AMP-activated protein kinase and hypoxic pulmonary vasoconstriction

Abstract

Hypoxic pulmonary vasoconstriction is a vital homeostatic mechanism that aids ventilation-perfusion matching in the lung, for which the underlying mechanism(s) remains controversial. However, our most recent investigations strongly suggest that hypoxic pulmonary vasoconstriction is precipitated, at least in part, by the inhibition of mitochondrial oxidative phosphorylation by hypoxia, an increase in the AMP/ATP ratio and consequent activation of AMP-activated protein kinase (AMPK). Unfortunately, these studies lacked the definitive proof that can only be provided by selectively blocking AMPK-dependent signalling cascades. The aim of the present study was, therefore, to determine the effects of the AMPK inhibitor compound C upon: (1) phosphorylation in response to hypoxia of a classical AMPK substrate, acetyl CoA carboxylase, in rat pulmonary arterial smooth muscle and (2) hypoxic pulmonary vasoconstriction in rat isolated intrapulmonary arteries. Acetyl CoA carboxylase phosphorylation was increased approximately 3 fold in the presence of hypoxia (pO(2) = 16-21 mm Hg, 1 h) and 5-aminoimidazole-4-carboxamide riboside (AICAR; 1 mM; 4 h) and in a manner that was significantly attenuated by the AMPK antagonist compound C (40 microM). Most importantly, pre-incubation of intrapulmonary arteries with compound C (40 microM) inhibited phase II, but not phase I, of hypoxic pulmonary vasoconstriction. Likewise, compound C (40 microM) inhibited constriction by AICAR (1 mM). The results of the present study are consistent with the activation of AMPK being a key event in the initiation of the contractile response of pulmonary arteries to acute hypoxia.

Fig. 1. Phosphorylation of acetyl CoA carboxylase in response to hypoxia and AICAR is inhibited by the AMPK antagonist compound C

Bar chart shows the phosphorylated acetyl CoA carboxylase / acetyl CoA carboxylase (PACC / ACC) ratio measured in pulmonary artery smooth muscle lysates under control conditions (2h normoxia, 150-160 mm Hg), hypoxia (1h at 16-21 mmHg; following 1h normoxia) and in the presence of 1 mM AICAR (4h) with and without 40 μM compound C.

Fig. 2. Hypoxic pulmonary vasoconstriction is concentration-dependently reversed, and inhibited, by Compound C

Panel A shows a typical response of a rat IPA to acute hypoxia that was biphasic in nature, consisting of a transient phase I constriction, superimposed on a sustained phase II contractile response. Panel B shows a representative response of a time-matched IPA following pre-incubation (10 min) with compound C (40 μM). Panel C shows the mean effects of pre-incubation with compound C (40 μM) upon subsequent hypoxic responses in rat IPA (P<0.05 for 40 μM compound C versus control for all time-points except that immediately following the peak of phase I). Panel D shows the concentration-dependent inhibition of the sustained phase II constriction by compound C (10-30 μM)

Fig. 3. AICAR-induced pulmonary vasoconstriction is inhibited by Compound C

Panel A shows a typical response of a rat IPA to 1 mM AICAR, which consisted of a slowly-developing sustained constriction. Panel B shows the effects of pre-incubation with compound C (40 μM) upon the response to 1 mM AICAR in paired IPA.

Fig. 4. Thapsigargin-induced pulmonary vasoconstriction is not inhibited by Compound C

Typical response of a rat IPA, without endothelium, to 1 μM thapsigargin in the absence (+ 1 mM EGTA) and then presence of extracellular Ca²⁺ and the effect of subsequent application of compound C (40 μM) to the bathing solution.