SubSol-HIe is an AMPK-dependent hypoxia-responsive subnucleus of the nucleus tractus solitarius that coordinates the hypoxic ventilatory response and protects against apnoea in mice.

Functional magnetic resonance imaging (fMRI) suggests that the hypoxic ventilatory response is facilitated by the AMP-activated protein kinase (AMPK), not at the carotid bodies, but within a subnucleus (Bregma -7.5 to -7.1 mm) of the nucleus tractus solitarius that exhibits right-sided bilateral asymmetry. Here, we map this subnucleus using cFos expression as a surrogate for neuronal activation and mice in which the genes encoding the AMPK-α1 (Prkaa1) and AMPK-α2 (Prkaa2) catalytic subunits were deleted in catecholaminergic cells by Cre expression via the tyrosine hydroxylase promoter. Comparative analysis of brainstem sections, relative to controls, revealed that AMPK-α1/α2 deletion inhibited, with right-sided bilateral asymmetry, cFos expression in and thus activation of a neuronal cluster that partially spanned three interconnected anatomical nuclei adjacent to the area postrema: SolDL (Bregma -7.44 mm to -7.48 mm), SolDM (Bregma -7.44 mm to -7.48 mm) and SubP (Bregma -7.48 mm to -7.56 mm). This approximates the volume identified by fMRI. Moreover, these nuclei are known to be in receipt of carotid body afferent inputs, and catecholaminergic neurons of SubP and SolDL innervate aspects of the ventrolateral medulla responsible for respiratory rhythmogenesis. Accordingly, AMPK-α1/α2 deletion attenuated hypoxia-evoked increases in minute ventilation (normalised to metabolism), reductions in expiration time, and increases sigh frequency, but increased apnoea frequency during hypoxia. The metabolic response to hypoxia in AMPK-α1/α2 knockout mice and the brainstem and spinal cord catecholamine levels were equivalent to controls. We conclude that within the brainstem an AMPK-dependent, hypoxia-responsive subnucleus partially spans SubP, SolDM and SolDL, namely SubSol-HIe, and is critical to coordination of active expiration, the hypoxic ventilatory response and defence against apnoea.

Of Mice and Men and Plethysmography Systems: Does LKB1 Determine the Set Point of Carotid Body Chemosensitivity and the Hypoxic Ventilatory Response?

Our recent studies suggest that the level of liver kinase B1 (LKB1) expression in some way determines carotid body afferent discharge during hypoxia and to a lesser extent during hypercapnia. In short, phosphorylation by LKB1 of an as yet unidentified target(s) determines a set point for carotid body chemosensitivity. LKB1 is the principal kinase that activates the AMP-activated protein kinase (AMPK) during metabolic stresses, but conditional deletion of AMPK in catecholaminergic cells, including therein carotid body type I cells, has little or no effect on carotid body responses to hypoxia or hypercapnia. With AMPK excluded, the most likely target of LKB1 is one or other of the 12 AMPK-related kinases, which are constitutively phosphorylated by LKB1 and, in general, regulate gene expression. By contrast, the hypoxic ventilatory response is attenuated by either LKB1 or AMPK deletion in catecholaminergic cells, precipitating hypoventilation and apnea during hypoxia rather than hyperventilation. Moreover, LKB1, but not AMPK, deficiency causes Cheyne-Stokes-like breathing. This chapter will explore further the possible mechanisms that determine these outcomes.

AMPK facilitates the hypoxic ventilatory response through non-adrenergic mechanisms at the brainstem.

We recently demonstrated that the hypoxic ventilatory response (HVR) is facilitated by the AMP-activated protein kinase (AMPK) in catecholaminergic neural networks that likely lie downstream of the carotid bodies within the caudal brainstem. Here, we further subcategorise the neurons involved, by cross-comparison of mice in which the genes encoding the AMPK-α1 (Prkaa1) and AMPK-α2 (Prkaa2) catalytic subunits were deleted in catecholaminergic (TH-Cre) or adrenergic (PNMT-Cre) neurons. As expected, the HVR was markedly attenuated in mice with AMPK-α1/α2 deletion in catecholaminergic neurons, but surprisingly was modestly augmented in mice with AMPK-α1/α2 deletion in adrenergic neurons when compared against a variety of controls (TH-Cre, PNMT-Cre, AMPK-α1/α2 floxed). Moreover, AMPK-α1/α2 deletion in catecholaminergic neurons precipitated marked hypoventilation and apnoea during poikilocapnic hypoxia, relative to controls, while mice with AMPK-α1/α2 deletion in adrenergic neurons entered relative hyperventilation with reduced apnoea frequency and duration. We conclude, therefore, that AMPK-dependent modulation of non-adrenergic networks may facilitate increases in ventilatory drive that shape the classical HVR, whereas AMPK-dependent modulation of adrenergic networks may provide some form of negative feedback or inhibitory input to moderate HVR, which could, for example, protect against hyperventilation-induced hypocapnia and respiratory alkalosis.

LKB1 is the gatekeeper of carotid body chemosensing and the hypoxic ventilatory response.

The hypoxic ventilatory response (HVR) is critical to breathing and thus oxygen supply to the body and is primarily mediated by the carotid bodies. Here we reveal that carotid body afferent discharge during hypoxia and hypercapnia is determined by the expression of Liver Kinase B1 (LKB1), the principal kinase that activates the AMP-activated protein kinase (AMPK) during metabolic stresses. Conversely, conditional deletion in catecholaminergic cells of AMPK had no effect on carotid body responses to hypoxia or hypercapnia. By contrast, the HVR was attenuated by LKB1 and AMPK deletion. However, in LKB1 knockouts hypoxia evoked hypoventilation, apnoea and Cheyne-Stokes-like breathing, while only hypoventilation and apnoea were observed after AMPK deletion. We therefore identify LKB1 as an essential regulator of carotid body chemosensing and uncover a divergence in dependency on LKB1 and AMPK between the carotid body on one hand and the HVR on the other.

On a Magical Mystery Tour with 8-Bromo-Cyclic ADP-Ribose: From All-or-None Block to Nanojunctions and the Cell-Wide Web.

A plethora of cellular functions are controlled by calcium signals, that are greatly coordinated by calcium release from intracellular stores, the principal component of which is the sarco/endooplasmic reticulum (S/ER). In 1997 it was generally accepted that activation of various G protein-coupled receptors facilitated inositol-1,4,5-trisphosphate (IP3) production, activation of IP3 receptors and thus calcium release from S/ER. Adding to this, it was evident that S/ER resident ryanodine receptors (RyRs) could support two opposing cellular functions by delivering either highly localised calcium signals, such as calcium sparks, or by carrying propagating, global calcium waves. Coincidentally, it was reported that RyRs in mammalian cardiac myocytes might be regulated by a novel calcium mobilising messenger, cyclic adenosine diphosphate-ribose (cADPR), that had recently been discovered by HC Lee in sea urchin eggs. A reputedly selective and competitive cADPR antagonist, 8-bromo-cADPR, had been developed and was made available to us. We used 8-bromo-cADPR to further explore our observation that S/ER calcium release via RyRs could mediate two opposing functions, namely pulmonary artery dilation and constriction, in a manner seemingly independent of IP3Rs or calcium influx pathways. Importantly, the work of others had shown that, unlike skeletal and cardiac muscles, smooth muscles might express all three RyR subtypes. If this were the case in our experimental system and cADPR played a role, then 8-bromo-cADPR would surely block one of the opposing RyR-dependent functions identified, or the other, but certainly not both. The latter seemingly implausible scenario was confirmed. How could this be, do cells hold multiple, segregated SR stores that incorporate different RyR subtypes in receipt of spatially segregated signals carried by cADPR? The pharmacological profile of 8-bromo-cADPR action supported not only this, but also indicated that intracellular calcium signals were delivered across intracellular junctions formed by the S/ER. Not just one, at least two. This article retraces the steps along this journey, from the curious pharmacological profile of 8-bromo-cADPR to the discovery of the cell-wide web, a diverse network of cytoplasmic nanocourses demarcated by S/ER nanojunctions, which direct site-specific calcium flux and may thus coordinate the full panoply of cellular processes.

AMPK and the Need to Breathe and Feed: What's the Matter with Oxygen?

We live and to do so we must breathe and eat, so are we a combination of what we eat and breathe? Here, we will consider this question, and the role in this respect of the AMP-activated protein kinase (AMPK). Emerging evidence suggests that AMPK facilitates central and peripheral reflexes that coordinate breathing and oxygen supply, and contributes to the central regulation of feeding and food choice. We propose, therefore, that oxygen supply to the body is aligned with not only the quantity we eat, but also nutrient-based diet selection, and that the cell-specific expression pattern of AMPK subunit isoforms is critical to appropriate system alignment in this respect. Currently available information on how oxygen supply may be aligned with feeding and food choice, or vice versa, through our motivation to breathe and select particular nutrients is sparse, fragmented and lacks any integrated understanding. By addressing this, we aim to provide the foundations for a clinical perspective that reveals untapped potential, by highlighting how aberrant cell-specific changes in the expression of AMPK subunit isoforms could give rise, in part, to known associations between metabolic disease, such as obesity and type 2 diabetes, sleep-disordered breathing, pulmonary hypertension and acute respiratory distress syndrome.

The cell-wide web coordinates cellular processes by directing site-specific Ca2+ flux across cytoplasmic nanocourses.

Ca2+ coordinates diverse cellular processes, yet how function-specific signals arise is enigmatic. We describe a cell-wide network of distinct cytoplasmic nanocourses with the nucleus at its centre, demarcated by sarcoplasmic reticulum (SR) junctions (≤400 nm across) that restrict Ca2+ diffusion and by nanocourse-specific Ca2+-pumps that facilitate signal segregation. Ryanodine receptor subtype 1 (RyR1) supports relaxation of arterial myocytes by unloading Ca2+ into peripheral nanocourses delimited by plasmalemma-SR junctions, fed by sarco/endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b). Conversely, stimulus-specified increases in Ca2+ flux through RyR2/3 clusters selects for rapid propagation of Ca2+ signals throughout deeper extraperinuclear nanocourses and thus myocyte contraction. Nuclear envelope invaginations incorporating SERCA1 in their outer nuclear membranes demarcate further diverse networks of cytoplasmic nanocourses that receive Ca2+ signals through discrete RyR1 clusters, impacting gene expression through epigenetic marks segregated by their associated invaginations. Critically, this circuit is not hardwired and remodels for different outputs during cell proliferation.

AMPK breathing and oxygen supply.

Regulation of breathing is critical to our capacity to accommodate deficits in oxygen availability and demand during, for example, sleep and ascent to altitude. Key to this are two reflex responses, hypoxic pulmonary vasoconstriction (HPV), which aids ventilation-perfusion matching at the lungs, and the hypoxic ventilatory response (HVR) which accelerates ventilation. In 2004 I proposed that HPV might be mediated by the AMP-activated protein kinase, which governs cell autonomous metabolic homeostasis. Pharmacological evidence was presented in support of this view, and the hypothesis extended to incorporate a role for AMPK in regulating carotid body afferent input responses during hypoxia and thus the HVR. The present article reviews our subsequent findings on these matters and those of others, which provide strong support for the view that AMPK mediates HPV. AMPK is also critical to the HVR, but against our expectations it is not required for carotid body activation during hypoxia. Contrary to current consensus in this respect, our findings suggest that AMPK deficiency blocks the HVR at the level of the brainstem, even when afferent input responses from the carotid body are normal. We have therefore revised our hypothesis on the HVR, now proposing that AMPK integrates local hypoxic stress at defined loci within the brainstem respiratory network with an index of peripheral hypoxic status, namely afferent chemosensory inputs. Nevertheless, in general outcomes are consistent with the original hypothesis, that the role of AMPK has evolved, through natural selection, to extend to the regulation of breathing, and thus oxygen and energy (ATP) supply to the whole body.

The LKB1-AMPK-α1 signaling pathway triggers hypoxic pulmonary vasoconstriction downstream of mitochondria.

Hypoxic pulmonary vasoconstriction (HPV), which aids ventilation-perfusion matching in the lungs, is triggered by mechanisms intrinsic to pulmonary arterial smooth muscles. The unique sensitivity of these muscles to hypoxia is conferred by mitochondrial cytochrome c oxidase subunit 4 isoform 2, the inhibition of which has been proposed to trigger HPV through increased generation of mitochondrial reactive oxygen species. Contrary to this model, we have shown that the LKB1-AMPK-α1 signaling pathway is critical to HPV. Spectral Doppler ultrasound revealed that deletion of the AMPK-α1 catalytic subunit blocked HPV in mice during mild (8% O2) and severe (5% O2) hypoxia, whereas AMPK-α2 deletion attenuated HPV only during severe hypoxia. By contrast, neither of these genetic manipulations affected serotonin-induced reductions in pulmonary vascular flow. HPV was also attenuated by reduced expression of LKB1, a kinase that activates AMPK during energy stress, but not after deletion of CaMKK2, a kinase that activates AMPK in response to increases in cytoplasmic Ca2+ Fluorescence imaging of acutely isolated pulmonary arterial myocytes revealed that AMPK-α1 or AMPK-α2 deletion did not affect mitochondrial membrane potential during normoxia or hypoxia. However, deletion of AMPK-α1, but not of AMPK-α2, blocked hypoxia from inhibiting KV1.5, the classical "oxygen-sensing" K+ channel in pulmonary arterial myocytes. We conclude that LKB1-AMPK-α1 signaling pathways downstream of mitochondria are critical for the induction of HPV, in a manner also supported by AMPK-α2 during severe hypoxia.

AMPK-α1 or AMPK-α2 Deletion in Smooth Muscles Does Not Affect the Hypoxic Ventilatory Response or Systemic Arterial Blood Pressure Regulation During Hypoxia.

The hypoxic ventilatory response (HVR) is markedly attenuated by AMPK-α1 deletion conditional on the expression of Cre-recombinase in tyrosine hydroxylase (TH) expressing cells, precipitating marked increases in apnea frequency and duration. It was concluded that ventilatory dysfunction caused by AMPK deficiency was driven by neurogenic mechanisms. However, TH is transiently expressed in other cell types during development, and it is evident that central respiratory depression can also be triggered by myogenic mechanisms that impact blood supply to the brain. We therefore assessed the effect on the HVR and systemic arterial blood pressure of AMPK deletion in vascular smooth muscles. There was no difference in minute ventilation during normoxia. However, increases in minute ventilation during severe hypoxia (8% O2) were, if affected at all, augmented by AMPK-α1 and AMPK-α2 deletion in smooth muscles; despite the fact that hypoxia (8% O2) evoked falls in arterial SpO2 comparable with controls. Surprisingly, these mice exhibited no difference in systolic, diastolic or mean arterial blood pressure during normoxia or hypoxia. We conclude that neither AMPK-α1 nor AMPK-α2 are required in smooth muscle for the regulation of systemic arterial blood pressure during hypoxia, and that AMPK-α1 deficiency does not impact the HVR by myogenic mechanisms.

mTORC1 controls lysosomal Ca2+ release through the two-pore channel TPC2.

Two-pore segment channel 2 (TPC2) is a ubiquitously expressed, lysosomally targeted ion channel that aids in terminating autophagy and is inhibited upon its association with mechanistic target of rapamycin (mTOR). It is controversial whether TPC2 mediates lysosomal Ca2+ release or selectively conducts Na+ and whether the binding of nicotinic acid adenine dinucleotide phosphate (NAADP) or phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is required for the activity of this ion channel. We show that TPC2 is required for intracellular Ca2+ signaling in response to NAADP or to mTOR inhibition by rapamycin. In pulmonary arterial myocytes, rapamycin and NAADP evoked global Ca2+ transients that were blocked by depletion of lysosomal Ca2+ stores. Preincubation of cells with high concentrations of rapamycin resulted in desensitization and blocked NAADP-evoked Ca2+ signals. Moreover, rapamycin and NAADP did not evoke discernable Ca2+ transients in myocytes derived from Tpcn2 knockout mice, which showed normal responses to other Ca2+-mobilizing signals. In HEK293 cells stably overexpressing human TPC2, shRNA-mediated knockdown of mTOR blocked rapamycin- and NAADP-evoked Ca2+ signals. Confocal imaging of a genetically encoded Ca2+ indicator fused to TPC2 demonstrated that rapamycin-evoked Ca2+ signals localized to lysosomes and were in close proximity to TPC2. Therefore, inactivation of mTOR may activate TPC2 and consequently lysosomal Ca2+ release.

Genotoxic Damage Activates the AMPK-α1 Isoform in the Nucleus via Ca2+/CaMKK2 Signaling to Enhance Tumor Cell Survival.

Many genotoxic cancer treatments activate AMP-activated protein kinase (AMPK), but the mechanisms of AMPK activation in response to DNA damage, and its downstream consequences, have been unclear. In this study, etoposide activates the α1 but not the α2 isoform of AMPK, primarily within the nucleus. AMPK activation is independent of ataxia-telangiectasia mutated (ATM), a DNA damage-activated kinase, and the principal upstream kinase for AMPK, LKB1, but correlates with increased nuclear Ca2+ and requires the Ca2+/calmodulin-dependent kinase, CaMKK2. Intriguingly, Ca2+-dependent activation of AMPK in two different LKB1-null cancer cell lines caused G1-phase cell-cycle arrest, and enhanced cell viability/survival after etoposide treatment, with both effects being abolished by knockout of AMPK-α1 and α2. The CDK4/6 inhibitor palbociclib also caused G1 arrest in G361 but not HeLa cells and, consistent with this, enhanced cell survival after etoposide treatment only in G361 cells. These results suggest that AMPK activation protects cells against etoposide by limiting entry into S-phase, where cells would be more vulnerable to genotoxic stress.Implications: These results reveal that the α1 isoform of AMPK promotes tumorigenesis by protecting cells against genotoxic stress, which may explain findings that the gene encoding AMPK-α1 (but not -α2) is amplified in some human cancers. Furthermore, α1-selective inhibitors might enhance the anticancer effects of genotoxic-based therapies. Mol Cancer Res; 16(2); 345-57. ©2017 AACR.

Tissue Specificity: The Role of Organellar Membrane Nanojunctions in Smooth Muscle Ca2+ Signaling.

In this chapter we examine the importance of cytoplasmic nanojunctions-nanometer scale appositions between organellar membranes including the molecular transporters therein-to the cell signaling machinery, with specific reference to Ca2+ transport and signaling in vascular smooth muscle and endothelial cells. More specifically, we will consider the extent to which quantitative modeling may aid in the development of our understanding of these processes. Testament to the requirement for such approaches lies in the fact that recent studies have provided evermore convincing evidence in support of the view that cytoplasmic nanospaces may be as significant to the process of Ca2+ signaling as the Ca2+ transporters, release channels, and Ca2+-storing organelles themselves. Moreover, the disruption and/or dysfunction of cytoplasmic nanospaces may be central to the origin of certain diseases. By way of introduction, we provide a historical perspective on the identification of smooth muscle cell plasma membrane (PM)-sarcoplasmic reticulum (SR) nanospaces and the early evidence in support of their role in the generation of asynchronous Ca2+ waves. We then summarize how stochastic modeling approaches can aid and guide the development of our understanding of two basic functional steps leading to healthy smooth muscle cell contraction. We furthermore outline how more sophisticated and realistic quantitative stochastic modeling may be employed not only to test working hypotheses, but also to lead in their development in a manner that informs further experimental investigation. Finally, we consider more recently defined nanospaces such as the lysosome-SR junction, by way of demonstrating the importance of quantitative stochastic modeling to our understanding of signaling mechanisms.

The emerging role of AMPK in the regulation of breathing and oxygen supply.

Regulation of breathing is critical to our capacity to accommodate deficits in oxygen availability and demand during, for example, sleep and ascent to altitude. It is generally accepted that a fall in arterial oxygen increases afferent discharge from the carotid bodies to the brainstem and thus delivers increased ventilatory drive, which restores oxygen supply and protects against hypoventilation and apnoea. However, the precise molecular mechanisms involved remain unclear. We recently identified as critical to this process the AMP-activated protein kinase (AMPK), which is key to the cell-autonomous regulation of metabolic homoeostasis. This observation is significant for many reasons, not least because recent studies suggest that the gene for the AMPK-α1 catalytic subunit has been subjected to natural selection in high-altitude populations. It would appear, therefore, that evolutionary pressures have led to AMPK being utilized to regulate oxygen delivery and thus energy supply to the body in the short, medium and longer term. Contrary to current consensus, however, our findings suggest that AMPK regulates ventilation at the level of the caudal brainstem, even when afferent input responses from the carotid body are normal. We therefore hypothesize that AMPK integrates local hypoxic stress at defined loci within the brainstem respiratory network with an index of peripheral hypoxic status, namely afferent chemosensory inputs. Allied to this, AMPK is critical to the control of hypoxic pulmonary vasoconstriction and thus ventilation-perfusion matching at the lungs and may also determine oxygen supply to the foetus by, for example, modulating utero-placental blood flow.

From contraction to gene expression: nanojunctions of the sarco/endoplasmic reticulum deliver site- and function-specific calcium signals.

Calcium signals determine, for example, smooth muscle contraction and changes in gene expression. How calcium signals select for these processes is enigmatic. We build on the "panjunctional sarcoplasmic reticulum" hypothesis, describing our view that different calcium pumps and release channels, with different kinetics and affinities for calcium, are strategically positioned within nanojunctions of the SR and help demarcate their respective cytoplasmic nanodomains. SERCA2b and RyR1 are preferentially targeted to the sarcoplasmic reticulum (SR) proximal to the plasma membrane (PM), i.e., to the superficial buffer barrier formed by PM-SR nanojunctions, and support vasodilation. In marked contrast, SERCA2a may be entirely restricted to the deep, perinuclear SR and may supply calcium to this sub-compartment in support of vasoconstriction. RyR3 is also preferentially targeted to the perinuclear SR, where its clusters associate with lysosome-SR nanojunctions. The distribution of RyR2 is more widespread and extends from this region to the wider cell. Therefore, perinuclear RyR3s most likely support the initiation of global calcium waves at L-SR junctions, which subsequently propagate by calcium-induced calcium release via RyR2 in order to elicit contraction. Data also suggest that unique SERCA and RyR are preferentially targeted to invaginations of the nuclear membrane. Site- and function-specific calcium signals may thus arise to modulate stimulus-response coupling and transcriptional cascades.

Exome Sequencing and the Management of Neurometabolic Disorders.

BACKGROUND: Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS: To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient's clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS: We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS: Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children's Hospital Foundation and others.).

AMP-activated protein kinase inhibits Kv 1.5 channel currents of pulmonary arterial myocytes in response to hypoxia and inhibition of mitochondrial oxidative phosphorylation.

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 Kv 1.5 channels in pulmonary arterial myocytes. AMPK activation by 5-aminoimidazole-4-carboxamide riboside, A769662 or C13 attenuated Kv 1.5 currents in pulmonary arterial myocytes, and this effect was non-additive with respect to Kv 1.5 inhibition by hypoxia and mitochondrial poisons. Recombinant AMPK phosphorylated recombinant human Kv 1.5 channels in cell-free assays, and inhibited K(+) currents when introduced into HEK 293 cells stably expressing Kv 1.5. These results suggest that AMPK is the primary mediator of reductions in Kv 1.5 channels following inhibition of mitochondrial oxidative phosphorylation during hypoxia and by mitochondrial poisons. ABSTRACT: 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 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 (α2ß2γ1 or α1ß1γ1). Hypoxia and inhibitors of mitochondrial oxidative phosphorylation reduced AMPK-sensitive K(+) currents, which were also blocked by the selective Kv 1.5 channel inhibitor diphenyl phosphine oxide-1 but unaffected by the presence of the BKCa channel blocker paxilline. Moreover, recombinant human Kv 1.5 channels were phosphorylated by AMPK in cell-free assays, and K(+) currents carried by Kv 1.5 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 Kv 1.5 and/or an associated protein.

AMP-activated Protein Kinase Deficiency Blocks the Hypoxic Ventilatory Response and Thus Precipitates Hypoventilation and Apnea.

RATIONALE: Modulation of breathing by hypoxia accommodates variations in oxygen demand and supply during, for example, sleep and ascent to altitude, but the precise molecular mechanisms of this phenomenon remain controversial. Among the genes influenced by natural selection in high-altitude populations is one for the adenosine monophosphate-activated protein kinase (AMPK) α1-catalytic subunit, which governs cell-autonomous adaptations during metabolic stress. OBJECTIVES: We investigated whether AMPK-α1 and/or AMPK-α2 are required for the hypoxic ventilatory response and the mechanism of ventilatory dysfunctions arising from AMPK deficiency. METHODS: We used plethysmography, electrophysiology, functional magnetic resonance imaging, and immediate early gene (c-fos) expression to assess the hypoxic ventilatory response of mice with conditional deletion of the AMPK-α1 and/or AMPK-α2 genes in catecholaminergic cells, which compose the hypoxia-responsive respiratory network from carotid body to brainstem. MEASUREMENTS AND MAIN RESULTS: AMPK-α1 and AMPK-α2 deletion virtually abolished the hypoxic ventilatory response, and ventilatory depression during hypoxia was exacerbated under anesthesia. Rather than hyperventilating, mice lacking AMPK-α1 and AMPK-α2 exhibited hypoventilation and apnea during hypoxia, with the primary precipitant being loss of AMPK-α1 expression. However, the carotid bodies of AMPK-knockout mice remained exquisitely sensitive to hypoxia, contrary to the view that the hypoxic ventilatory response is determined solely by increased carotid body afferent input to the brainstem. Regardless, functional magnetic resonance imaging and c-fos expression revealed reduced activation by hypoxia of well-defined dorsal and ventral brainstem nuclei. CONCLUSIONS: AMPK is required to coordinate the activation by hypoxia of brainstem respiratory networks, and deficiencies in AMPK expression precipitate hypoventilation and apnea, even when carotid body afferent input is normal.

Modulation of the LKB1-AMPK Signalling Pathway Underpins Hypoxic Pulmonary Vasoconstriction and Pulmonary Hypertension.

Perhaps the defining characteristic of pulmonary arteries is the process of hypoxic pulmonary vasoconstriction (HPV) which, under physiological conditions, supports ventilation-perfusion matching in the lung by diverting blood flow away from oxygen deprived areas of the lung to oxygen rich regions. However, when alveolar hypoxia is more widespread, either at altitude or with disease (e.g., cystic fibrosis), HPV may lead to hypoxic pulmonary hypertension. HPV is driven by the intrinsic response to hypoxia of pulmonary arterial smooth muscle and endothelial cells, which are acutely sensitive to relatively small changes in pO2 and have evolved to monitor oxygen supply and thus address ventilation-perfusion mismatch. There is now a consensus that the inhibition by hypoxia of mitochondrial oxidative phosphorylation represents a key step towards the induction of HPV, but the precise nature of the signalling pathway(s) engaged thereafter remains open to debate. We will consider the role of the AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1), an upstream kinase through which AMPK is intimately coupled to changes in oxygen supply via mitochondrial metabolism. A growing body of evidence, from our laboratory and others, suggests that modulation of the LKB1-AMPK signalling pathway underpins both hypoxic pulmonary vasoconstriction and the development of pulmonary hypertension.