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AICAR decreases the activity of two distinct amiloride-sensitive Na⁺-permeable channels in H441 human lung epithelial cell monolayers

¹Centre for Ion Channels and Cell Signalling, Division of Basic Medical Sciences, Saint George's, University of London, London; and ²School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, United Kingdom

Submitted 18 June 2008 ; accepted in final form 19 August 2008

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Transepithelial transport of Na⁺ across the lung epithelium via amiloride-sensitive Na⁺ channels (ENaC) regulates fluid volume in the lung lumen. Activators of AMP-activated protein kinase (AMPK), the adenosine monophosphate mimetic AICAR, and the biguanide metformin decreased amiloride-sensitive apical Na⁺ conductance (G_Na+) in human H441 airway epithelial cell monolayers. Cell-attached patch-clamp recordings identified two distinct constitutively active cation channels in the apical membrane that were likely to contribute to G_Na+: a 5-pS highly Na⁺ selective ENaC-like channel (HSC) and an 18-pS nonselective cation channel (NSC). Substituting NaCl with NMDG-Cl in the patch pipette solution shifted the reversal potentials of HSC and NSC, respectively, from +23 mV to –38 mV and 0 mV to –35 mV. Amiloride at 1 µM inhibited HSC activity and 56% of short-circuit current (I_sc), whereas 10 µM amiloride partially reduced NSC activity and inhibited a further 30% of I_sc. Neither conductance was associated with CNG channels as there was no effect of 10 µM pimoside on I_sc, HSC, or NSC activity, and 8-bromo-cGMP (0.3–0.1 mM) did not induce or increase HSC or NSC activity. Pretreatment of H441 monolayers with 2 mM AICAR inhibited HSC/NSC activity by 90%, and this effect was reversed by the AMPK inhibitor Compound C. All three ENaC proteins were identified in the apical membrane of H441 monolayers, but no change in their abundance was detected after treatment with AICAR. In conclusion, activation of AMPK with AICAR in H441 cell monolayers is associated with inhibition of two distinct amiloride-sensitive Na⁺-permeable channels by a mechanism that likely reduces channel open probability.

5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; AMP-activated protein kinase; ENaC

Human H441 Clara-like airway epithelial cells, cultured as monolayers at air interface, exhibit a number of similarities to primary cultured human airway epithelial cells and in vivo human airway (2, 6, 20). Therefore, they have been used by ourselves and a number of other researchers as a model of absorptive human airway (24, 34, 36–38).

We have previously shown that the AMP mimetic drug 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside [AICAR; which is converted to (AICA)-riboside or ZMP on entering the cell] activated AMP-activated protein kinase (AMPK) and inhibited amiloride-sensitive transepithelial Na⁺ transport and apical Na⁺ entry through amiloride-sensitive Na⁺ channels in H441 lung epithelial monolayers (37, 38). The action of AICAR on Na⁺ transport was reversed by Compound C, an inhibitor of AMPK, supporting a role for AMPK in the regulation of amiloride-sensitive Na⁺ channels. Metformin also had a similar effect on transepithelial Na⁺ transport, but its effect on apical Na⁺ conductance (G_Na+) has not yet been described.

Two populations of Na⁺-permeable cation channels have been identified in the membrane of cultured H441 cells, alveolar type I and type II cells that could contribute to apical G_Na+ (9, 17, 18, 24), and these have been described as a highly Na⁺ selective channel (HSC) and a nonselective cation channel (NSC). The relative expression of these channels in alveolar type II is determined by culture conditions (17, 24). Whether both channels contribute to apical G_Na+ and which channels are targeted by activation of AMPK in H441 cell monolayers is unknown.

To address these questions, we have investigated the effect of metformin on apical G_Na+ and examined the properties of two cation channel currents present in the apical membrane of H441 cells grown as monolayers. In addition, the present work investigates the effect of AICAR activation of AMPK on the activity of these cation channels.

	MATERIALS AND METHODS

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Cell culture. H441 cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI-1640 medium supplemented with FBS (10%; Invitrogen, Paisley, UK), L-glutamine (2 mM), sodium pyruvate (1 mM), insulin (5 µg/ml^–1), transferrin (5 µg/ml^–1), sodium selinite (7 ng/ml^–1), and the antibiotics penicillin (100 U/ml^–1) and streptomycin (100 µg/ml^–1) (Invitrogen). Cells were seeded in 25-cm² flasks and incubated in a humidified atmosphere with 5% CO₂ at 37°C.

Measurement of short-circuit current. H441 cells were seeded on snapwell clear membranes (Snapwell, Corning, VWR, Lutterworth, UK) and cultured overnight. The following day (or when fully confluent), the serum in the medium was replaced with 4% charcoal-stripped serum and the additional supplement of T3 (10 nM) and dexamethasone (200 nM). Cells were cultured for at least 7 days at air interface as previously described (37). Snapwells supporting resistive monolayers of H441 cells (200 /cm²) were treated in culture with the AMPK activators AICAR (2 mM) or metformin (2 mM) for 1 and 4 h, respectively. In some experiments, monolayers were pretreated with the AMPK inhibitor Compound C (Calbiochem, 80 µM) before treatment with AICAR. All agents were suspended in culture medium or as a 1,000x concentrated stock in DMSO and stored at –20°C. Untreated cells were overlaid with culture medium alone or treated with vehicle control. Monolayers were mounted in Ussing chambers where the drug was circulated in a physiological salt solution (PSS; in mM): 117 NaCl, 25 NaHCO₃, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 2.5 CaCl₂, and 11 D-glucose (pH 7.3–7.4). The solution was maintained at 37°C, bubbled with 21% O₂ + 5% CO₂ premixed gas, and continuously circulated throughout the course of the experiment. The monolayers were first maintained under open-circuit conditions while the transepithelial potential difference (V_t) and resistance (R_t) were monitored and observed to reach a stable level. The cells were then short circuited by clamping V_t at 0 mV using a DVC-4000 voltage/current clamp, and the current required to maintain this condition (I_sc) 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 V_t could be measured and R_t calculated. Control and drug-treated monolayers were analyzed in parallel. Amiloride (10 µM) and L-cis diltiazem (50 µM) were suspended in PSS. Pimozide {1-[1-(4,4-bis[4-fluorophenyl]butyl)-4-piperidinyl]-4,3-dihydro-2H-benz-imidazol-2-one, 150 µM} was first suspended in DMSO before dilution in salt solution. Experimentally induced changes in I_sc resulting from pimozide were therefore compared with changes induced by similar concentrations of DMSO alone.

Apical Na⁺ conductance was measured as previously described (10, 37). Briefly, the physiological salt buffer was replaced in both baths with K⁺-gluconate solution (in mM): 117 K⁺-gluconate, 25 KHCO₃, 1.2 MgSO₄, 1.2 KH₂PO₄, 11.5 Ca-gluconate, and 11 D-glucose (pH 7.3–7.4) to reduce the Na⁺ concentration of the bathing solution (11.5 mM). Na⁺-K⁺-ATPase activity was inhibited with 1 mM ouabain, and the basolateral membrane was permeabilized with 75 µM nystatin. The concentration of Na⁺ in the apical chamber was then raised to 47 mM to create a gradient for Na⁺ influx across the apical membrane using a Na⁺ gluconate solution (in mM): 117 Na-gluconate, 25 NaHCO₃, 4.7 K⁺-gluconate, 1.2 MgSO₄, 1.2 KH₂PO₄, 2.5 Ca-gluconate, and 11 D-glucose (pH 7.3–7.4). Na⁺ flux across the apical membrane was then inhibited with 10 µM amiloride added to the apical bath. Liquid junction potentials arising from the use of these solutions were compensated for in our recordings (3, 10), and G_Na+ was estimated from the amiloride-sensitive apical current as previously described (37).

Single channel current recording. Single cation channel currents were recorded from H441 cell monolayers with an AXOpatch 200B patch-clamp amplifier (Axon Instruments, Union City, CA) at room temperature (20–25°C) using the cell-attached configuration of the patch-clamp technique. In experiments using cell-attached patches, to set the membrane potential at 0 mV, H441 monolayer cells were perfused with a bathing solution containing (mM): 145 KCl, 1.8 CaCl₂, 2 MgCl₂, 5.5 glucose, and 10 HEPES, pH 7.4 with KOH. In inside-out experiments, the bathing (intracellular) solution contained a similar solution except that 0.48 CaCl₂, 1 BAPTA (free Ca²⁺ set at 100 nM), 1 Na₂ATP, and 0.2 NaGTP were added. The standard patch pipette solution contained (mM): 145 NaCl, 1.8 CaCl₂, 2 MgCl₂, 5.5 glucose, and 10 HEPES, pH 7.4 with NaOH; with this solution, patch pipette resistances were 10–15 M. In ion substitution experiments, NaCl was replaced with equimolar concentrations of NMDG-Cl (145 mM NMDG/145 mM HCl), and to investigate the amiloride sensitivity of cation channel activity, 1 µM or 10 µM amiloride was included in the standard patch pipette solution. In some experiments, 10 mM NaCl was included in the bathing solution to provide a similar extracellular-to-intracellular Na⁺ concentration, although this had no appreciable effect on unitary conductance or reversal potential of the cation channel currents.

Cation channel currents were recorded and analyzed using pCLAMP software (version 9.0, Axon Instruments) whereby channel activity was sampled at between 1 and 2 kHz and low-pass filtered between 100 and 200 Hz, and then idealized channel openings were created using a 50% threshold method. Open times <3.332–6.664 ms (2x rise time of low-pass filter) were excluded to reduce the number of channel currents not reaching full amplitude. The amplitude of single channel currents was determined by plotting histograms of number of observations vs. channel current amplitudes. These were then were fitted with Gaussian curves, and the peak values were used as a measure of channel amplitude. It should be noted that these plots are not all-point histograms and therefore do not relate to the level of channel open or closed times. Open probability (NP_o) was calculated using the equation

where n = number of channels in the patch, O_n = time spent at each open level, and T = total recording time. To produce representative NP_o values and amplitude histograms, single channels were analyzed from at least 60 s of original data. Permeability ratios of NMDG to Na (P_NMDG/P_Na) were calculated from E_r = RT/zF In P_B [B]_o/P_A [A]_o. The ion channel inhibitors amiloride and pimozide were made up as described above.

Western blotting of apical biotinylated proteins. The apical membranes of cells grown as monolayers were biotinylated using 0.5 mg/ml S-S-biotin (Pierce) in borate buffer (mM) (85 NaCl, 4 KCl, 15 Na2B4O7, pH 9) for 20 min as previously described (37). Briefly, the reaction was stopped by adding 10% FBS in PSS (see above). Monolayers were then washed with ice-cold PSS, and cells were lysed with a solution containing 0.4% deoxycholate, 1% Triton X-100, 50 mM EGTA, 10 mM Tris·Cl, pH 7.4, and 1% protease inhibitors for 10 min at room temperature. Nonsolubilized protein was removed by centrifugation. Similar concentrations of protein from treated and untreated samples were incubated overnight at 4°C with immobilized streptavidin beads (Pierce). The following day, the beads were washed and the bound protein eluted in NuPAGE sample buffer (Invitrogen). Samples were heated at 95°C for 5 min, fractionated on SDS-PAGE gels, and immunoblotted with anti-ENaC NH₂-terminal, β-actin (Abcam, Cambridge, UK), ENaC COOH-terminal (sc22239; Santa Cruz Biotechnology), βENaC COOH-terminal (sc22242; Santa Cruz), or ENaC NH₂-terminal (ENACg31-A; Alpha Diagnostics). All primary antisera were used at 1:500 dilution except for anti-ENaC, which was used at 1:200. Immunostained proteins were visualized using ECL (Amersham Biosciences, Amersham, UK).

Reagents. All reagents were obtained from Sigma (Poole, UK) unless otherwise stated.

Data analysis and statistical procedures. Since the resistance and basal levels of ion transport across the monolayers varied between batches of cells, control experiments and treatments were carried out on a set of monolayers that were plated on the same day and cultured similarly for the same amount of time. Results are compiled from at least three independent sets of cells containing controls and treatments. Data are presented as means ± SE. Statistical analysis was carried out using one-way-ANOVA with a post hoc Gabriel pairwise test (22) or Student's t-tests where applicable; P values of <0.05 were considered significant.

	RESULTS

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AICAR and metformin inhibit apical G_Na+ in H441 cell monolayers. We have previously shown that treatment with AICAR for 1 h and metformin for 4 h decreased transepithelial amiloride-sensitive Na⁺ conductance but had no significant effect on R_t. Consistent with previous experiments, AICAR reduced apical amiloride-sensitive G_Na+ across H441 cell monolayers from 301 ± 47 to 149 ± 33 µS/cm^–2, P = 0.01, n = 3, a 49% inhibition (Fig. 1). Metformin also reduced apical conductance to 206 ± 33 µS/cm^–2, P = 0.05, n = 3, a 30% inhibition (Fig. 1). Neither treatment had a significant effect on R_t in these cells (control, 207 ± 41; AICAR, 163 ± 20; metformin, 196 ± 19 µS/cm², n = 3). These data expand on our previous observations to show that pharmacological activators of AMPK inhibit apical Na⁺ conductance (37, 38).

View larger version (8K): [in this window] [in a new window]   Fig. 1. Effect of AICAR and metformin on GNa+ in H441 cell monolayers. A: typical continuous apical current (Iap) traces from untreated control, 2 mM AICAR-, or 2 mM metformin-treated monolayers bathed in a potassium gluconate solution with 11.5 mM Na+ (see MATERIALS AND METHODS). Basolateral membranes were permeabilized with nystatin (75 µM), and the Na+ concentration of the apical bath raised to 50 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. B: apical amiloride-sensitive GNa+ determined from untreated control, AICAR-, and metformin-treated monolayers. Data are presented as means ± SE. *Significantly different from control, P 0.05, n = 3.

More than 95% of cell-attached patches recorded from apical membranes of H441 monolayer cells contained constitutively active channel activity, which was maintained throughout the duration of recording (up to 30 min). It was readily apparent that this constitutive channel activity often consisted of two distinct cation channel currents that were present in cell-attached patches at different frequencies. Figure 2A shows a representative recording of 58% of cell-attached patches that contained constitutive channel activity composed of cation channel currents that had a mean unitary current amplitude of –0.54 ± 0.3 pA, a mean number of unitary channel openings of 3.2 ± 0.3 per patch, and a mean NP_o (product of open-state probability and number of channels) of 1.29 ± 0.19 at –100 mV (n = 18, from >10 sets of cell monolayers, see MATERIALS AND METHODS). Figure 2B illustrates a typical trace from the remaining 42% of cell-attached patches that had a mean NP_o value of 2.23 ± 0.39 (n = 13). These patches contained cation channel currents similar to those described in Fig. 2A but also contained channel currents that had a much larger mean unitary amplitude of –1.71 ± 0.08 pA and a mean number of openings of 2.6 ± 0.3 per patch at –100 mV (n = 13). It should be noted that the larger amplitude cation channel currents were only observed in the presence of the smaller amplitude channel currents, and the observed frequency in patches was similar in all monolayers tested (n = 10). Thus, this channel was not associated with a subset of monolayers.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Properties of 2 distinct cation channels in cell-attached patches from apical membrane of H441 cell monolayers. A(i) and (ii): representative trace showing constitutive activity of cation channel currents in a cell-attached patch at –100 mV on different time scales that had unitary amplitudes of –0.5 pA and long openings. A(iii) amplitude histogram of the cation channel currents shown in A(i) that had 1 closed (C) and 2 open levels (O1 and O2) indicating the patch contained at least 2 channels. A(iv): mean current/voltage (I/V) relationship of these cation channel currents illustrating that in 145 mM NaCl, the channel currents had a slope conductance and reversal potential (Er) of 5 pS + 23 mV, respectively (each point from at least n = 5). In the presence of 145 mM NMDG-Cl, the I/V relationship had extrapolated Er of –38 mV (each point from at least n = 4). B(i) and (ii): a cell-attached patch that contained 2 distinct channel currents at –100 mV. B(iii) amplitude histogram of the cation channel currents shown in (i) that had 2 major peaks illustrating that the unitary amplitudes of O1 and O2 were –0.58 pA and –1.76 pA, respectively. The other peaks represent multiple openings of both types of channels. In B, mean I/V relationship shows that the larger amplitude channel currents had a slope conductance of 18 pS and an Er of +4 mV, and that in the presence of NMDG, the extrapolated Er was shifted to –35 mV (each point from at least n = 4).

To confirm that these constitutively active channel currents in H441 cell monolayers were cation conductances, we studied the properties of channel currents in cell-attached patches using a patch pipette solution containing the less permeant cation, NMDG-Cl, as the main cation charge carrier (see MATERIALS AND METHODS). With NMDG-Cl patch pipette solutions, constitutively active currents of both the 5- and 18-pS channel currents were observed at positive membrane potentials, whereas at negative potentials, little channel activity was observed. Moreover, extrapolating the mean I/V relationships for these channel currents indicated that E_r for the 5- and 18-pS channels were shifted to –38 and –35 mV, which corresponded to P_NMDG/P_Na permeability ratios of 0.09 and 0.21, respectively (Fig. 2, A and B).

The differences in the unitary conductances, E_r, and permeability ratios of P_NMDG/P_Na indicate that the apical membrane of H441 cell monolayers contains two distinct nonselective cation channels. Moreover, these properties suggest that the 5-pS channel has a high permeability to Na⁺ (HSC), and the 18-pS channel is a nonselective cation channel (NSC), and are similar to previously described ENaC and NSC (17, 24).

Differential sensitivity of HSCs and NSCs to inhibition by amiloride. It is well known that the cation channel blocker amiloride is a potent inhibitor of ENaC and ENaC-like conductances in epithelial cells (4). Therefore, we investigated the effect of amiloride on constitutive HSC and NSC activity in cell-attached patches and transepithelial I_sc. Figure 3, A and D, illustrates that including 1 µM amiloride in the patch pipette solution markedly reduced channel activity at –100 mV, in a patch that only contained HSC channels, after 10–20 s from obtaining a G seal and beginning recording with a mean degree of inhibition of 98 ± 1% (n = 7, from 5 sets of cell monolayers). Figure 3, B and D, illustrates that 1 µM amiloride had no effect on NSC activity at –100 mV (n = 5, from 4 sets of cell monolayers). However, Fig. 3, C and D, shows that inclusion of 10 µM amiloride in the patch pipette solution did inhibit NSC activity at –100 mV with a mean degree of inhibition of 63 ± 14% (n = 4, from 4 sets of cell monolayers). These data indicate that in H441 cell monolayers, NSCs are less sensitive to inhibition by amiloride than HSCs.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Differential sensitivity of highly Na+ selective channel (HSC) and nonselective cation channel (NSC) activity to amiloride in cell-attached patches from H441 cell monolayers. A: inclusion of 1 µM amiloride in the patch pipette solution produced a pronounced inhibition of HSC activity after 10–20 s at –100 mV. B is a typical trace showing that NSC activity at –100 mV was not inhibited by inclusion of 1 µM amiloride in the patch pipette solution, whereas C illustrates that 10 µM amiloride induced partial inhibition of NSC activity. D: mean data of relative NPo after application of 1 and 10 µM amiloride to the bath compared with normalized NPo for patches with HSC alone or HSC + NSC before application of amiloride (control). ***Significantly different from control P < 0.001.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Effect of amiloride on transepithelial short-circuit current (Isc) across H441 cell monolayers. A: typical trace showing changes in transepithelial Isc measured across H441 cell monolayers in response to application of amiloride (0.01–100 µM) and ouabain (1 mM). B: mean data showing transepithelial Isc from untreated monolayers (control) or those treated with 1 and 10 µM amiloride. Data are expressed as % control Isc. ***Significantly different from control P < 0.001, significantly different from 1 µM amiloride, P < 0.01, n = 6. C: amiloride concentration effect curve. Data were fitted with a sigmoid dose response curve with a Hill slope of –1.04 and an r2 value of 0.9.

View larger version (12K): [in this window] [in a new window]   Fig. 5. Effect of inhibitors of CNG channels on transepithelial Isc across H441 cell monolayers. A: transepithelial Isc measured across H441 cell monolayers that were untreated (control) or treated with 10 µM pimoside followed by 10 µM amiloride (pimoside + amiloride). B: transepithelial Isc measured across H441 cell monolayers that were untreated (control) or treated with 50 µM L-cis-diltiazem followed by 10 µM amiloride (L-cis + amiloride). C: transepithelial Isc measured across H441 cell monolayers that were untreated (control) or treated with 10 µM amiloride followed by 10 µM pimoside (amiloride + pimoside) or L-cis-diltiazem (amiloride+ L-cis). Data are expressed as % control Isc to normalize between experiments. *Significantly different from control.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Effect of an inhibitor and activator of CNG channels in cell-attached patches from H441 cell monolayers. A: inclusion of 10 µM pimoside (a CNG channel inhibitor) in the patch pipette solution had no effect on either HSC or NSC activity at –100 mV. B and C: bath application of 500 µM 8-bromo-cGMP (a CNG channel activator) had no effect on HSC or NSC activity, respectively, at –100 mV.

Figure 7 shows representative traces of constitutively active cation channel activity in three different cell-attached patches from the same set of H441 cell monolayers following various treatment protocols to test the effect of AMPK activation on channel activity. Figure 7, A and C, shows a control cell-attached patch containing both constitutively active HSC and NSC activity that had a mean NP_o value of 1.391 ± 0.382 (n = 6, 3 sets of cell monolayers) at –100 mV.

View larger version (24K): [in this window] [in a new window]   Fig. 7. Effect of AICAR, an activator of AMP-activated protein kinase (AMPK), and Compound C, an inhibitor of AMPK, on HSC and NSC in cell-attached patches from H441 cell monolayers. A(i) shows a control patch that contained constitutive HSC and NSC activity at –100 mV that was maintained for the duration of the recording. A(ii) illustrates a patch that contained infrequent activity of a small amplitude channel current following pretreatment with 2 mM AICAR for 1 h. A(iii) shows a patch that contained marked HSC and NSC activity after pretreatment with 80 µM Compound C for 30 min followed by incubation with 80 µM Compound C and 2 mM AICAR for a further 1 h. Note the scale bar refers to i, ii, and iii. B: mean cation channel activity in control patches (n = 6) in the presence of 2 mM AICAR alone (n = 12) and 80 µM Compound C and 2 mM AICAR (n = 6) at –100 mV (***P < 0.001). C: acute bath application of 2 mM AICAR produced a marked inhibition of both HSC and NSC activity at –100 mV after 5–10 min. D: bath application of 2 mM AICAR had no effect on HSC/NSC activity in an inside-out patch held at –100 mV.

Furthermore, Fig. 7C shows that an acute bath application of 2 mM AICAR reduced both HSC and NSC activity in a cell-attached patch at –100 mV with 2 mM AICAR significantly inhibiting mean NP_o from 2.11 ± 0.46 to 0.47 ± 0.25 (n = 4, <0.05) after 5–10 min. In control experiments, we also showed that bath application of 2 mM AICAR to the cytosolic surface of inside-out patches had no effect on either HSC activity alone (96 ± 6% of control activity vs. in presence of AICAR for 2 min, n = 3) or HSC/NSC activity (94 ± 7% of control activity vs. in presence of AICAR for 2 min, n = 3) indicating that AICAR is unlikely to be acting as a nonselective cation channel blocker (Fig. 7D).

These data indicate that the AMP mimetic, AICAR, has a pronounced inhibitory action on both constitutive HSC and NSC activity in the apical membrane of H441 monolayer cells. Moreover, this inhibitory effect of AICAR on channel activity could be reversed following pretreatment with the AMPK inhibitor, Compound C, which indicates that activation of AMPK mediates the AICAR-induced reduction of channel activity.

Effect of AICAR on ENaC proteins. A possible mechanism by which AMPK may inhibit channel activity is through a reduction of channel protein at the plasma membrane by either reducing insertion or increasing retrieval of channel proteins, respectively. In these series of experiments, we investigated changes in the apical abundance of ENaC proteins in H441 cell monolayers following pretreatment with AICAR. Western blotting of proteins from H441 cells with anti-ENaC antiserum directed against the NH₂ terminus of the protein identified a predominant protein of 65 kDa in the apical biotinylated fraction. Occasionally, a larger protein of 90 kDa was detected. However, this larger protein was predominantly detected in the nonbound fraction together with a 65-kDa protein and two smaller proteins of 57 and 37 kDa (Fig. 8). Using a COOH-terminal antibody, we also observed a predominant protein of 67 kDa in the biotinylated fractions that was less abundant in the nonbound fraction. In addition, there were 90- and 98-kDa proteins that were present in both protein fractions. Both 90- and 65-kDa proteins for ENaC have previously been described in lung (30). Consistent with our observations, the 65 kDa protein is generally acknowledged to be a cleavage product of the 90-kDa protein and is the predominant form in the apical membrane. In addition, 65-kDa ENaC proteins have been described with the use of NH₂-terminal and COOH-terminal antiserum. There is currently no clear explanation as to why this may occur, but in human lung cells, it is possible that both the NH₂-terminal and the COOH-terminal of ENaC could be subject to proteolytic cleavage (1, 14, 30, 31). Using antiserum directed against the COOH-terminal of βENaC, we detected a protein of 100 kDa in both apical and total protein preparations and a predominant protein of 88 kDa in both apical and total protein preparations with anti-ENaC antiserum consistent with that reported by others (13, 35). Interestingly, although ENaC was cleaved in other cell systems, we did not find evidence for cleaved products in our cells using the described antiserum (15). Importantly, we did not observe any change in the apical abundance of -, β-, or ENaC proteins (measured by densitometry) after pretreatment with 2 mM AICAR for 1 h (Fig. 8). Furthermore, we did not detect any change in the abundance of other protein bands that would be indicative of ENaC protein cleavage. To confirm that changes in protein abundance could be detected by this method, treatment with the proteosomal inhibitor MG132 (6 µM for 3 h), which has been shown to inhibit the recycling and degradation of ENaC subunits in renal A6 cells, significantly increased the abundance of -, β-, and ENaC subunits in the biotinylated protein fraction [Fig. 8, P < 0.05, n = 3, (26)]. Immunostaining for β-actin confirmed the lack of cytosolic protein contamination of biotinylated fractions (Fig. 8).

View larger version (21K): [in this window] [in a new window]   Fig. 8. Effect of AICAR on apical abundance of -, β-, and ENaC proteins in H441 cell monolayers. A: typical Western blots of apical biotinylated (bound-biotinylated) and non-biotinylated (nonbound) protein samples from H441 cell monolayers. Samples were untreated (control) or treated with AICAR or MG132. Proteins were immunostained for ENaC using anti NH2-terminal and COOH-terminal antisera, βENaC, ENaC, and β-actin. The position of protein size markers is shown at left and the sizes of predominant immunostained proteins at right of the images. Images shown for each antiserum are from the same immunoblot but may have been digitally reordered for consistency of presentation. B: densitometry analysis of immunostained proteins from apical biotinylated protein samples. Data are shown as means ± SE for n = 3 samples. *Significantly different from control P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
In these and previous experiments, we have shown that AICAR and metformin activated AMPK and significantly decreased amiloride-sensitive transepithelial Na⁺ transport measured as I_sc across H441 cell monolayers (37, 38). Metformin was much less potent than AICAR in reducing transepithelial Na⁺ transport, but the effect of both agents on I_sc was reversed by the AMPK inhibitor Compound C, implying that AMPK mediated the inhibitions of amiloride-sensitive I_sc. We have also previously shown that AICAR inhibited apical amiloride-sensitive G_Na+, and, in the present work, we show that metformin has a similar effect on apical G_Na+. These data further imply that activation of AMPK inhibits amiloride-sensitive Na⁺ channels present in the apical membrane of H441 cells.

H441 cell monolayers contain two distinctive constitutively active Na⁺-permeable cation channels. In the present study, we have identified two distinct constitutively active amiloride-sensitive cation channels in the apical membrane of H441 cell monolayers: a 5-pS channel and an 18-pS channel. Although we have not specifically investigated the cation permeability of these channels, their conductance and E_r values exhibit remarkable similarity to the highly Na⁺ selective channel (HSC, 5 pS) and a nonselective cation channel (NSC, 20 pS) previously described in cultured H441 and rat alveolar type I and II cells (11, 17, 18, 24). We found that the 5-pS channel was present in more than 95% of cell-attached patches tested, whereas the 18-pS channel was observed in 40% of patches but was not present on its own. The 18-pS channel was, however, observed in almost all sets of H441 cell monolayers tested, and, therefore, the expression of this conductance is likely to be of physiological importance. Interestingly, our data also suggest that HSCs are more sensitive to amiloride than NSCs with HSCs and NSCs probably having IC₅₀ values for amiloride of <1 µM and 10 µM, respectively, which are values in good agreement with previous studies (18, 24). We could inhibit HSC activity, but NSC activity was unchanged with 1 µM amiloride, indicating that NSC activity is not dependent on HSC activity. Moreover, as only 56% of amiloride-sensitive I_sc was inhibited with 1 µM amiloride, it appears that both channels are likely to contribute to G_Na+ in H441 cells. The relative expression of these two channel types in rat alveolar type II cells has been shown to be dependent on culture conditions where cultured at air interface and the presence of steroids promotes the expression of HSCs over NSCs (17). Consistent with these findings, we found a predominance of the 5-pS HSC in H441 cell monolayers that were grown at air interface in the presence of dexamethasone.

The cyclic nucleotide-gated cation channel (CNG) is a nonselective cation channel that has been described in lung epithelial cells (21, 32, 33, 39). Both pimoside and L-cis-diltiazem have been used successfully to block CNG activity in lung epithelial cells and tissue at concentrations similar to those used in this study (18, 19, 21, 29). However, we did not observe a significant decrease in transepithelial I_sc or NSC activity in cell-attached patches using these agents. In addition, we could not increase I_sc or NSC activity with the CNG channel activator 8-bromo-cGMP as observed in other cells and tissues (18, 21, 33). Moreover, the reported IC₅₀ for amiloride block of CNG channels has been reported to be 39–125 µM (23, 39). We determined that 10 µM amiloride elicited a 63% inhibition of NSC activity in H441 cells. Together, these data indicate that the NSCs we have identified in H441 cell monolayers are not CNG channels. An amiloride-sensitive NSC was described in rat alveolar type I and II cells that is thought to be composed of ENaC -subunits alone or by -subunits in association with other, as yet unidentified, subunits (16). We speculate that this channel may underlie the NSCs we describe in H441 cells.

Our data provide strong evidence that both constitutively active HSCs and NSCs contribute to apical G_Na+ in H441 cell monolayers. Therefore, it is likely that both these channels are involved in maintaining transepithelial Na⁺ conductance and have a role in regulating airway fluid volume, although the frequency of observation of HSCs indicates that they may have a greater contribution to G_Na+ than NSCs.

Both HSC and NSC activity in H441 cell monolayers is inhibited by AMPK. We found that pretreatment and acute (5–10 min) application with the AMP mimetic AICAR markedly inhibited the activity of both the 5- and 18-pS channels in H441 monolayer cells, which supports our current and previous observations of the effect of AICAR on G_Na+ (37). This inhibitory effect of AICAR on HSC/NSC activity is unlikely to due to a nonselective cation channel blocking action as channel activity in inside-out patches was not blocked by AICAR. Moreover, the inhibitory action of AICAR was reversed following pretreatment with the AMPK inhibitor Compound C, which indicates that the inhibitory effects on channel activity were mediated via activation of AMPK.

Proteins encoding -, β-, and ENaC were expressed in the apical membrane of H441 cells. In alveolar type II cells, coexpression of -, β-, and -subunits of the cloned amiloride-sensitive channel ENaC is required for the formation of highly Na⁺ selective channels, whereas it has been proposed that NSCs are composed of ENaC subunits alone or by association with other subunits (16). Mutant active AMPK and ZMP (the active form of AICAR) have been shown to inhibit HSCs formed from -, β-, and ENaC expressed in Xenopus oocytes (5, 8). However, the effect of activation of AMPK on NSC activity has not previously been described. Our data, therefore, raise new and important questions as to how AMPK regulates HSCs and NSCs and whether the mechanisms of action of AMPK are related to the subunit composition of these channels.

Mechanisms involved in AMPK-mediated inhibition of channel activity. Our data provide strong evidence that both HSCs and NSCs are inhibited by AICAR through an AMPK-mediated mechanism. To date, there is little information on the mechanisms by which AMPK inhibits Na⁺-permeable cation channels in epithelial cells. Work in Xenopus oocytes has indicated that constitutively active AMPK and ZMP decreased -, β-, and ENaC activity by increasing retrieval of channels from the plasma membrane (8). The removal and degradation of ENaC are known to require the binding of the ubiquitin ligase Nedd4-2 to proline-rich regions (PY motifs) in the COOH termini of the ENaC subunits, which promotes ENaC ubiquitination and degradation (12, 27). In HEK-293 cells expressing -, β-, and ENaC, activation of AMPK did not change association of Nedd4-2 with - and -subunits but increased its association with β-subunits (5), suggesting that altering the level of βENaC subunits in the plasma membrane could be sufficient to modulate channel function. However, in this and our previous work, we could not find any evidence to support such a mechanism since changes in the abundance of apical biotinylated -, β-, and ENaC proteins after treatment with AICAR could not be detected (25). In addition, we could not detect any changes in abundance of cleaved or full-length ENaC proteins, which could potentially affect activity of the channel (1, 14, 30, 31). Furthermore, antisense oliogonucleotides directed against β- and ENaC proteins in rat alveolar type II cells decreased the number of HSCs in excised apical membrane patches but increased the number of NSCs observed, indicating that β- and -subunits are important for the formation of HSCs but not NSCs (16). Thus, if activation of AMPK was decreasing βENaC in the membrane, we may have expected to observe an increase in the frequency of NSCs in cell-attached patches. Moreover, as current evidence supports only a potential role of ENaC in the formation of NSCs, it is doubtful whether a mechanism involving β-subunits would play a role in their inhibition by AMPK.

Plasmalemma ion channel activity is determined by the number of channels expressed in the membrane (N) and the open probability (P_o) of each individual channel. The present work provides compelling evidence that the level of expression of ENaC subunits does not alter following activation of AMPK, and, therefore, it is unlikely that an AMPK-induced mechanism involving changes in N is involved. An alternative mechanism to explain our data is that activation of AMPK with AICAR produces inhibition of HSC and NSC activity in H441 monolayer cells through changing P_o.

Our findings contrast with those described by Carattino et al. (8) and Bhalla et al. (5). There are two potential mechanisms that may ratify these differences. First, recent work in our laboratory (25) has shown that treatment with AICAR altered PIP₂ association with β- and ENaC subunits to inhibit Na⁺ transport in H441 cells. Second, progesterone increased binding of Nedd4-2 to β- and ENaC subunits expressed in oocytes, leading to a decreased P_o without changes in N (28). In H441 cell monolayers, at least for HSCs, which are thought to be composed of -, β-, and -subunits, either mechanism could precede increased channel retrieval. It would be interesting to investigate longer-term treatments of AICAR/metformin on apical ENaC subunit abundance in H441 cells to see if this is the case. Alternatively, there is significant evidence that pathways regulating ENaC retrieval differ between cells and overexpression systems (12, 27). Thus, it is possible that mechanisms that regulate endogenous ENaC function and activity in H441 monolayer cells may be different from those in Xenopus oocytes and HEK-293 cells.

In conclusion, we have shown for the first time that AICAR and associated activation of AMPK inhibit the constitutive activity of two amiloride-sensitive ENaC-related channels in the apical membrane of H441 cells, a 5-pS highly Na⁺-selective channel (HSC) and an 18-pS nonselective cation channel (NSC). Moreover, as we could not detect a change in the abundance of ENaC proteins in the apical membrane of H441 cell monolayers, we suggest that rapid activation of AMPK with AICAR inhibits HSC and NSC activity by decreasing channel P_o.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was funded by Biotechnology and Biological Sciences Research Council Project Grant BB/E013597/1 and Wellcome Trust Project Grant PG/07/079/23568.

	ACKNOWLEDGMENTS

 
We thank Prof. W. Large for help and support in obtaining the patch-clamp data and Lucy Ly for technical assistance with the cell culture.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. L. Baines, Centre for Ion Channels and Cell Signalling, Div. Basic Medical Sciences, St. George's, Univ. of London, London, SW17 0RE, United Kingdom (e-mail: d.baines{at}sgul.ac.uk)

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

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