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Does AMP-activated Protein Kinase Couple Inhibition of Mitochondrial Oxidative Phosphorylation by Hypoxia to Calcium Signaling in O2-sensing Cells? *

Does AMP-activated Protein Kinase Couple Inhibition of Mitochondrial Oxidative Phosphorylation by... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 50, pp. 41504 –41511, December 16, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Does AMP-activated Protein Kinase Couple Inhibition of Mitochondrial Oxidative Phosphorylation by Hypoxia to Calcium Signaling in O -sensing Cells? Received for publication, September 13, 2005, and in revised form, September 29, 2005 Published, JBC Papers in Press, September 30, 2005, DOI 10.1074/jbc.M510040200 ‡1 § ‡ ¶ ‡ A. Mark Evans , Kirsteen J. W. Mustard , Christopher N. Wyatt , Chris Peers , Michelle Dipp , Prem Kumar , ‡ § Nicholas P. Kinnear , and D. Grahame Hardie From the Division of Biomedical Sciences, School of Biology, Bute Building, University of St. Andrews, St. Andrews, Fife KY16 9TS, United Kingdom, Division of Molecular Physiology, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dow Street, DD1 5EH, United Kingdom, Institute for Cardiovascular Research, University of Leeds, Leeds LS2 9JT, United Kingdom, and Department of Physiology, The Medical School, University of Birmingham, Birmingham B15 2TT, United Kingdom Specialized O -sensing cells exhibit a particularly low threshold Specialized O -sensing cells within the body have evolved as vital 2 2 to regulation by O supply and function to maintain arterial pO homeostatic mechanisms that monitor O supply and alter respiratory 2 2 2 within physiological limits. For example, hypoxic pulmonary vaso- and circulatory function, as well as the capacity of the blood to transport constriction optimizes ventilation-perfusion matching in the lung, O . By these means, arterial pO is maintained within physiological 2 2 whereas carotid body excitation elicits corrective cardio-respira- limits. Two key systems involved are the pulmonary arteries and the tory reflexes. It is generally accepted that relatively mild hypoxia carotid body. Constriction of pulmonary arteries by hypoxia optimizes inhibits mitochondrial oxidative phosphorylation in O -sensing ventilation-perfusion matching in the lung (1), whereas carotid body cells, thereby mediating, in part, cell activation. However, the mech- excitation by hypoxia initiates corrective changes in breathing patterns anism by which this process couples to Ca signaling mechanisms via increased sensory afferent discharge to the brain stem (2). Although remains elusive, and investigation of previous hypotheses has gen- O -sensitive mechanisms independent of mitochondria may also play a erated contrary data and failed to unite the field. We propose that a role (3–5), it is generally accepted that relatively mild hypoxia inhibits rise in the cellular AMP/ATP ratio activates AMP-activated protein mitochondrial oxidative phosphorylation and that this underpins, at kinase and thereby evokes Ca signals in O -sensing cells. Co-im- least in part, cell activation (2, 6–10). Despite this consensus, the mech- munoprecipitation identified three possible AMP-activated protein anism by which inhibition of mitochondrial oxidative phosphorylation couples to discrete cell-specific Ca signaling mechanisms has kinase subunit isoform combinations in pulmonary arterial myo- cytes, with 121 predominant. Furthermore, their tissue-spe- remained elusive. Recently, the field has focused on the possible role of cific distribution suggested that the AMP-activated protein the cellular energy status (ATP) (7, 11), reduced redox couples (12), and kinase-1 catalytic isoform may contribute, via amplification of the reactive oxygen species (13–17), respectively, but extensive investiga- tion of these hypotheses has delivered conflicting data and failed to metabolic signal, to the pulmonary selectivity required for hypoxic pulmonary vasoconstriction. Immunocytochemistry showed AMP- unite the field since its inception in 1930 (18, 19). However, Sylvester activated protein kinase-1 to be located throughout the cytoplasm and colleagues (11) have suggested that previous assessment of the role of pulmonary arterial myocytes. In contrast, it was targeted to the of the energy state may have been limited by the lack of knowledge of the identity of the energy variable that might signal the response. plasma membrane in carotid body glomus cells. Consistent with these observations and the effects of hypoxia, stimulation of AMP- Recently, the AMP-activated protein kinase (AMPK) cascade has activated protein kinase by phenformin or 5-aminoimidazole-4- come to prominence as a sensor of metabolic stress that appears to be carboxamide-riboside elicited discrete Ca signaling mechanisms ubiquitous throughout eukaryotes (20, 21). AMPK is activated by many different metabolic stresses, including heat shock and metabolic poi- in each cell type, namely cyclic ADP-ribose-dependent Ca mobi- lization from the sarcoplasmic reticulum via ryanodine receptors in sons in hepatocytes (22), exercise in skeletal muscle (23), and ischemia pulmonary arterial myocytes and transmembrane Ca influx into and hypoxia in the heart (24). AMPK complexes are heterotrimers com- carotid body glomus cells. Thus, metabolic sensing by AMP-acti- prising a catalytic  subunit and regulatory  and  subunits (20), which monitor the cellular AMP/ATP ratio as an index of metabolic stress vated protein kinase may mediate chemotransduction by hypoxia. (20). Through the action of adenylate kinase, any increase in the cellular ADP/ATP ratio is converted into an increase in the AMP/ATP ratio (25). Binding of AMP to two sites in the  subunits triggers activation of the kinase via phosphorylation of the  subunit at Thr-172, an effect * This work was supported by The Wellcome Trust Grant 070772 and Biotechnology and Biological Sciences Research Council Grant 01/A/S/07453 (to A. M. E.) and by a con- antagonized by high concentrations of ATP (26, 27). This phosphoryl- tract for an Integrated Project from the European Commission (LSHM-CT-2004- ation is catalyzed by upstream kinases (AMPK kinases), the major form 005272) and the pharmaceutical companies supporting the Division of Signal Transduc- tion Therapy Unit at Dundee (AstraZeneca, Boehringer-Ingleheim, GlaxoSmithKline, of which is a complex between the tumor suppressor kinase, LKB1, and Merck & Co., Inc., Merck KgaA, and Pfizer) (to D. G. H.). 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. The abbreviations used are: AMPK, AMP-activated protein kinase; AICAR, 5-aminoimi- To whom correspondence should be addressed: Div. of Biomedical Sciences, School of dazole-4-carboxamide riboside; MOPS, 4-morpholinepropanesulfonic acid; ACC, Biology, Bute Bldg., University of St. Andrews, St. Andrews, Fife KY16 9TS, UK. Tel.: acetyl-CoA carboxylase; T, torr; HPV, hypoxic pulmonary vasoconstriction; cADPR, 44-1334-463579; Fax: 44-1334-463600; E-mail: [email protected]. cyclic ADP-ribose; SR, sarcoplasmic reticulum. 41504 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 50 •DECEMBER 16, 2005 This is an Open Access article under the CC BY license. 2 AMPK and Ca Signaling in O -sensing Cells two accessory subunits, STRAD and MO25 (28–30). Upon activation, with fluorescein isothiocyanate-conjugated secondary antibodies AMPK serves to maintain ATP levels by activating catabolic pathways (1:200; excitation 490 nm, emission 518 nm), washed five times with and by inhibiting non-essential ATP-consuming processes. phosphate-buffered saline, and attached to slides by mountant (2.4 g The primary targets for AMPK had previously been presumed to be Mowiol4–88,6gof glycerol, 2 ml of 0.2 M Tris-HCl, pH 8.5, 2.5% mainly involved in energy metabolism, but it is now recognized that 1,4-diazabicyclo(2.2.2.) octane) with 4,6-diamidino-2-phenylindole (1 AMPK can also target non-metabolic processes (20). Given that inhibi- g/ml; excitation 358 nm, emission 461 nm). For controls, the primary tion of mitochondrial oxidative phosphorylation by hypoxia would be antibody was omitted. Images were acquired using a Deltavision micro- expected to promote a rise in the AMP/ATP ratio (20), we considered scope system (Applied Precision) on an Olympus IX70 microscope the proposal (31) that AMPK activation may mediate, in part, pulmo- using a 60, 1.40 numerical aperture, oil immersion objective, and Pho- nary artery constriction and carotid body excitation by hypoxia. The tometric CH300 charge-coupled device camera. Single or multiple Z findings of the present investigation support this proposal. sections (0.2 m) were taken through a cell. Images were deconvolved and analyzed off-line via Softworx software (Applied Precision). 2 2 MATERIALS AND METHODS Ca Imaging—Intracellular Ca concentration was reported by Fura-2 fluorescence ratio (F340/F380 excitation; emission 510 nm). Cell Isolation—All experiments were performed under the United Emitted fluorescence was recorded at 22 °C, with a sampling frequency Kingdom Animals (Scientific Procedures) Act 1986. Pulmonary arteries of 0.05 Hz, to avoid photobleaching during long recording periods using were excised from male Wistar rats (150–300 g) after cervical disloca- a Hamamatsu 4880 charge-coupled device camera via a Zeiss Fluar tion and then placed in physiological salt solution A (PSS-A) (mM; 130 (pulmonary artery smooth muscle cells) or a Nikon Fluor (carotid body NaCl, 5.2 KCl, 1 MgCl , 1.7 CaCl , 10 glucose, 10 Hepes, pH 7.4). Arter- 2 2 glomus cells) 40, 1.3 numerical aperture, oil immersion lens and Leica ies were incubated overnight (4 °C) in Ca -free PSS-A with 0.05 mg 1 1 DMIRBE microscope. Background subtraction was performed on-line. ml papain and 1 mg ml bovine serum albumin. Thereafter 0.2 mM Analysis was via Openlab (Improvision) (32). Pharmacological agents 1,4-dithio-DL-threitol was added (22 °C, 1 h), the tissue washed three were applied extracellularly by a microsuperfusion system via a flow times in Ca -free PSS-A without enzyme, and smooth muscle cells pipe positioned close to the cell under investigation, as described previ- were isolated by trituration and stored at 4 °C (32). ously (32). Carotid bodies were excised from 9–12-day-old rats (anesthetized by Autofluorescence Measurements—Cells were excited by a Blue diode 4% halothane), placed in ice-cold phosphate-buffered saline containing 405-nm laser (pinhole 114.4 m; 25% power) via a Leica 63, 1.2 collagenase (0.05% w/v), trypsin (0.025% w/v), and 50 M Ca , incu- numerical aperture, water immersion objective on a Leica SP2 confocal bated at 37 °C (30 min), centrifuged (200  g, 5 min, 4 °C), and resus- system. Cell autofluorescence was recorded (0.05 Hz, 22 °C) as the aver- pended in Ham’s F-12 culture medium (84 units liter insulin, 100 IU 1 1 age of 8 Z scans. Acquisition and analysis was by Leica LCS 3D liter penicillin, 100 gml streptomycin, 10% heat-inactivated fetal Physiology. calf serum). Cells were isolated by trituration, plated onto poly-D-lysine- Tension Recording—Records were from third order pulmonary artery coated coverslips, and maintained in a humidified incubator (5% CO in branches (internal diameter 300–400 m; 2–3 mm in length) via small air) overnight before use (33). vessel myographs (AM10; Cambustion Biological, Cambridge, UK) as Western Blots and AMPK Activities—AMPK subunit protein expres- described previously (32). Experimental chambers were filled with sion was analyzed using precast 4–12% BisTris gels in MOPS buffer. PSS-B (in mM: 118 NaCl, 4 KCl, 1 MgSO , 1.2 NaH PO , 24 NaHCO ,2 Acetyl-CoA carboxylase (ACC) phosphorylation and total ACC protein 4 2 4 3 CaCl ,2MgCl , 5.6 glucose, pH 7.4) at 37 °C, and bubbled with 75% N , levels were analyzed using precast 3–8% Tris acetate gels in Tris acetate 2 2 2 20% O ,5%CO (normoxia, 150–160 T;) or 93% N ,2%O ,5%CO buffer. Proteins were transferred to nitrocellulose membranes using an 2 2 2 2 2 (hypoxia, 16–21 T) via a gas-mixing flowmeter (Columbus Xcell II blot module and probed with antibodies against AMPK subunits Instruments). (34). ACC phosphorylation and total ACC protein were measured via Isolated Carotid Body—Rats were anesthetized with 1–4% halothane dual labeling using phosphospecific antibodies against Ser-221 on in O (Pepper et al. (37)), killed, and exsanguinated. Left and right ACC2, with secondary anti-sheep antibodies conjugated to IR680 (1 mg carotid bifurcations were identified and removed, pinned on Sylgard ml ) and streptavidin conjugated to IR800. Fluorescence from the two (184) in a 0.2-ml chamber and perfused (3 ml min ) with PSS-C (mM; dyes was measured simultaneously using an Odyssey infrared imaging 125 NaCl, 3 KCl, 1.25 NaH PO ,5Na SO , 1.3 MgSO , 24 NaHCO , 2.4 system (Li-Cor Biosciences) (27). Isoform-specific AMPK activities 2 4 2 4 4 2 CaCl , 10 glucose, pH 7.4) at 37  1 °C. Perfusate was equilibrated to 40 were determined by immunoprecipitating 100 mg of tissue lysate with T pCO and 400 T pO via precision flow valves (Cole–Palmer). The antibodies raised against 1, 2, 1, 2, or 3 subunits bound to protein 2 2 sinus nerve was sectioned at the junction with the glossopharyngeal G-Sepharose beads and quantified using the AMARA peptide and nerve, extracellular recordings of afferent fiber spike activity recorded [- P]ATP substrates (35). on video tape, and action potentials sampled digitally (37) via LabVIEW Measurement of the AMP/ATP and ADP/ATP Ratio—Adenine nucleotide content of arterial smooth muscle lysates was determined by software (National Instruments Co.). Drugs and Chemicals—Secondary antibodies used were from Strat- capillary electrophoresis (36). Immunocytochemistry—Cells were placed onto poly-D-lysine-coated ech and Molecular Probes. Gels and buffers were from Invitrogen. Phenformin, ryanodine, 8-bromo-cyclic ADP-ribose (8-bromo- coverslips, fixed using ice-cold methanol (15 min), permeabilized by three 5-min washes with 0.3% Triton X-100 in phosphate-buffered cADPR), bovine fetal serum albumin, dithiothreitol, collagenase, tryp- sin, insulin, penicillin, poly-D-lysine, papain, and caffeine were from saline (pH 7.4), washed three times for 5 min each in blocking solution (1% bovine serum albumin, 4% goat serum, and 0.3% Triton X-100 in Sigma. Fura-2/AM was from Molecular Probes. 5-aminoimidazole-4- phosphate-buffered saline), and incubated overnight at 4 °C with anti- carboxamide riboside (AICAR) was from Calbiochem. Stock solutions bodies against the AMPK 1 subunit (1:500). Coverslips were washed of ryanodine and 8-bromo-cADPR were in Me SO, and minimum dilu- four times with blocking solution and incubated (1 h, 22 °C, in the dark) tion 1:1000 in PSS was without effect on preparations. DECEMBER 16, 2005• VOLUME 280 • NUMBER 50 JOURNAL OF BIOLOGICAL CHEMISTRY 41505 2 AMPK and Ca Signaling in O -sensing Cells to be 121, although other combinations are probably present (i.e. 122, 221, and 222). This contrasted markedly with the activities in the main pulmonary artery that feeds the lung and systemic (mesenteric) arteries, where the activity of the 1 isoform became pro- gressively lower, while that of the 2 isoform was similar in each tissue type (Fig. 1C). Thus, AMPK-1 activity is inversely related to pulmo- nary artery diameter, as is the magnitude of pulmonary artery constric- tion by hypoxia (39) and, significantly, the enzymes for the synthesis and metabolism of cADPR, a Ca -mobilizing messenger (40, 41) that has been identified as a primary mediator of HPV (42, 43). Most impor- tantly, perhaps, the AMPK-1 activity was much higher in second and third order branches of the pulmonary arterial tree when compared with systemic (mesenteric) arteries (Fig. 1C), which dilate rather than constrict in response to hypoxia (38). Once more, this correlated with the distribution of the enzyme activities for the synthesis and metabo- lism of cADPR. Thus, the differential arterial distribution of AMPK-1 catalytic activity, together with that of the enzymes for the synthesis and metabolism of cADPR, could provide via signal amplification the degree of pulmonary selectivity required for HPV, the critical and distinguish- ing characteristic of pulmonary arteries (1). Hypoxia Elicits an Increase in the AMP/ATP Ratio and Concomitant Activation of AMPK in Pulmonary Arterial Smooth Muscle—Previous studies (25) have shown that, through the action of adenylate kinase, any increase in the cellular ADP/AMP ratio is converted into a rise in the FIGURE 1. AMPK subunit isoforms in pulmonary versus systemic arterial smooth muscle and AMPK activation in pulmonary arterial smooth muscle by hypoxia. A, AMP/ATP ratio leading to consequent activation of AMPK (28–30). Western blot of AMPK-1, -2, -1, -2, -1, and -3 expression. Lanes 1 and 2, pulmonary Consistent with these findings, capillary electrophoresis analysis on pul- arterial smooth muscle; lane 3, rat liver. B, AMPK activity immunoprecipitated from pul- monary arterial smooth muscle with anti-1, -2, -1, -2, and -3 antibodies. C, AMPK monary arterial smooth muscle lysates (32 arteries, 8 animals) showed activity immunoprecipitated from pulmonary and systemic arterial smooth muscle with that the AMP/ATP ratio rose from 0.040 under normoxia (155–160 T, anti-1 and -2 antibodies. D, activation of AMPK in pulmonary arterial smooth muscle 2 h) to 0.083 under hypoxic conditions (16–21 T, 1 h; following 1 h by switching from normoxia (154 –160 T, 1 h) to hypoxia (16 –21 T, 1 h). E, Western blot of ACC phosphorylation in response to a switch from normoxia (154 –160 T) to hypoxia equilibration under normoxia), whereas the ADP/ATP ratio rose from (16 –21 T). Upper panels, blots; lower panels, mean  S.D. (n  3, 32 arteries from 8 0.183 to 0.259. This suggests, as one would expect (20, 21), that the animals). adenylate kinase reaction is close to equilibrium, because the AMP/ATP RESULTS ratio varies approximately as the square of the ADP/ATP ratio. Most significantly, immunoprecipitate kinase assays demonstrated that the The Tissue-specific Distribution of the AMPK-1 Isozyme May Afford rise in the AMP/ATP ratio was associated with a concomitant, 2-fold the Pulmonary Selectivity Required of a Mediator of Hypoxic Pulmonary increase in AMPK activity (n  3, 32 arteries, 8 animals) (Fig. 1D) and Vasoconstriction—To determine whether or not AMPK activates phosphorylation of a classical AMPK substrate, acetyl-CoA carboxylase O -sensing cells in response to hypoxia, we first focused on pulmonary (n  3, 32 arteries, 8 animals) (Fig. 1E, ACC). arterial smooth muscle. Hypoxic pulmonary vasoconstriction (HPV) is Hypoxia activated both AMPK-1 and AMPK-2 catalytic activity in the critical and distinguishing characteristic of pulmonary arteries (1). pulmonary arterial smooth muscle (Fig. 1D). However, consistent with In marked contrast, systemic arteries dilate in response to hypoxia to AMPK-1 playing a prominent role in regulating pulmonary arterial match tissue perfusion to metabolism (38). Thus, a mediator of hypoxic smooth muscle function, hypoxia increased AMPK-1 activity to a pulmonary vasoconstriction should offer some degree of pulmonary greater extent than it increased AMPK-2 activity (Fig. 1D). selectivity. We assessed, therefore, the relative activities of the various AMPK Activation Initiates cADPR-dependent Ca Release from isoforms of the catalytic  subunit and regulatory  and  subunits that Ryanodine-sensitive Sarcoplasmic Reticulum (SR) Stores in Pulmonary comprise AMPK in pulmonary and systemic arterial smooth muscle. Arterial Smooth Muscle Cells—Our previous studies have established Western blot analysis in combination with co-immunoprecipitation that cADPR-dependent Ca release from smooth muscle SR stores via analysis of pulmonary arterial smooth muscle lysates identified the pres- ryanodine receptors is required for the full expression of HPV (42, 43). ence of the 1, 2, 2, 1, and 3 subunits of AMPK (Fig. 1, A and B). Consistent with a role for AMPK in mediating such a response, immu- Our anti-2 antibodies were not sufficiently specific in Western blotting nocytochemistry showed the AMPK-1 catalytic subunit isoform to be to confirm the presence of the 2 subunit isoform. However, they did distributed throughout the cytoplasm in pulmonary arterial smooth not cross-react with anti-1or-2 antibodies, and immunoprecipitate muscle cells, with little staining associated with the plasma membrane kinase assays revealed that 2 accounted for 40% and 1 for 60% of the (Fig. 2A). Thus, we examined the possibility that AMPK activation, sim- total AMPK activity in pulmonary arterial smooth muscle lysates, with ilar to hypoxia, might mobilize SR Ca stores in a cADPR-dependent 3 accounting for an insignificant fraction (n  3, 32 arteries, 8 animals) (Fig. 1B). Furthermore, immunoprecipitated kinase assays using anti-1 manner. To this end, we employed the now classical method of studying AMPK activation. This requires the use of two drugs, phenformin (10 and -2 antibodies showed that 1 accounted for 80–90% and 2 only 10–20% of the total catalytic activity in smooth muscle lysates from mM) and AICAR (1 mM), each of which activate AMPK via discrete second and third order branches of the pulmonary arterial tree (n  3, mechanisms. Phenformin (a drug formerly used in the treatment of type 32 arteries, 8 animals) (Fig. 1C). The predominant AMPK subunit com- 2 diabetes) inhibits Complex I of the mitochondrial respiratory chain position in small pulmonary arterial smooth muscle is likely, therefore, (44, 45) and thereby activates AMPK by increasing the cellular AMP/ 41506 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 50 •DECEMBER 16, 2005 2 AMPK and Ca Signaling in O -sensing Cells ACC phosphorylation in the smooth muscle was also increased, the phosphorylated ACC/ACC ratio measuring 0.44  0.14 and 2.38  0.02 in the presence and absence of AICAR (1 mM; n  3; 32 arteries, 8 animals). Likewise, phenformin (10 mM) increased AMPK-1-associated activ- ity from 0.025  0.001 to 0.403  0.012 nmol of phosphate incorpo- rated/min/mg protein and AMPK-2-associated activity from 0.0096  0.001 to 0.126  0.006 nmol of phosphate incorporated/ min/mg protein (n  3; 32 arteries, 8 animals). A concomitant increase in ACC phosphorylation in the smooth muscle was also observed, the phosphorylated ACC/ACC ratio measuring 0.46  0.014 and 3.51  0.12 in the presence and absence of phenformin (10 mM; n  3; 32 arteries, 8 animals) As observed during hypoxia (17), AMPK activation by phenformin (10 mM) was associated with an increase in the NAD(P)H autofluores- cence in pulmonary arterial smooth muscle cells (n  24) (Fig. 2B). This is consistent with the fact that phenformin, similar to hypoxia (6–10), inhibits mitochondrial oxidative phosphorylation (44, 45). In marked contrast, AICAR (1 mM), which activates AMPK without effect on mito- chondrial function (46), had no effect on cellular NAD(P)H autofluo- rescence (n  12) (Fig. 2C). Despite their different modes of action, both phenformin (10 mM) and AICAR (1 mM) induced an increase in intra- cellular Ca concentration in isolated pulmonary arterial smooth mus- cle cells at room temperature (20–22 °C), as reported by the Fura-2 fluorescence ratio (F340/F380), which increased (mean  S.E.) by 0.09  0.02 (n  7) and by 0.10  0.01 (n  22), respectively (Fig. 2, D and E). In each case, the response, similar to that of hypoxia (32), remained unaffected upon removal of extracellular Ca (Fig. 2, D and E). Prior blocking of sarcoplasmic reticulum stores by preincubation of cells with ryanodine (10 M) and caffeine (10 mM) abolished the increase in intracellular Ca concentration produced by 1 mM AICAR (0.025  0.016, n  8) (Fig. 2, F and H) and 10 mM phenformin (0.024  0.006, n  7) (Fig. 2H). Most significantly, the SR Ca release FIGURE 2. AMPK activation elicits cADPR-dependent SR Ca release in pulmonary evoked due to AMPK activation by AICAR (1 mM) was also abolished arterial smooth muscle. A(i), bright field image of a pulmonary arterial smooth muscle (0.016  0.01, n  23) (Fig. 2, G and H) by blocking the Ca -mobiliz- cell. (ii), Z-section showing staining by antibodies to AMPK-1(green) and of the nucleus by 4,6-diamidino-2-phenylindole (blue). (iii), three-dimensional reconstruction. ing messenger cADPR (40, 41) using 8-bromo-cADPR (100 M), a selec- NAD(P)H autofluorescence in isolated pulmonary arterial smooth muscle cells with and tive cADPR antagonist (43). Consistent with this observation, 8-bromo- without phenformin (10 mM)(B) and AICAR (1 mM)(C). Effect on Fura-2 fluorescence ratio (F340/F380) in isolated pulmonary artery smooth muscle cells of phenformin (10 mM)(D) cADPR also blocked the increase in SR Ca release evoked by and AICAR (1 mM)(E) with and without extracellular Ca (1mM EGTA). Shown are phenformin (10 mM; 0.024  0.007, n  5) (Fig. 2H). Thus, AMPK acti- AICAR after preincubation with caffeine (10 mM) and ryanodine (10 M)(F) and preincu- bation with 8-bromo-cADPR (100 M)(G). H, mean increase in the Fura-2 fluorescence vation by AICAR or phenformin triggers cADPR-dependent SR Ca ratio  S.E. (n  5). Phen., phenformin. release via ryanodine receptors in pulmonary arterial smooth muscle cells, as does hypoxia (32, 42, 43, 47). AMPK Activation Elicits Pulmonary Artery Constriction via the ATP ratio (60). By contrast, AICAR is metabolized to yield the AMP Mechanisms That Underpin Hypoxic Pulmonary Vasoconstriction— mimetic ZMP (AICAR monophosphate) and thereby selectively acti- Subsequent experiments focused on the effects of AICAR to rule out vates AMPK without affecting the cellular AMP/ATP ratio (45, 46). any possible AMPK-independent actions that might result from inhibi- Consistent with the effects of hypoxia, both AICAR and phenformin tion of mitochondrial metabolism by phenformin. To ascertain whether increased AMPK activity in pulmonary arterial smooth muscle. How- or not AMPK plays a functional role in HPV, we conducted a detailed ever, longer incubation times (4 h) were required to allow for adequate comparison of the effects of hypoxia and AMPK activation by AICAR penetration of bundles of 32 intact arteries (second and third order on isolated pulmonary arteries at 37 °C. Consistent with the effects of branches; 2 cm length, 300 m to 1 mm internal diameter) due to the hypoxia (32, 42, 43, 47), AICAR (1 mM) induced a slow sustained con- pharmacokinetics of AICAR and phenformin. During this time period, striction of pulmonary artery rings (2–3 mm; 300–400 m internal basal AMPK activities declined relative to those reported above; impor- diameter) from 1.3  0.4 to 3.2  0.9 millinewton mm (n  4) (Fig. tantly, however, the relative level of AMPK-1 and AMPK-2 activities, respectively, remained consistent. Under normoxic conditions, AICAR 3A). This was reversed rapidly on washing, consistent with the active metabolite of AICAR, ZMP, being metabolized rapidly at 37 °C (46). (1 mM) increased AMPK-1-associated activity from (mean  S.D.) 0.18  0.02 to 0.56  0.03 nmol of phosphate incorporated/min/mg Removal of the pulmonary artery endothelium reduced the constriction protein and AMPK-2-associated activity from 0.043  0.005 to in response to AICAR (1 mM; n  4) (Fig. 3A) and hypoxia (16–21 T) by 0.12  0.005 nmol of phosphate incorporated/min/mg protein (n  3; 29 and 28%, respectively (Fig. 3F). Furthermore, the endothelium-de- 32 arteries, 8 animals). Consistent with AMPK activation by AICAR, pendent component of constriction by AICAR (1 mM; n  4) (Fig. 3, A DECEMBER 16, 2005• VOLUME 280 • NUMBER 50 JOURNAL OF BIOLOGICAL CHEMISTRY 41507 2 AMPK and Ca Signaling in O -sensing Cells (48). The aforementioned findings are, therefore, entirely consistent with the characteristics of pulmonary artery constriction by hypoxia and support the view that AMPK activation, similar to hypoxia, medi- ates pulmonary artery constriction via cADPR-dependent mobilization of smooth muscle SR Ca stores via ryanodine receptors (32, 42, 43). Notably, blockade of cADPR with 8-bromo-cADPR completely abol- ished the constriction of pulmonary arteries, with or without endothe- lium, by both AICAR (1 mM) and hypoxia (16–21 T) (Fig. 3F). It was significant, therefore, that preconstriction of pulmonary arteries by K -induced (20 mM) depolarization restored only the endothelium-de- pendent component of constriction by AICAR (1 mM) in the continued presence of 8-bromo-cADPR (Fig. 3E). This is consistent with the effects of 8-bromo-cADPR on HPV and reflects the fact that the endo- thelium-derived vasoconstrictor released by hypoxia does not elicit pul- monary artery constriction in the absence of SR Ca release by cADPR (32, 42, 43), because it sensitizes the contractile apparatus to the increase in cytoplasmic Ca concentration induced by hypoxia (49). AMPK Activation Elicits Transmembrane Ca Influx into Carotid Body Glomus Cells and Consequent Carotid Body Excitation and Affer- ent Fiber Discharge—The findings above strongly support our hypoth- esis that AMPK acts as the primary metabolic sensor in pulmonary arteries and is the primary effector of HPV. If, however, metabolic sens- ing by AMPK were to be a general mechanism of chemotransduction, then one would expect it to mediate the cell-specific Ca signaling mechanisms observed in other O -sensing cells. We, therefore, turned our attention to the carotid body glomus cell. These cells are also stim- ulated by hypoxia, but in this case, excitation is primarily mediated by voltage-gated Ca influx, leading ultimately to neurosecretion (10, 33, 37, 50–52) rather than by Ca release from intracellular stores. Immu- nocytochemistry showed that, in marked contrast to its cytoplasmic distribution in pulmonary arterial smooth muscle cells (Fig. 2A), the AMPK-1 catalytic subunit isoform was almost entirely restricted to the plasma membrane of carotid body glomus cells (Fig. 4A). Thus, the spatial localization of AMPK in carotid body glomus cells would be FIGURE 3. AMPK activation by AICAR replicates hypoxic pulmonary vasoconstric- consistent with the regulation of plasma membrane-delimited pro- tion. Shown are pulmonary artery constriction in response to AMPK activation by AICAR 2 cesses such as voltage-gated Ca influx. Consistent with this proposal (1 mM) with (left panel) and without (right panel) the endothelium. A, with extracellular 2 2 and with the effects of hypoxia on carotid body glomus cells (10), AMPK Ca . B, without extracellular Ca (1mM EGTA). C, after preincubation of pulmonary arteries with caffeine (10 mM) and ryanodine (10 M). D, after preincubation with 8-bro- activation by AICAR (1 mM) induced an increase in intracellular Ca mo-cADPR (300 M). E, after preincubation with 8-bromo-cADPR (300 M) and submaxi- concentration in acutely isolated carotid body glomus cells at 22 °C, as mal preconstriction by K (20 mM). F, mean constriction  S.E. (n  3) of isolated pulmo- nary artery rings by AICAR (1 mM, left panel) and hypoxia (right panel) under the the Fura-2 fluorescence ratio increased by 0.07  0.02 (n  11). In conditions described for A–E. E, endothelium; RC, ryanodine and caffeine; 8Br, marked contrast to our findings on pulmonary arterial smooth muscle, 8-bromo-cADPR. this increase in intracellular Ca concentration was abolished by removal of extracellular Ca (n  6) and attenuated by blockade of and B) and hypoxia (16–21 T) (32), respectively, was abolished upon 2 2 transmembrane Ca influx pathways with Cd (100 M; removal of extracellular Ca (Fig. 3F). In contrast, constriction medi- 0.009  0.006, n  6) (Fig. 4, B and D(i)). Consistent with the effects of ated by mechanisms intrinsic to the smooth muscle was not, although it AICAR, phenformin (10 mM) increased the fluorescence ratio by is notable that this component of constriction was attenuated in the 0.272  0.087 (n  7) in a manner that was reversed by the removal of absence of extracellular Ca (Fig. 3, A, B, and F). Thus, maintained extracellular Ca (0.011  0.012, n  7) (Fig. 4D(i)). Thus, we can con- smooth muscle constriction by AICAR (1 mM) and hypoxia (16–21 T), clude that both phenformin and AICAR, similar to hypoxia, trigger respectively, exhibits a dependence (50%) on transmembrane Ca transmembrane Ca influx into isolated carotid body glomus cells. influx. In this respect, it is of major significance that blockade of SR Ca We then investigated the effect of AICAR alone on sensory afferent stores with caffeine (10 mM) and ryanodine (10 M) or blockade of discharge from the isolated carotid body in vitro to rule out any possible cADPR with 8-bromo-cADPR (100 M) (Fig. 3) completely abolished AMPK-independent actions that might result from inhibition of mito- the constriction of pulmonary arteries, with or without endothelium, by chondrial metabolism by phenformin. Under these conditions and at both AICAR (1 mM) (Fig. 3, C, D, and F) and hypoxia (16–21 T) (Fig. 3F). Thus, the partial dependence of smooth muscle constriction on extra- 37 °C, AICAR (1 mM) induced a relatively rapid and reversible increase 2 2 in single fiber sensory afferent discharge from the isolated carotid body cellular Ca must be due to SR store depletion-activated Ca influx/ store refilling, which is not mediated by AMPK activation by AICAR nor from 0.22  0.03 to 2.8  0.56 spikes s (n  19). This too was abol- hypoxia per se. Consistent with this observation, hypoxia has been ished by the removal of extracellular Ca (0.11  0.03, n  5) and was 2 2 2 shown to initiate SR store depletion-activated Ca influx indirectly by attenuated by blockade of transmembrane Ca influx with Cd (100 first mobilizing SR Ca stores in pulmonary arterial smooth muscle M; 0.91  0.18, n  5) (Fig. 4, C and D(ii)). Thus, AMPK activation 41508 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 50 •DECEMBER 16, 2005 2 AMPK and Ca Signaling in O -sensing Cells FIGURE 4. AMPK activation by AICAR activates carotid body glomus cells by eliciting trans- membrane Ca influx as does hypoxia. A(i), bright field image of a fixed carotid body glomus cell. (ii), Z-section showing staining by antibodies against AMPK-1(green), tyrosine hydroxylase (red), and staining for the nucleus by 4,6-dia- midino-2-phenylindole (blue). (iii), three-dimen- sional reconstruction. B, effect of AMPK activation by AICAR on Fura-2 fluorescence ratio (F340/F380) in an isolated carotid body glomus cell with and without extracellular Ca (1mM EGTA) and Cd (100 M). C, frequency histogram (upper panel) and record of action potential spikes (lower panel) show effect of AICAR on multiple fiber-affer- ent discharge from the carotid body in vitro with and without extracellular Ca (1mM EGTA). D, mean  S.E. for experiments in B and C. E, pro- posed model for cell-specific Ca signaling by AMPK in response to hypoxia. RyR, ryanodine receptor. reproduced the precise excitatory effects of hypoxia on the carotid body consequent increase in afferent fiber discharge (10, 33, 37, 52). We pro- (10, 37, 52). pose, therefore, that AMPK may act as a primary metabolic sensor and effector in O -sensing cells. This novel role for AMPK may therefore DISCUSSION 2 unite for the first time the mitochondrial and Ca signaling hypotheses Previous studies (2, 6–10) have established that hypoxia promotes for chemotransduction by hypoxia (18, 19). Before we can be certain of Ca -dependent pulmonary artery constriction and carotid body sen- a role for AMPK in this process, however, we need to demonstrate that sory afferent discharge, in part, by inhibiting oxidative phosphorylation cell activation by hypoxia can be inhibited by selective blocking of by mitochondria. Our findings now suggest that this leads to a rise in the AMPK. This may be achieved once the selective AMPK antagonist cellular AMP/ATP ratio, consequent AMPK activation, and the initia- Compound C (53, 54) becomes freely available or by the use of siRNA, tion of cell-specific Ca signaling mechanisms in pulmonary arterial which has yet to be applied successfully in studies on these wild-type smooth muscle and carotid body glomus cells (Fig. 4E). Thus, the char- cells. acteristic functional response of each tissue type to AMPK activation If confirmed, this process of activation would be exquisitely sensitive mirrors the primary aspects of the observed response to hypoxia: (a) to metabolic stress by hypoxia because of the triple mechanism by constriction of pulmonary arteries by cADPR-dependent SR Ca which AMPK is activated; adenylate kinase converts any rise in the release in the smooth muscle cells with a secondary component of con- cellular ADP/ATP ratio into a rise in the AMP/ATP ratio (25), whereas striction driven by the pulmonary arterial endothelium (32, 42, 43) and AMP binding to the  subunits of AMPK not only promotes phospho- (b) transmembrane Ca influx into carotid body glomus cells and a rylation by LKB1 but also inhibits dephosphorylation by protein phos- DECEMBER 16, 2005• VOLUME 280 • NUMBER 50 JOURNAL OF BIOLOGICAL CHEMISTRY 41509 2 AMPK and Ca Signaling in O -sensing Cells phatases and causes allosteric activation of the phosphorylated enzyme icity, and neuronal apoptosis (59) and ischemic damage to cardiac (20). 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Does AMP-activated Protein Kinase Couple Inhibition of Mitochondrial Oxidative Phosphorylation by Hypoxia to Calcium Signaling in O2-sensing Cells? *

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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 50, pp. 41504 –41511, December 16, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Does AMP-activated Protein Kinase Couple Inhibition of Mitochondrial Oxidative Phosphorylation by Hypoxia to Calcium Signaling in O -sensing Cells? Received for publication, September 13, 2005, and in revised form, September 29, 2005 Published, JBC Papers in Press, September 30, 2005, DOI 10.1074/jbc.M510040200 ‡1 § ‡ ¶ ‡ A. Mark Evans , Kirsteen J. W. Mustard , Christopher N. Wyatt , Chris Peers , Michelle Dipp , Prem Kumar , ‡ § Nicholas P. Kinnear , and D. Grahame Hardie From the Division of Biomedical Sciences, School of Biology, Bute Building, University of St. Andrews, St. Andrews, Fife KY16 9TS, United Kingdom, Division of Molecular Physiology, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dow Street, DD1 5EH, United Kingdom, Institute for Cardiovascular Research, University of Leeds, Leeds LS2 9JT, United Kingdom, and Department of Physiology, The Medical School, University of Birmingham, Birmingham B15 2TT, United Kingdom Specialized O -sensing cells exhibit a particularly low threshold Specialized O -sensing cells within the body have evolved as vital 2 2 to regulation by O supply and function to maintain arterial pO homeostatic mechanisms that monitor O supply and alter respiratory 2 2 2 within physiological limits. For example, hypoxic pulmonary vaso- and circulatory function, as well as the capacity of the blood to transport constriction optimizes ventilation-perfusion matching in the lung, O . By these means, arterial pO is maintained within physiological 2 2 whereas carotid body excitation elicits corrective cardio-respira- limits. Two key systems involved are the pulmonary arteries and the tory reflexes. It is generally accepted that relatively mild hypoxia carotid body. Constriction of pulmonary arteries by hypoxia optimizes inhibits mitochondrial oxidative phosphorylation in O -sensing ventilation-perfusion matching in the lung (1), whereas carotid body cells, thereby mediating, in part, cell activation. However, the mech- excitation by hypoxia initiates corrective changes in breathing patterns anism by which this process couples to Ca signaling mechanisms via increased sensory afferent discharge to the brain stem (2). Although remains elusive, and investigation of previous hypotheses has gen- O -sensitive mechanisms independent of mitochondria may also play a erated contrary data and failed to unite the field. We propose that a role (3–5), it is generally accepted that relatively mild hypoxia inhibits rise in the cellular AMP/ATP ratio activates AMP-activated protein mitochondrial oxidative phosphorylation and that this underpins, at kinase and thereby evokes Ca signals in O -sensing cells. Co-im- least in part, cell activation (2, 6–10). Despite this consensus, the mech- munoprecipitation identified three possible AMP-activated protein anism by which inhibition of mitochondrial oxidative phosphorylation couples to discrete cell-specific Ca signaling mechanisms has kinase subunit isoform combinations in pulmonary arterial myo- cytes, with 121 predominant. Furthermore, their tissue-spe- remained elusive. Recently, the field has focused on the possible role of cific distribution suggested that the AMP-activated protein the cellular energy status (ATP) (7, 11), reduced redox couples (12), and kinase-1 catalytic isoform may contribute, via amplification of the reactive oxygen species (13–17), respectively, but extensive investiga- tion of these hypotheses has delivered conflicting data and failed to metabolic signal, to the pulmonary selectivity required for hypoxic pulmonary vasoconstriction. Immunocytochemistry showed AMP- unite the field since its inception in 1930 (18, 19). However, Sylvester activated protein kinase-1 to be located throughout the cytoplasm and colleagues (11) have suggested that previous assessment of the role of pulmonary arterial myocytes. In contrast, it was targeted to the of the energy state may have been limited by the lack of knowledge of the identity of the energy variable that might signal the response. plasma membrane in carotid body glomus cells. Consistent with these observations and the effects of hypoxia, stimulation of AMP- Recently, the AMP-activated protein kinase (AMPK) cascade has activated protein kinase by phenformin or 5-aminoimidazole-4- come to prominence as a sensor of metabolic stress that appears to be carboxamide-riboside elicited discrete Ca signaling mechanisms ubiquitous throughout eukaryotes (20, 21). AMPK is activated by many different metabolic stresses, including heat shock and metabolic poi- in each cell type, namely cyclic ADP-ribose-dependent Ca mobi- lization from the sarcoplasmic reticulum via ryanodine receptors in sons in hepatocytes (22), exercise in skeletal muscle (23), and ischemia pulmonary arterial myocytes and transmembrane Ca influx into and hypoxia in the heart (24). AMPK complexes are heterotrimers com- carotid body glomus cells. Thus, metabolic sensing by AMP-acti- prising a catalytic  subunit and regulatory  and  subunits (20), which monitor the cellular AMP/ATP ratio as an index of metabolic stress vated protein kinase may mediate chemotransduction by hypoxia. (20). Through the action of adenylate kinase, any increase in the cellular ADP/ATP ratio is converted into an increase in the AMP/ATP ratio (25). Binding of AMP to two sites in the  subunits triggers activation of the kinase via phosphorylation of the  subunit at Thr-172, an effect * This work was supported by The Wellcome Trust Grant 070772 and Biotechnology and Biological Sciences Research Council Grant 01/A/S/07453 (to A. M. E.) and by a con- antagonized by high concentrations of ATP (26, 27). This phosphoryl- tract for an Integrated Project from the European Commission (LSHM-CT-2004- ation is catalyzed by upstream kinases (AMPK kinases), the major form 005272) and the pharmaceutical companies supporting the Division of Signal Transduc- tion Therapy Unit at Dundee (AstraZeneca, Boehringer-Ingleheim, GlaxoSmithKline, of which is a complex between the tumor suppressor kinase, LKB1, and Merck & Co., Inc., Merck KgaA, and Pfizer) (to D. G. H.). 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. The abbreviations used are: AMPK, AMP-activated protein kinase; AICAR, 5-aminoimi- To whom correspondence should be addressed: Div. of Biomedical Sciences, School of dazole-4-carboxamide riboside; MOPS, 4-morpholinepropanesulfonic acid; ACC, Biology, Bute Bldg., University of St. Andrews, St. Andrews, Fife KY16 9TS, UK. Tel.: acetyl-CoA carboxylase; T, torr; HPV, hypoxic pulmonary vasoconstriction; cADPR, 44-1334-463579; Fax: 44-1334-463600; E-mail: [email protected]. cyclic ADP-ribose; SR, sarcoplasmic reticulum. 41504 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 50 •DECEMBER 16, 2005 This is an Open Access article under the CC BY license. 2 AMPK and Ca Signaling in O -sensing Cells two accessory subunits, STRAD and MO25 (28–30). Upon activation, with fluorescein isothiocyanate-conjugated secondary antibodies AMPK serves to maintain ATP levels by activating catabolic pathways (1:200; excitation 490 nm, emission 518 nm), washed five times with and by inhibiting non-essential ATP-consuming processes. phosphate-buffered saline, and attached to slides by mountant (2.4 g The primary targets for AMPK had previously been presumed to be Mowiol4–88,6gof glycerol, 2 ml of 0.2 M Tris-HCl, pH 8.5, 2.5% mainly involved in energy metabolism, but it is now recognized that 1,4-diazabicyclo(2.2.2.) octane) with 4,6-diamidino-2-phenylindole (1 AMPK can also target non-metabolic processes (20). Given that inhibi- g/ml; excitation 358 nm, emission 461 nm). For controls, the primary tion of mitochondrial oxidative phosphorylation by hypoxia would be antibody was omitted. Images were acquired using a Deltavision micro- expected to promote a rise in the AMP/ATP ratio (20), we considered scope system (Applied Precision) on an Olympus IX70 microscope the proposal (31) that AMPK activation may mediate, in part, pulmo- using a 60, 1.40 numerical aperture, oil immersion objective, and Pho- nary artery constriction and carotid body excitation by hypoxia. The tometric CH300 charge-coupled device camera. Single or multiple Z findings of the present investigation support this proposal. sections (0.2 m) were taken through a cell. Images were deconvolved and analyzed off-line via Softworx software (Applied Precision). 2 2 MATERIALS AND METHODS Ca Imaging—Intracellular Ca concentration was reported by Fura-2 fluorescence ratio (F340/F380 excitation; emission 510 nm). Cell Isolation—All experiments were performed under the United Emitted fluorescence was recorded at 22 °C, with a sampling frequency Kingdom Animals (Scientific Procedures) Act 1986. Pulmonary arteries of 0.05 Hz, to avoid photobleaching during long recording periods using were excised from male Wistar rats (150–300 g) after cervical disloca- a Hamamatsu 4880 charge-coupled device camera via a Zeiss Fluar tion and then placed in physiological salt solution A (PSS-A) (mM; 130 (pulmonary artery smooth muscle cells) or a Nikon Fluor (carotid body NaCl, 5.2 KCl, 1 MgCl , 1.7 CaCl , 10 glucose, 10 Hepes, pH 7.4). Arter- 2 2 glomus cells) 40, 1.3 numerical aperture, oil immersion lens and Leica ies were incubated overnight (4 °C) in Ca -free PSS-A with 0.05 mg 1 1 DMIRBE microscope. Background subtraction was performed on-line. ml papain and 1 mg ml bovine serum albumin. Thereafter 0.2 mM Analysis was via Openlab (Improvision) (32). Pharmacological agents 1,4-dithio-DL-threitol was added (22 °C, 1 h), the tissue washed three were applied extracellularly by a microsuperfusion system via a flow times in Ca -free PSS-A without enzyme, and smooth muscle cells pipe positioned close to the cell under investigation, as described previ- were isolated by trituration and stored at 4 °C (32). ously (32). Carotid bodies were excised from 9–12-day-old rats (anesthetized by Autofluorescence Measurements—Cells were excited by a Blue diode 4% halothane), placed in ice-cold phosphate-buffered saline containing 405-nm laser (pinhole 114.4 m; 25% power) via a Leica 63, 1.2 collagenase (0.05% w/v), trypsin (0.025% w/v), and 50 M Ca , incu- numerical aperture, water immersion objective on a Leica SP2 confocal bated at 37 °C (30 min), centrifuged (200  g, 5 min, 4 °C), and resus- system. Cell autofluorescence was recorded (0.05 Hz, 22 °C) as the aver- pended in Ham’s F-12 culture medium (84 units liter insulin, 100 IU 1 1 age of 8 Z scans. Acquisition and analysis was by Leica LCS 3D liter penicillin, 100 gml streptomycin, 10% heat-inactivated fetal Physiology. calf serum). Cells were isolated by trituration, plated onto poly-D-lysine- Tension Recording—Records were from third order pulmonary artery coated coverslips, and maintained in a humidified incubator (5% CO in branches (internal diameter 300–400 m; 2–3 mm in length) via small air) overnight before use (33). vessel myographs (AM10; Cambustion Biological, Cambridge, UK) as Western Blots and AMPK Activities—AMPK subunit protein expres- described previously (32). Experimental chambers were filled with sion was analyzed using precast 4–12% BisTris gels in MOPS buffer. PSS-B (in mM: 118 NaCl, 4 KCl, 1 MgSO , 1.2 NaH PO , 24 NaHCO ,2 Acetyl-CoA carboxylase (ACC) phosphorylation and total ACC protein 4 2 4 3 CaCl ,2MgCl , 5.6 glucose, pH 7.4) at 37 °C, and bubbled with 75% N , levels were analyzed using precast 3–8% Tris acetate gels in Tris acetate 2 2 2 20% O ,5%CO (normoxia, 150–160 T;) or 93% N ,2%O ,5%CO buffer. Proteins were transferred to nitrocellulose membranes using an 2 2 2 2 2 (hypoxia, 16–21 T) via a gas-mixing flowmeter (Columbus Xcell II blot module and probed with antibodies against AMPK subunits Instruments). (34). ACC phosphorylation and total ACC protein were measured via Isolated Carotid Body—Rats were anesthetized with 1–4% halothane dual labeling using phosphospecific antibodies against Ser-221 on in O (Pepper et al. (37)), killed, and exsanguinated. Left and right ACC2, with secondary anti-sheep antibodies conjugated to IR680 (1 mg carotid bifurcations were identified and removed, pinned on Sylgard ml ) and streptavidin conjugated to IR800. Fluorescence from the two (184) in a 0.2-ml chamber and perfused (3 ml min ) with PSS-C (mM; dyes was measured simultaneously using an Odyssey infrared imaging 125 NaCl, 3 KCl, 1.25 NaH PO ,5Na SO , 1.3 MgSO , 24 NaHCO , 2.4 system (Li-Cor Biosciences) (27). Isoform-specific AMPK activities 2 4 2 4 4 2 CaCl , 10 glucose, pH 7.4) at 37  1 °C. Perfusate was equilibrated to 40 were determined by immunoprecipitating 100 mg of tissue lysate with T pCO and 400 T pO via precision flow valves (Cole–Palmer). The antibodies raised against 1, 2, 1, 2, or 3 subunits bound to protein 2 2 sinus nerve was sectioned at the junction with the glossopharyngeal G-Sepharose beads and quantified using the AMARA peptide and nerve, extracellular recordings of afferent fiber spike activity recorded [- P]ATP substrates (35). on video tape, and action potentials sampled digitally (37) via LabVIEW Measurement of the AMP/ATP and ADP/ATP Ratio—Adenine nucleotide content of arterial smooth muscle lysates was determined by software (National Instruments Co.). Drugs and Chemicals—Secondary antibodies used were from Strat- capillary electrophoresis (36). Immunocytochemistry—Cells were placed onto poly-D-lysine-coated ech and Molecular Probes. Gels and buffers were from Invitrogen. Phenformin, ryanodine, 8-bromo-cyclic ADP-ribose (8-bromo- coverslips, fixed using ice-cold methanol (15 min), permeabilized by three 5-min washes with 0.3% Triton X-100 in phosphate-buffered cADPR), bovine fetal serum albumin, dithiothreitol, collagenase, tryp- sin, insulin, penicillin, poly-D-lysine, papain, and caffeine were from saline (pH 7.4), washed three times for 5 min each in blocking solution (1% bovine serum albumin, 4% goat serum, and 0.3% Triton X-100 in Sigma. Fura-2/AM was from Molecular Probes. 5-aminoimidazole-4- phosphate-buffered saline), and incubated overnight at 4 °C with anti- carboxamide riboside (AICAR) was from Calbiochem. Stock solutions bodies against the AMPK 1 subunit (1:500). Coverslips were washed of ryanodine and 8-bromo-cADPR were in Me SO, and minimum dilu- four times with blocking solution and incubated (1 h, 22 °C, in the dark) tion 1:1000 in PSS was without effect on preparations. DECEMBER 16, 2005• VOLUME 280 • NUMBER 50 JOURNAL OF BIOLOGICAL CHEMISTRY 41505 2 AMPK and Ca Signaling in O -sensing Cells to be 121, although other combinations are probably present (i.e. 122, 221, and 222). This contrasted markedly with the activities in the main pulmonary artery that feeds the lung and systemic (mesenteric) arteries, where the activity of the 1 isoform became pro- gressively lower, while that of the 2 isoform was similar in each tissue type (Fig. 1C). Thus, AMPK-1 activity is inversely related to pulmo- nary artery diameter, as is the magnitude of pulmonary artery constric- tion by hypoxia (39) and, significantly, the enzymes for the synthesis and metabolism of cADPR, a Ca -mobilizing messenger (40, 41) that has been identified as a primary mediator of HPV (42, 43). Most impor- tantly, perhaps, the AMPK-1 activity was much higher in second and third order branches of the pulmonary arterial tree when compared with systemic (mesenteric) arteries (Fig. 1C), which dilate rather than constrict in response to hypoxia (38). Once more, this correlated with the distribution of the enzyme activities for the synthesis and metabo- lism of cADPR. Thus, the differential arterial distribution of AMPK-1 catalytic activity, together with that of the enzymes for the synthesis and metabolism of cADPR, could provide via signal amplification the degree of pulmonary selectivity required for HPV, the critical and distinguish- ing characteristic of pulmonary arteries (1). Hypoxia Elicits an Increase in the AMP/ATP Ratio and Concomitant Activation of AMPK in Pulmonary Arterial Smooth Muscle—Previous studies (25) have shown that, through the action of adenylate kinase, any increase in the cellular ADP/AMP ratio is converted into a rise in the FIGURE 1. AMPK subunit isoforms in pulmonary versus systemic arterial smooth muscle and AMPK activation in pulmonary arterial smooth muscle by hypoxia. A, AMP/ATP ratio leading to consequent activation of AMPK (28–30). Western blot of AMPK-1, -2, -1, -2, -1, and -3 expression. Lanes 1 and 2, pulmonary Consistent with these findings, capillary electrophoresis analysis on pul- arterial smooth muscle; lane 3, rat liver. B, AMPK activity immunoprecipitated from pul- monary arterial smooth muscle with anti-1, -2, -1, -2, and -3 antibodies. C, AMPK monary arterial smooth muscle lysates (32 arteries, 8 animals) showed activity immunoprecipitated from pulmonary and systemic arterial smooth muscle with that the AMP/ATP ratio rose from 0.040 under normoxia (155–160 T, anti-1 and -2 antibodies. D, activation of AMPK in pulmonary arterial smooth muscle 2 h) to 0.083 under hypoxic conditions (16–21 T, 1 h; following 1 h by switching from normoxia (154 –160 T, 1 h) to hypoxia (16 –21 T, 1 h). E, Western blot of ACC phosphorylation in response to a switch from normoxia (154 –160 T) to hypoxia equilibration under normoxia), whereas the ADP/ATP ratio rose from (16 –21 T). Upper panels, blots; lower panels, mean  S.D. (n  3, 32 arteries from 8 0.183 to 0.259. This suggests, as one would expect (20, 21), that the animals). adenylate kinase reaction is close to equilibrium, because the AMP/ATP RESULTS ratio varies approximately as the square of the ADP/ATP ratio. Most significantly, immunoprecipitate kinase assays demonstrated that the The Tissue-specific Distribution of the AMPK-1 Isozyme May Afford rise in the AMP/ATP ratio was associated with a concomitant, 2-fold the Pulmonary Selectivity Required of a Mediator of Hypoxic Pulmonary increase in AMPK activity (n  3, 32 arteries, 8 animals) (Fig. 1D) and Vasoconstriction—To determine whether or not AMPK activates phosphorylation of a classical AMPK substrate, acetyl-CoA carboxylase O -sensing cells in response to hypoxia, we first focused on pulmonary (n  3, 32 arteries, 8 animals) (Fig. 1E, ACC). arterial smooth muscle. Hypoxic pulmonary vasoconstriction (HPV) is Hypoxia activated both AMPK-1 and AMPK-2 catalytic activity in the critical and distinguishing characteristic of pulmonary arteries (1). pulmonary arterial smooth muscle (Fig. 1D). However, consistent with In marked contrast, systemic arteries dilate in response to hypoxia to AMPK-1 playing a prominent role in regulating pulmonary arterial match tissue perfusion to metabolism (38). Thus, a mediator of hypoxic smooth muscle function, hypoxia increased AMPK-1 activity to a pulmonary vasoconstriction should offer some degree of pulmonary greater extent than it increased AMPK-2 activity (Fig. 1D). selectivity. We assessed, therefore, the relative activities of the various AMPK Activation Initiates cADPR-dependent Ca Release from isoforms of the catalytic  subunit and regulatory  and  subunits that Ryanodine-sensitive Sarcoplasmic Reticulum (SR) Stores in Pulmonary comprise AMPK in pulmonary and systemic arterial smooth muscle. Arterial Smooth Muscle Cells—Our previous studies have established Western blot analysis in combination with co-immunoprecipitation that cADPR-dependent Ca release from smooth muscle SR stores via analysis of pulmonary arterial smooth muscle lysates identified the pres- ryanodine receptors is required for the full expression of HPV (42, 43). ence of the 1, 2, 2, 1, and 3 subunits of AMPK (Fig. 1, A and B). Consistent with a role for AMPK in mediating such a response, immu- Our anti-2 antibodies were not sufficiently specific in Western blotting nocytochemistry showed the AMPK-1 catalytic subunit isoform to be to confirm the presence of the 2 subunit isoform. However, they did distributed throughout the cytoplasm in pulmonary arterial smooth not cross-react with anti-1or-2 antibodies, and immunoprecipitate muscle cells, with little staining associated with the plasma membrane kinase assays revealed that 2 accounted for 40% and 1 for 60% of the (Fig. 2A). Thus, we examined the possibility that AMPK activation, sim- total AMPK activity in pulmonary arterial smooth muscle lysates, with ilar to hypoxia, might mobilize SR Ca stores in a cADPR-dependent 3 accounting for an insignificant fraction (n  3, 32 arteries, 8 animals) (Fig. 1B). Furthermore, immunoprecipitated kinase assays using anti-1 manner. To this end, we employed the now classical method of studying AMPK activation. This requires the use of two drugs, phenformin (10 and -2 antibodies showed that 1 accounted for 80–90% and 2 only 10–20% of the total catalytic activity in smooth muscle lysates from mM) and AICAR (1 mM), each of which activate AMPK via discrete second and third order branches of the pulmonary arterial tree (n  3, mechanisms. Phenformin (a drug formerly used in the treatment of type 32 arteries, 8 animals) (Fig. 1C). The predominant AMPK subunit com- 2 diabetes) inhibits Complex I of the mitochondrial respiratory chain position in small pulmonary arterial smooth muscle is likely, therefore, (44, 45) and thereby activates AMPK by increasing the cellular AMP/ 41506 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 50 •DECEMBER 16, 2005 2 AMPK and Ca Signaling in O -sensing Cells ACC phosphorylation in the smooth muscle was also increased, the phosphorylated ACC/ACC ratio measuring 0.44  0.14 and 2.38  0.02 in the presence and absence of AICAR (1 mM; n  3; 32 arteries, 8 animals). Likewise, phenformin (10 mM) increased AMPK-1-associated activ- ity from 0.025  0.001 to 0.403  0.012 nmol of phosphate incorpo- rated/min/mg protein and AMPK-2-associated activity from 0.0096  0.001 to 0.126  0.006 nmol of phosphate incorporated/ min/mg protein (n  3; 32 arteries, 8 animals). A concomitant increase in ACC phosphorylation in the smooth muscle was also observed, the phosphorylated ACC/ACC ratio measuring 0.46  0.014 and 3.51  0.12 in the presence and absence of phenformin (10 mM; n  3; 32 arteries, 8 animals) As observed during hypoxia (17), AMPK activation by phenformin (10 mM) was associated with an increase in the NAD(P)H autofluores- cence in pulmonary arterial smooth muscle cells (n  24) (Fig. 2B). This is consistent with the fact that phenformin, similar to hypoxia (6–10), inhibits mitochondrial oxidative phosphorylation (44, 45). In marked contrast, AICAR (1 mM), which activates AMPK without effect on mito- chondrial function (46), had no effect on cellular NAD(P)H autofluo- rescence (n  12) (Fig. 2C). Despite their different modes of action, both phenformin (10 mM) and AICAR (1 mM) induced an increase in intra- cellular Ca concentration in isolated pulmonary arterial smooth mus- cle cells at room temperature (20–22 °C), as reported by the Fura-2 fluorescence ratio (F340/F380), which increased (mean  S.E.) by 0.09  0.02 (n  7) and by 0.10  0.01 (n  22), respectively (Fig. 2, D and E). In each case, the response, similar to that of hypoxia (32), remained unaffected upon removal of extracellular Ca (Fig. 2, D and E). Prior blocking of sarcoplasmic reticulum stores by preincubation of cells with ryanodine (10 M) and caffeine (10 mM) abolished the increase in intracellular Ca concentration produced by 1 mM AICAR (0.025  0.016, n  8) (Fig. 2, F and H) and 10 mM phenformin (0.024  0.006, n  7) (Fig. 2H). Most significantly, the SR Ca release FIGURE 2. AMPK activation elicits cADPR-dependent SR Ca release in pulmonary evoked due to AMPK activation by AICAR (1 mM) was also abolished arterial smooth muscle. A(i), bright field image of a pulmonary arterial smooth muscle (0.016  0.01, n  23) (Fig. 2, G and H) by blocking the Ca -mobiliz- cell. (ii), Z-section showing staining by antibodies to AMPK-1(green) and of the nucleus by 4,6-diamidino-2-phenylindole (blue). (iii), three-dimensional reconstruction. ing messenger cADPR (40, 41) using 8-bromo-cADPR (100 M), a selec- NAD(P)H autofluorescence in isolated pulmonary arterial smooth muscle cells with and tive cADPR antagonist (43). Consistent with this observation, 8-bromo- without phenformin (10 mM)(B) and AICAR (1 mM)(C). Effect on Fura-2 fluorescence ratio (F340/F380) in isolated pulmonary artery smooth muscle cells of phenformin (10 mM)(D) cADPR also blocked the increase in SR Ca release evoked by and AICAR (1 mM)(E) with and without extracellular Ca (1mM EGTA). Shown are phenformin (10 mM; 0.024  0.007, n  5) (Fig. 2H). Thus, AMPK acti- AICAR after preincubation with caffeine (10 mM) and ryanodine (10 M)(F) and preincu- bation with 8-bromo-cADPR (100 M)(G). H, mean increase in the Fura-2 fluorescence vation by AICAR or phenformin triggers cADPR-dependent SR Ca ratio  S.E. (n  5). Phen., phenformin. release via ryanodine receptors in pulmonary arterial smooth muscle cells, as does hypoxia (32, 42, 43, 47). AMPK Activation Elicits Pulmonary Artery Constriction via the ATP ratio (60). By contrast, AICAR is metabolized to yield the AMP Mechanisms That Underpin Hypoxic Pulmonary Vasoconstriction— mimetic ZMP (AICAR monophosphate) and thereby selectively acti- Subsequent experiments focused on the effects of AICAR to rule out vates AMPK without affecting the cellular AMP/ATP ratio (45, 46). any possible AMPK-independent actions that might result from inhibi- Consistent with the effects of hypoxia, both AICAR and phenformin tion of mitochondrial metabolism by phenformin. To ascertain whether increased AMPK activity in pulmonary arterial smooth muscle. How- or not AMPK plays a functional role in HPV, we conducted a detailed ever, longer incubation times (4 h) were required to allow for adequate comparison of the effects of hypoxia and AMPK activation by AICAR penetration of bundles of 32 intact arteries (second and third order on isolated pulmonary arteries at 37 °C. Consistent with the effects of branches; 2 cm length, 300 m to 1 mm internal diameter) due to the hypoxia (32, 42, 43, 47), AICAR (1 mM) induced a slow sustained con- pharmacokinetics of AICAR and phenformin. During this time period, striction of pulmonary artery rings (2–3 mm; 300–400 m internal basal AMPK activities declined relative to those reported above; impor- diameter) from 1.3  0.4 to 3.2  0.9 millinewton mm (n  4) (Fig. tantly, however, the relative level of AMPK-1 and AMPK-2 activities, respectively, remained consistent. Under normoxic conditions, AICAR 3A). This was reversed rapidly on washing, consistent with the active metabolite of AICAR, ZMP, being metabolized rapidly at 37 °C (46). (1 mM) increased AMPK-1-associated activity from (mean  S.D.) 0.18  0.02 to 0.56  0.03 nmol of phosphate incorporated/min/mg Removal of the pulmonary artery endothelium reduced the constriction protein and AMPK-2-associated activity from 0.043  0.005 to in response to AICAR (1 mM; n  4) (Fig. 3A) and hypoxia (16–21 T) by 0.12  0.005 nmol of phosphate incorporated/min/mg protein (n  3; 29 and 28%, respectively (Fig. 3F). Furthermore, the endothelium-de- 32 arteries, 8 animals). Consistent with AMPK activation by AICAR, pendent component of constriction by AICAR (1 mM; n  4) (Fig. 3, A DECEMBER 16, 2005• VOLUME 280 • NUMBER 50 JOURNAL OF BIOLOGICAL CHEMISTRY 41507 2 AMPK and Ca Signaling in O -sensing Cells (48). The aforementioned findings are, therefore, entirely consistent with the characteristics of pulmonary artery constriction by hypoxia and support the view that AMPK activation, similar to hypoxia, medi- ates pulmonary artery constriction via cADPR-dependent mobilization of smooth muscle SR Ca stores via ryanodine receptors (32, 42, 43). Notably, blockade of cADPR with 8-bromo-cADPR completely abol- ished the constriction of pulmonary arteries, with or without endothe- lium, by both AICAR (1 mM) and hypoxia (16–21 T) (Fig. 3F). It was significant, therefore, that preconstriction of pulmonary arteries by K -induced (20 mM) depolarization restored only the endothelium-de- pendent component of constriction by AICAR (1 mM) in the continued presence of 8-bromo-cADPR (Fig. 3E). This is consistent with the effects of 8-bromo-cADPR on HPV and reflects the fact that the endo- thelium-derived vasoconstrictor released by hypoxia does not elicit pul- monary artery constriction in the absence of SR Ca release by cADPR (32, 42, 43), because it sensitizes the contractile apparatus to the increase in cytoplasmic Ca concentration induced by hypoxia (49). AMPK Activation Elicits Transmembrane Ca Influx into Carotid Body Glomus Cells and Consequent Carotid Body Excitation and Affer- ent Fiber Discharge—The findings above strongly support our hypoth- esis that AMPK acts as the primary metabolic sensor in pulmonary arteries and is the primary effector of HPV. If, however, metabolic sens- ing by AMPK were to be a general mechanism of chemotransduction, then one would expect it to mediate the cell-specific Ca signaling mechanisms observed in other O -sensing cells. We, therefore, turned our attention to the carotid body glomus cell. These cells are also stim- ulated by hypoxia, but in this case, excitation is primarily mediated by voltage-gated Ca influx, leading ultimately to neurosecretion (10, 33, 37, 50–52) rather than by Ca release from intracellular stores. Immu- nocytochemistry showed that, in marked contrast to its cytoplasmic distribution in pulmonary arterial smooth muscle cells (Fig. 2A), the AMPK-1 catalytic subunit isoform was almost entirely restricted to the plasma membrane of carotid body glomus cells (Fig. 4A). Thus, the spatial localization of AMPK in carotid body glomus cells would be FIGURE 3. AMPK activation by AICAR replicates hypoxic pulmonary vasoconstric- consistent with the regulation of plasma membrane-delimited pro- tion. Shown are pulmonary artery constriction in response to AMPK activation by AICAR 2 cesses such as voltage-gated Ca influx. Consistent with this proposal (1 mM) with (left panel) and without (right panel) the endothelium. A, with extracellular 2 2 and with the effects of hypoxia on carotid body glomus cells (10), AMPK Ca . B, without extracellular Ca (1mM EGTA). C, after preincubation of pulmonary arteries with caffeine (10 mM) and ryanodine (10 M). D, after preincubation with 8-bro- activation by AICAR (1 mM) induced an increase in intracellular Ca mo-cADPR (300 M). E, after preincubation with 8-bromo-cADPR (300 M) and submaxi- concentration in acutely isolated carotid body glomus cells at 22 °C, as mal preconstriction by K (20 mM). F, mean constriction  S.E. (n  3) of isolated pulmo- nary artery rings by AICAR (1 mM, left panel) and hypoxia (right panel) under the the Fura-2 fluorescence ratio increased by 0.07  0.02 (n  11). In conditions described for A–E. E, endothelium; RC, ryanodine and caffeine; 8Br, marked contrast to our findings on pulmonary arterial smooth muscle, 8-bromo-cADPR. this increase in intracellular Ca concentration was abolished by removal of extracellular Ca (n  6) and attenuated by blockade of and B) and hypoxia (16–21 T) (32), respectively, was abolished upon 2 2 transmembrane Ca influx pathways with Cd (100 M; removal of extracellular Ca (Fig. 3F). In contrast, constriction medi- 0.009  0.006, n  6) (Fig. 4, B and D(i)). Consistent with the effects of ated by mechanisms intrinsic to the smooth muscle was not, although it AICAR, phenformin (10 mM) increased the fluorescence ratio by is notable that this component of constriction was attenuated in the 0.272  0.087 (n  7) in a manner that was reversed by the removal of absence of extracellular Ca (Fig. 3, A, B, and F). Thus, maintained extracellular Ca (0.011  0.012, n  7) (Fig. 4D(i)). Thus, we can con- smooth muscle constriction by AICAR (1 mM) and hypoxia (16–21 T), clude that both phenformin and AICAR, similar to hypoxia, trigger respectively, exhibits a dependence (50%) on transmembrane Ca transmembrane Ca influx into isolated carotid body glomus cells. influx. In this respect, it is of major significance that blockade of SR Ca We then investigated the effect of AICAR alone on sensory afferent stores with caffeine (10 mM) and ryanodine (10 M) or blockade of discharge from the isolated carotid body in vitro to rule out any possible cADPR with 8-bromo-cADPR (100 M) (Fig. 3) completely abolished AMPK-independent actions that might result from inhibition of mito- the constriction of pulmonary arteries, with or without endothelium, by chondrial metabolism by phenformin. Under these conditions and at both AICAR (1 mM) (Fig. 3, C, D, and F) and hypoxia (16–21 T) (Fig. 3F). Thus, the partial dependence of smooth muscle constriction on extra- 37 °C, AICAR (1 mM) induced a relatively rapid and reversible increase 2 2 in single fiber sensory afferent discharge from the isolated carotid body cellular Ca must be due to SR store depletion-activated Ca influx/ store refilling, which is not mediated by AMPK activation by AICAR nor from 0.22  0.03 to 2.8  0.56 spikes s (n  19). This too was abol- hypoxia per se. Consistent with this observation, hypoxia has been ished by the removal of extracellular Ca (0.11  0.03, n  5) and was 2 2 2 shown to initiate SR store depletion-activated Ca influx indirectly by attenuated by blockade of transmembrane Ca influx with Cd (100 first mobilizing SR Ca stores in pulmonary arterial smooth muscle M; 0.91  0.18, n  5) (Fig. 4, C and D(ii)). Thus, AMPK activation 41508 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 50 •DECEMBER 16, 2005 2 AMPK and Ca Signaling in O -sensing Cells FIGURE 4. AMPK activation by AICAR activates carotid body glomus cells by eliciting trans- membrane Ca influx as does hypoxia. A(i), bright field image of a fixed carotid body glomus cell. (ii), Z-section showing staining by antibodies against AMPK-1(green), tyrosine hydroxylase (red), and staining for the nucleus by 4,6-dia- midino-2-phenylindole (blue). (iii), three-dimen- sional reconstruction. B, effect of AMPK activation by AICAR on Fura-2 fluorescence ratio (F340/F380) in an isolated carotid body glomus cell with and without extracellular Ca (1mM EGTA) and Cd (100 M). C, frequency histogram (upper panel) and record of action potential spikes (lower panel) show effect of AICAR on multiple fiber-affer- ent discharge from the carotid body in vitro with and without extracellular Ca (1mM EGTA). D, mean  S.E. for experiments in B and C. E, pro- posed model for cell-specific Ca signaling by AMPK in response to hypoxia. RyR, ryanodine receptor. reproduced the precise excitatory effects of hypoxia on the carotid body consequent increase in afferent fiber discharge (10, 33, 37, 52). We pro- (10, 37, 52). pose, therefore, that AMPK may act as a primary metabolic sensor and effector in O -sensing cells. This novel role for AMPK may therefore DISCUSSION 2 unite for the first time the mitochondrial and Ca signaling hypotheses Previous studies (2, 6–10) have established that hypoxia promotes for chemotransduction by hypoxia (18, 19). Before we can be certain of Ca -dependent pulmonary artery constriction and carotid body sen- a role for AMPK in this process, however, we need to demonstrate that sory afferent discharge, in part, by inhibiting oxidative phosphorylation cell activation by hypoxia can be inhibited by selective blocking of by mitochondria. Our findings now suggest that this leads to a rise in the AMPK. This may be achieved once the selective AMPK antagonist cellular AMP/ATP ratio, consequent AMPK activation, and the initia- Compound C (53, 54) becomes freely available or by the use of siRNA, tion of cell-specific Ca signaling mechanisms in pulmonary arterial which has yet to be applied successfully in studies on these wild-type smooth muscle and carotid body glomus cells (Fig. 4E). Thus, the char- cells. acteristic functional response of each tissue type to AMPK activation If confirmed, this process of activation would be exquisitely sensitive mirrors the primary aspects of the observed response to hypoxia: (a) to metabolic stress by hypoxia because of the triple mechanism by constriction of pulmonary arteries by cADPR-dependent SR Ca which AMPK is activated; adenylate kinase converts any rise in the release in the smooth muscle cells with a secondary component of con- cellular ADP/ATP ratio into a rise in the AMP/ATP ratio (25), whereas striction driven by the pulmonary arterial endothelium (32, 42, 43) and AMP binding to the  subunits of AMPK not only promotes phospho- (b) transmembrane Ca influx into carotid body glomus cells and a rylation by LKB1 but also inhibits dephosphorylation by protein phos- DECEMBER 16, 2005• VOLUME 280 • NUMBER 50 JOURNAL OF BIOLOGICAL CHEMISTRY 41509 2 AMPK and Ca Signaling in O -sensing Cells phatases and causes allosteric activation of the phosphorylated enzyme icity, and neuronal apoptosis (59) and ischemic damage to cardiac (20). 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Dec 5, 2005

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