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
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ABSTRACT |
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cystic fibrosis transmembrane conductance regulator; bicarbonate; adenosine 3',5'-cyclic monophosphate; 2-hydroxyestradiol
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.
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MATERIALS AND METHODS |
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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
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.
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RESULTS |
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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 23 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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DISCUSSION |
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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.
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GRANTS |
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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.
* Y. Wang and C. S. Lam contributed equally to this work.
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