Regulation of CFTR channels by HCO3-sensitive soluble adenylyl cyclase in human airway epithelial cells

Yan Wang,* Chak Sum Lam,* Fan Wu, Wen Wang, Yuanyuan Duan, and Pingbo Huang

Department of Biology, Hong Kong University of Science and Technology, Kowloon, Hong Kong, People's Republic of China

Submitted 21 December 2004 ; accepted in final form 14 June 2005


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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CFTR channels conduct HCO3 in addition to Cl in airway epithelial cells. A defective HCO3-transporting function of CFTR may underlie the pathogenesis of cystic fibrosis. In the present study, we have investigated whether a HCO3-sensitive soluble adenylyl cyclase (sAC) is functionally coupled with CFTR and thus forms an autoregulatory mechanism for HCO3 transport in human airway epithelial Calu-3 cells. A reverse transcriptase-polymerase chain reaction showed that transcripts of both full-length and truncated sACs are present in Calu-3 cells. Truncated sAC protein is the predominant, if not the only, isoform expressed in Calu-3 cells. HCO3 stimulated a modest increase in cAMP production, and the increase was sensitive to 2-hydroxyestradiol (2-HE), a sAC inhibitor, but not to SQ22,536, a blocker of conventional transmembrane adenylyl cyclases. These results suggest that sAC is functional in Calu-3 cells. Adding 25 mM HCO3 to the bath stimulated CFTR-mediated whole cell currents in the absence, but not in the presence, of 2-HE. In cell-attached membrane patches, 25 or 50 mM HCO3 in the bath markedly increased the product of channel number and open probability of CFTR, and this activation was attenuated by 2-HE. These findings demonstrate that sAC signaling pathway is involved in the regulation of CFTR function in human airway epithelium and thereby provides a link between the level of intracellular HCO3/CO2 and the modulation of HCO3-conductive CFTR function by cAMP/PKA.

cystic fibrosis transmembrane conductance regulator; bicarbonate; adenosine 3',5'-cyclic monophosphate; 2-hydroxyestradiol


CFTR IS THE ANION CHANNEL defective in cystic fibrosis (CF), a lethal hereditary disease. CFTR is subject to complex and integrated phosphorylation/dephosphorylation regulation by multiple enzymes, including PKA, PKC, Src tyrosine kinase, AMP-dependent kinase, and phosphatases. Of the kinases activating CFTR, PKA appears to be the most important pathway and its activity is required for CFTR to function (1, 19, 24, 28). It is well established that the CFTR channel conducts Cl, and recently increasing evidence has suggested that CFTR conducts HCO3 as well and may be the major HCO3 pathway in the apical membrane of airway epithelial cells (6, 7, 15, 22). HCO3 is suggested to play a crucial role in the pH regulation of the airway surface liquid and thus in the defense mechanism of the lung, which involves several pH-dependent physiological processes. The defective HCO3 transport function of CFTR is thought to underlie the pathogenesis of CF (5, 16, 25).

Apart from the well-characterized conventional transmembrane adenylyl cyclases (TMACs), a soluble form of AC (sAC) activity had been described in mammalian cells more than two decades ago (20, 21). Early studies suggested that sAC is biochemically different from TMAC. Unlike TMAC, sAC has no apparent transmembrane domains and is insensitive to G protein regulation and forskolin stimulation. Interestingly, sAC is stimulated by the physiologically important ion HCO3 (3, 4, 14, 32). The sAC gene in the mouse was first cloned, and subsequently rat and human sAC genes were identified by performing RT-PCR and database searches, respectively (3). The mouse gene shares 91% identity with the rat gene and 74% identity with the human gene. Alternative RNA splicing of sAC genes in the mouse and the rat generates two distinct transcripts encoding a full-length sAC (FL-sAC) and a truncated sAC (T-sAC), respectively (14). Deletion of exon 11 (56 nt) in the truncated sAC transcript results in a reading frame shift and early termination of translation. The cDNA of the FL-sAC predicts a protein of 187 kDa (~1,600 amino acid residues, which vary among different species). T-sAC is a protein of ~50 kDa and contains almost exclusively the two catalytic domains. Both FL-sAC and T-sAC show HCO3-dependent adenylyl cyclase activity; however, T-sAC is 10 times more active than FL-sAC. sAC appears to be ubiquitous, and it is proposed that sAC serves as a universal HCO3/CO2 sensor (4, 30). sAC has been found to play a critical role in several important biological processes in various tissues, including sperm maturation (9, 13), activation of cAMP response element-binding protein (31), and recycling of V-ATPase (23).

To epithelial biologists, it is of obvious interest to examine the targets of sAC, a HCO3-sensitive signaling molecule, in HCO3-transporting epithelial cells. Most recently, Sun et al. (27) reported that sAC activated CFTR in bovine corneal endothelium and suggested that sAC coupling to CFTR forms an autoregulatory mechanism for HCO3 transport by CFTR. While intriguing, these data must be interpreted with caution. First, no specific approach was used to disrupt sAC function. Thus doubt persists regarding whether the CFTR activation they observed was mediated by sAC or by TMAC. Second, the authors used a fluorescent dye-based readout and several nonspecific inhibitors to assess CFTR activity. It is widely recognized that such an indirect, macroscopic approach cannot determine CFTR or even CFTR-dependent activity unless it is substantiated by parallel studies in CFTR-deficient cells. This is particularly important for the model cell system in the Sun et al. study, in which CFTR function was not fully established. Generally, claims about the modulation of CFTR per se need to be verified at the single-channel level. A better understanding of sAC regulation of CFTR calls for further studies with more direct and relevant measurement of CFTR activity. In addition, it is of interest to extend these studies to the airway epithelium, where CFTR and its HCO3 transport function play a crucial role in the innate defenses of the lung. In the present work, in which we used patch-clamp techniques and a specific blocker of sAC, we studied the regulation of CFTR by sAC in Calu-3 cells, a widely used cellular model of human airway epithelia.


    MATERIALS AND METHODS
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Cells. Human Calu-3 cells (HTB-55; American Type Culture Collection, Manassas, VA) were grown in Eagle's minimum essential medium (MEM; GIBCO-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum, 10% sodium pyruvate, and penicillin/streptomycin in an atmosphere of 95% air-5% CO2 at 37°C. Cells were passaged at a 1:3 dilution every 5–7 days. For cAMP assay, cells were grown to confluence on clear Transwells (Costar) with a resistance >1,000 {Omega}·cm2. For patch-clamp and other studies, cells were grown on plastic dishes and used for experiments after 3–7 days.

RT-PCR. A pair of sAC primers flanking the region deleted in T-sAC was constructed on the basis of the published cDNA sequence (14). The sense and antisense primers were 5'-CTGTATCCATCGGTGT-3' and 5'-TTGGTCTGTCGTTCTT-3', respectively. Total RNA was extracted from Calu-3 cells with the RNeasy Mini kit (Qiagen). RT-PCR was performed with the OneStep RT-PCR kit (Qiagen). PCR was performed for initial denaturation of one cycle at 94°C for 10 min, followed by 35 cycles of denaturation (94°C for 45 s), annealing (52°C for 45 s), and extension (72°C for 45 s), and a final extension for one cycle at 72°C for 10 min. At the end of the PCR amplification, products were analyzed in 1.5% agarose gels stained with ethidium bromide and visualized under UV light. The amplified fragments of correct size were excised from the agarose gel and purified using a gel extraction kit (Qiagen). The second-round PCR was conducted using the purified PCR products as the template and another pair of sAC primers (sense primer, 5'-ATTGTGACCTGCGACTC-3'; antisense primer, 5'-AGACTTGGCTGCTGTTAG-3'). PCR was performed for an initial denaturation of 1 cycle at 94°C for 4 min and 39 cycles at denaturation (94°C for 30 s), annealing (50°C for 30 s), and extension (72°C for 30 s) with Taq polymerase (Invitrogen). This was followed by a final extension step at 72°C for 10 min. PCR products of the correct size of two splice variants were excised for sequencing.

Generation and purification of sAC antibody. A rabbit polyclonal antiserum was raised against a synthetic peptide of 17 amino acids corresponding to the NH2 terminus of human sAC (NH2-MNTPKEEFQDWPIVRIA-COOH). The serum was affinity purified using SulfoLink kit (Pierce, Rockford, IL) with the antigen peptide.

Membrane and cytosol preparation. Cells were homogenized manually in a glass homogenizer in homogenization buffer containing 10 mM Tris·HCl, 1 mM EDTA, 200 mM sucrose, and 1x protease inhibitor cocktail (Complete Mini; Roche), pH 7.4. The nuclei and cell debris were removed from the homogenate by performing centrifugation at 900 g for 10 min at 4°C. The resulting supernatant was centrifuged at 100,000 g for 60 min at 4°C. Subsequently, supernatants were collected to yield cytosolic extracts, and pellets were collected to generate a particulate fraction.

Western blot analysis. The membrane pellet and cytosolic extracts were solubilized in the homogenization buffer plus 1% Triton X-100. Cytosolic membrane extracts and total cell lysates were subjected to SDS-PAGE. Proteins were then transferred onto polyvinylidene difluoride (PVDF) membrane. The membrane was incubated in TBST (0.1% Tween 20, 20 mM Tris·HCl, and 500 mM NaCl; pH 7.4) containing 5% nonfat dry milk for 2 h to block nonspecific binding sites. Next, the PVDF membrane was incubated overnight with the affinity-purified rabbit anti-sAC serum with or without 4 mg/ml antigen peptide or a monoclonal antibody against the CFTR COOH terminus (R&D Systems). The membrane was washed three times with TBST and incubated for 1 h with a horseradish peroxidase (HRP)-conjugated secondary antibody diluted 1:10,000 in TBST containing 5% nonfat dry milk. After being washed several times with TBST, bound antibodies were detected using enhanced chemiluminescence (Pierce).

cAMP assay. Cells were incubated in 25 mM HEPES-buffered, air-equilibrated, HCO3-free MEM, pH 7.2, for 3 h at 37°C, followed by 10-min incubation with the mucosal addition of 40 mM NaHCO3 or 10 µM forskolin as positive control or 40 mM NaNO3 as a negative control. NaHCO3 solution was made in the assay buffer immediately before starting the assay and was buffered to pH 7.2 with predetermined amounts of HEPES (40 mM NaHCO3 + 8 mM HEPES), and NaHCO3-containing reaction mixtures were placed in an atmosphere of 95% air-5% CO2. Papaverine (200 µM) and 300 µM 8-(p-sulfophenyl)theophylline (8-SPT) were present in all reactions. Papaverine is a nonspecific phosphodiesterase inhibitor, and 8-SPT was used to block the autocrine stimulation of A2B adenosine receptors and thus reduce cAMP background (11). The cells were then lysed in 0.1 N HCl, and cAMP was measured using a cAMP immunoassay kit (Assay Designs).

Whole cell voltage-clamp studies. CFTR-mediated whole cell Cl current was recorded as previously described previously (12). Briefly, the pipette solution contained (in mM) 40 Tris-Cl, 100 Tris-gluconate, 2 MgCl2, 5 HEPES, 1 EGTA, and 0.1 CaCl2, 1 MgATP, and 0.2 LiGTP, pH 7.4, with Tris. Ca2+ activity was buffered to ~40 nM. The bath solution contained (in mM) 150 Tris-Cl, 2 MgCl2, 1 CaCl2, 5 HEPES, 30 sucrose, 10 D-glucose, pH 7.4, with Tris. Patch pipettes had a resistance of ~3 M{Omega} with these solutions. In a standard protocol, voltage was clamped to –40 mV and stepped to 60 mV every 10 s as current was recorded. Current-voltage (I-V) plots were obtained every 2 min from the current responses to step pulses from –100 to 100 mV in 20-mV increments. The whole cell conductance was calculated by the slope of a linear fit of the I-V relationship and was used as readout of CFTR channel activity as previously established (12). After the whole cell recording configuration was obtained, basal current was recorded for 6 min in the presence or absence of 20 µM 2-hydroxyestradiol (2-HE). Freshly made 25 mM NaHCO3 (buffered with 5 mM HEPES) was then added to the bath, and whole cell current was recorded for 8 min. The bath solution was not gassed with 5% CO2, and the pH change of the HCO3-containing bath solution was minimal in an 8-min period (<0.02 pH units). HCO3-stimulated slope conductance was calculated from the I-V plot with the highest slope conductance. Reversal potentials were calculated using Vpipette at zero current with correction for the junction potential of the pipette and bath solution (–5 mV).

Cell-attached recording. The procedures were essentially the same as previously described (12), with some modification. The bath contained (in mM) 150 Tris-Cl, 1 CaCl2, 2 MgCl2, 10 D-glucose, 5 HEPES, pH 7.4 maintained with Tris. The pipette solution contained 160 mM Tris-Cl and 30 mM sucrose at pH 7.4 adjusted with Tris. After the formation of a cell-attached seal, CFTR single-channel activity was recorded for 5 min at Vpipette of –60 mV in the presence or absence of 20 µM 2-HE. Subsequently, freshly made 25 or 50 mM NaHCO3 (buffered to pH 7.4 with HEPES) was added into the bath, and the activity of CFTR was recorded for 5 min. In our experiments, CFTR channels were identified by typical burstlike openings and flickery kinetics at the positive holding potential and the slope conductance of ~7.0 pS in excised patches. The identity of these channels has previously been confirmed to be CFTR on the basis of extensive work by others and by us (12, 13).

Data acquisition and analysis of patch-clamp studies. Data were acquired using an Axopatch 200B amplifier and Axon DigiData 1322A with Axon pClamp 9 software. Single-channel currents were filtered at 100 Hz and sampled at 10 KHz. The product of CFTR channel number (n) and open probability (Po) was calculated as CFTR channel activity (nPo). nPo was calculated from the 100-s segment recorded before addition of NaHCO3 or from the 100-s segment (of 300 s) with the highest nPo after addition of NaHCO3. Whole cell current was acquired at 500 Hz and filtered at 100 Hz. All data were analyzed using Clampfit 9.0 software.

Statistics. All of the data are expressed as means ± SE. Statistical analysis was performed with GraphPad Prism 4.0 software. Unless indicated otherwise, Student's t-test was used for statistical analysis. P < 0.05 was considered as statistically significant.

Reagents. Oligonucleotides were obtained from Proligo (Singapore). All other reagents were obtained from Sigma unless indicated otherwise.


    RESULTS
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RT-PCR experiments were performed to detect the expression of the two sAC transcripts in Calu-3 cells. Initial RT-PCR trials with several different pairs of primers closely flanking the deletion region generated two PCR bands with slightly different mobility (Fig. 1A, left, results shown are of one pair of these primers), which were expected to correspond to FL-sAC and T-sAC transcripts, respectively (14). DNA sequencing confirmed that the upper band was a FL-sAC transcript but failed to identify T-sAC transcript in the lower band because of overwhelming nonspecific DNA sequences. To circumvent this problem, the lower band in the first round of RT-PCR was excised, extracted, and used as a template for another round of PCR with a different pair of primers (see MATERIALS AND METHODS). The second round of RT-PCR generated two bands (Fig. 1A, right), in which the lower band had the predicted size of the T-sAC transcript and the upper band represented the contamination of FL-sAC transcripts in the excised template. DNA sequencing confirmed the identity of the PCR products of two bands to be FL-sAC and T-sAC transcripts (Fig. 2). These findings demonstrate that there are two different transcripts of sAC in human cells as well (14).



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Fig. 1. A: soluble adenylyl cyclase (sAC) mRNA expression in Calu-3 cells. Two rounds of PCR were performed to generate two clear, specific bands at the predicted sizes of 266 and 210 bp (see text and MATERIALS AND METHODS). B: immunoblotting of sAC in whole cell lysate using the affinity-purified antiserum in the absence (left) or presence (right) of 4 mg/ml antigen peptide; the other conditions in the left and right images were identical. The presence of the antigen peptide abolished the detection of sAC. Shown is one of total four similar experiments. C: top, immunoblotting of sAC; bottom, immunoblotting of CFTR as control for particulate fraction. TL, total lysate; P, particulate fraction; S, supernatant. The fraction ratio of TL, P, and S was 1:5:1. The particulate fraction composed ~7% of total sAC and 100% of total CFTR as quantified using densitometry.

 


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Fig. 2. Comparison of the full length (FL)-sAC and total (T)-sAC splice variants in human and mouse (14). The nucleotide sequences spanning exon 11 (deleted in T-sAC), together with the deduced amino acid sequence of FL-sAC and T-sAC splice variants, are shown. As seen in mouse and rat species (14), the human T-sAC transcript lacked 56 nt corresponding to the entire exon 11, and a translation stop codon is predicted to occur 4 bp downstream of the deleted region. Underlined nucleotides in the FL-sAC are the nucleotides deleted in T-sAC. Regarding the amino acid sequence of the mouse FL-sAC, only amino acids different from human are shown. FL-hsAC and T-hsAC, human full-length and truncated sAC, respectively; FL-msAC and T-msAC, mouse full-length and truncated sAC, respectively. The asterisk indicates the stop codon.

 
With the use of anti-sAC antiserum, a protein migrating coincident with the T-sAC positive control mouse kidney tissue (5) was found in the whole cell lysate of Calu-3 cells (Fig. 1B). No reliable band of FL-sAC was detected in Calu-3 cells, although it was detected in the mouse testis tissue as a predominant band (Fig. 1B). The predominant presence of T-sAC in Calu-3 cells confirmed the results in a previous study (26) and is in line with observations in many other cells (4, 30). Consistent with its apparent lack of transmembrane domains, a major portion of sACs was present in the cytosol (Fig. 1C). Interestingly, a fraction of T-sAC (~7%) (Fig. 1C) were present in the particulate fraction, suggesting that sAC could be membrane associated as well.

A unique feature differentiating sAC from TMACs is HCO3 sensitivity. Exploiting this characteristic, we tested whether sAC is functional in Calu-3 cells by determining HCO3-sensitive adenylyl cyclase activity. The addition of 40 mM NaHCO3/5% CO2 (buffered to pH 7.2 with HEPES) resulted in a small yet highly reproducible intracellular cAMP increase (0.30 ± 0.02-fold over control, n = 5; P = 0.0008) (Fig. 3). The cAMP increase resulted from HCO3 instead of Na+ because adding 40 mM NaNO3 had virtually no effect on cAMP increase (–0.13 ± 0.10-fold over the control, n = 3; P > 0.1). These data support the presence of functional sAC in Calu-3 cells. However, caution should be taken when interpreting the effect of HCO3 of physiological concentration, because HCO3 concentration and pH have reciprocal effects. Although NaHCO3 solution was buffered with HEPES to avoid extracellular pH change resulting from HCO3 addition, we found that adding HCO3 solution resulted in a transient drop in intracellular pH (~0.13 to 0.20 pH units, which recovered in 2–3 min; n = 3) as measured using pH-sensitive fluorescent dye seminaphthorhodafluor-5F 5-(and-6)-carboxylic acid acetoxymethyl ester acetate (S-23922; Molecular Probes), which, although small, could potentially contribute to the observed cAMP change by affecting TMAC activity (18). To confirm that HCO3-stimulated cAMP increase indeed resulted from sAC, we used a sAC inhibitor, 2-HE. Braun first reported 2-HE to block sAC in 1990 (2), and this finding was recently verified by using purified recombinant rat sAC (23). However, little is known concerning its specificity and possible effect on TMACs. To clarify this issue, we evaluated the effect of 2-HE on HCO3- and forskolin-stimulated cAMP production and included a TMAC inhibitor, SQ22,536, as a control. HCO3-stimulated cAMP increase was completely eliminated by 2-HE but was not affected by SQ22,536, although SQ22,536 partially reduced the basal cAMP production (Fig. 3, top). It was noted that basal cAMP level was not effected by 2-HE, suggesting that sAC was dormant in the absence of HCO3 under the basal conditions. In contrast, forskolin-stimulated cAMP increase, reflecting TMAC activity, was attenuated by SQ22,536 (P = 0.002 vs. forskolin stimulation in the control) but was not affected by 2-HE (Fig. 3, bottom). These data together demonstrate that sAC is functional in Calu-3 cells and also validate that 2-HE could serve as a specific sAC inhibitor.



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Fig. 3. Analysis of HCO3-stimulated (top) and forskolin-stimulated (bottom) cAMP production in Calu-3 cells. Intracellular cAMP concentration is expressed as the relative increase over the basal cAMP level in the control. Data summarize five independent experiments with each performed in triplicate or quadruplicate and are expressed as means ± SE. SQ, SQ22,536. The concentrations of 2-hydroxyestradiol (2-HE) and SQ22,536 were 20 and 100 µM, respectively. *P < 0.001 vs. basal cAMP level.

 
Next, we used direct functional assays of CFTR to test the notion that sAC is coupled with CFTR. We began to assess the effect of HCO3 on CFTR-mediated Cl current in whole cell voltage-clamp studies. The addition of 25 mM NaHCO3 into the bath stimulated whole cell mean current density from 90.9 ± 18.0 to 138.8 ± 26.6 pA·pF–1·100 mV–1 (n = 9), and slope conductance increased 54.4 ± 9.6% compared with before the addition of HCO3 (P = 0.005) (Fig. 4). The reversal potentials of basal currents were 25.6 ± 1.6 mV, which are close to ECl (–31.9 mV) and consistent with CFTR-mediated Cl current. Given a HCO3/Cl selectivity of 0.1 (17), adding 25 mM HCO3 to the bath is predicted to shift equilibrium potential only from ECl –31.9 mV to –30.5 mV, a difference too small to measure. This may explain our observation that the reversal potentials of HCO3-stimulated currents (26.5 ± 1.5 mV) were not significantly different from those of basal currents. In the presence of glibenclamide, a CFTR channel blocker, 25 mM NaHCO3 was not able to stimulate whole cell mean current density from 88.6 ± 20.7 to 98.8 ± 26.2 pA·pF–1·100 mV–1 (n = 3), and slope conductance increased 12.0 ± 11.1% compared with before addition of HCO3 (P = 0.19). The Cl permeability, glibenclamide sensitivity, and linear I-V relationship together suggest that NaHCO3-stimulated current is conducted by CFTR channels. Cells treated with 2-HE had virtually no increase in current in response to 25 mM NaHCO3 (mean current density from 91.5 ± 6.7 to 99.0 ± 13.5 pA·pF–1·100 mV–1, and slope conductance increased 5.0 ± 7.5%) (n = 3; P = 0.28), implicating sAC being physiologically coupled to CFTR activity. An alternative explanation could be that 2-HE directly binds to and blocks CFTR or PKA, because 2-HE may have other effects (8). This possibility seems less likely because adding 2-HE had no effect on forskolin-stimulated whole cell current. Forskolin (10 µM) stimulated whole cell mean current density from 71.2 ± 12.1 to 152.3 ± 42.1 pA·pF–1·100 mV–1 (n = 7), and slope conductance increased 106.3 ± 24.9% compared with before the addition of forskolin (P = 0.0002). In the presence of 2-HE, forskolin stimulated whole cell mean current density from 73.2 ± 17.7 to 136.3 ± 37.7 pA·pF–1·100 mV–1 (n = 6), slope conductance increased 98.2 ± 20.9% compared with before the addition of forskolin (P = 0.0007). The forskolin-stimulated slope conductance increases with or without 2-HE were not significantly different (Fig. 4C).



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Fig. 4. HCO3 activation of CFTR-mediated Cl current in whole cell voltage-clamp studies. A: families of currents in response to voltage steps before and after 25 mM NaHCO3 in the absence of 2-HE. One typical experiment is shown. Voltages were stepped from a holding potential of –40 mV to a voltage range from –100 to +100 mV in 20-mV increments. Each clamp voltage level was maintained for 850 ms, for which time currents were averaged and used for plotting current-voltage (I-V) relationships. B: plots of I-V data displayed in A. Vp, holding potential of the pipette. C: summary data of HCO3- and forskolin-stimulated slope conductance change in the absence and presence of 20 µM 2-HE or 400 µM glibenclamide. Slope conductance of whole cell Cl current was calculated as described in MATERIALS AND METHODS. Slope conductance of Cl current after the addition of HCO3 (HCO3-stimulated in A) or forskolin was normalized to the slope conductance of Cl current before the addition of HCO3 (basal in A) or forskolin, and the percentage increase is plotted. NaHCO3, n = 9; NaHCO3 + 2-HE, n = 3; NaHCO3 + glibenclamide, n = 3; forskolin, n = 7; forskolin + 2-HE, n = 6. Glib, glibenclamide. The slope conductance increases stimulated by NaHCO3 alone or in the presence of 2-HE or glibenclamide were significantly different. P < 0.05 (one-way ANOVA with Tukey's post hoc test). The forskolin-stimulated slope conductance increases with or without 2-HE were not significantly different. P > 0.05 (one-way ANOVA with Tukey's post hoc test).

 
To determine the functional coupling of sAC and CFTR more rigorously, we examined the effect of HCO3 on CFTR single-channel activity in cell-attached membrane patches. Adding 25 mM NaHCO3 (buffered to pH 7.4 with predetermined HEPES) in the bath robustly stimulated CFTR channels in cell-attached patches (nPo increased from 0.23 ± 0.06 to 0.83 ± 0.29, n = 22; P = 0.02). The stimulation did not seem to result from osmolarity or an electrolyte effect of NaHCO3 (29), because no effect on CFTR was observed with the addition of either 25 mM NaCl (nPo changed from 0.07 ± 0.03 to 0.04 ± 0.03, n = 4; P = 0.23) or 35 mM NaNO3 (nPo changed from 0.68 ± 0.40 to 0.66 ± 0.41, n = 5; P = 0.24). HEPES (5 mM Na+-HEPES) used to buffer the solution had no effect on CFTR either (nPo increased from 0.06 ± 0.04 to 0.055 ± 0.032, n = 3; P = 0.40). More important, the activation was attenuated by the sAC inhibitor 2-HE (nPo increased from 0.28 ± 0.06 to 0.43 ± 0.10, n = 13, P = 0.08, Fig. 5), suggesting that sAC regulates CFTR activity. Adding 2-HE had no effect on basal nPo of CFTR channels (from 0.15 ± 0.03 to 0.20 ± 0.06, n = 22; P = 0.12), consistent with no effect of 2-HE on the forskolin-stimulated whole cell current. Adding HCO3 had no effect on the amplitude of CFTR single-channel current (Fig. 5) as expected on the basis of little predicted effect of adding 25 mM HCO3 on the resting potential of Calu-3 cells. Increasing the concentration of NaHCO3 to 50 mM did not seem to cause any further stimulation of CFTR channel (without 2-HE, nPo changed from 0.13 ± 0.06 to 0.68 ± 0.24, n = 6, P = 0.042; with 2-HE, nPo changed from 0.16 ± 0.06 to 0.20 ± 0.05, n = 8, P = 0.26) (Fig. 5D), indicating that external 25 mM NaHCO3 elicited maximal stimulation.



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Fig. 5. HCO3 activated CFTR in cell-attached membrane patches. A and B: representative single-channel traces. NaHCO3 (25 mM) was added into the bath in the absence (A) and presence (B) of 20 µM 2-HE. Pipette voltage was held at –60 mV. Numbers and dashes denote active channel numbers. C: CFTR channel number (n) and open probability (Po) calculated as CFTR channel activity (nPo) histogram of A and B. D: summary data of experiments with 25 mM HCO3 stimulation [Control, n = 22; 2-HE, n = 13 (*P = 0.03)] and with 50 mM HCO3 stimulation [Control, n = 6; 2-HE, n = 8 (*P = 0.042)].

 

    DISCUSSION
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 DISCUSSION
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In summary, our data together demonstrate that sAC is coupled to the regulation of CFTR function in Calu-3 cells. This coupling ties the level of intracellular HCO3/CO2 to the modulation of HCO3-conductive CFTR function by cAMP/PKA. With a more direct and relevant functional assay at the single-channel level and a sAC-specific inhibitor, our study has confirmed and extended the previous study in bovine corneal endothelium (27). It is not clear whether coupling of sAC and CFTR is present in other HCO3-transporting tissues. Conceivably, distinct biochemical properties of sAC may cause the underappreciation of its importance in HCO3-transporting and other biological processes because activators of G proteins and forskolin are two means primarily used to manipulate cAMP signaling.

Consistent with previous studies in various cell types (4, 26, 30), we found that T-sAC protein is the predominant isoform in Calu-3 cells (Fig. 1B). In contrast, RT-PCR studies showed an apparent dominance of the product corresponding to FL-sAC mRNA in the first round of RT-PCR using total RNA as the template (Fig. 1A, left) but failed to identify the T-sAC transcript because of overwhelming nonspecific products. Using the lower band as the template, which supposedly contained the RT-PCR product corresponding to T-sAC and contamination of the upper band (FL-sAC), the product corresponding to FL-sAC gene was still predominant in the second round of PCR (Fig. 1A, right), suggesting a relative scarceness of the T-sAC mRNA and/or overwhelming contamination of the upper band (FL-sAC). Because the RT-PCRs of both FL-sAC and T-sAC were performed in the same tube, tube-to-tube variation and other factors affecting amplification efficiency were negated. Thus only the quantity and the heterogeneity of the FL-sAC and T-sAC RNA templates could account for the different abundance of the PCR products. The RNA templates of the FL-sAC and T-sAC are highly homologous; however, it has been reported that even a small difference in the composition of RNA templates might result in a three- to fourfold change in the efficiency of reverse transcription (10). An early study in the rat demonstrated that the abundance ratio of the FL-sAC to T-sAC in RT-PCR experiments was in good agreement with the data of RNA protection analysis (14), suggesting that the heterogeneity of the rat FL-sAC and T-sAC RNA templates does not have a significant impact on the RT-PCR amplification and that the RT-PCR data reflect the actual abundance of the mRNA of FL-sAC and T-sAC. Whether this finding is also true for the human sAC needs further confirmation.

The overlapping regulatory properties of the TMAC isoforms and the lack of isoform-specific agonists and antagonists remain a major hurdle for functionally distinguishing TMAC isoforms, particularly in studies of the human cells, in which knockout of a specific adenylyl cyclase isoform is not practicable. sAC possesses a peculiar regulatory property, i.e., the sensitivity to HCO3, which can be exploited to distinguish sAC from other adenylyl cyclases. However, because HCO3 concentration and pH in a solution are interdependent, adding HCO3 might result in pH alteration. In the present work, possible pH change resulting from the addition of HCO3 was carefully minimized. Yet, the minimal pH change could still complicate the data interpretation. To distinguish sAC from other adenylyl cyclases rigorously, we used a sAC blocker, 2-HE. 2-HE has been found to block both purified native and recombinant rat sAC (2, 23), but whether 2-HE has any effect on TMACs is not clear. In our studies, we found that 2-HE had no effect on forskolin-stimulated cAMP production, reflecting the TMAC activity; in contrast, 2-HE completely blocked HCO3-stimulated cAMP production (Fig. 3). These data suggest that 2-HE could be used as a specific blocker to distinguish sAC from TMACs. Subsequently, we used 2-HE to disrupt sAC function to test whether sAC is functionally coupled with CFTR. We have demonstrated that 2-HE could attenuate the HCO3-stimulated CFTR activity in both whole cell voltage-clamp and cell-attached single-channel studies (Figs. 4 and 5), consistent with the finding that sAC regulates CFTR function. We also conducted control experiments to rule out the possible effect of 2-HE on the CFTR channel itself and/or PKA. We found that 2-HE had no effect on forskolin-stimulated CFTR-mediated whole cell current, arguing against any effect of 2-HE on CFTR itself and/or PKA (Fig. 5). This finding is further supported by the observation that 2-HE had no effect on basal CFTR single-channel activity (see RESULTS). Taking all of these results together, we conclude that sAC is functionally coupled to CFTR. Nevertheless, pharmacological reagents such as 2-HE have nonspecific effects (8). Even with carefully designed controls as described in our studies, conclusions drawn from these studies are inherently indirect and need to be substantiated further by evidence using specific molecular approaches to manipulating sAC activity, such as RNA interference.

Interestingly, HCO3 induced reasonably robust activation of CFTR while generating a rather modest cAMP increase compared with forskolin (Fig. 3). This is reminiscent of adenosine stimulation of CFTR in Calu-3 cells (11), suggesting localized cAMP signaling from sAC to CFTR. In addition, a fraction of sAC was distributed in the membrane fraction in Calu-3 cells (Fig. 2), consistent with several previous studies indicating that so-called soluble adenylyl cyclase could be membrane associated and compartmentalized in certain intracellular organelles (14, 18, 30, 31). It will be interesting to test whether sAC resides with CFTR in an apical microdomain in Calu-3 cells.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by Hong Kong University Grant Committee Direct Allocation Grant DAG02/03.SC06 and Research Grant Council Grants HKUST6275/03M and HKUST6468/05M. Y. Wang and F. Wu were supported by postdoctoral matching funds from the Hong Kong University of Science and Technology.


    ACKNOWLEDGMENTS
 
We thank Drs. L. R. Levin and J. Buck for generously providing monoclonal anti-sAC antibody R21 at the initial stage of this study and Candy Lee for technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. Huang, Dept. of Biology, Hong Kong Univ. of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, People's Republic of China (e-mail: bohuangp{at}ust.hk)

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.

* Y. Wang and C. S. Lam contributed equally to this work. Back


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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