Depression of cell metabolism and proliferation by membrane-permeable and -impermeable modulators: role for AMP-to-ATP ratio

Abstract

The metabolic and developmental depression commonly observed during natural states of dormancy, such as diapause and quiescence, is typically accompanied by an increase in the intracellular ratio of AMP to ATP. We investigated the impact of artificially increasing the AMP-to-ATP ratio in mouse macrophages. Evidence is presented here that the P2X7receptor channel can be used as an effective means to load cells with membrane-impermeable compounds. Intracellular loading of adenosine-5′- O-thiomonophosphate (AMPS), a nonhydrolyzable analog of 5′-AMP and potent activator of AMP-activated protein kinase, significantly depresses metabolism and proliferation of macrophages. The intracellular effective AMP-to-ATP ratio obtained (the sum of AMPS plus endogenous 5′-AMP) was 0.073, well above that reported to activate AMP-activated protein kinase in vitro. Optimizing both the conditions under which the P2X7receptor channel is opened and the duration of opening facilitates high analog uptake and ∼98% survivorship. An advantage to AMPS is its minimal impact on other components of the nucleotide pool, most notably the unchanged concentration of ADP. An alternative way to shift the effective AMP-to-ATP ratio is by incubation with the membrane-permeable compound 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), which is phosphorylated intracellularly to form the 5′-AMP analog ZMP. Despite a rapid intracellular accumulation of AICAR, conversion to ZMP was slow and inefficient. Furthermore, AICAR incubation increased cellular ADP, and, although cell proliferation was depressed, the overall cellular energy flow was unchanged. The rapid action of AMPS avoids upregulation of compensatory metabolic pathways and may provide a viable approach for promoting cell stasis.