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TOXICOLOGY
Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan
Received February 7, 2006; accepted March 24, 2006.
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Abstract |
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-dependent anion transporter (NBC1/AE2) inhibitor]-sensitive ISC in the control, whereas the up-regulation evidently favored bumetanide-sensitive ISC in the basolateral presence of H2O2. The FK-induced NKCC1 augmentation after exposure to basolateral H2O2 was counteracted by cytochalasin D, an inhibitor of microfilament function, but not by charybdotoxin, a blocker of the intermediate conductance Ca2+-activated K+ channel, whose activation could be related to NKCC1-mediated Cl- secretion. These observations suggest that basolaterally but not apically applied H2O2 potentiates subsequent cAMP-mediated Cl- secretion by an increase in Cl- uptake via basolateral NKCC1, whose sensitivities to cAMP/protein kinase A are up-regulated, overcoming the H2O2-induced inhibition of cAMP-mediated apical anion conductance. The basolateral membrane-specific effects of H2O2 may be relevant to the basolateral cytoskeleton, which is believed to interact with NKCC1.
). It is well known that ROS damage tissue via direct oxidation of protein, DNA, or lipids (Okayama, 2005). Because of the toxicological effects of ROS, their production by inflammatory cells during episodes of infection and inflammation is responsible for the pathogenesis of a number of respiratory diseases, including bronchial asthma, cystic fibrosis, and chronic obstructive pulmonary disease (Ricciardolo et al., 2006). These ROS-related airway diseases share aspects of mucous congestive diseases (Kellerman, 2002), in which excessive and tenacious mucus secretion causes airway obstruction, and the resultant dysfunction of mucociliary clearance is involved in the morbidity and mortality of these diseases (Rogers, 2005). In vivo, bronchial gland cells contribute to maintenance of effective mucociliary clearance by regulating salt and water secretion via the vectorial ion transport system, thereby forming low-viscosity mucus (Shimura, 2000). Thus, the relationship between ROS and airway ion transport is of considerable interest. The purpose of this work is to elucidate this point, using polarized Calu-3 cells, which may be a model of human airway submucosal gland serous cells (Shen et al., 1994). This cell line expresses high levels of the cystic fibrosis transmembrane conductance regulator (CFTR), a representative anion exit pathway, on the apical membrane (Haws et al., 1994) and several anion uptake transporters on the basolateral membrane (Loffing et al., 2000). In the present study, we examined the effects of oxidant stress caused by H2O2 on cAMP-dependent anion secretion in polarized human airway serous cell epithelia and found the paradoxical phenomena that the oxidative stimuli applied from the apical or basolateral membrane had opposite effects on cAMP-activated anion secretion. |
Materials and Methods |
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). The cells were fed by replacement of the basolateral medium every 48 h. The cells seeded on the filters normally reached complete confluence, which was confirmed by microscopic observations, in 7 days. Over 13 days, the surface of the monolayers on the filter became clouded day by day. Thus, we determined to carry out our experiments after 7 to 13 days in culture.
Bioelectric Studies. Snapwell inserts on which Calu-3 cells had grown confluent were rinsed with physiological saline solution (PSS) and transferred to modified Ussing chambers (EasyMount Chamber, Physiologic Instruments, San Diego, CA) that contained PSS at 37°C. The PSS was composed of 115 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose, 10 mM Hepes, and 25 mM NaHCO3. The pH of the solution was adjusted to 7.4 (at 37°C) using NaOH before the addition of NaHCO3. The pH of the solution was kept at 7.4 when gassed with a mixture of 5% CO2 and 95% O2. The monolayers were continuously measured under a short-circuited condition, using a high-impedance millivoltmeter that could function as a voltage clamp with automatic fluid resistance compensation (VCC MC2, Physiologic Instruments). Transepithelial resistance (Rt) was determined by short-circuit current (ISC) changes (
ISC) in response to an imposed voltage pulses (V = 4 mV) with 0.5-s duration, using the equation of Ohm's law (Rt = V/ISC). The ISC value represents the net charge movement across the monolayer.
Permeabilized Monolayers. To investigate apical membrane Cl- conductance (GCl), the basolateral membrane was permeabilized with the pore-forming antibiotic nystatin (100 µM) to which the cells were pre-exposed for 30 to 35 min. This level of nystatin was determined as the concentration at which bumetanide, an inhibitor of the basolateral Na+-K+-2Cl- cotransporter, had no effect on the ion current (Devor et al., 1999). This procedure avoids the complexities associated with basolateral ion transporters and permits analyses of GCl. GCl was estimated as the apical membrane Cl- current (ICl) in the apical-to-basolateral Cl- concentration gradient under short-circuit conditions. The Cl- concentration gradient was established by replacing NaCl with equimolar Na-gluconate in the basolateral PSS. Changes in ICl reflect those of the CFTR-mediated Cl- current because Cl- conductance on the apical membrane is exclusively mediated by CFTR in Calu-3 cells without any contribution of Ca2+-activated Cl- channel (Haws et al., 1994; Wine et al., 1994). In this basolateral solution, CaCl2 was increased to 4 mM to compensate for the Ca2+-chelating capacity of the gluconate (Devor et al., 1999).
cAMP Assay. Confluent Calu-3 cells on the permeable supports were exposed to forskolin (FK) (10 µM) for 15 min in the presence of H2O2 and its absence using a cAMP Biotrack enzyme immunoassay kit (Amersham, Arlington, IL). The concentrations of cAMP ([cAMP]i) in the samples were determined, according to the manufacturer's instructions. The cAMP levels were expressed as femtomole/well.
Chemicals. FK, 8-bromo-cAMP (8-Br-cAMP), DNDS, NPPB, bumetanide, indomethacin, nystatin, pyruvate, and cytochalasin D (Cyto-D) were obtained from Sigma-Aldrich Co. NS-398 and SC-560 were purchased from Cayman Chemicals (Ann Arbor, MI). H2O2 and charybdotoxin (ChTx) were products of Wako Chemical (Tokyo, Japan) and Peptide Institute Inc. (Osaka, Japan), respectively. Stock solutions of 8-Br-cAMP, DNDS, pyruvate, and ChTx were prepared by dissolving them in distilled water. All of the other drugs were dissolved in dimethyl sulfoxide. Nystatin stock solution (100 mM) was made and sonicated for 30 s just before use.
Analysis of Results. Numerical data are presented as mean ± S.E.M., where n refers to the number of experiments. Statistical differences were determined by Student's t test. A value of p < 0.05 was considered statistically significant.
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cm2, respectively (n = 181). Previous studies have reported that relatively high concentrations of H2O2, ranging from 0.1 to 5 mM, were required to induce barrier dysfunction and anion secretion because airway epithelial cells, including Calu-3, have a strong antioxidant defense capacity (Waters et al., 1997; Zhao and Davis, 1998; Cowley and Linsdell, 2002). As shown in Fig. 1, A and B, the Rt of cells decreased with clearly defined nadirs immediately after exposure to oxidative stimulation of H2O2 (5 mM) from either the apical (A) or basolateral (B) aspect of the membrane, and this parameter gradually returned to its basal level. In a polarized epithelial monolayer, an irreversible increase in monolayer conductance is suggestive of cell damage and loss of viability (Alvarez et al., 1998; Ito et al., 2001). The reversible changes in monolayer Rt observed in the present study indicate that the cells are tolerant of H2O2 at this concentration. Concomitant with the Rt changes, the corresponding peak values in ISC were 73.0 ± 3.6 (n = 5, to apical H2O2) and 49.0 ± 3.3 µA/cm2 (n = 9, to basolateral H2O2), respectively (p < 0.01) (Fig. 1, C and D). These bioelectric changes seem likely to include predominantly anion transport because both of them were markedly reduced to 24.0 ± 1.3 (n = 4, p < 0.01), and 22.8 ± 2.3 µA/cm2 (n = 4, p < 0.01) by the presence of NPPB (100 µM), a Cl- channel blocker. Previous investigations have shown that airway epithelial cells release cyclooxygenase (COX) products in response to ROS-related stimuli (Matyas et al., 2002). Indomethacin, a COX inhibitor, is generally believed to suppress endogenous production of prostaglandins and thus intracellular cAMP (Mall et al., 1998). As shown in Fig. 1, E through G, the H2O2-induced effects were diminished to 27.2 ± 2.2 (n = 4, p < 0.01) and 21.7 ± 1.2 µA/cm2 (n = 4, p < 0.01) by pretreatment with indomethacin (10 µM). Similar results were obtained when we used SC-560 (1 µM, a COX-1 inhibitor) and NS-398 (10 µM, a COX-2 inhibitor), resulting in a suppressed peak ISC in response to apical [27.5 ± 1.8 (n = 4, p < 0.01) and 25.6 ± 1.2 µA/cm2 (n = 4, p < 0.01)] and basolateral [20.1 ± 2.0 (n = 4, p < 0.01) and 17.6 ± 3.4 µA/cm2 (n = 4, p < 0.01)] application of H2O2 (Fig. 1G).
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Despite the analogous electrical properties in epithelial responses to H2O2 applied from either aspect, careful comparison revealed a difference between them. Namely, although the acute ISC increments in response to apical H2O2 were larger than those to basolateral H2O2, as described above, sustained ISC levels 20 min after apical addition of H2O2 remained lower than those after its basolateral addition [0.3 ± 0.8 (n = 5) versus 6.8 ± 1.0 µA/cm2 (n = 9), p < 0.01].
Effects of Apically and Basolaterally Applied H2O2 on Subsequent FK-Elicited ISC. As has been shown in previous investigations, the cells responded to cAMP-related agents, such as FK (10 µM, an adenylate cyclase activator), with anion secretion that reflects ISC changes (Fig. 2A) (Devor et al., 1999; Ito et al., 2004a). Next, we examined the effects of H2O2 on the subsequent ISC changes in response to FK. Surprisingly, despite the similarity of apical and basolateral H2O2-induced ISC, subsequent FK-elicited responses behaved differently, depending on to which side the oxidative stimuli were applied. Namely, FK-induced responses, which were composed of rapid and subsequent sustained components (Fig. 2A), were attenuated by the apical presence of H2O2 (5 mM, Fig. 2B), whereas they were contrastingly augmented by H2O2 applied from the basolateral side (Fig. 2C). Concretely, the peak values of the ISC caused by FK (71.3 ± 2.3 µA/cm2, n = 10) were diminished to 41.2 ± 6.4 µA/cm2 (n = 5, p < 0.01) and potentiated to 101.7 ± 5.5 µA/cm2 (n = 9, p < 0.01) by apical and basolateral oxidant stimuli, respectively. As shown in Fig. 2D, the H2O2-induced down-regulation and up-regulation of the FK-induced responses occurred in a concentration-dependent fashion in opposite directions from each other.
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Effects of H2O2 on Intracellular cAMP Production. Based on the data in Fig. 2, we naturally assumed that the ISC responses to FK would be correlated with the changes in [cAMP]i. However, this was not so. As shown in Fig. 3, FK(10 µM)-induced [cAMP]i elevation, which had increased from 423.9 ± 61.5 (n = 14, the control) to 14,464.3 ± 3153.1 fmol/well (n = 14, p < 0.01, compared with the control) 15 min after its application, was inhibited by the apical presence of H2O2 [5 mM, 1282.3 ± 184.0 fmol/well (n = 8, p < 0.01) compared with FK group without H2O2]. However, we here observed the paradoxical situation that the FK-induced [cAMP]i elevation was also hindered by the oxidative stimuli from the basolateral side [3926.1 ± 826.1 fmol/well (n = 8, p < 0.05) compared with the FK group without H2O2], inconsistent with the movement of ISC (see Fig. 2C). Neither inhibitory effect of H2O2 seems likely to be caused by leakage of cAMP from the cells as a result of oxidative damage of the plasma membrane, because apical and basolateral H2O2 did not significantly affect the basal levels of [cAMP]i, which were 384.2 ± 34.0 (n = 6) and 403.8 ± 60.0 fmol/well (n = 6), respectively (Fig. 3).
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Effects of H2O2 on FK-Induced Apical Cl- Conductance. Figure 4 shows the apical membrane Cl- conductance (GCl), which was estimated as apical membrane Cl- current (ICl) after establishment of an apical-basolateral Cl- gradient and permeabilization of the basolateral membrane with nystatin (100 µM). As shown in Fig. 4A, application of FK (10 µM) caused development of the inward ICl (ICl = 61.3 ± 4.5 µA/cm2, n = 4). As correlated with the ISC data (see Fig. 2), the addition of H2O2 from either side of the membrane caused inward ICl development and decay (Fig. 4, B and C). In the apical presence of H2O2, the FK-induced ICl changes were markedly suppressed (ICl = 14.0 ± 3.5 µA/cm2, n = 5, p < 0.01), consistent with the data in Figs. 2B and 3. Paradoxically, however, the basolateral presence of H2O2, as shown in Fig. 4C, inhibited the FK-induced ICl development (ICl = 24.1 ± 4.4 µA/cm2, n = 5, p < 0.01), inconsistent with the data in Fig. 2C but consistent with the data in Fig. 3. Current pharmacological approaches to prevent the burden of oxidative stress include pyruvate and cell-permeable superoxide dismutase mimetics (Cuzzocrea et al., 2001). The antioxidant effects of pyruvate are produced by a direct nonenzymatic reaction with H2O2, which produces acetate, CO2, and H2O, and restoration of the balance between reduced and oxidized glutathione (Leon et al., 2004). Figure 4, D and E, shows that preincubation with pyruvate (5 mM) counteracted the inhibition of ICl development as a result of the apical [ICl = 55.4 ± 2.8 µA/cm2 (n = 4, p < 0.01) compared with the values in the absence of pyruvate] and basolateral presence of H2O2 [ICl = 61.4 ± 4.7 µA/cm2 (n = 4, p < 0.01) compared with the values in the absence of pyruvate]. In addition, acute ICl changes caused by apical and basolateral H2O2 seem to be up-regulated by the presence of pyruvate.
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8-Br-cAMP-Induced Responses in the Presence of H2O2. The aspect-specific effects of H2O2 were reproduced in ISC responses to the cell-permeable cAMP analog 8-Br-cAMP (1 mM) (Fig. 5, A-C). The ISC responses to 8-Br-cAMP (59.6 ± 4.4 µA/cm2, n = 12) (Fig. 5A) were down-regulated to 40.8 ± 1.5 µA/cm2 (n = 7, p < 0.01) (Fig. 5B) and up-regulated to 84.9 ± 3.6 µA/cm2 (n = 12, p < 0.01) (Fig. 5C) by apical and basolateral pretreatment, respectively, with H2O2. These observations suggest that cAMP production via adenylate cyclase is not necessarily a primary target of H2O2 in the intricate responses. Similar to the results in Fig. 4, 8-Br-cAMP-augmented ICl (ICl = 48.5 ± 1.9 µA/cm2, n = 6) (Fig. 5D) was prevented by preincubation with H2O2 from either the apical (ICl = 10.1 ± 1.4 µA/cm2, n = 4, p < 0.01) (Fig. 5E) or basolateral surface (21.9 ± 3.0 µA/cm2, n = 4, p < 0.01) (Fig. 5F).
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), NBC1 (the DNDS-sensitive 
cotransporter) (Inglis et al., 2002), and AE2 (the DNDS-sensitive 
exchanger) (Loffing et al., 2000; Inglis et al., 2002). Indeed, FK application similarly increased both bumetanide- and DNDS-sensitive ISC from 0.7 ± 0.1 (n = 7) to 11.2 ± 0.8 µA/cm2 (n = 11) and from 0.5 ± 0.1 (n = 7) to 9.9 ± 0.9 µA/cm2 (n = 11), respectively, without H2O2 (Fig. 6A), whereas the cAMP-mediated up-regulation evidently favored bumetanide-sensitive ISC [34.1 ± 2.5 µA/cm2 (n = 8, p < 0.01) compared with the value without H2O2] in the basolateral presence of H2O2 (5 mM), which inhibited DNDS-sensitive ISC (7.0 ± 2.6 µA/cm2, n = 8) (Fig. 6B). The data of these observations are summarized in Fig. 6, C and D.
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Mechanisms Underlying Up-Regulated cAMP-Mediated NKCC1-Mediated Anion Transport under Basolateral H2O2. The airway epithelial cells secrete Cl- via NKCC1 in response to activation of human intermediate conductance Ca2+-activated K+ channels (KCNN4), so that anion secretion induced by 1-ethyl-2-benzimdazolinone (a KCNN4 activator) and thapsigargin (a cytosolic Ca2+ mobilizing agent) is markedly inhibited by either bumetanide, a NKCC1 inhibitor, or ChTx, a KCNN4 channel blocker (Devor et al., 1999; Ito et al., 2004a,b). NKCC1 is also activated by cAMP/protein kinase A (PKA)-mediated phosphorylation (Haas and Forbush, 2000; Matthews, 2002); this mechanism fails to involve KCNN4 activation because of the lower sensitivity of ChTx to cAMP-mediated anion secretion (Ito et al., 2002, 2004a). This was confirmed by the results shown in Fig. 7A. Although exposure to basolateral H2O2 further potentiated the bumetanide-sensitive component in FK-induced ISC from 12.4 ± 1.0 (n = 7) to 37.4 ± 1.5 µA/cm2 (n = 13), preincubation with ChTx did not affect the potentiation (Fig. 7B). Namely, bumetanide-sensitive ISC was augmented from 10.2 ± 0.9 (n = 7) to 36.0 ± 2.9 µA/cm2 (n = 7) in the basolateral presence of ChTx and H2O2 (Fig. 7E).
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). Thus, we hypothesized that the cytoskeletal remodeling induced by oxidative stress around the basolateral membrane could be attributed to the augmentation of cAMP/PKA-dependent NKCC1 activity. To test this hypothesis, the effects of H2O2 on FK-induced responses were observed in the presence of Cyto-D (10 µM), which is conventionally used to disrupt microfilament function (Matthews et al., 1997). After the addition of Cyto-D, we observed gradual increases in ISC in response to imposed voltage pulses, indicating an increase in monolayer permeability in the presence of this chemical. Nevertheless, the bumetanide-sensitive ISC under the FK-stimulated condition was maintained at approximately 76% even 60 min after the addition of Cyto-D (9.4 ± 0.9 µA/cm2, n = 7) (Fig. 7C). The presence of Cyto-D rather offsets the bumetanide-sensitive component increased by exposure to basolateral H2O2 (15.1 ± 1.3 µA/cm2, n = 7) (Fig. 7D). These data are summarized in Fig. 7E.
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Discussion |
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). As shown in the present study, the marked inhibition of apical or basolateral H2O2-induced ISC by indomethacin suggests that H2O2-induced CFTR-mediated anion secretion seems likely to be, at least in part, mediated by intrinsic prostaglandins depending on COX activity. Furthermore, in the responses, both subtypes of COX seem likely to be involved because ISC responses to H2O2 application were equally suppressed by a COX-2 inhibitor (NS-398, Ki of COX-1/COX-2 = 75/1.77 µM) (Barnett et al., 1994) and a COX-1 inhibitor (SC-560, Ki of COX-1/COX-2 = 9 nM/6.5 µM) (Smith et al., 1998). Although the responses to apical and basolateral H2O2 seem likely to be commonly relevant to COX signals, we found that the ISC responses to apical H2O2 were larger than those to basolateral H2O2. These observations allow us to speculate that polarization of the cells produces laterality between the apical and basolateral membrane in the membrane-located COX activity.
Despite the apparent similarity in the responses to the oxidative stimuli from either side of the membrane, the result that FK-induced anion secretion is inhibited by the apical presence of H2O2 but potentiated by its basolateral presence led us to hypothesize that H2O2 induces different signal transductions in each membrane. The effect of apical H2O2 is reasonable because the ISC changes correlated with FK-induced ICl and [cAMP]i elevation. In the hindrance of cAMP production, oxidative stress may operate primarily at the level of the plasma membrane on the functioning of adenylate cyclase by altering the state of phosphorylation (See et al., 2001). However, the effect of H2O2 is also observed in the apical ICl in response to 8-Br-cAMP, a cell-permeable cAMP analog, suggesting that oxidative stress to either aspect hinders activation of CFTR by hindrance of the channel gating and cAMP synthesis. Regarding the mechanisms underlying dysfunction of CFTR under oxidative insults, previous studies have shown that redox reagents alter the kinetics of CFTR gating such that reducing conditions speed up gating and increase the open probability, whereas oxidizing conditions slow down CFTR gating, probably through cysteine residues located on the nucleotide-binding domains of CFTR (Harrington et al., 1999; Harrington and Kopito, 2002). Alternatively, AMP-activated protein kinase, which is activated by oxidative stress and consequently phosphorylates CFTR to inhibit its conductance, may also be involved in the mechanisms (Walker et al., 2003). Naturally, because FK-induced and 8-Br-cAMP-induced ISC were potentiated by the presence of basolateral H2O2, we first assumed that these cAMP-related parameters, such as cAMP production and cAMP-elicited ICl, were correlated with the up-regulated ISC changes. Unexpectedly, however, we found that these parameters were inhibited by basolateral H2O2, inconsistent with the behavior of ISC. These observations led us to conclude that H2O2 stimulation from either side hindered the cAMP/PKA signal transduction (cAMP synthesis process and CFTR activation) from the cytosolic side and simultaneously allowed us to deduce the presence of a specific pathway, which is localized around the basolateral membrane.
Anion secretion is the end result of coordinated activities of several different anion transporters. This event requires not only the activity of the apical anion channel but also basolateral transporters (Devor et al., 1999; Ito et al., 2004b). The apical CFTR, which is well accepted as a common pathway for and Cl- export (Devor et al., 1999), displays no less than 
60% of maximum conductance at rest, so that the Calu-3 cell is fully capable of anion secretion even under the cAMP-unstimulated state (Moon et al., 1997). Thus, rather than CFTR as an anion exit pathway, anion uptake across the basolateral membrane is thought to be the rate limiter that largely determines the overall secretion capacity, as is the case of other polarized epithelia (Matthews, 2002). In epithelial cells, anion entry across the basolateral membrane chiefly depends on the activity of basolateral anion transporters, such as NBC1, NKCC1, and AE2 (Loffing et al., 2000; Liedtke et al., 2002). Thus, a possible explanation for the seemingly paradoxical results is that H2O2-induced up-regulation of anion uptake across the basolateral membrane would compensate for the down-regulation of CFTR activation of anion exits across the apical membrane. Estimation of the anionic composition of the FK-elicited ISC revealed that both bumetanide (an NKCC1 inhibitor)- and DNDS (an NBC1/AE2 inhibitor)-sensitive components are similarly increased in the control, whereas the increase evidently favored the bumetanide-sensitive ISC in the presence of basolateral H2O2 (see Fig. 5, A and B); this suggests that cAMP-activated ISC potentiated by the presence of basolateral H2O2 mirrored the augmentation of Cl- current mediated through NKCC1, whose sensitivity to PKA may be up-regulated by the basolaterally localized effect of the oxidative stress.
Previous studies (Devor et al., 1999; Ito et al., 2004b) reported that the switch between secretion and Cl- secretion is determined by the basolateral membrane potential regulated by a ChTx-sensitive Ca2+-activated K+ channel, KCNN4. Namely, when the basolateral membrane is hyperpolarized by KCNN4 activation, the driving force for entry via NBC1 (the transporter), which carries electrogenically negative charges into the cell, is reduced, whereas the driving force for Cl- entry across the electroneutral NKCC1 (the Na+-K+-2Cl- cotransporter) is up-regulated. Because the hyperpolarization simultaneously provides a driving force for anion export across the apical CFTR, the activation of the KCNN4 would cause a large Cl- secretion (Moon et al., 1997; Devor et al., 1999). To exclude the possibility of NKCC1 up-regulation by way of KCNN4 activation, we observed the effect of basolateral oxidative stress on the FK-induced responses in the presence of ChTx, but it made no significant difference in the oxidant-induced modulation.
It is now been established that a complex cortical meshwork of cytoskeleton proteins localizes adjacent to the cytosolic faces of the plasma membrane, where it is uniquely placed to interact with a variety of transmembrane proteins such as NKCC1 (Matthews, 2002). For NKCC1 regulation, cAMP may induce the surface recruitment of membrane proteins to form a regulatory complex with NKCC1 (D'Andrea et al., 1996). Furthermore, several lines of evidence have shown that cAMP-dependent signals themselves are transduced to NKCC1, at least in part, by dynamic remodeling of F-actin microfilaments within the cortical submembranous cytoskeleton (Shapiro et al., 1991; Matthews, 2002). It has been shown that exposure to H2O2 remodels actin structures that take the form of microfilaments associated with cortical F-actin in Calu-3 cells (Boardman et al., 2004). Thus, it is most conceivable that the oxidant-induced remodeling of the cytoskeleton would help the PKA-induced reorganization of the submembranous cytoskeleton linked to NKCC1, resulting in selective augmentation of bumetanide-sensitive ISC. Indeed, the oxidant-induced up-regulation of the NKCC1-mediated ISC in response to FK was markedly suppressed by Cyto-D. Considering the results obtained from the present study, we propose the hypothetical scheme of H2O2-elicited effects on cAMP-dependent anion secretion shown in Fig. 8.
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Airway epithelial cells are exposed to oxidative stress not only through inhalation of ozone and other environmental oxidants from the apical side but also through intrinsic ROS from the basolateral side because formation of ROS takes places constantly in every cell during the metabolic process (Ricciardolo et al., 2006). Especially in acute and chronic airway inflammations, activated phagocytic cells, such as neutrophils, eosinophils, monocytes, and macrophages, are recruited in the subepithelial sites of the respiratory tract, and they generate and release large amounts of ROS (Ricciardolo et al., 2006). In contrast to the basolateral membrane, the apical membrane is fully protected by antioxidant substances such as vitamin C and glutathione at very high concentrations in the human airway (Kelly et al., 1999; Dauletbaev et al., 2001). Therefore, the responses shown in our study may serve to compensate for basolateral ROS-induced disturbance of mucociliary clearance to help clear infections before consequent tissue damage can be initiated. The site-dependent effects of H2O2 that we have shown here should provide new insight into a variety of epithelial biology and toxicology in which oxidant stress is implicated.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: ROS, reactive oxygen species; H2O2, hydrogen peroxide; CFTR, cystic fibrosis transmembrane conductance regulator; PSS, physiological saline solution; Rt, transepithelial resistance; ISC, short-circuit current; GCl, apical membrane Cl- conductance; ICl, apical membrane Cl- current; FK, forskolin; 8-Br-cAMP, 8-bromo-cAMP; DNDS, 4,4'-dinitrostilbene-2,2'-disulfonic acid; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoate; Cyto-D, cytochalasin D; NS-398, N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide; SC-560, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole; ChTx, charybdotoxin; COX, cyclooxygenase; NKCC1, Na+-K+-2Cl- cotransporter; NBC1, cotransporter; AE2, 
exchanger; KCNN4, human intermediate conductance Ca2+-activated K+ channels; PKA, protein kinase A.
Address correspondence to: Yasushi Ito, Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Tsurumai-cho, Showaku, Nagoya, 466-8550, Japan. E-mail: itoyasu{at}med.nagoya-u.ac.jp
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, a significant difference (p < 0.01) from the values of the control response to apical (*) and basolateral (
(p < 0.05), significant increases and decreases, respectively, compared with the values in each FK group without H2O2 stress.
