AMP-activated protein kinase couples mitochondrial inhibition by hypoxia to cell-specific Ca~(2+) signalling mechanisms in oxygen-sensing cells

摘要

It is generally accepted that relatively mild hypoxia inhibits mitochondrial oxidative phosphorylation in O_2-sensing cells, and thereby mediates, in part, cell activation. However, the mechanism by which this process is coupled to discrete, cell-specific Ca~(2+) signalling mechanisms remains elusive. We considered the possibility that hypoxia may increase the cellular ATP/AMP ratio, increase the activity of AMP-activated protein kinase (AMPK) and thereby evoke Ca~(2+) signals in O_2-sensing cells. Co-immunoprecipitation identified a1/(beta)2(gamma)1 as the primary AMPK isozyme in pulmonary arterial smooth muscle, whilst the tissue-specific distribution of AMPK activities and their activation by hypoxia suggested that the AMPK-al catalytic subunit isoform is key to the regulation of O_2-sensing cells. Strikingly, 3D reconstruction of immunofluorescence images showed AMPK-al to be located throughout the cytoplasm of pulmonary arterial smooth muscle cells and, by contrast, targeted to the plasma membrane in carotid body glomus cells. Consistent with these observations Ca~(2+) imaging, tension recording and electrophysiology demonstrated that AMPK, like hypoxia, activates each cell type via discrete Ca~(2+) signalling mechanisms: cyclic ADP-ribose-dependent Ca~(2+) mobilization from the sarcoplasmic reticulum via ryanodine receptors in pulmonary arterial smooth muscle cells and voltage-gated Ca~(2+) influx into carotid body glomus cells. Thus, metabolic-sensing by AMPK underpins the cell-specific response of O_2-sensing cells to hypoxia.