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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, People's Republic of China
Submitted 21 November 2007 ; accepted in final form 25 March 2008
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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phospholipase C; cystic fibrosis transmembrane conductance regulator; anion secretion; mucociliary clearance
Extracellular adenosine is a ubiquitous signaling molecule and modulates a wide array of biological processes via cell surface adenosine receptors. In airway epithelia, extracellular adenosine primarily comes from the metabolism of ATP release (33, 34), which is triggered by a variety of stimuli such as mechanical stress, osmotic challenge, or inflammation/tissue damage. Adenosine regulates distinct Cl– channels, mucin secretion, and ciliary mobility (7, 18, 30, 32, 47), and it is thought to play a key role in airway surface liquid homeostasis and mucociliary clearance (18, 27, 30, 49).
Apical adenosine modulates anion secretion by activating CFTR via cAMP/PKA signaling in intestinal and airway epithelial cells including Calu-3 cells (3, 6, 18, 42, 43). Intriguingly, the potent stimulation of anion secretion by adenosine is not correlated with its modest intracellular cAMP elevation (3, 5, 6, 18, 42), so it has long been surmised that other signaling pathways may be also involved. Recently, the uncorrelated effects have been partly attributed to a highly localized cAMP signaling from adenosine receptors to CFTR (1, 18, 19, 41, 45) and an additional phospholipase A2 (PLA2) signaling (2, 8, 28). Adenosine receptors also couple with PLC/Ca2+ signaling and PKC activation; whether or not this signaling pathway plays a part in the anion secretion and contributes to the uncorrelated efficacies of adenosine in anion secretion and in cAMP production remains to be determined. The aim of the present study was to investigate the role of adenosine-coupled PLC/Ca2+ signaling in anion secretion in Calu-3 cells.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Cell Culture
Human Calu-3 cells (American Type Culture Collection, Manassas, VA) were grown in Eagle's minimum essential medium (GIBCO-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum and 1% sodium pyruvate in an atmosphere of 95% air-5% CO2 at 37°C. For Ussing chamber studies, cells were seeded at a density of 
106 cells/cm2 on Costar snapwell inserts (0.4-µm pore size and 1.13-cm2 surface area). The cells were grown submerged in culture medium overnight, and the medium on the apical side was then removed to establish an air interface, which improves the cells’ differentiation and polarization. Cells were used for experiments 15–22 days after plating with a resistance >500 
·cm2.
Ussing Chamber Studies
Calu-3 epithelial monolayers grown on Costar snapwell inserts were mounted in Ussing chambers (Easy Mount, Physiologic Instruments, San Diego, CA). The transepithelial voltage was clamped to zero using a multichannel voltage-current clamp (VCC MC6; Physiologic Instruments), and the epithelial monolayers were allowed to reach a steady basal state for 15 to 30 min before each experiment. Unless indicated otherwise, all test compounds were added in both mucosal and serosal bath solutions. Data were sampled and recorded using Acquire & Analyze software (Physiologic Instruments), and peak current responses to test compounds were measured and plotted as 
I. Several different protocols were used.
Short-circuit current. Unpermeabilized cells were used in these studies. Both the mucosal and serosal baths contained Krebs-bicarbonate-Ringer solution (KBR; in mM: 140 Na+, 120 Cl–, 5.2 K+, 25 HCO3–, 2.4 H2PO4–, 0.4 HPO42–, 1.1 Ca2+, 1.2 Mg2+, and 5.2 glucose). The solution had pH 7.4 when gassed with 95% O2-5% CO2 at 37°C in the chambers. For ion substitution experiments, the bicarbonate-free bath contained (in mM) 140 Na+, 145 Cl–, 5.2 K+, 2.4 H2PO4–, 0.4 HPO42–, 1.1 Ca2+, 1.2 Mg2+, 10 HEPES, 5.2 glucose, and pH 7.4 adjusted with NaOH and was gassed with air.
Apical chloride current. To assess apical Cl– current (ICl) in isolation, the basolateral membrane was permeabilized with 20- to 30-min treatment of 20 µM amphotericin B, and a serosal-to-mucosal Cl– gradient was imposed with low Cl– KBR (in mM: 140 Na+, 4.5 Cl–, 5.2 K+, 25 HCO3–, 2.4 H2PO4–, 0.4 HPO42–, 1.1 Ca2+, 1.2 Mg2+, 115 gluconate, and 5.2 mannitol) on the mucosal side and KBR on the serosal side. Complete permeabilization of the basolateral membrane was evidenced by recording a current consistent with the serosal-to-mucosal flow of negative charge (13).
Basolateral potassium current. To examine basolateral K+ current (IK) in isolation, the apical membrane was permeabilized with 10 µM amphotericin B for 20–30 min, and a mucosal-to-serosal K+ gradient was established with modified, chloride-free KBR solutions on the mucosal and serosal sides. NaCl in KBR in the mucosal and serosal bath solution was replaced by equimolar K-gluconate and Na-gluconate, respectively. The KCl, CaCl2, and MgCl2 in the KBR on both sides were replaced with K-gluconate, Ca-gluconate, and Mg-gluconate, respectively, and calcium was raised to 4 mM to offset the Ca2+-buffering capacity of gluconate (13). Successful permeabilization of the apical membrane was evidenced by the recording of a current consistent with the mucosal-to-serosal flow of positive charge.
Single-Channel Recording Calu-3 cells were plated onto 35-mm cell culture plates 18–48 h before use. The patch-clamp experiments were conducted at 22°C, on isolated cells or cells at the edge of cell islands to record basolateral channels. For cell-attached recording configurations, both the bath and pipette solutions contained (in mM) 135 Na-gluconate, 5 K-gluconate, 1 Mg(gluconate)2, 2 Ca(gluconate)2, 10 glucose, and 10.5 HEPES. For excised, inside-out patches, the bath solution contained (in mM) 145 K-gluconate, 5 KCl, 1 MgCl2, 1 EGTA, 0.78 CaCl2 (free Ca2+ = 400 nM), 10 HEPES, and pH 7.2 (adjusted with KOH). In some experiments, CaCl2 was omitted to make a Ca2+-free bath solution. Switching between Ca2+-containing and Ca2+-free bath solutions was carried out using a fast-step perfusion system (SF-77B, Warner Instrument). The pipette solution contained (in mM) 140 K-gluconate, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and pH 7.2 (adjusted with KOH). Data were acquired using an Axopatch 200B amplifier and an Axon DigiData1322A with Axon pClamp9 software. Single-channel currents were filtered at 200 Hz and sampled at 1 kHz.
The product of channel number (N) and open probability (Po) was taken as channel activity. A control NPo was calculated from the 100 s of recording before adenosine exposure. For each test condition, NPo was calculated from the 100-s segment (among 200–300 s of recording) with the highest NPo.
RT-PCR The primers for A2A, A2B, A1, and A3 adenosine receptors were 5'-GCCATCGCCATTGACCGCTAC-3'(sense)/5'-GCAGTCGGGGCAGAAGAAAGT-3'(antisense), 5'-CTCTTCCTCGCCTGCTTCGT-3'(sense)/5'-GGGCAGAACACACCCAAAGAA-3'(antisense), 5'-CTTGCCTCGTGCCCCTTGGT-3'(sense)/5'-CGCTCCACCGCACTCAGATTGTT-3'(antisense), and 5'-CAGGTGCATTTTATGGACGGGAGT-3'(sense)/ 5'-AATAATACGTTGTCCCCAAGTCAGG-3'(antisense), respectively. Total RNA in Calu-3 cells was isolated with the RNeasy Mini kit (Invitrogen). The RT-PCRs were performed with the OneStep RT-PCR kit (Qiagen). The PCRs involved initial denaturation for 5 min at 95°C, followed by various cycles of denaturation (45 s at 95°C), annealing (45 s at 60°C), and extension (1 min at 72°C), and one final cycle of extension at 72°C for 10 min. The number of cycles performed for the A2A, A2B, A1, and A3 receptors were 35, 30, 42, and 42, respectively. β-Actin was used as an internal positive control. The identities of the PCR products were confirmed by DNA sequencing.
Statistical Analysis All the data are expressed as means ± SE. Statistical analysis was performed with Student's t-test, and P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Isc of 21 µA/cm2. U-73122, a specific inhibitor of PLC-β, attenuated 34% of the Isc (Fig. 1, A and B). The U-73122-insensitive Isc is underlaid by the activation of CFTR through Gs/adenylyl cyclase (AC)/cAMP signaling pathway, as has been previously demonstrated (8, 18).
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Isc by 35% (Fig. 1B). 2-Aminoethoxydiphenyl borate (2-APB), a reagent that blocks inositol 1,4,5-trisphosphate-dependent increase in intracellular Ca2+, showed a similar effect (46% inhibition; Fig. 1B). These data point to a role for Ca2+ in adenosine-induced Isc, reinforcing the notion that PLC signaling is involved in adenosine-induced Isc. It should be noted that the amplitude of the agonist-induced Isc varied from batch to batch of cells, as has previously been observed in other laboratories (11, 26), so control and treatment experiments were always performed in parallel with cells from the same batch.
Adenosine-Induced Isc Was Dependent On Neither PKC Nor Cross Talk of Ca2+ and cAMP Signaling
Isc is a complex readout of several ion channels and transporters, including CFTR in the apical domain and Ca2+-activated potassium (KCa) channels, Na/K-2Cl cotransporters, and Na+/HCO3– cotransporters in the basolateral domain (11, 13, 26). Since PKC, a downstream effector of PLC/Ca2+ signaling, potentiates the PKA activation of CFTR (24), we reasoned that the PKC activation of CFTR may account for the PLC/Ca2+-dependent component of the adenosine-induced Isc. However, bisindolylmaleimide II (BIM II), an inhibitor of conventional and novel PKC, failed to inhibit adenosine-induced Isc (control 26.6 ± 2.2 vs. BIM II 26.9 ± 2.1 µA/cm2, n = 6, P = 0.93), ruling out a role for PKC in the PLC/Ca2+-dependent component of adenosine-induced Isc. Furthermore, up to 2 µM PMA failed to evoke any significant Isc (data not shown), suggesting that acute PKC activation generally may not be important for anion secretion in Calu-3 cells.
Ca2+ activates cAMP/PKA signaling through several cross talk mechanisms including modulating ACs and phosphodiesterases. So, in principle, adenosine-induced PLC/Ca2+ signaling could regulate CFTR or other membrane transporters through cross talk with cAMP/PKA signaling. Direct test of this possibility is precluded, however, by the fact that adenosine regulates CFTR via Gs/AC/cAMP signaling (8, 18). Therefore, thapsigargin (TG) and A-23187 were used to address this question indirectly. TG depletes intracellular Ca2+ stores and produces a sustained elevation of cytosolic Ca2+ by selectively inhibiting Ca2+-ATPase in the endoplasmic reticulum; A-23187 is a Ca2+ ionophore that allows extracellular Ca2+ to flow into the cytosol. Neither TG (1 µM) nor A-23187 (5 µM) had any significant effect on intracellular cAMP accumulation (data not shown, n = 3). More importantly, PKA inhibitor H-89 (20 µM) failed to block the Isc induced by 1 µM TG (control 62.4 ± 9.1 vs. H-89 61.8 ± 4.9 µA/cm2, n = 3, P = 0.95). These results indicate that Ca2+ is generally dissociated from cAMP/PKA signaling with respect to anion secretion in Calu-3 cells, arguing against the idea that adenosine-induced PLC/Ca2+ signaling regulates CFTR or other membrane transporters through cross talk with cAMP/PKA signaling.
The PLC/Ca2+ Signaling Targeted to the Basolateral Ca2+-Activated K+ Channel Because the data just described appear to rule out ipsilateral CFTR as a target of apical adenosine-induced PLC/Ca2+ signaling (also see Fig. 3, C and D), contralateral KCa channels were then considered as a potential target. The opening of KCa channels hyperpolarizes the membrane potential and increases the electrochemical driving force for anion exit of the apical membrane (13).
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Isc induced by 10 µM mucosal adenosine by 47% (Fig. 2, A and B). To rule out the possibility that the effect of clotrimazole resulted from its nonspecific action on cytochrome P-450 enzymes (51), TRAM-34 {1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole}, an analog of clotrimazole, which blocks KCa channels but not cytochrome P-450 (51), was also tested. TRAM-34 (1 µM) also attenuated the adenosine-induced Isc by 31% (Fig. 2B). These data together point to a possible role for the KCa channels in adenosine-induced Isc.
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IK (50% inhibition). This basolateral IK was also inhibited by BAPTA-AM (37% inhibition), 2-APB (45% inhibition), clotrimazole (50% inhibition), and TRAM-34 (49% inhibition), consistent with the idea that apical adenosine activates basolateral KCa channels through PLC/Ca2+. Moreover, this basolateral IK seems to require neither PKC nor PKA activation because it was insensitive to BIM II and H-89 (Fig. 3B), complementing the data in the last section, which suggest a dissociation of the adenosine-induced PLC/Ca2+ signaling from PKC and PKA in the anion secretion.
A little unexpectedly, 20 µM clotrimazole or 1 µM TRAM-34 suppressed only 50% of the adenosine-induced basolateral IK (Fig. 3B). In contrast, 20 µM clotrimazole or 1 µM TRAM-34 blocked 80–100% of the basolateral IK induced by 1 µM serosal TG (100% inhibition, control 44.3 ± 3.1 vs. clotrimazole 0.3 ± 0.1 µA/cm2, n = 7, P < 0.000001; 77% inhibition, control 33.6 ± 5.6 vs. TRAM-34 7.7 ± 2.9 µA/cm2, n = 5, P = 0.007), or by 200 µM 1-EBIO (83% inhibition, control 25.8 ± 1.8 vs. clotrimazole 4.5 ± 0.9 µA/cm2, n = 6, P = 0.0004), all of which presumably solely reflect the activity of KCa channels. In addition, 20 µM clotrimazole nearly completely blocked KCa single channels in patch-clamp studies (Fig. 4). Thus, the clotrimazole- or TRAM-34-insensitive component in the adenosine-induced basolateral IK seems to largely reflect the activation of additional potassium channels rather than an incomplete inhibition of KCa channels (see more in DISCUSSION).
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ICl was substantially blocked by H-89 (Fig. 3D), consistent with the adenosine regulation of apical CFTR through cAMP/PKA signaling. In contrast, U-73122 or clotrimazole had virtually no effect (Fig. 3, C and D). These results confirm the specificity of the effects of U-73122 and clotrimazole on the adenosine-induced Isc and place their target exclusively in the basolateral domain, in good accordance with the data in the last section, which indirectly argue against the involvement of apical CFTR. Adenosine Activated KCa Single Channels To further determine whether adenosine activates KCa channels, patch-clamp techniques were used to test the effect of adenosine on the KCa single channels. In resting cells, KCa channels were rarely evident in cell-attached patches, but they were robustly activated after a membrane patch was excised and exposed to a bath containing 400 nM Ca2+ (Fig. 4A), and the channel displayed a current-voltage relationship and potassium selectivity (Fig. 5) resembling the KCa channel previously reported in Calu-3 cells (13). The channel was silenced by removal of Ca2+ from the bath solution or by addition of clotrimazole (Fig. 4, A and B), while it was activated by 1-EBIO (data not shown). These biophysical and pharmacological characteristics identify this channel as the hIK1 channel previously described (12, 16, 17). The activation of the KCa channel by 1 µM TG further verified its Ca2+ sensitivity in a cell-attached configuration (Fig. 4C). In cell-attached membrane patches, addition of 10 µM adenosine to the bath activated the KCa channel, and 100 µM adenosine triggered a more robust activation, which was sensitive to clotrimazole (Fig. 4, D–F). These single-channel data further confirmed that adenosine receptors are coupled to the activation of KCa channels.
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Adenosine Modulated KCa Channels Via A2B Adenosine Receptors Next, we sought to identify the adenosine receptor subtype(s) in the activation of KCa channels. Previous studies have suggested that Calu-3 cells express A2A and A2B but not A1 and A3 receptors (8, 47). RT-PCR experiments confirmed that Calu-3 cells express the transcripts of A2A and A2B receptors (Fig. 6A). However, in contrast with findings of the previous study (47), A1 transcripts was also detected after a larger number of PCR cycles (Fig. 6A).
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IK by enprofylline, an A2B-selective antagonist (Fig. 6, C and D). Although these experiments did not distinguish clotrimazole/TRAM-34-sensitive from -insensitive IK, A2B receptors can be considered as playing a major role in the clotrimazole/TRAM-34-sensitive IK, because the portion of clotrimazole/TRAM-34-sensitive IK was substantial (50%) (Fig. 3B). It is less likely that such a substantial portion would be mainly contributed by adenosine receptor subtypes other than A2B, because the A2B subtype plays an overwhelmingly predominant role in eliciting total basolateral IK (Fig. 6B). In another series of experiments, the adenosine analogs were tested on apical ICl in basolaterally permeabilized epithelia. The rank order potency (NECA > AB-MECA >CPA > CGS-21680) also suggested a major role of A2B receptors in apical CFTR chloride channel activation (Fig. 6E). The predominant role for A2B receptors in the regulation of both apical CFTR chloride channels and basolateral potassium channels is in good agreement with the previous observation that A2B receptor is the major adenosine receptor subtype involved in transepithelial ion transport in intact Calu-3 cells (8, 18).
Adenosine Induced Secretion of Both Chloride and Bicarbonate It has been proposed that Calu-3 cells secrete bicarbonate or chloride depending on the mode of stimulation (13, 26): a rise of cAMP leads to bicarbonate secretion by solely opening apical CFTR channels, whereas Ca2+ elevation results in a predominant Cl– secretion by activating basolateral potassium channels and hyperpolarizing the cell membrane. This model would predict that apical adenosine causes a mixed secretion of bicarbonate and chloride because of its dual modulation of apical CFTR by cAMP signaling and basolateral KCa channels by PLC/Ca2+ signaling. To test this prediction, the portions of chloride and bicarbonate in the adenosine-induced anion secretion were assessed.
Bumetanide, a blocker of the basolateral Na/K-2Cl cotransporter that supplies chloride for transepithelial secretion, had no effect on basal Isc (data not shown) as has previously been described (39), but it decreased the plateau of the adenosine-induced Isc by 36% (Fig. 7, A and C), indicating that the adenosine-induced anion secretion contains 30% chloride secretion. The addition of DNDS (a blocker of sodium-bicarbonate cotransporters) and acetazolamide (a carbonic anhydrase inhibitor) caused a further suppression of the Isc (Fig. 7A), suggesting a bicarbonate secretion. Consistent with this, removal of bicarbonate from the mucosal and serosal bath solutions substantially reduced the adenosine-induced Isc (Fig. 7, D and E). These results together suggest that apical adenosine induced a mixed secretion of chloride and bicarbonate and further confirm that apical adenosine regulates KCa channels. In marked contrast, forskolin-induced Isc was blocked by DNDS and acetazolamide but not by bumetanide (Fig. 7, B and C), confirming the previous observations that forskolin predominantly evokes a bicarbonate secretion (13, 26).
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Adenosine, through A1 receptors and Ca2+ signaling, seems to regulate potassium current in airway epithelial cell types that lack CFTR (36, 48). However, the identity and sideness of the channel underlying the potassium current were not resolved. In the present study, KCa channels were identified as the target of adenosine receptors. In addition, we demonstrated that apical A2B adenosine receptors modulated contralateral KCa channels. It should be pointed out that for characterizing the basolateral KCa channel at single-channel level, we patched isolated cells or cells at the edge of cell islands to gain access to the basolateral membrane (see EXPERIMENTAL PROCEDURES), on the basis of the assumption that these cells are not fully polarized. To our knowledge, although not ideal, this is the best available approach.
The activation of contralateral KCa channels by apical adenosine implies that Ca2+ signaling transits the cell. Indeed, previous studies have suggested that a significant portion of Ca2+ moves from the stimulated to the contralateral membrane in polarized airway epithelial cells (31, 35), although the regulation of Ca2+ release and influx was membrane restricted (31) and Ca2+ diffusion was largely restricted by the mitochondria, which act as physical barriers (35). In contrast, cAMP signaling activated by apical adenosine seems to be more tightly confined to the apical membrane in Calu-3 cells [(1, 18); also, see below]. In our case, the portion of Ca2+ transiting the cell appeared to play a significant role in the adenosine regulation of anion secretion, rather than being a trivial, undesirable signaling "leakage."
Might Adenosine Activate Other Basolateral Potassium Channels? Our data suggest that apical adenosine may activate basolateral potassium channels other than KCa (see Fig. 3B and the text). Since cAMP-activated KCNQ1 potassium channels have been observed in the basolateral domain in Calu-3 cells (9), we used clofilium, a widely used blocker of KCNQ1, to determine whether or not apical adenosine activates basolateral KCNQ1 through cAMP. Clofilium proved to be ineffectual, however, because it blocked Ca2+ activated K+ current in Ussing chamber studies (9) and KCa single channels in our hands (data not shown). Indeed, clofilium seems to block many other types of potassium channels (44). Alternatively, we used MDL-12330A to disrupt AC activities. However, MDL-12330A had no effect on the adenosine-induced basolateral potassium current while markedly blocking the adenosine-induced Isc in intact cells (data not shown), suggesting that adenosine-triggered cAMP signaling is constrained in the apical domain, as has previously been proposed (18). In addition, KCa is regulated not only by Ca2+ but also by cAMP/PKA and other signaling molecules (16, 17). The adenosine activation of KCa channels, however, was insensitive to PKA blocker H-89 (Fig. 3B), further supporting the proposed apical confinement of adenosine-coupled cAMP signaling.
We also attempted to investigate whether PLA2 mediates the adenosine activation of other potassium channels, because Cobb et al. (8) and others (28) have suggested that adenosine receptors regulate CFTR and possibly potassium channels through PLA2 with an undetermined mechanism in Calu-3 cells. They found that adenosine-induced halide efflux was inhibited by chlorpromazine (CPZ), a phenothiazine derivative blocking cPLA2. Similarly, we found that CPZ attenuated adenosine or NECA-induced Isc by 30% (data not shown). However, CPZ also blocks various membrane receptors and ion channels and reportedly inhibits basolateral KCa channels in Calu-3 cells (21). This direct effect on basolateral KCa channels, also observed by us in Ussing chamber and single-channel studies (data not shown), compromises the utility of CPZ as a tool for determining whether adenosine activates potassium channels by PLA2 signaling. Since our present data suggest adenosine regulation of basolateral KCa channels, the previously observed CPZ effect on adenosine-induced Isc in Calu-3 cells (8, 28) may be at least partly attributable to its direct effect on basolateral KCa channels rather than PLA2.
So, adenosine seems to activate additional potassium channels through an unidentified signaling pathway(s). It should be stressed that this was observed in apically permeabilized cells. Whether or not it takes place in intact cells remains to be determined.
Contributions of KCa and Other Potassium Channels to the Adenosine-Induced Anion Secretion in Intact Cells
Because clotrimazole or TRAM-34 blocked 80–100% of 1-EBIO or TG-induced IK, which presumably solely reflects the activity of KCa channels, clotrimazole or TRAM-34-sensitive Isc may well represent the contribution of KCa activation. Our results thus suggest that activation of KCa by PLC/Ca2+ contributed a significant portion (31–47%) of the adenosine-induced anion secretion (Fig. 2). In principle, the PLC/Ca2+ signaling should dictate chloride secretion, but their quantitative correlation is a complex driving force issue involving several membrane transporters (13, 26). The bumetanide experiments in the present study suggest that it led to 30% chloride secretion (Fig. 7).
As discussed above, a considerable portion (50%) of adenosine-induced basolateral IK was clotrimazole/TRAM-34-insensitive permeabilized cells (Fig. 3B). If this clotrimazole/TRAM-34-insensitive channel(s) is also activated by adenosine in intact cells and contributes to the Isc as effectively as KCa channels, the activation of total basolateral potassium channels (including KCa channels) would contribute much more than the 31–47% (contributed by KCa channels) to Isc shown in Fig. 2. If so, a predominant chloride secretion would be expected, which apparently does not fit well with the bumetanide data suggesting an approximate 30% chloride secretion (Fig. 7). Thus, the clotrimazole-insensitive channel may contribute little to the Isc in intact cells.
Mixed Secretion of Chloride and Bicarbonate On the basis of the proposed anion secretion model for Calu-3 cells (13, 26), adenosine may be expected to induce a mixed secretion of chloride and bicarbonate as a result of its dual regulation of CFTR and KCa channels. Indeed, the effect of 20 µM bumetanide indicates an approximate 30% chloride secretion (Fig. 7, A and C). However, 20 µM bumetanide may not be maximal because it blocked only 70% of 1-EBIO-induced isotopic Cl– flux (13). A more accurate assessment of chloride secretion portion requires further examination using isotope efflux techniques (13).
A recent study, apparently conflicting with the proposed anion secretion model for Calu-3 cells, found that in single submucosal glands of pig, carbachol and forskolin elicited secretion with approximately equal portions of chloride and bicarbonate (25). The authors attributed this unexpected discrepancy to the cell heterogeneity of the glands and suggested that secretion from serous cells, which are modeled by Calu-3 cells, could be modified by other cell types in the glands. While this may be true, the present results potentially offer an additional explanation: carbachol/Ca2+ and cAMP stimulate ATP release in airway epithelia cells including Calu-3 cells (4, 37, 50), although the effect of cAMP on ATP release is still debatable; ATP is converted into adenosine by ecto-nucleotidases, and elevation of extracellular adenosine in turn evokes a mixed secretion of chloride and bicarbonate and alters the anion composition in the secretion induced by carbachol and forskolin. The question, then, is why such an effect, if operative, has not been observed in the previous studies (13, 26). Conceivably, the effect of the ATP released by secretagogues on ion secretion may have been diluted or eliminated by large lumen volume in the Ussing chambers used in these studies (13, 26) or even in an improved "virtual gland" method (20). Irokawa et al. (20) suggested that the ratio of cell surface to apical lumen volume is 20 and 6,000 times greater in human submucosal gland tubules than in the "virtual gland" and Ussing chambers, respectively. The effect of released ATP is perhaps only evident when the apical lumen volume stays put and remains small (18, 22, 49).
Interestingly, direct activation of KCa channels by 1-EBIO or chlorzoxazone appears to trigger a robust apical ATP release in Calu-3 cells (22). This mechanism may play a part in carbachol-induced ATP release since carbachol activates KCa channels. In addition, this mechanism, put together with our present observation, potentially provides a positive feedback loop in ATP release in vivo: released ATP is converted into adenosine that activates KCa, and activation of KCa subsequently further promotes ATP release.
The Role of PKC in the Anion Secretion PKC does not by itself seem to activate CFTR, but it is required for acute stimulation of CFTR by PKA (24). Consistent with this idea, manipulation of basal PKC phosphorylation through PKC activator or inhibitor pretreatment could alter the subsequent activation of CFTR by PKA (15). Halide efflux studies in unpolarized Calu-3 cells have suggested that pretreatment with chelerythrine markedly reduces the activation of CFTR by cAMP-elevating reagents (29). In the Ussing chamber studies in the present study, however, pretreatment with BIM II had no effect on the activation of CFTR by adenosine that also couples with cAMP signaling (see the text). The discrepancy may be due to different PKC dephosphorylation rates of CFTR in these studies as a result of cell culture and experimental conditions, because the effect of PKC inhibitors on PKA activation of CFTR is only evident if there is active PKC dephosphorylation. On the other hand, acute activation of PKC by phorbol ester alone has variable effect on CFTR activation (15). In this work, up to 2 µM PMA failed to induce any significant change in Isc (data not shown), suggesting that acute PKC activation is not important for CFTR regulation in Calu-3 cells.
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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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.
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REFERENCES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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0.019. Unless indicated otherwise, test compounds were added to both the mucosal and serosal bath solutions in this and the other figures (see EXPERIMENTAL PROCEDURES).
, n = 4) or after replacement of 100 meq K+ with Na+ in the pipette solution (
, n = 4).
