HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 11, 8092-8098, March 16, 2007

This Article Abstract Full Text (PDF) All Versions of this Article: 282/11/8092    most recent M608742200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wyatt, C. N. Articles by Evans, A. M. Search for Related Content PubMed PubMed Citation Articles by Wyatt, C. N. Articles by Evans, A. M. Social Bookmarking                      What's this?

AMP-activated Protein Kinase Mediates Carotid Body Excitation by Hypoxia^*

Christopher N. Wyatt¹, Kirsty J. Mustard², Selina A. Pearson^¶3, Mark L Dallas^||4, Lucy Atkinson^||4, Prem Kumar^¶, Chris Peers^||4, D. Grahame Hardie², and A. Mark Evans¹⁵

From the Division of Biomedical Sciences, School of Biology, Bute Building, University of St. Andrews, St. Andrews, Fife. KY16 9TS, Division of Molecular Physiology, College of Life Sciences, Sir James Black Centre, University of Dundee, Dow Street, DD1 5EH, ^¶Department of Physiology, The Medical School, University of Birmingham, Birmingham B15 2TT, and the ^||School of Medicine, University of Leeds, Leeds LS2 9JT, United Kingdom

Received for publication, September 11, 2006 , and in revised form, December 14, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Early detection of an O₂ deficit in the bloodstream is essential to initiate corrective changes in the breathing pattern of mammals. Carotid bodies serve an essential role in this respect; their type I cells depolarize when O₂ levels fall, causing voltage-gated Ca²⁺ entry. Subsequent neurosecretion elicits increased afferent chemosensory fiber discharge to induce appropriate changes in respiratory function (1). Although depolarization of type I cells by hypoxia is known to arise from K⁺ channel inhibition, the identity of the signaling pathway has been contested, and the coupling mechanism is unknown (2). We tested the hypothesis that AMP-activated protein kinase (AMPK) is the effector of hypoxic chemotransduction. AMPK is co-localized at the plasma membrane of type I cells with O₂-sensitive K⁺ channels. In isolated type I cells, activation of AMPK using 5-aminoimidazole-4-carboxamide riboside (AICAR) inhibited O₂-sensitive K⁺ currents (carried by large conductance Ca²⁺-activated (BK_Ca) channels and TASK (tandem pore, acid-sensing potassium channel)-like channels, leading to plasma membrane depolarization, Ca²⁺ influx, and increased chemosensory fiber discharge. Conversely, the AMPK antagonist compound C reversed the effects of hypoxia and AICAR on type I cell and carotid body activation. These results suggest that AMPK activation is both sufficient and necessary for the effects of hypoxia. Furthermore, AMPK activation inhibited currents carried by recombinant BK_Ca channels, whereas purified AMPK phosphorylated the subunit of the channel in immunoprecipitates, an effect that was stimulated by AMP and inhibited by compound C. Our findings demonstrate a central role for AMPK in stimulus-response coupling by hypoxia and identify for the first time a link between metabolic stress and ion channel regulation in an O₂-sensing system.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Chronic and intermittent deficits in O₂ supply to the body precipitate a variety of pathologies including dementia (3) and pulmonary hypertension (4). To develop effective therapies, it is necessary to understand the homeostatic mechanisms that monitor O₂ supply to the body and elicit corrective changes in respiratory and circulatory function to maintain O₂ levels. O₂-sensitive ion channels, which were first identified in the carotid body type I cell, play a pivotal role in this respect and have now been reported in a diverse range of highly specialized O₂-sensing tissues (5). Within the carotid body, clusters of type I cells lie in presynaptic contact with afferent sensory fibers, whose discharge increases in proportion to the degree of systemic arterial O₂ deficit, providing information concerning blood O₂ levels to the central respiratory centers (1, 2). This occurs subsequent to hypoxic inhibition of type I cell K⁺ channels, membrane depolarization, voltage-gated Ca²⁺ influx (6), and consequent neurotransmitter release. For many years, there has existed compelling evidence that mitochondria serve an important role in O₂ sensing by type I cells (2, 7). Indeed, mitochondrial inhibitors can both mimic the cellular effects of hypoxia and occlude type I cell O₂ sensitivity (8, 9). These effects, and the downstream cellular responses, are manifest at relatively high tissue O₂ levels, which would not be limiting in other, non-specialized cells (10). Thus, type I cell metabolism may be the key to the mechanism by which hypoxia is coupled to neurosecretion. We therefore investigated the potential role in this process of AMPK,⁶ a key sensor of metabolic stress that is universally expressed in eukaryotes. When cells are exposed to metabolic stresses, a rise in the cellular ADP/ATP ratio is partly compensated by the adenylate kinase reaction, which precipitates a much larger increase in the AMP/ATP ratio, although the fall in ATP may be negligible (11, 12). AMPK is activated via multiple mechanisms that make the system acutely sensitive to relatively small changes in the AMP/ATP ratio (13). Although it was originally proposed to regulate energy balance within single cells (14), it has recently become clear that AMPK also performs this function at the level of the whole animal, through its regulation of hypothalamic neurones that drive food intake and by its ability to stimulate energy expenditure in peripheral tissues (15). The hypothesis that AMPK also underpins the response of other physiological systems to stimuli that affect metabolism, e.g. excitation of the carotid body by hypoxia (16), has therefore become attractive. This is particularly so given that our previous study established that physiologically relevant levels of hypoxia increased the AMP/ATP ratio in O₂-sensing cells, leading to concomitant activation of AMPK activity that was primarily associated with heterotrimers containing the AMPK1 catalytic subunit isoform (16). Furthermore, this study suggested that activation of AMPK by 5-aminoimidazole-4-carboxamide riboside (AICAR) induces transmembrane calcium influx into carotid body type I cells and thereby increases afferent fiber discharge from the isolated carotid body (16). However, we have yet to determine whether this is achieved by the same mechanisms that are engaged by hypoxia. Here we demonstrate that AMPK elicits carotid body activation by mimicking precisely the effects of hypoxia on carotid body type I cells and that inhibition of AMPK reverses these effects.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
All experiments were performed under the United Kingdom Animals (Scientific Procedures) Act 1986.

Carotid Body Type I Cell Isolation—Neonatal rats (10–21 days) were killed, and carotid bodies were rapidly removed and placed in ice-cold, oxygenated phosphate-buffered saline without Ca²⁺ or Mg²⁺ (Sigma). These were then enzymically dissociated and cultured as described previously (9).

HEK 293 Cell Culture—HEK 293 cells that express human brain -BK_Ca channels (17) were cultured as described previously (18). The co-expressed and subunits were KCNMA1 (GenBank^TM accession number U11717 [GenBank] ) and KCNMB1 (GenBank accession number U42600 [GenBank] ), respectively.

Immunocytochemistry—Type I cells were fixed, immunostained, and visualized as described previously (16). The primary antibodies were selective for the AMPK1 subunit (1:500, sheep polyclonal, D. G. Hardie, Dundee, Scotland, raised against peptide residues 344–358 of rat AMPK1) and the BK_Ca subunit (1:100, mouse monoclonal, BD Transduction Laboratories, raised against peptide residues 995–1113 of human brain BK_Ca). Images were deconvolved and analyzed off-line via Softworx (Applied Precision) and Volocity (Improvision).

Carotid Body Electrophysiology—Recordings were made using the amphotericin perforated-patch configuration of the whole-cell patch clamp technique. Data were acquired using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) controlled by Clampex 6.0 or Fetchex 6.0 software (Axon Instruments). Currents were digitized at 6.6 kHz and filtered at 1–2 kHz, and membrane potential recordings were digitized at 0.1 kHz and filtered at 1 kHz. Experiments were performed at 35–36 °C. Cells were bathed in solution A of composition (in mM): 4.5 KCl; 140 NaCl; 2.5 CaCl₂; 1 MgCl₂; 11 glucose; 20 HEPES, adjusted to pH 7.4. Pipette solution was (in mM): 55 K₂SO₄; 30 KCl; 5 MgCl₂; 1 EGTA; 10 glucose; 20 HEPES, adjusted to pH 7.2. Pipette solution contained amphotericin B (240 µg ml^-1, Sigma). Liquid junction potential was 6 mV. Results were not leak-subtracted, seal resistance was typically >5 gigaohms, access resistance was typically 15–20 megaohms and was not compensated for, and holding currents were typically less than 5 pA. All data were analyzed using Clampfit and Fetchan programs (pClamp6, Axon Instruments).

HEK 293 Cell Electrophysiology—Recordings were made using the conventional whole-cell configuration of the patch clamp technique. Data were acquired using a Multiclamp 700A amplifier (Axon Instruments) controlled by Clampex 9.0 software (Axon Instruments). Data were digitized at 5 kHz and filtered at 1 kHz. All experiments were performed at 22 °C. Cells were bathed in a solution of composition (in mM): 5 KCl; 150 NaCl; 2 CaCl₂; 2 MgCl₂; 10 glucose; 10 HEPES, adjusted to pH 7.4. Pipette solution was (in mM): 140 KSCN; 5 EGTA; 1 MgCl₂; 0.5 CaCl₂; 10 HEPES; 3 MgATP; 0.3 NaGTP, adjusted to pH 7.25. Liquid junction potential was 6.5 mV. Results were leak-subtracted, seal resistance was typically >7 gigaohms, access resistance was typically 8–12 megaohms and series resistance was compensated by 60–80%, and holding currents were 20–50 pA. All data were analyzed using Clampfit 9 (Axon Instruments).

Ca²⁺ Imaging—Cells were incubated for 30 min (22–24 °C, dark) in a Ca²⁺-free solution (in mM): 4.5 KCl; 140 NaCl; 1 MgCl₂; 11 glucose; 1 EGTA; 20 HEPES, adjusted to pH 7.4, containing 5 µM FURA-2AM (Molecular Probes), and then allowed to equilibrate for 20 min in solution A (35–36 °C). Hypoxic solutions were generated by gassing solution A with 100% N₂, pO₂ was monitored using an ISO2 oxygen meter (World Precision Instruments, Stevenage, UK). Changes in [Ca²⁺]_i were monitored using alternate excitation wavelengths of 340 and 380 nm (F₃₄₀ and F₃₈₀, 20-ms exposure). Emitted fluorescence was recorded as described previously (16) at 0.05 Hz with background subtraction carried out online.

Isolated Carotid Body—Rats were anesthetized with 1–4% halothane in O₂, and carotid bodies with attached carotid sinus nerve were excised as described previously (19). Extracellular recordings of single or few-fiber afferent fiber action potentials were recorded on videotape and sampled digitally via Spike2 software (Cambridge Electronic Design, Cambridge, UK) for analysis of discharge frequency.

Co-immunoprecipitation—Cells were harvested in phosphate-buffered saline (PBS) and centrifuged at 1000 x g, after which the pellet was homogenized in 1 ml of ice-cold buffer A (50 mM Tris-HCl (pH 7.4), 140 mM KCl, 1 mM EGTA, 1 mM MgCl₂, 1 mM phenylmethanesulfonyl fluoride, and 50 µg/ml aprotinin). This was followed by centrifugation (1000 x g,5 min, 4 °C). The pellet was then solubilized in 0.5 ml of ice-cold buffer B (5 mM Tris-HCl (pH 7.4), 500 mM NaCl, 1.5% v/v Triton X-100, with 1 mM phenylmethanesulfonyl fluoride and 50 µg/ml aprotinin). Antibodies (either anti-BK_Ca subunits (5 µg) or anti-AMPK1 subunits (6 µg), as used for immunocytochemistry) were added to solubilized suspensions and incubated overnight at 4 °C before the addition of 50 µl of protein G beads. Following further incubation (>4 h, 4 °C, with rotation), the beads were pelleted at 7000 x g (5 min) and washed three times in 1 ml of ice-cold buffer C (5 mM Tris-HCl (pH 7.4), 20 mM NaCl, 0.5% (v/v) Triton X-100, with 1 mM phenylmethanesulfonyl fluoride and 50 µg/ml aprotinin).

Pelleted immunoprecipitates were taken up into 50 µl of sample buffer (62.5 mM Tris-HCl (pH 6.8), 10% v/v glycerol, 0.2% w/v SDS 5% v/v -mercaptoethanol, 0.02% bromphenol blue), heated for 3 min at 100 °C, and loaded onto 10% (w/v) SDS-polyacrylamide gels and separated at 100 V for 1 h. Proteins were transferred to polyvinylidene difluoride membrane at 250 mA for 1 h, and membranes were blocked overnight with 10% (w/v) nonfat powdered milk in PBS at 4 °C. After washing membranes three times in PBS/0.05% Tween 20, they were incubated with anti-AMPK1 antibody (1:500) or anti-BK_Ca antibody (1:250) in PBS/0.5% nonfat powdered milk for 1 h at 21 °C. Membranes were then washed extensively with PBS before incubation with anti-mouse or anti-sheep peroxidase-linked secondary antibody (1:10,000 in PBS, Pierce) for 1 h. Proteins were visualized using enhanced chemiluminescence (Amersham Biosciences, Amersham, UK). Molecular masses were estimated using calibrated prestained standards (Bio-Rad, Hemel Hempstead, UK).

Purification of BK_Ca Subunit from HEK293 Cells—BK_Ca was immunoprecipitated from HEK-293 cells stably expressing the channel (20). Culture dishes (4 x 15 cm) of cells were grown to 90% confluency. Cells were washed once in 10 ml of ice-cold PBS and then lysed in situ by the addition of 2 ml of lysis buffer (50 mM Tris-HCl, pH 7.4 at 4 °C, 150 mM NaCl, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM benzamidine, 0.1 mM phenylmethanesulfonyl fluoride, 5 µg/ml soybean trypsin inhibitor, 2% Triton X-100, 0.25 M mannitol). Lysate was then precleared by incubating on a roller mixer with 2 ml of protein G-Sepharose (Amersham Biosciences, 50% slurry in lysis buffer) for 2 h at 4 °C followed by centrifugation (230 x g, 5 min). BK_Ca was subsequently immunoprecipitated from the supernatant using 20 µg of anti-BK_Ca antibody (BD Transduction Laboratories) prebound to 40 µl of protein G-Sepharose. After 2 h at 4 °C, the precipitate was collected by centrifugation (230 x g, 5 min). The immunoprecipitate was washed five times in lysis buffer containing 1 M NaCl followed by five times washing in lysis buffer without NaCl.

Phosphorylation of BK_Ca Subunit Using Purified Rat Liver AMPK—Immunopurified BK_Ca was washed six times in assay buffer (50 mM sodium-Hepes, pH 7.2, 1 mM dithiothreitol, 0.02% (w/v) Brij-35) containing 1 M NaCl followed by eight washes in assay buffer without NaCl. The immune complexes were incubated for 30 min at 30 °C with 4.5 µg/ml recombinant PP1 in assay buffer. They were washed five times in assay buffer containing 1 M NaCl followed by five further washes in assay buffer. They were then incubated for 30 min at 30 °C with 5 mM MgCl₂ and 200 µM [-³²P]ATP (300 cpm/pmol) in 50 mM sodium-Hepes, pH 7.2, 1 mM dithiothreitol, 0.02% (w/v) Brij-35, with or without purified rat liver AMPK (Hawley et al. (34)) and with or without AMP (200 µM) or compound C (100 µM). The reactions were stopped by the addition of lithium dodecyl sulphate loading dye and reducing agent (Invitrogen). The samples were analyzed by SDS-PAGE using the 3–8% gradient Tris acetate system (Invitrogen) and gels stained with Colloidal Blue staining kits (Invitrogen). Destained gels were dried and subjected to autoradiography. The BK_Ca band was excised, and the incorporation of [³²P]phosphate was determined by Cerenkov counting. The amount of BK_Ca subunit was estimated by running known amounts of bovine serum albumin on the same gel, comparing intensities of Coomassie Blue-stained bands by densitometry and assuming that the same amount of dye bound per µg of protein.

Identification of the BK_Ca Subunit by Mass Spectrometry—A gel slice was excised and washed successively for 15 min in water, CH₃CN + 100 mM NH₄HCO₃ (50:50 v/v), and finally in 20 mM NH₄HCO₃/CH₃CN (50:50 v/v). The gel slice was dehydrated in water + CH₃CN (50:50 v/v) for 5 min. The supernatant was dried in a SpeedVac concentrator and resuspended in 20 µl of 12.5 µg/ml trypsin (Roche Applied Science, modified sequencing grade) in 20 mM NH₄HCO₃/0.1% n-octylglucoside. Digestion occurred overnight at 30 °C on a shaking platform. An equal volume of CH₃CN was then added to the digest and incubated for 30 min at 30 °C on a shaking platform. An aliquot (10%) of the peptide digest was then analyzed by tandem matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (Applied Biosystems 4700 Proteomics Analyzer). The mass spectrometry and tandem mass spectrometry data generated were used to search the Celera Discovery System human data base (Applied Biosystems) using the Mascot search algorithm (35). This confirmed the identity of the 125-kDa band as the BK_Ca subunit (SwissProt accession number KCNMA1).

Statistics—All data are presented as means ± S.E. of the mean. Statistical significance was assessed using two-tailed Bonferroni-Dunn post hoc test (in vitro carotid body) or Student's paired or unpaired t test (all other data) as appropriate. p < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Consistent with the hypothesis that AMPK serves a key role in O₂ sensing by the carotid body, we found that in isolated carotid body type I cells, the AMPK1 catalytic subunit isoform and O₂-sensitive BK_Ca channels co-localize at the plasma membrane (Fig. 1A, panels i–iv). Thus, 75.1 ± 4.4% of the total AMPK1 staining was located within 1 µm of the type I cell plasma membrane (n = 14), where it co-localized with O₂-sensitive BK_Ca channels (21), with 71.0 ± 2.6% of staining for the pore-forming BK_Ca subunit coinciding with that for the AMPK1 catalytic subunit isoform (Pearson's correlation 0.65 ± 0.05, n = 5; Fig. 1, A, panels iii and iv). Furthermore, BK_Ca channel currents were selectively inhibited in acutely isolated type I cells by 5-aminoimidazole-4-carboxamide riboside (AICAR), a nucleoside that is taken up by cells and metabolized to the AMP mimetic, ZMP, thus activating AMPK in the absence of ATP depletion (22). Analysis of current-voltage relationships for macroscopic K⁺ currents showed that AICAR (1 mM, 10 min) reversibly inhibited voltage-sensitive K⁺ currents throughout their activation range, e.g. by 40.0 ± 12.2% during a voltage step from -80 to +30 mV (Fig. 1B; n = 5, p = 0.041). Selective blockade of BK_Ca channels by iberiotoxin (100 nM) inhibited type I cell macroscopic K⁺ currents by 60.3 ± 6.8% at +30 mV (n = 7, p = 0.0069) and, in the presence of the toxin, AICAR had no further inhibitory effect on residual voltage-gated K⁺ currents (Fig. 1C; n = 3). Rat carotid body type I cells also express O₂-sensitive, voltage-independent leak K⁺ channels (23) in addition to BK_Ca channels, although the molecular identity of these TASK-like channels is not known. AICAR also inhibited this Ba²⁺-sensitive, voltage-independent leak conductance (measured during voltage ramps from -100 mV to -40 mV) by 33.0 ± 2.3%, from 258 ± 24 pS to 171 ± 11 pS (Fig. 1D; n = 4, p = 0.008). Furthermore, in the continued presence of Ba²⁺, AICAR did not significantly inhibit the residual currents (Fig. 1E; n = 5). These data indicate that AMPK activation, like hypoxia (21, 23), selectively inhibits both the O₂-sensing BK_Ca currents and TASK-like leak K⁺ currents in rat carotid body type I cells, without affecting O₂-insensitive delayed rectifier K⁺ currents.

View larger version (40K): [in this window] [in a new window]   FIGURE 1. AMPK1 and BKCa subunits co-localize and activation of AMPK inhibits O2-sensitive K+ currents in type I cells. A, panel i, bright field image of a type I cell. panel ii, Z-section with immunostaining for AMPK1 and nuclear staining by 4',6-diamidino-2-phenylindole (DAPI). panel iii, Z-section with immunostaining for BKCa subunit and DAPI. panel iv, three-dimensional reconstruction showing AMPK1 and BKCa co-localization. B, AICAR inhibits macroscopic current. Data are mean ± S.E. The insets are example currents ± AICAR. C, AICAR inhibits the iberiotoxin (IBTX)-sensitive current. Data are mean ± S.E. The insets are example currents, control, +IBTX, and +IBTX and AICAR. D, Ba2+ and AICAR inhibit leak K+ currents. E, AICAR does not inhibit residual currents in the continued presence of Ba2+.

Recordings from type I cells are not necessarily a reliable indicator of a functional response at the level of the intact carotid body (26, 27). We therefore investigated the role of AMPK in mediating afferent chemosensory fiber discharge from the carotid body in vitro. Consistent with our findings on isolated carotid body type I cells, 1 mM AICAR caused a 7-fold increase in spontaneous, single afferent fiber discharge frequency (Fig. 3A). AICAR at 100 or 300 µM was without effect upon basal discharge. The excitatory effect of AICAR at 1 mM was significantly inhibited by around 50% after preincubation with compound C (40 µM) for 10–20 min (Fig. 3A). After 30–40 min of preincubation with compound C, basal discharge was reduced to essentially zero, and the response to AICAR was abolished. Basal carotid body single or few fiber chemoafferent discharge varied slightly between preparations, reflecting individual fiber excitability, but in all cases, discharge increased in response to hypoxia (Fig. 3B). Hypoxia-augmented afferent chemosensory fiber discharge frequency was also inhibited by preincubation with compound C. At 10–20 min, the degree of inhibition was around 50% (Fig. 3C). Despite a reduction in basal discharge following sustained incubation with compound C, a hypoxic response was retained, albeit reduced by about 90% at 70 min. That this was not simply due to "run down" of the preparation was confirmed by control studies, which showed that the hypoxia response was largely retained for up to 120 min following repeated stimulation trials. The mean data are shown in Fig. 3D. Thus, AMPK activation mediates a large component of carotid body type I cell activation by hypoxia, and this is sufficient to trigger an increase in afferent chemosensory fiber discharge that can be substantially blocked by compound C.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Activation of AMPK mimics hypoxia in type I cells and its inhibition reverses the effect of hypoxia. A, depolarization induced by AICAR (1 mM). B, Ni2+ (5 mM) attenuates the AICAR (1 mM)-induced rise in the Fura-2 F340/F380 ratio. C, AMPK antagonist compound C (40 µM) attenuates the hypoxia (14–21 mmHg)-induced rise in the Fura-2 F340/F380 ratio. D, compound C (40 µM) attenuates the AICAR-induced rise in Fura-2 F340/F380 ratio. E, K+ (50 mM) causes a robust rise in Fura-2 F340/F380 ratio in the presence of compound C (40 µM).

View larger version (34K): [in this window] [in a new window]   FIGURE 3. AMPK inhibition attenuates hypoxia- and AICAR-induced increases in carotid sinus nerve discharge. A, AICAR (1 mM) reversibly increases few-fiber carotid sinus nerve afferent discharge, a stimulatory effect that was attenuated by preincubation with compound C (40 µM, after 10 min of preincubation). The top trace shows a discharge frequency histogram (1-s intervals), and the lower trace shows extracellular action potentials. B, hypoxia increases chemoafferent discharge. Traces show (from top): discharge frequency histogram in 1-s intervals, superfusate pO2 at the carotid body surface; and extracellular action potentials. Note that pO2 levels recorded in the superfusate at the surface of the carotid body are related to but do not represent the precise tissue level of pO2 to which the afferent nerve is responding. This would vary with depth from the surface and can be calculated as -1 mmHg/µm. Carotid bodies are maintained at a superfusate pO2 of 400 mmHg to ensure adequate oxygenation to the core between hypoxic episodes, and maximal responses are normally observed as superfusate pO2 falls below 150 mmHg. C, compound C (40 µM; after 20 min of preincubation) reduces the effect of hypoxia on few fiber-afferent discharge (traces as for B). D, panel i, mean data ± S.E. for the experiment in A. Panel ii, mean data ± S.E. for experiments in B and C. * p < 0.0001 versus control, p < 0.002 versus AICAR alone or hypoxia alone. Com C, compound C.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (29K): [in this window] [in a new window]   FIGURE 4. AMPK activation inhibits BKCa channels expressed in HEK 293 cells by direct phosphorylation. A, AICAR (1 mM) inhibits BKCa currents; this is attenuated by compound C (comp C). Data are mean ± S.E. B, co-immunoprecipitation with Western blot detection; no association of AMPK1 with BKCa was detected. Upper blots show results using anti-BKCa antibodies, revealing AMPK1 in the supernatant (S) rather than the immunoprecipitate (IP), in extracts from cells treated with or without AICAR (1 mM). Lower blots show results using anti-AMPK1 antibodies, revealing BKCa only in the supernatant. C, Coomassie Blue-stained gel and autoradiogram demonstrating phosphorylation of BKCa subunits in the presence of AMP (200 µM) with or without AMPK (5 units/ml). D, as C, except the AMPK was reduced to 1 units/ml and the reaction was performed in the presence and absence of AMP (200 µM) or compound C (100 µM).

Thus, our results suggest that activation of AMPK is necessary as well as sufficient for carotid body activation by hypoxia. We also provide a plausible mechanism for the inhibitory effects on the BK_Ca channels by showing that AMPK directly phosphorylates the pore-forming subunit in immunoprecipitates. As expected for an effect of AMPK, phosphorylation was stimulated by AMP and inhibited by compound C, and the stoichiometry approached 1 mole of phosphate/mole of subunit, showing that the extent of phosphorylation was significant. We propose, therefore, that the AMPK system acts as the primary metabolic sensor as well as the effector of Ca²⁺ signaling in response to hypoxia in carotid body type I cells. This mechanism can unite the previous mitochondrial and membrane hypotheses for chemotransduction by hypoxia (2).

Recently, O₂ sensing by BK_Ca channels has been proposed to arise via O₂-dependent CO production from the closely associated enzyme, heme oxygenase-2 (HO-2 (32)). Clearly, when this enzyme is active (i.e. in the presence of its substrates, including O₂), channel activity is markedly enhanced due to CO production by HO-2. However, the requirement of this mechanism of channel regulation for O₂ sensing in the intact carotid body has not been evaluated. Furthermore, the importance of HO-2 in O₂ sensing has more recently been questioned since BK_Ca channels have been shown to be suppressed by hypoxia in excised membrane patches (33). Collectively, these data suggest that there exist multiple means by which hypoxia might inhibit BK_Ca channels. However, unlike our proposed mechanism, other mechanisms cannot account for findings that inhibitors of mitochondrial oxidative phosphorylation prevent carotid body activation by hypoxia (8, 9). Furthermore, no other mechanism has been so comprehensively tested with respect to its involvement in O₂ sensing at the level of ion channel activity, membrane potential, [Ca²⁺]_i regulation, and afferent nerve discharge. On the basis of our present data, therefore, we propose that AMPK is the physiologically relevant mediator of O₂ sensing by the carotid body. Furthermore, we propose that the targeting of AMPK1 complexes to the plasma membrane of carotid body type 1 cells, the acute sensitivity of AMPK to changes in the AMP/ATP ratio, and a reliance of these cells on mitochondrial oxidative phosphorylation for ATP production underpin the activation of type I cells by relatively small, physiological changes in arterial pO₂. Thus, in addition to its ubiquitous role in regulating metabolism in response to metabolic stress at the cellular level, the AMPK signaling pathway has become adapted to mediate the responses of physiological systems that are key to meeting the metabolic needs of the whole body, including hypoxia-response coupling in O₂-sensing cells.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a Wellcome Trust Project Grant.

² Supported by a Wellcome Trust Programme Grant.

³ Supported by a University of Birmingham, Division of Medical Sciences Ph. D. studentship.

⁴ Supported by the British Heart Foundation, the Alzheimer's Research Trust and the Alzheimer's Society.

⁵ To whom correspondence should be addressed. Tel.: 44-1334-463579; Fax: 44-1334-463600; E-mail: ame3{at}st-andrews.ac.uk.

⁶ The abbreviations used are: AMPK, adenosine monophosphate-activated protein kinase; TASK, tandem pore, acid-sensing potassium channel; BK_Ca, large conductance voltage and Ca²⁺-activated potassium channel; AICAR, 5-aminoimidazole-4-carboxamide riboside; pO₂, partial pressure of oxygen; PBS, phosphate-buffered saline.

⁷ S. A. Pearson and P. Kumar, unpublished data.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Gonzalez, C., Almaraz, L., Obeso, A., and Rigual, R. (1994) Physiol. Rev. 74, 829-898[Free Full Text]
2. Prabhakar, N. R. (2006) Exp. Physiol. 91, 17-23[Abstract/Free Full Text]
3. Desmond, D. W., Moroney, J. T., Sano, M., and Stern, Y. (2002) Stroke 33, 2254-2260[Abstract/Free Full Text]
4. Voelkel, N. F. (1986) Am. Rev. Respir. Dis. 133, 1186-1195[Medline] [Order article via Infotrieve]
5. Lopez-Barneo, J., del Toro, R., Levitsky, K. L., Chiara, M. D., and Ortega-Saenz, P. (2004) J. Appl. Physiol. 96, 1187-1195; discussion 1170-1182[Abstract/Free Full Text]
6. Weir, E. K., Lopez-Barneo, J., Buckler, K. J., and Archer, S. L. (2005) N. Engl. J. Med. 353, 2042-2055[Free Full Text]
7. Mills, E., and Jobsis, F. F. (1972) J. Neurophysiol. 35, 405-428[Free Full Text]
8. Mulligan, E., Lahiri, S., and Storey, B. T. (1981) J. Appl. Physiol. 51, 438-446[Abstract/Free Full Text]
9. Wyatt, C. N., and Buckler, K. J. (2004) J. Physiol. (Lond.) 556, 175-191[Abstract/Free Full Text]
10. Duchen, M. R., and Biscoe, T. J. (1992) J. Physiol. (Lond.) 450, 13-31[Abstract/Free Full Text]
11. Wyatt, C. N., Kumar, P., Aley, P., Peers, C., Hardie, D. G., and Evans, A. M. (2006) Adv. Exp. Med. Biol. 580, 191-196; discussion 351-199[Medline] [Order article via Infotrieve]
12. Hardie, D. G. (2004) J. Cell Sci. 117, 5479-5487[Abstract/Free Full Text]
13. Hardie, D. G., Salt, I. P., Hawley, S. A., and Davies, S. P. (1999) Biochem. J. 338, 717-722
14. Hardie, D. G., and Carling, D. (1997) Eur. J. Biochem. 246, 259-273[Medline] [Order article via Infotrieve]
15. Kahn, B. B., Alquier, T., Carling, D., and Hardie, D. G. (2005) Cell Metab. 1, 15-25[CrossRef][Medline] [Order article via Infotrieve]
16. Evans, A. M., Mustard, K. J., Wyatt, C. N., Peers, C., Dipp, M., Kumar, P., Kinnear, N. P., and Hardie, D. G. (2005) J. Biol. Chem. 280, 41504-41511[Abstract/Free Full Text]
17. Ahring, P. K., Strobaek, D., Christophersen, P., Olesen, S. P., and Johansen, T. E. (1997) FEBS Lett. 415, 67-70[CrossRef][Medline] [Order article via Infotrieve]
18. Lewis, A., Peers, C., Ashford, M. L., and Kemp, P. J. (2002) J. Physiol. (Lond.) 540, 771-780[Abstract/Free Full Text]
19. Pepper, D. R., Landauer, R. C., and Kumar, P. (1995) J. Physiol. (Lond.) 485, 531-541[Abstract/Free Full Text]
20. Hartness, M. E., Brazier, S. P., Peers, C., Bateson, A. N., Ashford, M. L., and Kemp, P. J. (2003) J. Biol. Chem. 278, 51422-51432[Abstract/Free Full Text]
21. Peers, C. (1990) Neurosci. Lett 119, 253-256[CrossRef][Medline] [Order article via Infotrieve]
22. Corton, J. M., Gillespie, J. G., Hawley, S. A., and Hardie, D. G. (1995) Eur. J. Biochem. 229, 558-565[Medline] [Order article via Infotrieve]
23. Buckler, K. J. (1997) J. Physiol. (Lond.) 498, 649-662[Abstract/Free Full Text]
24. Wyatt, C. N., and Peers, C. (1995) J. Physiol. (Lond.) 483, 559-565[Abstract/Free Full Text]
25. Zhou, G., Myers, R., Li, Y., Chen, Y., Shen, X., Fenyk-Melody, J., Wu, M., Ventre, J., Doebber, T., Fujii, N., Musi, N., Hirshman, M. F., Goodyear, L. J., and Moller, D. E. (2001) J. Clin. Investig. 108, 1167-1174[CrossRef][Medline] [Order article via Infotrieve]
26. Donnelly, D. F. (1999) Respir. Physiol. 115, 151-160[CrossRef][Medline] [Order article via Infotrieve]
27. Donnelly, D. F. (1996) J. Appl. Physiol. 81, 657-664[Abstract/Free Full Text]
28. Gadalla, A. E., Pearson, T., Currie, A. J., Dale, N., Hawley, S. A., Sheehan, M., Hirst, W., Michel, A. D., Randall, A., Hardie, D. G., and Frenguelli, B. G. (2004) J. Neurochem. 88, 1272-1282[CrossRef][Medline] [Order article via Infotrieve]
29. McQueen, D. S., and Ribeiro, J. A. (1981) Br. J. Pharmacol. 74, 129-136[Medline] [Order article via Infotrieve]
30. Vandier, C., Conway, A. F., Landauer, R. C., and Kumar, P. (1999) J. Physiol. (Lond.) 515, 419-429[Abstract/Free Full Text]
31. Conde, S. V., and Monteiro, E. C. (2006) Adv. Exp. Med. Biol. 580, 179-184; discussion 351-179[Medline] [Order article via Infotrieve]
32. Williams, S. E., Wootton, P., Mason, H. S., Bould, J., Iles, D. E., Riccardi, D., Peers, C., and Kemp, P. J. (2004) Science 306, 2093-2097[Abstract/Free Full Text]
33. McCartney, C. E., McClafferty, H., Huibant, J. M., Rowan, E. G., Shipston, M. J., and Rowe, I. C. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 17870-17876[Abstract/Free Full Text]
34. Hawley, S. A., Davison, M., Woods, A., Davies, S. P., Beri, R. K., Carling, D., and Hardie, D. G. (1996) J. Biol. Chem. 271, 27879-27887[Abstract/Free Full Text]
35. Perkins, D. N., Pappin, D. J., Creasy, D. M., and Cottrell, J. S. (1999) Electrophoresis 20, 3551-3567[CrossRef][Medline] [Order article via Infotrieve]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				H. Klein, L. Garneau, N. T. N. Trinh, A. Prive, F. Dionne, E. Goupil, D. Thuringer, L. Parent, E. Brochiero, and R. Sauve Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells Am J Physiol Cell Physiol, February 1, 2009; 296(2): C285 - C295. [Abstract] [Full Text] [PDF]

				D. D'Agostino, E. Mazza Jr., and J. A. Neubauer Heme oxygenase is necessary for the excitatory response of cultured neonatal rat rostral ventrolateral medulla neurons to hypoxia Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2009; 296(1): R102 - R118. [Abstract] [Full Text] [PDF]

				K. R. Olson Hydrogen sulfide and oxygen sensing: implications in cardiorespiratory control J. Exp. Biol., September 1, 2008; 211(17): 2727 - 2734. [Abstract] [Full Text] [PDF]

				K. R. Olson, M. J. Healy, Z. Qin, N. Skovgaard, B. Vulesevic, D. W. Duff, N. L. Whitfield, G. Yang, R. Wang, and S. F. Perry Hydrogen sulfide as an oxygen sensor in trout gill chemoreceptors Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2008; 295(2): R669 - R680. [Abstract] [Full Text] [PDF]

				D. Heitzmann and R. Warth Physiology and Pathophysiology of Potassium Channels in Gastrointestinal Epithelia Physiol Rev, July 1, 2008; 88(3): 1119 - 1182. [Abstract] [Full Text] [PDF]

				D. Zheng, A. Perianayagam, D. H. Lee, M. D. Brannan, L. E. Yang, D. Tellalian, P. Chen, K. Lemieux, A. Marette, J. H. Youn, et al. AMPK activation with AICAR provokes an acute fall in plasma [K+] Am J Physiol Cell Physiol, January 1, 2008; 294(1): C126 - C135. [Abstract] [Full Text] [PDF]

				R. Varas, C. N. Wyatt, and K. J. Buckler Modulation of TASK-like background potassium channels in rat arterial chemoreceptor cells by intracellular ATP and other nucleotides J. Physiol., September 1, 2007; 583(2): 521 - 536. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 282/11/8092    most recent M608742200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wyatt, C. N. Articles by Evans, A. M. Search for Related Content PubMed PubMed Citation Articles by Wyatt, C. N. Articles by Evans, A. M. Social Bookmarking                      What's this?