Pflügers Arch – Eur J Physiol (1997) 434:227–233

© Springer-Verlag 1997

O R I G I NA L A RT I C L E

&roles:Naomi Niisato · Yoshinori Marunaka

Regulation of Cl– transport by IBMX in renal A6 epithelium

&misc:Received: 9 September 1996 / Received after revision: 14 January 1997 / Accepted: 28 February 1997

& bstract We studied regulation of Cl– transport by p.1:A cAMP and Ca2+ in renal epithelial A6 cells. Stimulation of A6 cells by 1 mM 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of phosphodiesterase), which increased cytosolic cAMP, elicited biphasic increases in short-circuit current (Isc), i.e., a transient phase followed by a sustained one. Apical application of 5-nitro-2-(3phenylpropylamino)-benzoate (NPPB, a Cl– channel blocker) markedly and dose-dependently inhibited the IBMX-induced Isc. Pretreatment with nifedipine (100 µM, a Ca2+ channel blocker) or 1,2-bis (o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA/AM, 10 µM, a Ca2+ chelator) partially but markedly inhibited the Isc. On the other hand, a cAMP-dependent protein kinase inhibitor, H89 (0.5 µM for 1 h), also reduced the IBMX-induced Isc to a level similar to that following nifedipine or BAPTA pretreatment. Nifedipine had no synergistic effects on the IBMX-induced Isc in cells treated with H89. Ionomycin (a Ca2+ ionophore) could mimic the transient increase dose dependently, and H89 did not block the ionomycininduced Isc. Taken together, our observations suggest that: (1) part of the IBMX-stimulated Cl– release is regulated by an increased cytosolic Ca2+ through nifedipinesensitive Ca2+ influx; (2) cAMP-dependent phosphorylation may be required for elevation of the cytosolic Ca2+ concentration but not for activation of Cl– channels, which are directly activated by cytosolic Ca2+; and (3) the IBMX-induced sustained Cl– release requires cAMP elevation in addition to an increase in the cytosolic Ca2+ concentration. &kwd:Key words IBMX · cAMP · Protein kinase · NPPB · Ca2+ · Nifedipine · BAPTA · Short-circuit current&bdy: N. Niisato · Y. Marunaka (✉) Laboratory for Cellular and Molecular Physiology, Division of Respiratory Research, Hospital for Sick Children Research Institute, University of Toronto Faculty of Medicine, Toronto, Ontario M5G 1X8, Canada&/fn-block:

Introduction Ion transport is regulated by multiple intracellular signaling molecules which are activated by various extracellular stimuli. Continuous transepithelial Cl– secretion requires activation of ion channels and carriers, which may be regulated by common or different signaling pathways [13]. Activation of Cl– channels in the apical membrane by intracellular second messengers of hormones, such as cAMP or Ca2+, is responsible for Cl– secretion [11]. Agonists such as arginine vasopressin (AVP), forskolin or TPA (12-O-tetradecanoylphorbol 13-acetate) stimulate Cl– secretion via an increase in cAMP or Ca2+ [6, 27], which activates cAMP-dependent protein kinase (PKA) or protein kinase C (PKC). Thus, transepithelial Cl– secretion is regulated directly or indirectly by Ca2+, and PKA- or PKC-mediated, phosphorylation-dependent mechanisms. The Ca2+-induced activation of Cl– secretion [36] usually results from activation of apical Cl– channels [39] and, in some cases, activation of basolateral K+ channels [26] also contributes to the activation of Cl– secretion. Xenopus laevis renal epithelial cell line A6 cell is a useful model with which to study transepithelial ion transport [8–10, 12, 14, 15, 24, 25, 28, 30, 34, 35, 37, 40], and AVP and forskolin have been shown to stimulate transepithelial Cl– transport via cAMP-dependent pathways in A6 cells [3, 7, 30, 37, 40]. We have shown that the apical membrane of A6 cells has two types of Cl– channels, with single-channel conductances of 3 and 8 pS respectively [20]. The 3-pS and 8-pS Cl– channels are diversely regulated by AVP and cAMP [22, 23, 33]: the 3-pS Cl– channel is activated by cytosolic Ca2+, while the activity of the 8-pS Cl– channel is independent of cytosolic Ca2+. We have some information about the regulation of apical Cl– channels at the single-channel current level; however, little information is available about the cAMP-mediated regulation of Cl– secretion at cell and tissue levels. In this study, we report stimulatory mechanisms of IBMX-induced short-circuit current (Isc) generated by Cl– secretion.

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Materials and methods Solutions The solution used in the present study for measurement of Isc contained (in mM): 95 NaCl, 3.5 KCl, 1 CaCl 2, 1 MgCl2, 5 glucose, 10 N-[2-hydroxyethyl]-piperazine-N′-[2-ethanesulfonic acid] (HEPES), and 25 NaHCO3. The pH of the solution was adjusted to 7.4 (before addition of NaHCO 3) using NaOH. The solution was circulated by airlift equilibrated with 5% CO 2, 21% O2 and 74% N 2. Chemicals Medium NCTC-109, fetal bovine serum, penicillin and streptomycin were purchased from Gibco (Grand Island, N.Y.). 3-Isobutyl1-methylxanthine (IBMX), nifedipine, protein kinase inhibitor (H7) and quinine were purchased from Sigma (St Louis, Mo., USA). Ionomycin, 1,2-bis (o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA/AM) and protein kinase inhibitor (H89) were obtained from Calbiochem (San Diego, Calif., USA). 5-Nitro-2-(3-phenylpropylamino)-benzoate (NPPB) was kindly gifted from Professor R. Greger (Germany). All compounds were of reagent grade. Cell culture A6 cells were derived from Xenopus laevis kidney and purchased from American Type Culture Collection (ATCC). Briefly, A6 cells (passage 72–80) were grown on plastic flasks in medium modified for amphibian cells and supplemented with 10% fetal bovine serum (osmolality = 255 mosmol/kg H2O). The flasks were kept in a humidified incubator at 26°C with 4% CO2 in air. Upon passage, cells were seeded onto polycarbonate porous membranes attached to the bottom of plastic cups (6.5 mm Transwell filter; Tissue culture-treated Transwell; Costar, Cambridge, Mass., USA) at density of 1×105 cells/well and maintained as above. The cells cultured for 9–13 days were used for Iscexperiments. The culture medium was an NCTC medium modified for amphibian cells with 100 mM NaCl, 20 mM NaHCO3, and a pH of 7.4. In addition, the following components were present: 10% fetal bovine serum (Gibco), 100 µg/ml streptomycin, and 100 U/ml penicillin (Irvine Scientific, Santa Ana, Calif., USA) were present. No supplemental aldosterone was added to the cell culture media. Electrophysiology For measurement of Isc, monolayers grown on polycarbonate porous membranes were rinsed with the solution and were transferred to a modified Ussing chamber (Jim′s Instrument, Iowa City, Iowa, USA) designed to hold the intact filter cup (6.5-mm Transwell filter) connected to a VCC-600 four-electrode voltage-clamp (Physiological Instrument, San Diego, Calif., USA). A positive current represents a net flow of cation from the apical to basolateral solutions or a net flow of anion from basolateral to apical solutions. The bathing solution was kept at room temperature and stirred with bubbling with 5% CO2/21% O2/74% N2. To measure the transepithelial resistance (Rt), a 1.0-µA pulse (1 s duration) was imposed across the epithelium every 10 s. Transepithelial potential (PD) was measured with a pair of calomel electrodes immersed in saturated KCl and bridged to the Ussing chamber by a pair of polyethylene tubes filled with 2% agarose-containing 2 M KCl solution. The volume of both the apical and basolateral compartments was 5 ml. cAMP assay Intracellular concentrations of cAMP in A6 cells exposed to 1 mM IBMX for the indicated time period were measured. Before or af-

ter the cells were stimulated with 1 mM IBMX for indicated time, the reaction was stopped by addition of ice-cold 6% trichloroacetic acid and the cells were kept on ice for 30 min. After centrifugation at 2000 g for 15 min at 4°C, the supernatants were washed 4 times with water-saturated diethyl ether. The lower aqueous extracts were lyophilized. For cAMP assay, the samples (dried extracts) were dissolved with an appropriate volume of assay buffer. The concentrations of cAMP in the samples were determined with a commercially available cAMP enzyme immunoassay kit (Amersham, Arlington, Ill., USA). The data are shown as the mean ± SE.

Results cAMP-induced biphasic Isc To investigate the cAMP-mediated regulation of transepithelial ion transport, we measured Isc in response to IBMX. Basolateral application of 1 mM IBMX, an inhibitor of phosphodiesterase which increases intracellular cAMP levels, elicited biphasic increases in Isc, i.e., a transient increase (transient phase) followed by a sustained one (sustained phase) in Isc (Fig. 1A). The transient increase in Isc reached a peak about 3 min after application of 1 mM IBMX and the subsequent sustained increase was maintained at a steady level over 50 min.

Fig. 1 Response of short-circuit current (Isc) to: A 3-isobutyl-1methylxanthine (IBMX), B 8-bromo-cAMP or C forskolin. IBMX (1 mM, n = 8), 8-bromo-cAMP (5 mM, n = 4) or forskolin (50 µM, n = 6) was added to basolateral bathing solution at time 0&ig.c:/f

Fig. 2 Effects of IBMX on the cytosolic cAMP concentration. Cytosolic cAMP concentration was measured at 3 or 30 min after addition of 1 mM IBMX to monolayered A6 cells. Data shown as control were measured without addition of IBMX, n = 4&ig.c:/f

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Fig. 3A, B Effects of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) on IBMX-induced Isc. A NPPB (100 µM) was added to apical solution at 15 min before addition of IBMX (squares, control without NPPB addition, n = 8; circles, NPPB, n = 4). IBMX (1 mM) was added to basolateral solution at time 0. B Dose-dependent effect of NPPB on IBMX-induced Isc. Various concentrations of NPPB were added to apical solution 15 min before addition of IBMX. The values of Isc were measured at 3 (hatched bars) or 30 (solid bars) min after addition of IBMX (0 µM NPPB, n = 8; 20, 50 and 100 µM NPPB, n = 4)&ig.c:/f

The peak in the transient phase was approximately sevenfold larger than the basal level. Other agents which increase the intracellular concentration of cAMP could mimic this response of Isc to IBMX, i.e., basolateral application of 8-bromo-cAMP (5 mM) or forskolin (50 µM) also elicited biphasic increases in Isc (Fig. 1B, C). On the other hand, IBMX increased the intracellular cAMP concentration with time. The intracellular cAMP concentration had increased by about twofold the basal level at 3 min and fivefold at 30 min after its application (Fig. 2). These observations suggest that IBMX induces biphasic increases in Isc through an increase in the intracellular cAMP concentration.

Effects of Cl– channel blocker on IBMX-stimulated Isc Apical pretreatment with NPPB, which is a potent Cl– channel blocker, significantly inhibited the IBMX-stimulated Isc (Fig. 3A). NPPB showed dose-dependent inhibition of the Isc in a range between 20 µM and 100 µM and was more effective in the transient phase than that in the sustained phase (Fig. 3B). These observations are consistent with our previous observations that A6 cells have 3pS and 8-pS Cl– channels in the apical membrane [19, 21–23], which are blocked by NPPB [33]. These observations suggest that NPPB-sensitive Cl– release across the apical membrane through NPPB-sensitive Cl– channels mainly generates the IBMX-induced Isc.

Fig. 4A, B Effects of ionomycin on Isc. A NPPB (100 µM, circles) was added to apical solution 15 min before addition of 1 µM ionomycin. Squares show control experiments without NPPB treatment. Ionomycin of 1 µM was added to apical solution at time 0, n = 6. B Ionomycin-induced peak values of Isc, n = 6&ig.c:/f Table 1 Effects of ionomycin (1 µM) on transepithelial potentials, resistances and currents.&/tbl.c:&

Ionomycin (n = 6) Base Peak Sustained

PD (mV)

Rt (kΩ cm2)

Isc (µA/cm2)

−1.87±0.37 −13.63±1.38 −1.50±0.41

5.35±0.50 2.40±0.27 3.97±0.55

0.34±0.05 6.06±0.93 0.35±0.07

&/tbl.:

([Ca2+]c), induced a transient increase in Isc which reached a peak level after about 2 min and rapidly declined to the basal level (squares in Fig. 4A) and the peak value of ionomycin-induced Isc was dependent on the concentration of ionomycin (Fig. 4B). Pretreatment with 100 µM NPPB completely attenuated the ionomycin-induced Isc (circles in Fig. 4A). These results indicate that ionomycin generates Cl– secretion due to activation of NPPB-sensitive Cl– channels by increasing [Ca2+]c. Although 1 µM ionomycin caused a transient increase in Isc, it could not induce a sustained phase (Fig. 4A). Ionomycin decreased Rt (Table 1), suggesting that Ca2+ increases conductance. The Ca2+-induced decrease in Rt was observed even at 30 min (sustained phase) after addition of ionomycin (Table 1), suggesting that ionomycin was still effective; namely, the failure of ionomycin to induce a sustained increase in Isc was not due to a transient effect of ionomycin on conductance, which was still larger than the basal level at 30 min after addition of ionomycin. These observations suggest that the increase in [Ca2+]c can mimic the transient phase of IBMX-stimulated Isc and that the maintenance of the sustained phase may require an increase in cytosolic cAMP in addition to an increase in [Ca2+]c.

Effects of cytosolic Ca2+ on IBMX-induced Isc

Effects of Ca2+ channel blocker and Ca2+ chelator on IBMX-stimulated Isc

Since our previous reports [19, 21–23] indicate that the apical membrane of A6 cells has Ca2+-activated Cl– channels, we studied effects of cytosolic Ca2+ on the IBMX-induced Isc. Apical addition of 1 µM ionomycin, a Ca2+ ionophore to increase the cytosolic Ca2+ concentration

cAMP-dependent phosphorylation is known to cause activation of Ca2+ channels [31, 42] which causes an increase in [Ca2+]c. Since IBMX may stimulate Cl– secretion through Ca2+-activated Cl– channels by increasing [Ca2+]c due to Ca2+ influx through Ca2+ channels, we

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Fig. 5A, B Effects of nifedipine (a Ca2+ channel blocker) and BAPTA/AM (a Ca2+ chelator) on IBMX-induced Isc. A Nifedipine (100 µM, triangles, n = 8) and BAPTA/AM (10 µM, circles, n = 4) were added to both solutions 15 and 60 min, respectively, before addition of 1 mM IBMX (squares, control; n = 8). IBMX (1 mM) was added to basolateral solution at time 0. B The values of Isc were shown at 3 (hatched bars) or 30 (solid bars) min after addition of IBMX under each condition (control, n = 8; nifedipine, n = 8; BAPTA, n = 4)&ig.c:/f

studied effects of a Ca2+ channel blocker on IBMX-stimulated Isc. Bilateral pretreatment with 100 µM nifedipine, a voltage-dependent Ca2+ channel blocker, for 15 min diminished the IBMX-stimulated Isc (Fig. 5A). The transient peak was markedly inhibited by 65% (Fig. 5B), whereas the pretreatment with nifedipine had less effect on the sustained phase (only by 30%) than on the transient phase (Fig. 5B). This observation suggests that the IBMX-stimulated Isc is at least partially due to an increase in Ca2+ influx through nifedipine-sensitive Ca2+ channels. We next chelated cytosolic Ca2+ by pretreatment with 10 µM BAPTA/AM, a permeable Ca2+ chelator, for 1 h. The pretreatment with BAPTA/AM resulted in marked inhibition of the IBMX-induced transient phase but had a smaller effect on the sustained phase, similar to nifedipine (Fig. 5). These observations suggest that an increase in [Ca2+]c, which would be induced by IBMX, is an important factor for the generation of Isc. Based on these results, it appears that the transient phase of the IBMX-induced Isc is largely mediated by a Ca2+dependent pathway, which may be caused by Ca2+ influx, and that the sustained phase is also regulated by a Ca2+-dependent pathway but to a lesser extent than the transient phase. NPPB still decreased the IBMX-stimulated Isc obtained from cells treated with nifedipine or BAPTA (data not shown). On the other hand, in the presence of NPPB, nifedipine had no further effect on the IBMX-stimulated Isc (data not shown). These observations suggest that the NPPB-sensitive component of the IBMX-stimulated Isc consists of Ca2+-dependent and Ca2+-independent Cl– secretion. Effects of protein kinase inhibitors on IBMX-stimulated Isc Phosphorylation is the major signal of the cAMP-dependent pathway. To clarify whether the IBMX-induced Isc

Fig. 6A, B Effects of protein kinase inhibitors on IBMX-induced Isc. A After pretreatment with 0.5 µM H89 (circles; n = 4) and 50 µM H7 (triangles; n = 8) for 1 and 4 h respectively, 1 mM IBMX (squares, control; n = 8) was added to basolateral solution at time 0. B The values of Isc were shown at 3 (hatched bars) or 30 (solid bars) min after addition of IBMX under each condition (control, n = 8; H89, n = 4; H7, n = 8)&ig.c:/f

Fig. 7 Effects of nifedipine (a Ca2+ channel blocker) and H89 (a protein kinase A inhibitor) on IBMX-induced Isc. After pretreatment with 100 µM nifedipine (circles, n = 8) for 15 min, 0.5 µM H89 (diamonds, n = 4) for 1 h or both 100 µM nifedipine and 0.5 µM H89 (triangles, n = 4), IBMX (1 mM) was added to basolateral solution at time 0. Squares show the time course of the Isc without any treatment (control; n = 8)&ig.c:/f

is mediated through cAMP-dependent phosphorylation, we examined the effects of H89 (an inhibitor of PKA) on the IBMX-stimulated Isc. Pretreatment with 0.5 µM H89 for 1 h markedly inhibited the transient phase by 68% (Fig. 6). The sustained phase of IBMX-induced Isc in cells pretreated with 0.5 µM H89 was diminished by 57%. It was found that 50 µM H7 also inhibited the Isc (Fig. 6). Nifedipine (100 µM) did not have a synergistic effect on the transient or sustained phases of Isc in cells pretreated with 0.5 µM H89 (Fig. 7). These findings suggest that nifedipine-sensitive Ca2+ influx is increased through PKA-dependent phosphorylation pathways.

Effects of protein kinase inhibitors (H89 and H7) on ionomycin-induced Isc Next we studied whether the stimulatory action of Ca2+ on Cl– channels requires PKA-mediated phosphorylation. In cells pretreated with H89 (0.5 µM for 1 h) or H7 (50 µM for 4 h), ionomycin (1 µM) still stimulated Isc to a level similar to that of the control (without H89 pre-

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Discussion

Fig. 8A–C Effects of protein kinase inhibitors on ionomycin-induced Isc. A The time course of 1 µM ionomycin-induced Isc (n = 6). B After pretreatment with 0.5 µM H89 for 1 h, 1 µM ionomycin was added to apical solution at time 0 (n = 4). C After pretreatment with 50 µM H7 for 4 h, 1 µM ionomycin was added to apical solution at 0 time (n = 4)&ig.c:/f

Fig. 9 A model of IBMX-induced Cl– secretion. (AC Adenylate cyclase, PDE phosphodiesterase. * Ca2+ increases the open probability of the 3-pS Cl– channel, # cAMP increases the number of 8pS Cl– channels in the apical membrane)&ig.c:/f

treatment) (Fig. 8). These results suggest that activation of the Cl– channel by cytosolic Ca2+ does not require phosphorylation and that the phosphorylation-dependent process is required to increase [Ca2+]c via a nifedipinesensitive pathway.

IBMX increases the concentration of cytosolic cAMP by inhibiting phosphodiesterase. The activity of phosphodiesterase is dependent on [Ca2+]c [2]; in some cells the activity of phosphodiesterase is increased by a decrease in [Ca2+]c and cytosolic Ca2+ may be required for the inhibition of phosphodiesterase by IBMX. These observations suggest that nifedipine or BAPTA decrease the IBMX-induced Isc by reducing the cytosolic cAMP concentration. To rule out this possibility, we applied forskolin to increase the cytosolic cAMP concentration. Our preliminary observation indicates that nifedipine diminishes the forskolin-induced (50 µM) Isc and the IBMXinduced Isc similarly. Although there is still some possibility that the IBMX-induced increase in cytosolic cAMP concentration is diminished by nifedipine, the stimulatory action of cAMP on Isc would be partially mediated through Ca2+ influx. We observed only a very small amount of Isc generated by Na+ absorption in the present study. The small amount of amiloride-sensitive Isc (Na+ transport) may be due to the osmolality of the bathing solution and cell culture media, as shown by Wills et al. [38]; in particular, the osmolality of cell culture media used in the study by Wills and her colleagues was 200 mosmol/kg H2O, whereas the osmolality of cell culture media in our present study was 255 mosmol/kg H2O. Our preliminary observation suggests that hyperosmolality of cell culture media diminishes the amount of amiloride-sensitive Isc. Therefore, the small amount of amiloride-sensitive Isc (Na+ transport) may be mainly due to the high osmolality (255 mosmol/kg H2O) of the cell culture media. Furthermore, our previous report [29] indicates that most of the IBMX-induced Isc, measured under conditions that were the same as those of the present study, was due to Cl– secretion but not Na+ absorption. Our previous report [29] suggests that removal of apical Na+ or application of amiloride to the apical solution had no significant effect on the IBMX-induced Isc. Accordingly, although there was a small amount of the amiloride-sensitive Isc in the present study, the presence of the amiloride-sensitive Isc could not affect the conclusion of the present study. To study whether cAMP regulates the ion transport by stimulating phosphorylation of channel proteins, we used protein kinase inhibitors H89 and H7. H89 is one of the most specific inhibitors for PKA [4, 17]: the IC50 of H89 for PKA is 0.048 µM, which is much lower than that for PKC (31.7 µM) and cGMP-dependent protein kinase (PKG, 0.48 µM). The observation of the behaviour of cells pretreated with H89 indicates that cAMP-dependent phosphorylation contributes to the generation of IBMXinduced Isc. A large part of the Isc in the sustained phase was sensitive to H89, as was the transient phase. We also examined the effect of H7, which is a potent inhibitor of PKA, PKG, and PKC. H7 also inhibited the IBMX-induced Isc to almost the same extent as did H89. The IC50 of H7 for PKA is 3.0 µM, which is very similar to those values for PKC and PKG [18]. We tested the effect of H7

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on Isc to find out more about the involvement of other kinases, in particular PKC, in the generation of IBMXinduced Isc. Our data suggest that PKA-mediated but not PKC-mediated Cl– transport generates the IBMX-induced Isc. The basal Isc was also diminished by H89 and H7, suggesting that part of the basal Isc is also regulated in a phosphorylation-dependent manner. The effect of cytosolic Ca2+ on the Cl– channel may be mediated by Ca2+/calmodulin-dependent protein kinase [32]. We tested the effect of a Ca2+/calmodulin-dependent protein kinase inhibitor, KN93 (10 µM for 60 min; Calbiochem), on the ionomycin-induced Isc: KN93 did not inhibit the ionomycin-induced Isc. This observation suggests that the Ca 2+-activated Isc is not mediated through Ca2+/calmodulin-dependent protein kinase. Most of the IBMX-induced increase in Isc was generated through NPPB-sensitive Cl– channels. Our previous reports indicate that the apical membrane of A6 cells has at least two types of NPPB-sensitive Cl– channels [19, 21–23, 33]: the 3-pS Cl– channel is activated by cytosolic Ca2+, but the 8-pS Cl– channel is not. These observations suggest that IBMX activates the 3-pS Cl– channel by increasing [Ca2+]c, generating the transient phase of Isc in a manner dependent on [Ca2+]c. On the other hand, it is implied that the IBMX-induced sustained phase of Isc is generated by PKA/Ca2+-dependent and -independent components. The PKA/Ca2+-dependent component of the IBMX-induced sustained phase of Isc is thought to be generated by Cl– release through the 3-pS Cl– channel, while the 8-pS Cl– channel in the apical membrane is thought to contribute to the PKA/cytosolic Ca2+-independent component of the IBMX-induced sustained phase of Isc. Our previous observations [33] suggest that 8-bromo-cAMP increases the number of 8-pS Cl– channels in a PKA-independent manner. Therefore, the PKA/Ca2+-independent portion of the IBMX-induced sustained phase of Isc may be due to the cAMP-induced increase in the number (or density) of 8-pS Cl– channels in the apical membrane. These observations suggest that the IBMX-induced NPPB-sensitive Cl– secretion consists of two components: (1) the Ca2+-dependent component through the 3-pS Ca2+-activated channel, and (2) the Ca2+-independent component through the 8-pS Cl– channel, whose number in the apical membrane is increased by cAMP [33]. Ionomycin mimicked only the transient phase of the IBMX-induced Isc. Our previous report [29] suggests that IBMX can activate Cl– accumulation across the basolateral membrane mediated by the Na+/K+/2Cl– cotransporter and by the Na+/HCO3– symport-coupled Cl–/HCO3– exchanger. An increase in [Ca2+]c would not be sufficient for the generation of the IBMX-induced sustained Isc, which would require an increase in cAMP to activate Cl– accumulation into the cytosolic space via the aforementioned cotransporter and exchanger in a phosphorylation-dependent manner [1, 5, 16, 41].

&p.2:Acknowledgements This work was supported by a Grant-in-Aid from the Kidney Foundation of Canada to Y. Marunaka and partly supported by Grants-in-Aid from the Medical Research Council of Canada (Group Grant, Project 9 and MA13494), the Ontario Thoracic Society (Block Term Grant), MPG Research Fund to Y. Marunaka, and the Ministry of Education, Science and Culture of Japan (no. 07044298). Y. Marunaka is a Scholar of the Medical Research Council of Canada.

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