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¹ Division of Biomedical Sciences, School of Biology, Bute Building, University of St Andrews, St Andrews, Fife KY16 9TS, UK

Abstract

In order to maintain tissue partial pressure of oxygen (P_O2) within physiological limits, vital homeostatic mechanisms monitor O₂ supply and respond to a fall in P_O2 by altering respiratory and circulatory function, and the capacity of the blood to transport O₂. Two systems that are key to this process in the acute phase are the pulmonary arteries and the carotid bodies. Hypoxic pulmonary vasoconstriction is driven by mechanisms intrinsic to the pulmonary arterial smooth muscle and endothelial cells, and aids ventilation–perfusion matching in the lung by diverting blood flow from areas with an O₂ deficit to those that are rich in O₂. By contrast, a fall in arterial P_O2 precipitates excitation–secretion coupling in carotid body type I cells, increases sensory afferent discharge from the carotid body and thereby elicits corrective changes in breathing patterns via the brainstem. There is a general consensus that hypoxia inhibits mitochondrial oxidative phosphorylation in these O₂-sensing cells over a range of P_O2 values that has no such effect on other cell types. However, the question remains as to the identity of the mechanism that underpins hypoxia–response coupling in O₂-sensing cells. Here, I lay out the case in support of a primary role for AMP-activated protein kinase in mediating chemotransduction by hypoxia.

(Received 15 May 2006; accepted after revision 26 May 2006; first published online 1 June 2006)
Corresponding author A. M. Evans: Division of Biomedical Sciences, School of Biology, Bute Building, University of St Andrews, St Andrews, Fife KY16 9TS, UK. Email: ame3{at}st-andrews.ac.uk

Investigations on O₂-sensing cells have consistently shown that inhibitors of the mitochondrial electron transport chain may mimic and/or occlude chemotransduction by hypoxia at the pulmonary artery or carotid body (Mills & Jobsis, 1972; Rounds & McMurtry, 1981; Duchen & Biscoe, 1992a,b; Leach et al. 2000; Wyatt & Buckler, 2004). Moreover, one consistent finding irrespective of the type of O₂-sensing cell under study is that relatively mild hypoxia inhibits mitochondrial oxidative phosphorylation, as indicated by depolarization of the mitochondrial membrane potential and/or an increase in cellular ß-NAD(P)H levels (Archer et al. 1986; Duchen & Biscoe, 1992a,b; Youngson et al. 1993; Shigemori et al. 1996; Leach et al. 2001). This occurs over a range of partial pressure of oxygen (P_O2) values that elicits no such response in other cell types (Duchen & Biscoe, 1992b) and has therefore been proposed to underpin, at least in part, chemotransduction by hypoxia. The sole argument against this view has been that the affinity of cytochrome c oxidase for O₂ is too high to allow for the inhibition of mitochondrial oxidative phosphorylation by physiological levels of hypoxia (60–20 mmHg), despite the fact that this parameter may vary dramatically in a cell- and tissue-specific manner that is dependent on both metabolic state and rate (Gnaiger et al. 1998).

AMP-activated protein kinase underpins hypoxic pulmonary vasoconstriction

Regulation of the AMP/ATP ratio by hypoxia in pulmonary arterial smooth muscle.  An obvious consequence of the attenuation of mitochrondrial oxidative phosphorylation by hypoxia would be a fall in the cellular energy status, but investigations on pulmonary arterial smooth muscle revealed that cellular ATP levels remained remarkably stable in the presence of hypoxia, although a fall in phosphocreatine levels was noted (Buescher et al. 1991; Leach et al. 2000). Despite the stability of the cellular ATP levels, we found that physiological hypoxia precipitated an increase in the AMP/ATP ratio (Evans et al. 2005, 2006a,b). This is consequent on the action of adenylate kinase, which catalyses the reaction that converts two molecules of ADP to ATP + AMP in order to aid ATP supply. Therefore, the cellular AMP/ATP ratio is predicted to vary as the square of the ADP/ATP ratio (Hardie & Hawley, 2001), which was the case with respect to the increase in AMP/ATP ratio by hypoxia in pulmonary arterial smooth muscle (Evans et al. 2005). These findings are entirely consistent with the proposal that AMP-activated protein kinase (AMPK) may mediate chemotransduction by hypoxia (Evans, 2004), because it is activated by an increase in the AMP/ATP ratio and its primary function is to promote catabolic pathways in order to maintain ATP supply, whilst switching off non-essential ATP-consuming (anabolic) pathways (Hardie et al. 2003).

Activation by hypoxia of AMPK in pulmonary arterial smooth muscle.  AMP-activated protein kinase is a serine threonine kinase comprising a catalytic subunit and regulatory ß and subunits, and has come to prominence as a metabolic fuel gauge which monitors the cellular AMP/ATP ratio as an index of metabolic stress. AMP-activated protein kinase is activated in response to a variety of metabolic stresses that either increase cellular ATP consumption or reduce ATP supply via mitochondrial oxidative phosphorylation (Hardie et al. 2003). In pulmonary arterial smooth muscle, the rise in the AMP/ATP ratio in response to hypoxia was associated with a concomitant twofold increase in AMPK activity and phosphorylation of a classical AMPK substrate, acetyl CoA carboxylase (Evans et al. 2005). This is likely to be mediated by the binding of AMP to two Bateman domains on the subunit of AMPK (Scott et al. 2002; Kemp, 2004; Hawley et al. 1995), which has been shown to trigger activation of the kinase by: (1) allosteric regulation via the subunit; (2) permitting phosphorylation of the subunit at Thr172 by an upstream kinase that is a complex between the tumour suppressor kinase LKB1 and two accessory proteins, STRAD and MO25 (Hawley et al. 2003; Woods et al. 2003; Shaw et al. 2004, 2005); and (3) inhibiting dephosphorylation of AMPK. In the absence of metabolic stress, each of these processes is antagonized by high concentrations of ATP, for which the Bateman domains on the subunit have lower affinity than they do for AMP. Thus, AMPK is regulated by a triple mechanism that is exquisitely sensitive to very small changes in the AMP/ATP ratio (Hardie & Hawley, 2001).

Could reactive oxygen species (ROS) regulate AMPK in response to metabolic stress?.  Consistent with the proposal that ROS may mediate chemotransduction by hypoxia (for review see Guzy & Schumacker 2006), several studies have suggested that ROS, and in particular H₂O₂, may activate AMPK (Choi et al. 2001; Halse et al. 2003; Leon et al. 2004; Nagata et al. 2004; Toyoda et al. 2004). The physiological significance of these findings is, however, open to question. For example, other studies have suggested that apoptotic effects of H₂O₂ and AMPK activation may be additive in mouse neuroblastoma cells (Jung et al. 2004), that AMPK activation may reduce stimulated H₂O₂ release by neutrophils (Alba et al. 2004) and, most recently, that AMPK activation by metabolic interventions that increase ROS (under normoxia) in clone 9 cells (from cell line "clone 9") is mediated by an increase in the AMP/ATP ratio and not by ROS (Jing & Ismail-Beigi, 2006).

A discrete AMPK subunit isoform combination may underpin hypoxic pulmonary vasoconstriction (HPV).  In 1946, von Euler and Liljestrand demonstrated that hypoxia without hypercapnia induced constriction within the pulmonary circulation and proposed that HPV might aid ventilation–perfusion matching, by diverting blood flow away from poorly ventilated areas of the lung (von Euler & Liljestrand, 1946). This is now recognized as the critical and distinguishing characteristic of pulmonary arteries; systemic arteries dilate in response to tissue hypoxaemia (Roy & Sherrington, 1890). In this respect, it is important to note that while AMPK is ubiquitously expressed throughout eukaryotic cells, at least 12 different heterotrimers may be formed from multiple isoforms (Cheung et al. 2000) of the catalytic subunit (1 and 2) and regulatory ß (ß1 and ß2) and subunits (1–3); splice variants of which may add to the diversity. Thus, the selective expression of a particular AMPK isozyme(s) could offer in some way the pulmonary selectivity required for HPV. Consistent with this view, the 1 catalytic subunit isoform (80–90% of total activity) and the 1ß21 heterotrimer (> 50% of total activity) may predominate in pulmonary arterial smooth muscle from second and third order branches of the pulmonary arterial tree; although significant activity may be carried by three other AMPK subunit isoform combinations, namely 1ß22, 2ß21 and 2ß22 (Evans et al. 2005).

Also noteworthy is the findng that the activity associated with the 1 catalytic subunit isoform was much lower ( 50%) in smooth muscle from the main pulmonary artery when compared to second and third order branches. Thus, AMPK-1 activity is inversely related to pulmonary artery diameter, as is the magnitude of pulmonary artery constriction by hypoxia (Kato & Staub, 1966). Most importantly, perhaps, AMPK-1 activity was at least fourfold higher in second and third order branches of the pulmonary arterial tree when compared with systemic (mesenteric) arteries, which dilate in response to hypoxia. In marked contrast, AMPK-2-associated activity was comparable between these vascular beds.

The distribution of the AMPK-1 subunit in pulmonary arterial smooth muscle.  Our investigations on the cellular distribution of the AMPK-1 subunit isoform in isolated pulmonary arterial smooth muscle cells revealed that associated catalytic activity is likely to be targeted to sites throughout the cytoplasm, whilst being excluded from the nucleus and from the plasma membrane.

AMPK activation mobilizes intracellular Ca⁺ stores in pulmonary arterial smooth muscle.  To determine the impact of AMPK activation on pulmonary artery function, we utilized 5-aminoimidazole-4-carboxamide riboside (AICAR), which is metabolized to yield the AMP-mimetic AICAR monophosphate (ZMP) and thereby selectively activates AMPK without affecting the cellular AMP/ATP ratio (Corton et al. 1995; Owen et al. 2000). In isolated pulmonary arterial smooth muscle cells AICAR, like hypoxia (Dipp et al. 2001; Evans et al. 2005), activated AMPK and evoked an increase in intracellular Ca²⁺ concentration that was resistant to removal of extracellular Ca²⁺. Furthermore, prior block of sarcoplasmic reticulum (SR) Ca²⁺ release via ryanodine receptors (RyRs) by pre-incubation of cells with ryanodine and caffeine abolished the increase in intracellular Ca²⁺ concentration induced by each stimulus. Importantly, SR Ca²⁺ release in response to AMPK activation was also abolished upon blocking the Ca²⁺-mobilizing messenger, cyclic adenosine diphosphate-ribose (cADPR), with 8-bromo-cADPR, a selective cADPR antagonist (Sethi et al. 1997; Walseth et al. 1997). Thus, AMPK activation triggers cADPR-dependent SR Ca²⁺ release via RyRs in isolated pulmonary arterial smooth muscle cells, as does hypoxia (Dipp & Evans, 2001; Dipp et al. 2001; Wilson et al. 2001). The significance of this finding lies in the fact that we had previously shown that the enzyme activities (ADP-ribosyl cyclase) for the synthesis and metabolism of cADPR are inversely related to pulmonary artery diameter, as is the magnitude of HPV, that the level of ADP-ribosyl cyclase activities too confer a degree (> 10-fold) of pulmonary selectivity relative to systemic arteries, that cADPR accumulation in pulmonary arterial smooth muscle is augmented by hypoxia and that cADPR-dependent SR Ca²⁺ release is a prerequisite for the full expression of HPV (Dipp & Evans, 2001; Wilson et al. 2001). Thus, via signal amplification, AMPK and ADP-ribosyl cyclase may provide the pulmonary selectivity required for HPV. However, the precise mechanism by which AMPK elicits cADPR-dependent SR Ca²⁺ release remains to be determined. This may result, for example, from: (1) phosphorylation by AMPK of ADP-ribosyl cyclase and a consequent increase in cADPR accumulation; and/or (2) increased sensitivity of the Ca²⁺ release process to cADPR due to phosphorylation by AMPK of RyRs or an intermediate cADPR-binding protein.

It should also be noted that there is evidence to suggest that inhibition of mitochondrial metabolism by hypoxia may modulate cADPR accumulation in pulmonary arterial smooth muscle via mechanisms independent of AMPK. Thus, an increase in ß-NADH levels may augment cADPR synthesis and/or inhibit cADPR metabolism by ADP-ribosyl cyclase (Wilson et al. 2001).

AMPK activation induces pulmonary artery constriction.  Consistent with the time course of the maintained phase of pulmonary artery constriction by hypoxia (Dipp & Evans, 2001), AICAR induced a slow, sustained and reversible constriction of pulmonary artery rings (Evans et al. 2005). Removal of the pulmonary artery endothelium reduced the constriction in response to hypoxia and AICAR by approximately 30%. Furthermore, the endothelium-dependent component of constriction by AICAR and hypoxia was abolished upon removal of extracellular Ca²⁺, and therefore requires Ca²⁺ influx into the endothelium. In contrast, constriction mediated by mechanisms intrinsic to the smooth muscle was not abolished. However, smooth muscle constriction was abolished by blocking the mobilization of SR Ca²⁺ stores via RyRs with ryanodine and caffeine and by blocking Ca²⁺ mobilization in response to cADPR with 8-bromo-cADPR. Thus, both AMPK activation and hypoxia mediate maintained constriction of pulmonary artery smooth muscle by cADPR-dependent mobilization of SR Ca²⁺ stores via RyRs. It should be noted, however, that maintained smooth muscle constriction by AICAR and hypoxia, respectively, exhibited a partial dependence on transmembrane Ca²⁺ influx. In this respect, it is of major significance that block of SR Ca²⁺ stores with caffeine and ryanodine or blockade of cADPR with 8-bromo-cADPR completely abolished the constriction of pulmonary arteries, with or without endothelium, by both AICAR and hypoxia. Thus, the partial dependence of smooth muscle constriction on extracellular Ca²⁺ must be determined by SR Ca²⁺ release-activated Ca²⁺ influx, as suggested by the investigations of others (Kang et al. 2003; Ng et al. 2005; Wang et al. 2005). However, it seems likely that this process of store-release-activated Ca²⁺ influx is consequent on the mobilization of SR Ca²⁺ stores rather than being directly regulated by hypoxia (Kang et al. 2003; Ng et al. 2005) or AMPK activation per se (Evans et al. 2005).

Strong support for the above mechanism comes from our most recent studies, which have shown that the AMPK antagonist, compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]-pyrimidine) (Zhou et al. 2001), inhibits HPV, (T.P. Robertson, E.A. Blanco, K.J.W. Mustard, T.H. Lewis, D.G. Hardie & A.M. Evans, unpublished observations). It seems likely, therefore, that AMPK activation mediates pulmonary artery constriction by hypoxia.

AMPK and carotid body excitation by hypoxia

If AMPK were to play a central role in chemotransduction by hypoxia in all O₂-sensing cells, we would expect its activation to mimic precisely the effects of hypoxia irrespective of any cell-specific variation in signalling mechanism. To this end, we studied the role of AMPK in carotid body excitation by hypoxia. Carotid body type I cells monitor systemic arterial P_O2. Upon exposure to hypoxia, voltage-gated Ca²⁺ influx into type I cells, rather than ER Ca²⁺ release (Buckler & Vaughan-Jones, 1994), initiates neurosecretion (Fidone et al. 1988; Gonzalez et al. 1994; Chen et al. 1997) and thereby increases sensory afferent discharge to the brainstem. This process is driven by the inhibition of what have been termed O₂-sensitive K⁺ channels (Lopez-Barneo et al. 1988; Peers, 1990; Stea & Nurse, 1991; Lopez-Lopez & Gonzalez, 1992; Wyatt & Peers, 1992, 1995; Buckler, 1997, 1999; Buckler et al. 2000). Consistent with this, immunofluorescence imaging revealed that the AMPK-1 catalytic subunit isoform expressed in carotid body type I cells was almost entirely (75%) restricted to a volume within 1 µm of the plasma membrane. Thus, the spatial localization of AMPK 1 in carotid body type I cells is consistent with it targeting plasma membrane-delimited processes. This is contrary to our observations on pulmonary arterial smooth muscle cells, in which AMPK-1 appeared to be absent from the plasma membrane and targeted to structures throughout the cytoplasm. It is possible, therefore, that different heterotrimeric subunit combinations may determine, in part, the targeting of AMPK to these discrete cellular compartments.

Consistent with AMPK-1 being targeted to the plasma membrane in carotid body type I cells, AMPK activation by AICAR, like hypoxia, induced a reversible depolarization of the membrane potential (Wyatt et al. 2006). Perhaps most significantly, like hypoxia (Wyatt & Peers, 1995; Buckler et al. 2000), AMPK activation (Wyatt et al. 2006) elicited depolarization of rat carotid body type I cells by inhibiting O₂-sensitive K⁺ currents carried by the TASK (TWIK-related acid-sensitive potassium)-like leak K⁺ channels and large-conductance Ca²⁺-activated K⁺ channels (BK_Ca), but not by inhibiting the O₂-insensitive voltage-gated K⁺ channel (Kv) current. Consequently, an increase in the intracellular Ca²⁺ concentration was evoked via Ni²⁺- and Cd²⁺-sensitive voltage-gated Ca²⁺ influx pathways, which ultimately led to an increase in sensory afferent discharge from the isolated carotid body (Evans et al. 2005; Wyatt et al. 2006). Thus, AMPK is likely to mediate the excitatory effects of hypoxia on isolated carotid body type I cells and on the carotid body in vitro. Once more, this view gains strong support from our most recent investigations, which have shown that the AMPK antagonist, compound C, reverses the increase in intracellular Ca²⁺ concentration evoked by hypoxia and AICAR in isolated carotid body type I cells and the increase in sensory afferent discharge elicited from the isolated carotid body by each of these stimuli (C.N. Wyatt, K.J.W. Mustard, S.A. Pearson, M.L. Dallas, L. Atkinson, P. Kumar, C. Peers, D.G. Hardie & A.M. Evans, unpublished observations).

Could AMPK activation regulate gene expression associated with pathophysiologies of chronic and intermittent hypoxia?

The development of pathophysiologies (e.g. pulmonary hypertension) associated with chronic and intermittent hypoxia may be determined, at least in part, by the transcription factor hypoxia-inducible factor 1 (HIF-1); a heterodimer composed of an and a constitutively expressed ß subunit (for review see Semenza 2006). Here too the diversity of AMPK subunit isoform combinations may come into play. Thus, the AMPK-2 but not the AMPK-1 catalytic subunit isoform may increase HIF-1 expression and HIF-1 activity (Hwang et al. 2004) and thereby regulate associated transcriptional activity and target gene expression in response to prolonged hypoxia (Salt et al. 1998; Lee et al. 2003; Neurath et al. 2006). Therefore, AMPK-1 may underpin the regulation of primary cell function by hypoxia, whilst AMPK-2 may regulate O₂-dependent gene expression in response to chronic and intermittent hypoxia.

Summary

Our findings suggest that inhibition of mitochondrial oxidative phosphorylation by hypoxia leads to a rise in the cellular AMP/ATP ratio, and consequent activation of AMPK-1-associated catalytic activity in pulmonary arterial smooth muscle and carotid body type I cells. The reliance of O₂-sensing cells on mitochondria for ATP supply and a higher level of activity of the AMPK-1 catalytic subunit isoform may determine, at least in part, the relative sensitivity of such cells to physiological hypoxia. Thereafter, different heterotrimeric subunit combinations may target AMPK to discrete cellular compartments in pulmonary arterial smooth muscle cells and carotid body type-I cells, respectively. Thus, the characteristic response of each tissue type to hypoxia may be mediated by AMPK, namely: (1) constriction of pulmonary arteries by cADPR-dependent SR Ca²⁺ release in the smooth muscle cells, with a secondary component of constriction driven by the pulmonary arterial endothelial cells; and (2) inhibition of TASK-like and BK_Ca currents, leading to depolarization, voltage-gated Ca²⁺ influx into rat carotid body type I cells and a consequent increase in afferent fibre discharge (Fig. 1). Alternative proposed mechanisms, such as haemeoxygenase-2-dependent regulation of BK_Ca channels (Williams et al. 2004; for review see Kemp 2006) may contribute to carotid body type I cell activation by hypoxia, but cannot explain the finding that inhibitors of mitochondrial oxidative phosphorylation can mimic and/or occlude carotid body type I cell excitation by hypoxia and HPV. I propose, therefore, that in addition to maintaining the cellular energy state, AMPK acts as the primary metabolic sensor and effector of chemotransduction by hypoxia in O₂-sensing cells.

View larger version (28K): [in this window] [in a new window]   Figure 1.  AMPK-dependent regulation of O2-sensing cells Schematic diagram illustrating the role of AMPK in HPV and carotid body activation by hypoxia. Physiological hypoxia inhibits mitochondrial oxidative phosphorylation, which leads to an increase in the AMP/ATP ratio, AMPK activation and consequent activation of cell-specific Ca2+ signalling mechanisms in pulmonary arterial smooth muscle cells (cADPR-dependent SR Ca2+ release) and carotid body type I cells (voltage-gated Ca2+ influx). This figure is modified from Figure 4E in A. M. Evans etal. (2005) J Biol Chem 280, 41504–41511.

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Acknowledgements

This work was supported by the Wellcome Trust.

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