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Phenformin and 5-aminoimidazole-4-carboxamide-1-D-ribofuranoside(AICAR) activation of AMP-activated protein kinase inhibits transepithelial
http://www.100md.com 《生理学报》 2005年第15期
     1 Department of Basic Medical Sciences, Physiology, St Georges' Hospital Medical School, University of London, Cranmer Terrace, Tooting, London SW17 0RE, UK

    2 Division of Molecular Physiology, Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK

    Abstract

    Active re-absorption of Na+ across the alveolar epithelium is essential to maintain lung fluid balance. Na+ entry at the luminal membrane is predominantly via the amiloride-sensitive Na+ channel (ENaC) down its electrochemical gradient. This gradient is generated and maintained by basolateral Na+ extrusion via Na+,K+-ATPase an energy-dependent process. Several kinases and factors that activate them are known to regulate these processes; however, the role of AMP-activated protein kinase (AMPK) in the lung is unknown. AMPK is an ultra-sensitive cellular energy sensor that monitors energy consumption and down-regulates ATP-consuming processes when activated. The biguanide phenformin has been shown to independently decrease ion transport processes, influence cellular metabolism and activate AMPK. The AMP mimetic drug 5-aminoimidazole-4-carboxamide-1-D-ribofuranoside (AICAR) also activates AMPK in intact cells. Western blotting revealed that both the 1 and 2 catalytic subunits of AMPK are present in Na+ transporting H441 human lung epithelial cells. Phenformin and AICAR increased AMPK activity in H441 cells in a dose-dependent fashion, stimulating the kinase maximally at 5–10 mM (P = 0.001, n = 3) and 2 mM (P < 0.005, n = 3), respectively. Both agents significantly decreased basal ion transport (measured as short circuit current) across H441 monolayers by approximately 50% compared with that of controls (P < 0.05, n = 4). Neither treatment altered the resistance of the monolayers. Phenformin and AICAR significantly reduced amiloride-sensitive transepithelial Na+ transport compared with controls (P < 0.05, n = 4). This was a result of both decreased Na+,K+-ATPase activity and amiloride-sensitive apical Na+ conductance. Transepithelial Na+ transport decreased with increasing concentrations of phenformin (0.1–10 mM) and showed a significant correlation with AMPK activity. Taken together, these results show that phenformin and AICAR suppress amiloride-sensitive Na+ transport across H441 cells via a pathway that includes activation of AMPK and inhibition of both apical Na+ entry through ENaC and basolateral Na+ extrusion via the Na+,K+-ATPase. These are the first studies to provide a cellular signalling mechanism for the action of phenformin on ion transport processes, and also the first studies showing AMPK as a regulator of Na+ absorption in the lung.
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    Introduction

    The regulation of Na+ absorption across lung epithelia is fundamentally important at both early developmental stages and throughout adult life (O'Brodovich, 1991). Transepithelial movement of Na+ provides an osmotic driving force for water movement from the luminal side of the epithelium to the interstitium. Na+ enters the epithelial cell via the amiloride-sensitive Na+ channel ENaC, located at the luminal membrane, and is then extruded basolaterally by the Na+,K+-ATPase, which modulates its activity to maintain a low intracellular Na+ concentration.
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    This process of active re-absorption consumes cellular energy, and as a measure of the importance of the Na+,K+-ATPase, it has been estimated that in resting humans, the pump hydrolyses roughly 25% of all cytoplasmic ATP (Lingrel & Kuntzweiler, 1994). Enzyme kinetic studies have shown that the Michaelis-Menton constant (Km) of the Na+,K+-ATPase for ATP is very low, indicating that only a drastic depletion of ATP would alter its activity (Boldyrev et al. 1991). Despite this, it has been well documented that epithelial Na+,K+-ATPase activity is impaired in response to hypoxia (Planes et al. 1996), even when cellular nucleotide levels are not likely to be significantly altered (Frederich et al. 2005). Therefore, it seems logical that in times of cellular stress (e.g. hypoxia, glucose deprivation) a more sensitive cellular response is activated capable of modulating the activity of the Na+,K+-ATPase accordingly.
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    AMP-activated protein kinase (AMPK) has been proposed to be an extremely sensitive sensor of cellular energy status, responding to subtle changes in the intracellular ADP: ATP ratio which has a much larger effect on the AMP: ATP ratio (Hardie & Hawley, 2001). Once activated by AMP binding and phosphorylation by an upstream kinase, AMPK phosphorylates and down-regulates key biosynthetic pathways/processes that consume ATP and up-regulates those that generate ATP (Hardie, 2003).
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    AMPK is also activated in intact cells by the biguanides; metformin (Zhou et al. 2001), which is commonly used in clinical practice to treat disease states associated with insulin resistance, and by its more potent analogue phenformin (Hawley et al. 2003). The biguanides are inhibitors of Complex I of the mitochondrial respiratory chain (Owen et al. 1995; El-Mir et al. 2000), indicating that they may activate AMPK by increasing the intracellular AMP: ATP ratio, although changes in cellular nucleotides have been difficult to detect, at least in the case of metformin (Fryer et al. 2002; Hawley et al. 2002). Since agents that inhibit the respiratory chain would be likely to have side-effects unrelated to AMPK activation, several groups have used the adenosine analogue 5-aminoimidazole-4-carboxamide-1-D-ribofuranoside (AICAR) to activate AMPK in intact cells. AICAR is taken up into the cell by adenosine transporters (Gadalla et al. 2004) and converted by adenosine kinase into the monophosphorylated nucleotide, ZMP, which mimics all of the effects of AMP on the AMPK system (Corton et al. 1995).
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    Although a recently identified target of AMPK is the cystic fibrosis transmembrane conductance regulator (CFTR) (Hallows et al. 2003), no reports exist as to whether transepithelial Na+ transport is regulated by AMPK in the lung. However, phenformin has been shown to inhibit ion transport processes in other tissues (Saito & Yoshida, 1984). Therefore, we hypothesized that the effects of phenformin are mediated via activation of the AMPK pathway and that this kinase is a regulator of Na+ absorption, the primary ion transport process in absorptive lung epithelia. Using both phenformin and AICAR as pharmacological tools, we have investigated the possibility that AMPK can be activated and modulate transepithelial Na+ transport in the human lung epithelial cell line H441.
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    Methods

    Cell culture

    H441 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (ISL, UK), 2 mM L-glutamine, 1 mM sodium pyruvate, 5 μg ml–1 insulin, 5 μg ml–1 transferrin, 10 nM sodium selinite and antibiotics (penicillin/streptomycin). Cells were seeded in 25 cm2 flasks and incubated in a humidified atmosphere of 5% CO2–95% O2 at 37°C.
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    Western blotting

    For analysis of endogenous expression of AMPK catalytic subunits (1 and 2), protein was prepared from H441 cells and rat liver by homogenization (positive control; Woods et al. 1996) in an ice-cold solution of tissue lysis buffer (100 mM Tris pH 6.8, 1 mM EDTA pH 8.0, 10% v/v glycerol) and 1% protease inhibitor cocktail (Sigma, UK). Total protein lysates were assayed for protein content (Bio-Rad, UK) and 100 μg of each was heated to 95°C for 10 min in the presence of sample buffer and reducing agent (Invitrogen, UK; according to the manufacturers' instructions). Denatured samples were subjected to electrophoresis on pre-cast SDS-polyacrylamide gels (4–12% Bis-Tris, Invitrogen, UK). Fractionated proteins were transferred to nitrocellulose membranes and immunostained with anti1/-2 AMPK antiserum (Professor D. G. Hardie, Dundee, UK), using standard techniques.
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    Measurement of AMPK activity

    H441 cells were treated with increasing concentrations of phenformin (0.1–10 mM) or AICAR (0.1–5 mM), to measure the concentration-dependent relationship to AMPK activity. The concentration ranges and treatment time used for both agents were based on a previous report on the use of these compounds to activate AMPK (Sakamoto et al. 2004). AMPK activity was measured as the rate at which radiolabelled phosphate was transferred from ATP to the SAMS peptide by AMPK. The SAMS peptide is derived from the sequence surrounding the major AMPK phosphorylation site on acetyl-CoA carboxylase (ACC). Phosphorylation of ACC was used as an indicator of AMPK activity in the intact cell since AMPK is the only kinase capable of phosphorylating this enzyme (Sakamoto et al. 2005). Cell lysates (10 μg) were incubated with 5 μl of protein G–Sepharose bound with an equimolar mixture of AMPK 1 and 2 antibodies for 2 h at 4°C. The beads were washed with 2 x 1 ml of ice-cold immunoprecipitation (IP) buffer (mM): Tris, 50 pH 7.4; NaCl, 150; NaF, 50; Na+ pyrophosphate, 5; EDTA, 1; EGTA, 1; dithiothreitol (DTT), 1; benzamidine, 0.1; phenylmethylsulphonylfluoride (PMSF), 0.1; 5 μg ml–1 soybean trypsin inhibitor (SBTI) and 1% v/v Triton X-100 (non-ionic surfactant)], then with 5 x 1 ml of ice-cold IP buffer containing 1 M NaCl. Thereafter the beads were washed 3 x 1 ml with assay buffer (50 mM Hepes pH 7.4, 1 mM DTT and 0.02% v/v Brij-35 (non-ionic surfactant). AMPK activity in the immunoprecipitates was determined by phosphorylation of the SAMS peptide as previously described (Davies et al. 1989). Western blotting of H441 cell lysates (10 μg) with a phosphospecific ACC antibody conjugated to IR680 fluorescent dye was used as a further indicator of AMPK activity. In addition, as cellular ACC is naturally biotinylated, blots were probed with streptavidin conjugated to IR800 fluorescent dye to control for protein loading. The membranes were analysed using an Odyssey IR imager.
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    Functional experiments

    Confluent flasks of H441 cells were seeded at 1: 12 confluent density on snapwell clear membranes (Costar Transwells; Corning, VWR, UK) and cultured overnight. The following day, the serum in the medium was replaced with 4% charcoal stripped serum (CSS) and the supplementations also included thyroxine (T3) 10 nM and dexamethasone (200 nM). Cells were cultured for 6 days at air interface. Snapwells supporting resistive monolayers of H441 cells (> 500 cm–2) were pre-treated in culture (apically and basolaterally) for 1 h with phenformin (0.1–10 mM) or 2 mM AICAR. Both drugs were solubilized in culture medium and untreated cells overlaid with vehicle control. Since the resistance and basal levels of ion transport across the monolayers varied considerably between batches of cells, all controls and treatments were plated on the same day from the same batch. Results are compiled from at least three sets of paired data. Monolayers were mounted in Ussing chambers where the drug was circulated in a physiological salt solution (mM): NaCl, 117; NaHCO3, 25; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; D-glucose, 11 (equilibrated with 5% CO2 to pH 7.3–7.4). The solution was maintained at 37°C, bubbled with 21% O2 + 5% CO2 pre-mixed gas, and continuously circulated throughout the course of the experiment. Control and drug-treated monolayers were analysed in parallel. The monolayers were firstly maintained under open circuit conditions whilst the transepithelial potential difference (Vt) and resistance (Rt) were monitored and observed to reach a stable level. The cells were then short circuited by clamping Vt at 0 mV using a DVC-4000 voltage/current clamp and the current required to maintain this condition (Isc) was measured and recorded using a PowerLab computer interface. Every 30 s, throughout each experiment, the preparations were returned to open circuit conditions for 3 s so that the spontaneous Vt could be measured and Rt calculated.
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    To determine the effects of phenformin or AICAR on amiloride-sensitive transepithelial Na+ transport, 10 μM amiloride (EC50: 0.7 μM; D. Baines, M. Dockrell, M. Hazell, S. Dunford and A. Wollhead, unpublished material) was added to the solution perfusing the apical side of the monolayer.

    To measure the activity of the basolateral pump, monolayers were apically permeabilized using 75 μM nystatin followed by blockade with 1 mM basolateral ouabain (Ramminger et al. 2000). To measure apical current, cells were firstly bathed in physiological salt solution to measure spontaneous transepihelial Isc. The physiological salt buffer was then replaced in both baths with aliquots of potassium gluconate solution (composition (mM): potassium gluconate, 121.7; KHCO3, 25; MgSO4, 1.2; KH2PO4, 1.2; calcium gluconate, 11.5; D-glucose, 11 (equilibrated with 5% CO2 to pH 7.3–7.4) (a dilution of 8.1: 91.9 physiological salt solution: potassium gluconate solution)) so that the Na+ concentration of the bathing solution was reduced to 11.5 mM. When Isc had reached a new stable level pump currents were inhibited with 1 mM ouabain and the basolateral membrane was permeabilized with 75 μM nystatin. Using a sodium gluconate solution (composition (mM): sodium gluconate, 117; NaHCO3, 25; potassium gluconate, 4.7; MgSO4, 1.2; KH2PO4, 1.2; calcium gluconate, 2.5; D-glucose, 11 (equilibrated with 5% CO2 to pH 7.3–7.4) diluted 91.9: 8.1 with physiological salt solution), the concentration of Na+ in the apical chamber was then raised to 55 mM to create a gradient for Na+ influx across the apical membrane. Amiloride (10 μM) was added to the apical bath to inhibit currents and GNa was then esimated from the amiloride-sensitive apical current (Iap) using the equation GNa = Iap/VNa where VNa is the driving force for Na+ entry where transepithelial potential difference (Vt) = 0 and the equilibrium potential for Na+ (ENa) = 41.8 mV (Collet et al. 2002; Ramminger et al. 2004). At the end of each experiment the cell monolayers were rinsed in ice-cold PBS and harvested into the following buffer (mM): Tris, 50 (pH 7.4); NaCl, 150; NaF, 50; sodium pyrophosphate, 5; EDTA, 1; EGTA, 1; DTT, 1; 1% v/v Triton X-100 and 1% protease inhibitor cocktail (Sigma, UK). H441 cell suspensions were then stored at –80°C until required.
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    Statistical analysis was carried out using Student's paired t tests where P values of < 0.05 were considered significant. Results are presented as mean ± S.E.M.

    Results

    Expression of AMPK catalytic subunit proteins in H441 cells

    Western blotting of cell extracts showed specific immunostained products for the two catalytic subunits of AMPK (1 and 2) in H441 cells and liver homogenate at 63 kDa (Fig. 1).
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    Typical Western blot of proteins extracted from H441 cells and liver homogenate (positive control). Blots were immunostained with anti-1 and 2 AMPK antisera. Protein products of 63 kDa, corresponding to 1 and 2 proteins, are indicated by the arrow.

    Effects of increasing concentrations of phenformin or AICAR on AMPK activity in H441 cells

    In order to test whether AMPK was active in H441 cells and whether its activity could be modified as seen in other lung epithelial cell types (Hallows et al. 2003), cells were treated with phenformin or AICAR. We found H441 cells to have low levels of endogenous AMPK activity that could be stimulated by increasing concentrations of phenformin or AICAR (Figs 2 and 3A). Activity of the kinase doubled after treatment with both agents at a concentration of 0.5 mM (phenformin, 0.2 ± 0.002–0.4 ± 0.008 nmol min–1 mg–1; AICAR, 0.1 ± 0.003–0.2 ± 0.02 nmol min–1 mg–1, P = 0.005, n = 3), and was stimulated maximally after treatment with 5–10 mM phenformin (0.5 ± 0.01 nmol min–1 mg–1, P < 0.001, n = 3) and 2 mM AICAR (0.3 ± 0.05 nmol min–1 mg–1, P = 0.005, n = 3), respectively. This dose-dependent rise in AMPK activity correlated with increased phosphorylation of acetyl-CoA carboxylase (ACC) as shown by Western blotting using a phosphospecific antibody against the major AMPK site, with no change in the amount of total ACC, determined by probing the same blots with streptavidin (Figs 2 and 3B and C).
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    H441 cells were treated in culture for 1 h with 0.1–10 mM phenformin. A, AMPK activity was measured as the rate of phosphorylation of the SAMS peptide (nmol min–1 mg–1). B, ACC phosphorylation; representative Western blot of phosphorylated ACC (using a phosphospecific antibody) from H441 cell protein as an indicator of intracellular AMPK activity. C, total ACC protein; representative Western blot of total ACC from H441 cell protein (visualised by streptavidin binding). Results are shown as mean ± S.E.M. *Significantly different from control, **significantly different from 1 mM (P < 0.05, n = 3).
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    H441 cells were treated in culture for 1 h with 0.1–5 mM AICAR. A, AMPK activity was measured as the rate of phosphorylation of the SAMS peptide (nmol min–1 mg–1). B, ACC phosphorylation; representative Western blot of phosphorylated ACC (using a phosphospecific antibody) from H441 cell protein as an indicator of intracellular AMPK activity. C, total ACC protein; representative Western blot of total ACC from H441 cell protein (visualised by streptavidin binding). Results are shown as mean ± S.E.M. *Significantly different from control, **significantly different from 1 mM (P < 0.05, n = 3).
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    Effects of phenformin or AICAR on the electrical properties of H441 cell monolayers

    Treatment with 10 mM phenformin (Fig. 4A and E) or 2 mM AICAR (Fig. 5A and E) resulted in at least 50% reduction in spontaneous short circuit current (Isc) compared with that of controls (P < 0.05, n = 4) (Table 1). This was not the result of differences in the resistive properties between treated and untreated monolayers (P > 0.5, n = 4) (Table 1).
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    A, spontaneous short circuit current (ISC) was measured at the start of each experiment. *Significantly different from control, P < 0.05, n = 4. B, amiloride-sensitive Isc was calculated after apical application of 10 μM amiloride (Iamiloride). *Significantly different from control (P < 0.05, n = 4). C, Na+,K+-ATPase activity was determined by apical permeabilization with 75 μM nystatin then basolateral blockade with 1 mM ouabain (Iouabain). *Significantly different from control (P < 0.05, n = 4). Results are shown as mean ± S.E.M.D, AMPK activity of monolayers was determined from control and 10 mM phenformin-treated cells. ***Significantly different from control (P = 0.001, n = 3). E, typical continuous Isc traces from control and phenformin-treated monolayers. Arrows indicate the point of application of each drug.
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    A, spontaneous short circuit current (ISC) was measured at the start of each experiment. *Significantly different from control (P < 0.05, n = 4). B, amiloride-sensitive ISC was calculated after apical application of 10 μM amiloride (Iamiloride). *Significantly different from control (P < 0.05, n = 4). C, Na+,K+-ATPase activity was determined by apical permeabilization with 75 μM nystatin then basolateral blockade with 1 mM ouabain (Iouabain). *Significantly different from control (P = 0.05, n = 4). D, AMPK activity of monolayers was determined from control and 2 mM AICAR-treated cells. *Significantly different from control (P < 0.05, n = 4). Results are shown as mean ± S.E.M.E, typical continuous ISC traces from control and AICAR-treated monolayers. Arrows indicate the point of application of each drug.
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    Effects of phenformin or AICAR on the amiloride-sensitive component of transepithelial Isc

    Amiloride-sensitive transepithelial Isc (Iamiloride) was calculated from the fall in Isc following apical application of amiloride. Treatment with either 10 mM phenformin (Fig. 4B and E) or 2 mM AICAR (Fig. 5B and E) reduced the amiloride-sensitive component of Isc (Iamiloride) by at least 50% compared with that of untreated controls (P < 0.05, n = 4) (Table 1).
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    Effects of phenformin or AICAR on Na+, K+-ATPase activity

    Na+,K+-ATPase activity was determined firstly by apical permeabilization with nystatin, which allowed ions to flood into the cell, increasing the turnover of the pump. Once this reached a maximal level, attributable to the ability of the pump to remove Na+ from the cell, basolateral blockade with ouabain resulted in current decline to baseline levels allowing ouabain-sensitive current (Iouabain) to be calculated. This value was reduced by > 80% and 50% in monolayers treated with 10 mM phenformin (Fig. 4C and E) or 2 mM AICAR (Fig. 5C and E), respectively, when compared with that of controls (P < 0.05, n = 4) (Table 1).
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    Effects of 10 mM phenformin or 2 mM AICAR on AMPK activity in H441 cell monolayers

    At the end of each functional experiment, monolayers were assayed for AMPK activity to establish any correlation with functional response. Compared with controls, AMPK activity rose 3.6-fold (0.5 ± 0.1 to 1.8 ± 0.2 nmol min–1 mg–1, P = 0.001, n = 3) with phenformin and 2.4-fold (0.1 ± 0.01 to 0.3 ± 0.05 nmol min–1 mg–1, P < 0.005, n = 3) with AICAR (Figs 4D and 5D, respectively; Table 1).
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    Effects of increasing concentrations of phenformin on H441 cell monolayers

    H441 cell monolayers were exposed to increasing concentrations of phenformin ranging from 0.1 to 10 mM (Fig. 6A). At 0.1 mM phenformin caused a significant reduction in Na+,K+-ATPase activity from 28.0 ± 3.5 to 11.3 ± 4.7 μA cm–2 (P < 0.05, n = 3), without affecting the amiloride-sensitive transepithelial Isc (Iamiloride) in these cells (untreated, 32.7 ± 4.9 μA cm–2; treated, 28.7 ± 13.7 μA cm–2, P > 0.5, n = 3). However, 0.5 mM phenformin significantly decreased both Iamiloride as well as Na+ extrusion via the pump (Iouabain) (P < 0.05, n = 3). At 5 mM phenformin reduced Iamiloride and Iouabain to an even greater extent (Iamiloride, 32.7 ± 4.9 μA cm2 (untreated) to 7.8 ± 3.2 μA cm2 (treated); and Iouabain, 28.0 ± 3.5 μA cm2 (untreated) to 4.7 ± 1.0 μA cm2 (treated), P < 0.05, n = 3). There was no further significant change in function in monolayers treated with 10 mM phenformin (P > 0.5, n = 3) (Fig. 6A), indicating that, functionally, the detrimental effects of this drug on transepithelial Na+ were maximal at 5 mM. These findings significantly correlated with the AMPK activity seen within this concentration range, where no further increase was observed at concentrations in excess of 5 mM phenformin (Fig. 2A).
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    A, amiloride-sensitive ISC (Iamiloride) was calculated after apical application of 10 μM amiloride. Significantly different from control *P < 0.05, **P < 0.005, n = 3, ; not significantly different from 5 mM (P > 0.05, n = 3). Na+,K+-ATPase activity (Iouabain) was determined by apical permeabilization with 75 μM nystatin then pump blockade with 1 mM ouabain. Significantly different from control *P < 0.05, **P < 0.005, n = 3, ; not significantly different from 5 mM (P > 0.05, n = 3). Results are shown as mean ± S.E.M.B, AMPK activity was plotted against Iamiloride. C, AMPK activity was plotted against Iouabain. r2 values are shown at the top right of each graph.
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    Association between AMPK activity and Isc in H441 cell monolayers

    There was a significant relationship between AMPK activity and transepithelial Na+ transport in H441 cell monolayers. We obtained correlation values of r2 equal to 0.95 and 0.98 for AMPK activity plotted against Iamiloride and Iouabain, respectively (Fig. 6B and C). However, the relationship between AMPK activity and Iouabain and AMPK and transepithelial Na+ current Iamiloride was different. As pump currents generally exceeded transepithelial Na+ currents in our preparation and as apical entry of Na+ is considered the rate-limiting step in transepithelial Na+ transport we speculated that the differences could be reflective of AMPK-induced changes to the apical conductive pathway.
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    Effect of phenformin and AICAR on amiloride-sensitive apical Na+ conductance (GNa)

    Phenformin (5 mM) significantly reduced amiloride-sensitive apical Na+ conductance (GNa) of H441 cells from 207 ± 6 to 63 ± 9 μS cm–2, P < 0.05, n = 3 (Fig. 7A and C). Similarly 2 mM AICAR also reduced apical conductance from 238 ± 12 to 120 ± 3 μS cm–2, P < 0.01, n = 3 (Fig. 7B and D).

    A, apical Na+ conductance was determined in control and 5 mM phenformin-treated cells. **Significantly different from control, P < 0.01, n = 3. B, apical Na+ conductance was determined in control and 2 mM AICAR-treated cells. *Significantly different from control, P < 0.05, n = 3. Typical continuous apical current (Iap) traces from control and 5 mM phenformin- (C) or control and 2 mM AICAR-treated cells (D) bathed in a potassium gluconate solution with 11.5 mM Na+ (see Methods). Basolateral membranes were permeabilised with nystatin (75 μM) and the Na+ concentration of the apical bath raised to 55 mM to create a driving force for Na+ influx (Na+). Amiloride was then added to the apical bath (amiloride) to determine the amiloride-sensitive GNa. Arrows indicate the point of application of each drug.
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    Discussion

    AMPK mediates its effects by phosphorylation and inhibition of several important rate-limiting biosynthetic enzymes, thereby acting to preserve cellular ATP levels during metabolic imbalance (Hardie & Carling, 1997). Phenformin, a known activator of AMPK, has previously been reported to inhibit Na+ transport in epithelial tissue (Saito & Yoshida, 1984). Therefore, we hypothesized that activation of AMPK would decrease amiloride-sensitive Na+ transport in H441 cells since it is an ATP-consuming process and that the mechanism of phenformin action on ion transport would involve activation of AMPK. In support of our hypotheses, we have demonstrated that the two catalytic subunits of AMPK (1 and 2) are endogenously expressed in H441 cells. Dose-dependent activation of AMPK with phenformin or AICAR, leads to a significant reduction in transepithelial Na+ transport by a mechanism that includes a reduction in ouabain-sensitive Na+,K+-ATPase activity and amiloride-sensitive apical conductance via the amiloride-sensitive sodium channel ENaC. Furthermore, exposure of H441 cell monolayers to increasing concentrations of phenformin showed a significant correlation between short circuit current (Isc) and AMPK activity. Whilst AMPK activation has been described to have functional implications in the transport of ions in lung submucosal cells (Hallows et al. 2003) and kidney epithelial cells (Carrattino et al. 2005), this is the first description that both Na+ entry and extrusion pathways are regulated targets of AMPK in intact lung epithelial cells. Furthermore, our studies show that activation of AMPK present at physiological levels within the cell (rather than by exogenous over-expression of mutant active AMPK protein or by co-expression with ion channel proteins in oocytes) can have a significant inhibitory effect on Na+ transport processes.
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    We have shown that both phenformin and AICAR activate AMPK to a similar degree (2- to 4-fold maximal stimulation) in H441 cells, consistent with their effects in other tissues (Sakamoto et al. 2004). Phenformin is thought to target complex I of the respiratory chain, thus compromising the ATP level of the cell and raising the AMP: ATP ratio (El-Mir et al. 2000; Owen et al. 2000). Since H441 cells are a tumour-derived cell line, they may be less dependent than primary cells on the respiratory chain to generate ATP, which might explain the need for such a high concentration of phenformin to stimulate the kinase maximally. On the other hand, AICAR activates AMPK by being converted into an AMP mimetic (ZMP) without effect on the cellular levels of AMP, ADP or ATP (Corton et al. 1995; Henin et al. 1995). Therefore, as both agents elicited similar effects on AMPK activity and ion transport, it is unlikely that phenformin elicits its effect by compromising ATP availability. As an AMP mimetic drug, it is possible that AICAR could mediate changes in ion transport by activating adenosine receptors present in airway epithelial cells. A2B receptors have been reported to be present in mouse trachea and human bronchiolar epithelial cells (Lazarowski et al. 2004; Kornerup et al. 2005). However, in mouse trachea, adenosine activation of these receptors caused an increase in Isc and in human airway cells adenosine decreased the volume of airway surface liquid which would be consistent with a decrease in Cl– secretion and/or an increase in Na+ absorption. In our cells, AICAR had the opposing effect which would infer that the action of AICAR was not via adenosine receptors. We also observed that the downstream reduction in transepithelial Na+ transport was more profound in phenformin-treated cells compared with those treated with AICAR. A possible explanation for this could be additional non-specific effects of this drug. In contrast to AICAR, phenformin has been shown to alter the fluidity and surface charge of phospholipid membranes (Schafer, 1976), which could have additional effects on Na+,K+-ATPase and ENaC function (Boldyrev et al. 1991).
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    The role of kinases such as the serum- and glucocorticoid-inducible kinase (SGK), protein kinase A (PKA) and mitogen activated protein kinase (MAPK), in the regulation of ion transport, has been studied using H441 cells as a model of human lung epithelium (Baines et al. 2005; McDonald & Welsh, 1995; Naray-Fejes-Toth et al. 2000; Thomas et al. 2004; Ramminger et al. 2004). Consistent with these studies, H441 cells formed monolayers with transepithelial resistance (Rt) > 500 cm–2 in the culture conditions used in this study. Furthermore, we have previously shown that these cells exhibit amiloride-sensitive Na+ currents that constitute > 90% of total ion transport and exhibit an EC50 for amiloride of 0.7 μM (D. Baines, M. Dockrell, M. Hazell, S. Duntard & A. Woollhead, unpublished observations). Our finding that phenformin and AICAR decreased spontaneous, amiloride-sensitive Isc (Iamiloride), Na+,K+-ATPase activity (Iouabain) and GNa, has not previously been described in lung epithelial cells (Fig. 8). In frog skin, phenformin modulated Na+ transport and similarly inhibited both amiloride-sensitive Na+ entry and basolateral Na+ flux (Saito & Yoshida, 1984) but the pathway by which phenformin elicited this effect was not described. We now show that the mechanism of action of phenformin on Na+ transport processes includes activation of AMPK. However, whilst we found 0.5 mM phenformin inhibited transepithelial Na+ transport in lung cells, Saito and coworkers found that concentrations up to 2 mM stimulated Na+ transport in frog skin. This disparity may reflect regulatory differences between the tissues and/or experimental techniques between the two studies (Saito & Yoshida, 1984). Consistent with our observations, phenformin has also been shown to inhibit Na+, K+-ATPase activity in isolated rat liver membranes (Luly et al. 1977). There is very little reported work on the effect of AICAR on ion transport in lung epithelial cells, although it has been reported to down-regulate CFTR activity in airway submucosal cells (Hallows et al. 2003). We also cannot exclude effects of phenformin or AICAR on K+ current flow which is required for transepithelial transport (Fig. 8). However, in the permeabilization studies there was free movement of K+ ions across the permeabilized membranes indicating that both the pump activity and apical entry pathways were decreased independently of K+ current flow.
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    Na+ entry at the luminal membrane is predominantly via the amiloride-sensitive Na+ channel (ENaC) down its electrochemical gradient. This gradient is generated and maintained by basolateral Na+ extrusion via the ATP-consuming Na+,K+-ATPase. Phosphorylation of the pump as a result of ATP hydrolysis to ADP, catalyses the transport of 3 intracellular Na+ ions to the interstitium in exchange for 2 K+ ions. K+ is recycled across the basolateral membrane. Addition of 10 μM amiloride to the luminal side of the epithelium blocks Na+ entry through ENaC. Basolateral application of 1 mM ouabain blocks Na+,K+-ATPase function. Treatment with phenformin or AICAR results in suppression of transepithelial Na+ transport via a pathway that includes activation of AMPK and inhibition of both apical Na+ entry through ENaC and basolateral Na+ extrusion via the Na+,K+-ATPase.
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    Whilst earlier studies have investigated the effects of these agents on ion transport processes, there has been no previous correlation between transepithelial Na+ transport and AMPK activation. We have now shown that there is a significant correlation between AMPK activity and Iamiloride and Iouabain. Exactly how AMPK down-regulates amiloride-sensitive Na+ transport by inhibiting Na+ entry and Na+,K+-ATPase activity is as yet unknown although a similar phenomenon has been described where hypoxia decreases both pump activity and the apical Na+ entry pathways (Ramminger et al. 2000). Decreased pump activity could increase the intracellular Na+ concentration reducing the driving force for Na+ entry into H441 cells, and raised intracellular Na+ has been reported to decrease the conductance of ENaC (Cook et al. 2002). The significant effects on Na+ transport processes we describe after a 1 h incubation with phenformin and AICAR indicate that these effects are unlikely to be mediated by transcriptional changes. Furthermore, as we have detected inhibitory effects of phenformin on Iamiloride after a 10 min treatment (data not shown), our findings support a post-transcriptional mechanism for AMPK action. The effect of AMPK on apical GNa we describe is corroborated by a recent study where activation of AMPK co-expressed with ENaC in oocytes or mutant active AMPK over-expressed in renal epithelial cells lead to a decrease in Na+ transport (Carattino et al. 2005). We have demonstrated ENaC protein in H441 cells by Western blotting and mRNAs encoding all three subunits of ENaC are expressed in these cells (Ramminger et al. 2004). Expression of Na+, K+-ATPase proteins is tissue specific but it is generally considered that 1 and 1 Na+,K+-ATPase proteins are present in lung epithelial cells (Orlowski & Lingrel, 1988). There are putative consensus sequences for AMPK phosphorylation on , and ENaC, 1 and 1 Na+, K+-ATPase subunit proteins. However, Carattino et al. (2005) were unable to show an association of AMPK with ENaC subunit proteins, phosphoryation of ENaC proteins by AMPK or a change in open probability (Po) as a mechanism for Na+ transport inhibition. They inferred that activation of AMPK may therefore alter the number of ENaC channels at the apical membrane. No mechanisms of action for AMPK on Na+, K+-ATPase has been described, although a previous study has demonstrated that protein kinase C (PKC)-mediated phosphorylation of the pump decreases its activity by removing it from the basolateral membrane via internalization (Chibalin et al. 1999). Thus, AMPK may act by altering insertion and retrieval of ENaC and pump subunit proteins in the membrane. We are currently investigating these possibilities. Furthermore, the effect of chronic application of phenformin and AICAR and the effect of sustained AMPK activation on lung epithelial ion transport (which could evoke genomic changes) have yet to be investigated.
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    In conclusion, we have shown that the effects of phenformin on ion transport are primarily mediated by activation of AMPK. Furthermore, we have shown for the first time that activation of endogenous AMPK in a physiological lung cell system decreases amiloride-sensitive transepithelial Na+ transport by decreasing both Na+ entry and extrusion pathways. We propose that as a metabolic sensor in cells, AMPK may be important in fine tuning both Na+,K+-ATPase and amiloride-sensitive Na+ channel (ENaC) activity thereby providing a link between cellular metabolism and amiloride-sensitive Na+ transport in the lung. Understanding the role of AMPK in the regulation of epithelial Na+ transport could also have important clinical implications for the use of current therapies which are known activators of AMPK.
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    Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J et al. (2001). Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 108, 1167–1174., http://www.100md.com(Alison M Woollhead, John )