our protocol in enriched spinal cultures results in a 40% decrease in ATP within 2 min and a fall to one-third of control values by 15 min. The overall pool size of the total adenine nucleotides is decreased 46% by 15 min and does not completely recover after 5 min of reenergization. The ATP/ADP ratio declines to one-third of control values during deenergization and returns to control values after 5 min in control buffer. Such a loss of the total adenylate pool closely mimics that seen in vivo during ischemia and provides an in vitro model system in which the effects of the combination of this means of cellular injury with others (e.g., excitotoxins) may be examined.
Stokes
B T
BT
Department of Physiology, Ohio State University, Columbus 43210.
Altschuld
R A
RA
Hohl
C M
CM
Li
Q
Q
Anderson
D K
DK
D000227:Adenine Nucleotides / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D004734:Energy Metabolism / Q000502:physiology*; D051379:Mice; D009474:Neurons / Q000378:metabolism*
D000227:Adenine Nucleotides