Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 mu M using metabolic inhibitors during both normal oxygenation (95% O-2-5% CO2, normoxia) and limited oxygenation ( 95% N-2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol circle min(-1)circle mg protein(-1) in normoxic hearts and from 5 to 55 pmol circle min(-1)circle mg protein(-1) in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity (A(0.5)) was 3 +/- 1 mu M for hypoxic hearts and 28 +/- 13 mu M for normoxic hearts. The A(0.5) for alpha 2-isoform AMPK activity was 2 +/- 1 mu M for hypoxic hearts and 13 +/- 8 mu M for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr(172) residue of the AMPK alpha-subunit. In potassium-arrested hearts perfused with variable O-2 content, alpha-subunit Thr(172) phosphorylation increased at O-2 <= 21% even though [AMP] was < 0.3 mu M. Thus hypoxia or O-2 <= 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr(172) by an upstream kinase or reduction in the A(0.5) for [AMP].