1Division of Gastroenterology,2Department of Surgery, and 3Division of Pulmonary Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2C2
Submitted 17 February 2003 ; accepted in final form 15 July 2003
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ABSTRACT |
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interleukin-10; colitis; cystic fibrosis transmembrane conductase regulator; inflammatory bowel disease
Recently, AMP-activated protein kinase (AMPK) has emerged as a sensor of energy stores within the cell (32, 34). AMPK is a serine/threonine kinase that exists as a heterotrimer composed of a catalytic -subunit and regulatory
- and
-subunit (12, 13). AMPK activity increases during conditions of metabolic stress as a result of an elevated intracellular AMP/ATP ratio (36, 44). Intracellular metabolic stress is seen under varied conditions, including heat shock (17), hypoglycemia (37, 53, 58), hypoxia (49, 66, 69), ischemia (33, 40), and oxidant exposure (21, 41). Activation of AMPK involves an allosteric mechanism by which AMP binds to AMPK, in addition to a phosphorylation of a threonine residue on the catalytic subunit of AMPK catalyzed by an upstream kinase, AMPK kinase (AMPKK) (60). AMPK responds to alterations in cellular energy changes by regulating both ATP-consuming and ATP-generating pathways (34, 36). Studies have shown AMPK activation to regulate numerous pathways, including the inhibition of fatty acid, triglyceride and sterol synthesis (22, 24), and the stimulation of glucose uptake (25, 53, 57), glycolysis (40, 49), and fatty acid oxidation (35). In addition, AMPK activation has also been linked with changes in the expression of various different genes (46).
In the gut, the cystic fibrosis transmembrane conductance regulator (CFTR) is the primary channel responsible for chloride secretion (27). CFTR requires the hydrolysis of ATP for activity and has been shown to physically interact with AMPK, with activation of AMPK resulting in an inhibition of CFTR in epithelial cells (29, 30). Thus we hypothesized that the activation of AMPK in response to metabolic stress may be responsible for the dysfunction in chloride secretion seen under conditions of chronic inflammation (2, 6, 28).
In the present study, we examined the role of AMPK in regulating epithelial chloride secretion under chronic inflammatory conditions in colonic tissue from IL-10-deficient mice and in vitro in human colonic T84 epithelial monolayers. Results obtained in this study suggest that the hyporesponsiveness observed under conditions of chronic inflammation is related to AMPK activation.
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MATERIALS AND METHODS |
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Homozygous IL-10-deficient mice, generated on a 129 Sv/Ev background, and wild-type 129 Sv/Ev controls were housed behind a barrier under specific pathogen-free conditions. The mice had ad libitum access to autoclaved 9% fat rodent blocs and sterile filtered water. The facility's sanitation was verified by Health Sciences Lab Animal Services at the University of Alberta (Edmonton, AB, Canada). To demonstrate the role of AMPK in the defect in chloride secretion seen in the IL-10-deficient mice, colonic tissue from IL-10-deficient mice was treated in vitro with 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-ylpyyrazolo(1,5-a)pyrimidine (compound C; 75 µM). Compound C is a specific potent reversible inhibitor of AMPK that is competitive with ATP in the absence of AMP (73). Compound C was kindly provided by Dr. G. Zhou (Merck & Rahway). Colons from 129 Sv/Ev wild-type mice were treated in vitro with the AMPK-specific activator 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR; 1 mM). AMPK is activated pharmacologically by 5-amino-4-imidazolecarboxamide riboside monophosphate (ZMP), which mimics the effects of AMP on the AMPK cascade (60). All experiments were performed according to the institutional guidelines for the care and use of laboratory animals in research and with the permission of the local ethics committee.
Epithelial Chloride Secretion
Mice were killed by cervical dislocation, and a segment of proximal colon was removed. Colonic tissue was mounted in Lucite chambers exposing mucosal and serosal surfaces to 10 ml of oxygenated Krebs buffer (in mM: 115 NaCl, 8 KCl, 1.25 CaCl2, 1.2 MgCl2, 2 KH2PO4, 25 NaHCO3; pH 7.35). When a Cl--free buffer was used, 115 mM Na+-gluconate replaced NaCl, 1.2 mM MgSO4 replaced MgCl2, and 1.2 mM Ca2+-gluconate replaced CaCl2. The -free buffer used was similar except that 125 mM Na-gluconate was used to replace all Cl-, and
was replaced by 10 mM HEPES buffer titrated to pH 7.4 with 1 M Tris base. Acetazolamide (10-3 M) was added to the
-free buffer to inhibit endogenous
production. The
-free buffer was gassed with 100% O2. The buffers were maintained at 37°C by a heated water jacket and circulated by CO2/O2. Fructose (10 mM) was added to the serosal and mucosal sides. The spontaneous transepithelial potential difference (PD) was determined, and the tissue was clamped at 0 V by continuously introducing an appropriate short-circuit current (Isc) with an automatic voltage clamp (model DVC 1000; World Precision Instruments, New Haven, CT) except for 510 s every 5 min when PD was measured by removing the voltage clamp. Tissue ion conductance (G) was calculated from PD and Isc according to Ohm's law. PD is expressed as millivolts, Isc as microamperes per square centimeter, and G as millisiemens per centimeter squared. Baseline Isc and G were measured after a 20-min equilibration period. Increases in Isc were induced by the addition of the adenylate cyclase-activating agent forskolin (10-8 to 10-4 M) to the serosal surface. Epithelial responsiveness was defined as the maximal increase in Isc to occur within 5 min of exposure to secretagoges.
For the measurement of basal chloride fluxes, the tissue was clamped at 0 V by continuously introducing an appropriate Isc with an automatic voltage clamp, except for 510 s every 5 min when PD was measured by removing the voltage clamp. Tissue pairs were matched for conductance and discarded if conductance varied by > 20%. After 5 µCi 36Cl was added to the serosal side after mounting, the tissue was allowed to equilibrate for 20 min. Unidirectional flux from serosal-to-mucosal surface was determined for paired tissues by measuring four consecutive 5-min fluxes before the addition of forskolin (10-4 M) and two 5-min fluxes after the addition of the secretagog.
Chloride flux in response to forskolin is reported as the difference among the averaged values of the four consecutive 5-min fluxes before the addition of the secretagog and the averaged values of the two consecutive 5-min fluxes post-stimulation.
AMPK Activity Assay
AMPK enzyme activity was assayed in an enriched colonic mucosal homogenate (20). After death, proximal colons were excised from mice and opened longitudinally over ice under sterile conditions. Mucosa was removed by scraping and was homogenized in 300 µl enzyme-enrichment homogenization buffer (in mM): 50 Tris · HCl, 250 mannitol, 1 EDTA, 1 EGTA, 50 NaF, 5 Na4P2O7 · 10 H2O, 1 PMSF, 1 DTT, and 10% glycerol, 0.1% Triton X-100, and 1 µl/ml protease inhibitor cocktail (Sigma, St. Louis, MO) by using a glass-stem tissue homogenizer. Subsequently, the crude homogenate was centrifuged at 15 000 g for 20 min, after which the supernatant was vortexed for 10 min after the addition of polyethylene glycol 6000 (model PEG 6000; Fluka) to a final concentration of 2.5%. Samples were then centrifuged at 10,000 g for 10 min, after which the pellet was discarded, and the supernatant was vortexed again for 10 min after the addition of PEG 6000 to a final concentration of 6%. Samples were centrifuged at 10,000 g for 10 min, after which the supernatant was discarded, while the pellet was washed once in a volume of 6% PEG 6000/homogenization buffer before resuspension in 50 µl enzyme-enrichment resuspension buffer (in mM): 50 Tris · HCl, 1 EDTA, 1 EGTA, 50 NaF, 5 Na4P2O7 · 10 H2O, 1 PMSF, 1 DTT, and 10% glycerol, 0.1% Triton X-100, and 1 µl/ml protease inhibitor cocktail (Sigma). Protein concentrations were determined by using the Bradford method, and each sample was diluted to a final protein concentration of 1 mg/ml in the same resuspension buffer. Before beginning the assay, 2 µg of the enzyme was added to each of three different condition assay buffers, one containing saturating amounts of AMP and the synthetic AMPK-target SAMS peptide (in mM: 80 HEPES buffer, 160 NaCl, 1.6 EDTA, 100 EDTA, and 200 µM SAMS peptide, 200 µM AMP, 16% glycerol, 0.1% Triton X-100), and two identical to the previous buffer, excepting the addition of AMP, and AMP and SAMS peptide, respectively. All three of the assay conditions included saturating levels of ATP (1 µCi 32P-g-ATP, 200 µM unlabeled ATP, 5 mM MgCl2). The assay was initiated by the addition of enzyme to the reaction tube, followed by a 5-s vortexing, and a 5-min incubation at 30°C. After the 5-min incubation, the reaction mixture was revortexed and spotted on P81 Whatman filter paper (Sigma), briefly allowed to dry, and washed three times in 1% perchloric acid before a single wash in acetone. After sufficient time to allow the filter papers to air dry, they were immersed in a scintillant-fluor cocktail and the activity of each sample was measured in a Beckman scintillation counter. The activity of the AMPK enzyme is reported as the difference (in picomole ATP incorporated) between the activity of the AMP-saturated sample and the sample devoid of AMP.
Brush-Border Membrane Vesicle Isolation
Animals were killed by cervical dislocation, and the colon was removed and flushed with ice-cold PBS. The colon was opened longitudinally, and the mucosa was collected by scraping with a glass slide. The mucosal scraping was homogenized for 60 s in 200 µl brush-border membrane vesicle homogenizing buffer (300 mM mannitol, 12 mM Tris · HCl, pH 7.1) by using a glass-stem homogenizer. Posthomogenization, the homogenate was diluted with 1 ml ice-cold water, before adding 12 µl of 1 M MgCl2. The homogenate was then left to stand on ice for 40 min. After the 40-min incubation, the homogenate was centrifuged at 8,000 g for 15 min at 4°C. The supernatant was decanted, held on ice, and then resuspended in 1 ml brush-border membrane vesicle homogenization buffer by gentle repeat pipetting before adding 12 µl of 1 M MgCl2 and left to stand on ice for 15 min. After the 15-min incubation, the resuspended homogenate was centrifuged at 8,000 g for 15 min at 4°C. Again, the supernatant was decanted and then combined with the previously obtained supernatant before centrifuging at 27,000 g for 30 min at 4°C. The pellet was washed once in 1 ml brush-border membrane vesicle resuspension buffer before its final resuspension in 50 µl Mono-Q buffer (50 mM -glycerophosphate, 1 mM EGTA, 2 mM MgCl2, 0.5% Triton-X 100, pH 7.2) and subsequent analysis by Western blotting.
Cell Culture Studies
T84 cells at passages 3034 were grown as monolayers in a 1:1 mixture of Dulbecco-Vogt modified Eagle's medium and Ham's F-12 medium supplemented with 15 mM Na+-HEPES buffer, pH 7.5, 14 mM NaHCO3, and 5% newborn calf serum. For subculture, a cell suspension was obtained from confluent monolayers by exposing the monolayers to 0.25% trypsin and 0.9 mM EDTA in Ca+- and Mg+-free PBS. Cells were seeded at a density of 1 x 106 cells/1.13 cm2 polycarbonate tissue culture-treated filter and maintained at 37°C in a 5% CO2 atmosphere. Cultures were refed daily with fresh media.
To qualitatively determine whether the T84 cells had reached confluence, formed tight junctions, and established cell polarity, the electrical conductance and the spontaneous potential across the monolayer were determined by using an EVOM voltohmmeter and an STX-2 electrode set (World Precision Instruments, Sarasota, FL). To determine the effect of sustained activation of AMPK, monolayers were exposed to AICAR (1 mM) for 48 h and then mounted in Ussing chambers for measurement of Isc, PD, and basal and forskolin-stimulated chloride fluxes.
AMPK is activated through a phosphorylation mechanism (13). To examine the effect of epithelial exposure to oxidants on AMPK phosphorylation, monolayers were treated with H2O2 (1 mM serum-free media) or peroxynitrite (0.1 mM serum-free media) for 0, 5, 10, 15, 30, or 60 min before collection by scraping (over ice). Phosphorylation was examined by Western blotting. To examine the effect of epithelial exposure to oxidants on AMPK activity and chloride flux, monolayers were treated with H2O2 (2.5 mM) for 30 min. After the 30-min incubation, the media were changed (serum free, minus oxidants). Cells were harvested by scraping over ice, and AMPK activity was assessed by enzymatic assay. Chloride flux was measured in Ussing chambers, and inhibition of AMPK activity was confirmed by enzymatic assay.
To determine the effect of inhibiting AMPK activity in the presence of H2O2, monolayers were preincubated with compound C at a concentration of 75 µM (73) before the addition of H2O2 (2.5 mM).
Western Blot Analysis
T84 cells were harvested from 6- or 12-well plates by scraping and were suspended in 0.5 ml Mono-Q buffer. After collection, cell suspensions were sonicated on ice for 15 s. Protein concentrations were determined by using the Bradford method, and samples were diluted to the equivalent concentrations. Duplicate samples were separated by SDS-PAGE and either Coomassie-stained (to ensure even loading of lanes) or transferred onto PVDF membrane (Millipore). Membranes were blocked for 2 h with 3% skim milk-TTBS (20 mM Tris, 0.5 M NaCl, 0.05% Tween 20, pH 7.4) and incubated overnight at 4°C with anti-phospho-AMPK antibody (1:1,000 dilution, rabbit anti-mouse phospho-AMPK).
Brush-border membrane vesicles prepared from animals were subjected to a single freeze-thaw cycle and subsequently sonicated on ice for 25 s. PVDF membranes were prepared the same as for T84 cell samples and incubated overnight at 4°C with anti-CFTR antibody (1:1,000 dilution, mouse anti-human CFTR, cross-reactive to murine CFTR) for CFTR blots. Membranes were then washed three times with water and incubated for 2 h with goat anti-rabbit or goat anti-mouse secondary antibodies (1:3,000 dilution; Bio-Rad, Hercules, CA), followed by two washes with TTBS. Autoradiography was performed on Kodak X-OMAT AR film by using a chemiluminescence kit (Lumi-light, Amersham).
Flow Cytometry
Single cell suspensions from T84 monolayers were fixed with 5% formalin for 5 min. The fixation reaction was stopped by adding PBS/1% BSA. Cells were blocked with PBS/0.1% saponin/5% dried milk for 24 h. Fixed cells were incubated with primary MAb anti-CFTR (COOH terminus, Cat. no. 250301; Genzyme) for 1 h followed by the secondary antibody, goat IgM/FITC (model AM4708; Biosource) for a second hour.
Statistical Analysis
Data are expressed as means ± SE, and statistical analyses were performed by using the statistical software Sigma-Stat (Jandel, San Rafael, CA) Differences among mean values were evaluated by analysis of variance or paired t-test where appropriate. Specific differences were tested by using the Student-Newman-Keuls test.
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RESULTS |
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Previous studies have shown that under conditions of chronic inflammation, intestinal tissue becomes hyporesponsive to secretagoges (6, 59, 63). When housed under conventional conditions, IL-10-deficient mice develop a patchy chronic colitis that is limited to the colon (7). To determine whether proximal colonic segments from IL-10-deficient mice demonstrated hyporesponsiveness to secretagoges, Isc was measured in response to increasing doses of the adenylate cyclase inducing agent, forskolin. As seen in Fig. 1, colons from IL-10-deficient mice had a significantly reduced Isc response to increasing doses of forskolin, compared with responses seen in wild-type mice. Measurement of unidirectional chloride fluxes confirmed the increase in serosal-to-mucosal movement of chloride in colons from wild-type mice and the absence of such a response in IL-10-deficient mice (Fig. 2).
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As expected, the delta Isc response to forskolin in wild-type mice was largely associated with Cl- and (10, 55). Removal of chloride resulted in a
55% reduction (P < 0.05; Fig. 3) in forskolin-stimulated Isc, and simultaneous removal of both Cl- and
resulted in virtually no Isc response. These findings are in agreement with other studies showing that the rodent colon is dominated by electroneutral absorption of sodium; thus the Isc measured is primarily due to chloride and bicarbonate movement (10, 55).
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Basal electrical parameters in the colon were similar between wild-type (PD: 1.6 ± 0.3 mV; Isc: 28.0 ± 3.7 µA/cm2) and IL-10-deficient mice (PD: 1.2 ± 0.3 mV; Isc: 21.6 ± 2.5 µA/cm2). Basal conductance (G) was also similar and did not change significantly in response to forskolin in either wild-type (17.6 ± 1.1 vs. 22.7 ± 3.3 mS/cm2) or IL-10-deficient mice (22.9 ± 3.7 vs. 18.9 ± 2.2 mS/cm2), indicating that the changes in chloride serosal-to-mucosal fluxes were not due to increased paracellular movement of chloride.
Under inflammatory conditions, the levels of prostaglandins would be expected to be increased (70); thus in the IL-10-deficient mice, the failure to respond to forskolin may have been related to already elevated intracellular cAMP levels. To determine whether the secretory defect seen in the IL-10-deficient mice was due to elevated cAMP levels, colonic tissue was incubated with indomethacin to reduce prostanoid activity and then stimulated with forskolin. As seen in Table 1, the presence of indomethacin did not alter the hyporesponsiveness seen in the IL-10-deficient mice. These data would suggest that the secretory defect in the proximal colon of IL-10-deficient mice was not due to elevated epithelial cAMP levels.
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CFTR Brush-Border Membrane Localization
In the colon, both calcium and cAMP-mediated chloride secretion are primarily mediated by CFTR (27). Previous studies (19, 51, 65) have suggested that activation of CFTR by cAMP can involve an increased number of transporters being inserted in the brush-border membrane, either by increasing the rate of exocytosis or decreasing the rate of endocytosis of CFTR-containing membrane vesicles. Examination of isolated brush-border vesicles from wild-type and IL-10-deficient mice by Western blotting techniques demonstrated a reduction in CFTR expression associated with the brush-border membrane in IL-10-deficient mice (Fig. 4). This would suggest that the inability of IL-10-deficient mice to respond to forskolin was associated with reductions of CFTR associated with the brush-border membrane fraction.
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AMPK Activity Assay
AMPK is activated in response to metabolic stress and acts to phosphorylate and inhibit several biosynthetic enzymes, thereby preserving cellular ATP levels during periods of metabolic depletion (17). We have previously shown that chronic inflammation in the IL-10-deficient mouse is associated with high levels of peroxynitrite, suggesting enhanced nitrosative stress (40). Thus to determine whether AMPK activity was upregulated in inflamed colons in IL-10-deficient mice, colonic tissue was examined in IL-10-deficient mice at 2 wk of age (in the absence of any inflammation) and at 6 wk of age (after inflammation develops) and compared with age-matched controls. At 2 wk of age, there was no significant difference in the activity of the enzyme between the two groups; however, by 6 wk of age, a significant increase in enzymatic activity was evident in the IL-10-deficient group (Fig. 5). This data would suggest that the activity of AMPK was upregulated in IL-10-deficient mice under conditions of colonic inflammation.
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Effect of AMPK Inhibition on Colonic Chloride Secretion
Recent studies (29, 30) have shown that AMPK interacts with CFTR and inhibits its activity. Having shown that colonic tissue from IL-10-deficient mice demonstrated enhanced levels of AMPK activity and also was unresponsive to forskolin stimulation, we carried out a series of experiments to determine whether inhibition of AMPK activity would restore cAMP-mediated chloride secretion. Colons from IL-10-deficient mice were treated in vitro with compound C, and chloride secretion was assessed. Compound C is a potent reversible inhibitor of AMPK that is competitive with ATP and does not exhibit significant inhibition of other kinases structurally related to AMPK (73). As seen in Fig. 6, colonic tissue from IL-10-deficient mice did not respond to forskolin with an increase in serosalto-mucosal chloride flux. In contrast, colonic tissue from IL-10-deficient mice pretreated with compound C demonstrated a complete restoration in serosal-to-mucosal chloride flux to levels seen in wild-type mice. There was no significant effect of forskolin on conductance in either colons from IL-10 mice (26.4 ± 2.4 vs. 30.1 ± 2.9 mS/cm2) or in colons treated with compound C (21.3 ± 2.8 vs. 23.3 ± 2.2 mS/cm2).
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Effect of AMPK Activation on Colonic Chloride Secretion
To further correlate the deficiency in chloride secretion seen in the IL-10 gene-deficient mouse with AMPK activity, healthy colonic tissue from wild-type mice was treated in vitro with AICAR, and chloride flux was measured (Fig. 6). Incubation with the AMPK activator AICAR results in accumulation of the monophosphorylated derivative ZMP, the active intracellular form of AICAR, within the cell (18). ZMP mimics both activating effects of AMP on AMPK; that is, a direct allosteric activation, and promotion of phosphorylation by AMPKK (60). While activating AMPK, AICAR does not perturb the cellular concentration of ATP or its metabolites ADP or AMP (60). As seen in Fig. 6, AICAR-treated colonic tissue from normal healthy mice functionally resembled tissue from IL-10-deficient mice, in that the tissue did not respond to forskolin. There was no significant effect of forskolin on conductance in either colons from wild-type mice (27.2 ± 1.6 vs. 31.8 ± 2.5 mS/cm2) or in colons treated with AICAR (22.9 ± 1.8 vs. 23.8 ± 2.3 mS/cm2). These data support the hypothesis that activation of AMPK in colonic epithelial tissue occurs under conditions of chronic inflammation and that this activation is linked with decreases in cAMP-mediated chloride secretion.
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Cell Culture Experiments |
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Treatment of T84 monolayers with AICAR. Under conditions of chronic inflammation, AMPK activity may be elevated for significant periods of time. To examine the effects of chronic activation of AMPK on epithelial function, T84 epithelial monolayers were incubated with AICAR for 48 h, and monolayer resistance, Isc, and chloride fluxes were assessed. The presence of AICAR significantly increased conductance while reducing Isc, PD, and Isc response to forskolin (Table 2). In addition, serosal-to-mucosal flux of chloride in response to forskolin was significantly reduced in the presence of AICAR (Table 2). This was associated with decreased levels of CFTR in the apical membrane as measured by flow cytometry (Fig. 9). These data suggest that long-term activation of AMPK has significant consequences on several aspects of ionic epithelial function.
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DISCUSSION |
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Diarrhea is commonly seen clinically in patients with inflammatory bowel disease (11). Although it was initially suggested that inflammation-associated diarrhea occurred as a result of inflammatory mediators acting as secretagoges and stimulating chloride secretion (14), recent evidence appears to refute this concept (52). Indeed, the fluid accumulation and diarrhea seen in patients with inflammatory bowel disease may occur as a result of decreased Na+ absorption and Cl- secretory processes rather than a stimulation of ion secretion (62, 63, 64). Data from this study support this concept and further implicate activation of AMPK as the intracellular mediator responsible for these transport abnormalities.
In the IL-10-deficient mouse, colonic inflammation is associated with increased secretion of proinflammatory cytokines and high levels of nitrosative stress (42). Previous studies in animal models of colitis (6, 25, 45) and cell culture models (15, 23, 62) have shown that exposure of epithelial cells to either nitrogen- or oxygen-derived species or proinflammatory cytokines such as IFN- and TNF-
results in the induction of a hyporesponsive chloride secretory state. In our study, although basal chloride secretion was similar between wild-type mice and IL-10-deficient mice, adult IL-10-deficient mice with established colitis exhibited an inability to respond to forskolin with an upregulation of colonic chloride secretion. Previous studies (43) have shown a similar pattern, with basal electrogenic ion transport remaining unchanged in a rat model of colitis, whereas the tissue response to phosphodiesterase inhibitor IBMX was significantly reduced. Inflamed human intestinal tissue has also shown a similar lack of response to theophylline compared with control tissue (39).
Chloride secretion through CFTR is a major determinant of mucosal hydration throughout the intestinal tract (4, 45). CFTR plays an important role in colonic absorption and secretion of salt and water and appears to be critical for determining the overall rate of transepithelial ion transport (27). CFTR is activated in epithelial cells through hormone and neurotransmitter-induced cAMP-mediated signaling (27). Thus in that forskolin acts to stimulate chloride secretion through an activation of the adenylate cyclase pathway and a subsequent rise in intracellular cAMP levels, the failure of tissue to respond to forskolin-stimulation could be attributed to already elevated cAMP levels in colonocytes. This may occur as a consequence of increased levels of inflammatory mediators in the mucosa, including leukotriene B4, prostaglandin E2, IL-1, and reactive oxygen metabolites (70), all of which have been shown to increase cAMP levels (4). However, in our study, pretreatment of colonic tissue with indomethacin to inhibit prostanoid activity had no effect on the hyporesponsiveness of tissue to forskolin, arguing against already elevated intracellular cAMP as being the mechanism behind the failure of chronically inflamed tissue to respond to forskolin. In addition, previous studies have shown enterocyte cAMP levels to be decreased under chronic inflammatory conditions, as opposed to increased, which occurs under acute inflammatory conditions (59).
CFTR belongs to the family of ATP-binding cassette proteins and requires ATP binding and hydrolysis for activity (56). It has been proposed that the ATP requirement for CFTR activity allows for the coupling of CFTR with cellular energetics (32). Recently, Hallows et al. (29, 31) have shown that AMPK and CFTR colocalize in epithelial cells, and AMPK phosphorylation of CFTR in vitro inhibits cAMP-activated CFTR conductance in Xenopus oocytes (31) and T84 cells (29). Our data, both in whole animals and in cell culture, support the concept of AMPK being an endogenous inhibitor of CFTR activity. AMPK was upregulated in colons from IL-10-deficient mice only under inflammatory conditions; thus the upregulation was not due to the lack of IL-10 per se. Furthermore, pharmacological inhibition of AMPK activity resulted in restoration of tissue responsiveness to forskolin. Finally, in wild-type mice, stimulation of AMPK with AICAR produced a profound inhibition of CFTR activity.
Mechanism(s) underlying AMPK regulation of CFTR channel activity under chronic inflammatory conditions may involve either a downregulation of CFTR protein expression, an inhibition of protein maturation (5), increased degradation (5), or an inhibition of CFTR protein insertion into the brush-border membrane during stimulus of secretion (1, 38). In addition, CFTR activity can be regulated through a phosphorylation mechanism involving cAMP and protein kinase A (PKA) (61, 67). Activation of PKA by rises in intracellular cAMP results in the phosphorylation of the cytoplasmic domain of CFTR and a resultant activation of gating by a destabilization of channel closed states (72). Indeed, the regulation of CFTR channel activity and/or apical membrane expression is complex and occurs at several different levels. Numerous proteins have been shown to interact with CFTR, including syntaxin 1A (16), the postsynaptic density-95/discs large/zona occludens-1 domain-containing proteins NHERF and CAP70 (54), and the m2-subunit of the AP-2 adaptor protein complex (71). The catalytic subunit of AMPK has also been shown to bind to a region near the COOH terminus of CFTR and result in an AMPK-dependent phosphorylation in vitro (31). Thus although PKA-mediated phosphorylation is the most well-characterized mechanism for modifying CFTR activity, AMPK phosphorylation may have an equally important role in regulating chloride secretion. Supporting this concept are recent studies (30) in human lung epithelial cells, which demonstrated that pharmacological activation of AMPK resulted in an inhibition of single-channel gating rather than a reduction of CFTR expression in the apical membrane. However, whether this occurs by a direct phosphorylation of CFTR by AMPK remains to be shown, because AMPK may also induce its effects through modifying the activity of either PKA or other kinases/phosphatases involved in the regulation of CFTR expression or activity. Indeed, AMPK phosphorylates and inhibits numerous rate-limiting biosynthetic enzymes to maintain cellular ATP stores during metabolic stress (32).
In this study, the levels of CFTR protein appear to be reduced in IL-10-deficient mice, although whether this involves only a reduction of CFTR channels in the apical membrane or also a reduction in intracellular protein, remains to be clarified. The use of brush-border vesicles from mice colon cannot differentiate between CFTR in certain endosomal compartments, and CFTR expressed in brush-border membranes. Thus the reduction observed in brush-border membranes from IL-10-deficient mice may also have been due to an overall reduction of CFTR within the cell. However, in that basal levels of chloride secretion were similar in wild-type and IL-10-deficient mice, and, furthermore, inhibition of AMPK with compound C resulted in the rapid restoration of chloride secretion, it would appear that the amount of CFTR present in colonocytes in IL-10-deficient mice was sufficient to manifest chloride secretion once the inhibitory effect of AMPK was removed. Whether this involved increased insertion of CFTR into the brush-border membrane or an alteration in the phosphorylation state of CFTR remains to be clarified. Conflicting results have been obtained regarding the effect of inflammation on CFTR expression. Indeed, both downregulation and no change in expression have been reported (9, 15, 59). These differences may be related to either the model system studied, or the length of time of colitis. Further experiments will be necessary to resolve this issue.
In conclusion, the role of AMPK in mediating the chloride transport abnormalities seen under conditions of chronic inflammation would appear to be one of suppression. This inhibition of chloride secretion by AMPK would serve to reduce CFTR-dependent ATP utilization, to preserve intracellular levels of ATP. Furthermore, such an association between AMPK and CFTR would allow for the efficient coupling of intracellular metabolism with the maintenance of transcellular epithelial ion transport.
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DISCLOSURES |
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FOOTNOTES |
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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.
Present address of M. Kulka, Lab of Allergic Disorders, National Institute of Allergy and Infectious Diseases, 10 Center Drive, MSC 1881, Bethesda, MD 20892-1881.
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REFERENCES |
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