AMP‐activated protein kinase inhibits K_v1.5 channel currents of pulmonary arterial myocytes in response to hypoxia and inhibition of mitochondrial oxidative phosphorylation

Abstract

Key points Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage‐gated potassium channels (Kv) in pulmonary arterial smooth muscle by hypoxia, although the precise molecular mechanisms have been unclear.AMP‐activated protein kinase (AMPK) has been proposed to couple inhibition of mitochondrial metabolism by hypoxia to acute hypoxic pulmonary vasoconstriction and progression of pulmonary hypertension.Inhibition of complex I of the mitochondrial electron transport chain activated AMPK and inhibited Kv1.5 channels in pulmonary arterial myocytes.AMPK activation by 5‐aminoimidazole‐4‐carboxamide riboside, A769662 or C13 attenuated Kv1.5 currents in pulmonary arterial myocytes, and this effect was non‐additive with respect to Kv1.5 inhibition by hypoxia and mitochondrial poisons.Recombinant AMPK phosphorylated recombinant human Kv1.5 channels in cell‐free assays, and inhibited K+ currents when introduced into HEK 293 cells stably expressing Kv1.5.These results suggest that AMPK is the primary mediator of reductions in Kv1.5 channels following inhibition of mitochondrial oxidative phosphorylation during hypoxia and by mitochondrial poisons. AbstractProgression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage‐gated potassium channels (Kv) in pulmonary arterial smooth muscle cells that is mediated by the inhibition of mitochondrial oxidative phosphorylation. We sought to determine the role in this process of the AMP‐activated protein kinase (AMPK), which is intimately coupled to mitochondrial function due to its activation by LKB1‐dependent phosphorylation in response to increases in the cellular AMP:ATP and/or ADP:ATP ratios. Inhibition of complex I of the mitochondrial electron transport chain using phenformin activated AMPK and inhibited Kv currents in pulmonary arterial myocytes, consistent with previously reported effects of mitochondrial inhibitors. Myocyte Kv currents were also markedly inhibited upon AMPK activation by A769662, 5‐aminoimidazole‐4‐carboxamide riboside and C13 and by intracellular dialysis from a patch‐pipette of activated (thiophosphorylated) recombinant AMPK heterotrimers (α2β2γ1 or α1β1γ1). Hypoxia and inhibitors of mitochondrial oxidative phosphorylation reduced AMPK‐sensitive K+ currents, which were also blocked by the selective Kv1.5 channel inhibitor diphenyl phosphine oxide‐1 but unaffected by the presence of the BKCa channel blocker paxilline. Moreover, recombinant human Kv1.5 channels were phosphorylated by AMPK in cell‐free assays, and K+ currents carried by Kv1.5 stably expressed in HEK 293 cells were inhibited by intracellular dialysis of AMPK heterotrimers and by A769662, the effects of which were blocked by compound C. We conclude that AMPK mediates Kv channel inhibition by hypoxia in pulmonary arterial myocytes, at least in part, through phosphorylation of Kv1.5 and/or an associated protein.

Tetraethylammonium, glibenclamide, and 4‐aminopyridine modulate post‐occlusive reactive hyperemia in non‐glabrous human skin with no roles of NOS and COX

“…Interestingly, we found that K V channel blockade by 4‐aminopyridine augmented PORH Amplitude in non‐glabrous forearm skin (Figure B), suggesting that K V channels may partly restrict PORH. Since hypoxia reportedly inhibits K V channels thereby causing vasoconstriction, arterial occlusion‐induced local hypoxia might have caused a similar response in the present study, explaining why K V channel blockade augmented PORH Amplitude. However, given the Area under the curve was unaffected by 4‐aminopyridine (Figure E), the restricting effect of K V channels, if any, appears to occur at the beginning of the PORH response only.…”

Nitric oxide activates AMPK by modulating PDE3A in human pulmonary artery smooth muscle cells

“…Whereas NO acts as both a target and an effector of the AMPK pathway in endothelial cells (Fisslthaler & Fleming, 2009;Suzuki et al, 2008), less is known about the mechanism by which NO regulates AMPK in smooth muscle cells. Moreover, data on the role of AMPK in vascular proliferation and pulmonary hypertension are conflicting (Agard et al, 2009;Dean, Nilsen, Loughlin, Salt, & MacLean, 2016;Ibe et al, 2013;Moral-Sanz et al, 2016;Salt & Hardie, 2017;Song et al, 2016). Several studies have shown that AMPK promotes SMC proliferation and the development of pulmonary hypertension, which was reversed with AMPK inhibition (Ibe et al, 2013;Moral-Sanz et al, 2016).…”

“…Moreover, data on the role of AMPK in vascular proliferation and pulmonary hypertension are conflicting (Agard et al, 2009;Dean, Nilsen, Loughlin, Salt, & MacLean, 2016;Ibe et al, 2013;Moral-Sanz et al, 2016;Salt & Hardie, 2017;Song et al, 2016). Several studies have shown that AMPK promotes SMC proliferation and the development of pulmonary hypertension, which was reversed with AMPK inhibition (Ibe et al, 2013;Moral-Sanz et al, 2016). Conversely, other studies have shown that AMPK activation reduced SMC proliferation and vascular remodeling and may be protective against the development of hypoxia-induced pulmonary hypertension (Agard et al, 2009;Dean et al, 2016;Song et al, 2016).…”

AMPK breathing and oxygen supply

“…Furthermore, as noted above, the fact that AMPK is a serine threonine kinase suggested the capacity for regulation of processes outside of metabolism such as ion channel activity ( Figure 1), which our findings (Evans et al, 2005c;Ikematsu et al, 2011;Ross et al, 2011) and those of others have since confirmed. For example, AMPK may phosphorylate and "inactivate" the pore forming alpha subunit of multiple Ca 2+activated potassium channels (KCa1.1 and KCa3.1) (Klein et al, 2009;Ross et al, 2011), the voltage-gated potassium channel Kv1.5 (Andersen et al, 2015;Mia et al, 2012;Moral-Sanz et al, 2016) and the ATP-inhibited KATP channel (Kir6.2) (Chang et al, 2009), or may phosphorylate and "activate" Kv2.1 alpha subunits . Thereby AMPK has the potential to increase or decrease cell excitability, in a manner determined by both the cell-specific expression of its subunits and members of the ion channel superfamily, providing the capacity for system-level adjustments to whole-body metabolic status (Evans, 2006a).…”