AMP-activated protein kinase activator A-769662 increases intracellular calcium and ATP release from astrocytes in an AMPK-independent manner

AIM: Astrocytes are the main sources of extracellular ATP (eATP) within the brain, which functions as a gliotransmitter, capable of modulating neuronal and astrocytic activity. These cells play an important role in regulating energy homeostasis partly via astrocyte-derived ATP. Given the role of AMPK in regulating intracellular ATP levels, we hypothesised that AMPK may alter ATP release from astrocytes.

METHODS: Measurements of ATP release were made from human U373 astrocytoma cells, primary mouse hypothalamic (HTAS) and cortical astrocytes (CRTAS) and wild type and AMPK α1/α2 null mouse embryonic fibroblasts (MEFs). Cells were treated with drugs known to modulate AMPK activity: A-769662, AICAR and metformin, for up to 3 hours. Intracellular calcium was measured using Fluo4 and Fura-2 calcium-sensitive fluorescent dyes.

RESULTS: In U373 cells, A-769662 (100 μM) increased AMPK phosphorylation, whereas AICAR and metformin (1 mM) induced a modest increase or had no effect, respectively. Only A-769662 increased extracellular ATP (eATP) levels, which was partially blocked by AMPK inhibitor Compound C. A-769662-induced increases in eATP were preserved in AMPK α1/α2 null MEF cells. A-769662 increased intracellular calcium in U373, HTAS and CRTAS cells and chelation of intracellular calcium using BAPTA-AM reduced A-769662-induced eATP levels. A-769662 also increased ATP release from a number of other central and peripheral endocrine cell types.

CONCLUSIONS: AMPK is required to maintain basal eATP levels but is not required for A-769662-induced increases in eATP. A-769662 (>50 μM) enhanced intracellular calcium levels leading to ATP release in an AMPK and purinergic receptor independent pathway.