MENZE, M.A.*; HAND, S.C.; Louisiana State University, Baton Rouge; Louisiana State University, Baton Rouge: Altering AMP:ATP ratio in mammalian cells to depress metabolic activity
Metabolic depression during states of dormancy like diapause and quiescence is often accompanied by an increase in the intracellular ratio of AMP to ATP. Depending on the magnitude of change, elevating this ratio stimulates the AMP-activated protein kinase (AMPK), which is an ultra sensitive senor of the cellular energy status. The activated AMPK in turn phosphorylates key regulatory proteins, thereby depressing biosynthesis and cell proliferation. We have explored mechanisms to modulate AMP:ATP ratio in mammalian cells to test the hypothesis that metabolism will be depressed and tolerance to environmental insult enhanced. Mouse 3T3 fibroblasts were incubated with 2 mM 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) for 60 min. This membrane permeable compound is phosphorylated in vivo to form ZMP (an AMP analogue and weak activator of AMPK), which then accumulates intracellular as verified by HPLC. When compared to untreated cells, ZMP led to a 13% reduction in cell proliferation after 48 h and a 19.4% reduction in overall metabolic rate as measured by microcalorimetry. When AICAR treatment is combined with glucose removal and glycolytic inhibition, heat dissipation was depressed by up to 80%. To modulate AMP:ATP ratio directly, we opened the ATP-sensitive P2X7 membrane receptor channel in mouse macrophages (J774) by adding extracellular ATP, which permeabilizes cells to small solutes. Loading 5�-AMP through this pore increased the AMP:ATP ratio from 0.025 ± 0.011 (SD) to 0.277 ± 0.156, but only transiently. Prolonged elevation of AMP:ATP required loading adenosine 5�-phosphorothioate (AMPS), which is resistance to metabolic modification. A higher tolerance is observed to cellular dehydration after preconditioning with AICAR and trehalose. [DARPA grant N00173-01-1-G011]
