[HCO3-]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells
© Sun et al; licensee BioMed Central Ltd. 2004
Received: 31 October 2003
Accepted: 29 April 2004
Published: 29 April 2004
Bicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO3- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl- permeability. Here, we examined whether HCO3- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO3- and the stimulation of CFTR in BCECs.
RT-competitive PCR indicated that sAC mRNA expression in BCECs is dependent on [HCO3-] and incubation time in HCO3-. Immunoblots showed that 10 and 40 mM HCO3- increased sAC protein expression by 45% and 87%, respectively, relative to cells cultured in the absence of HCO3-. Furthermore, 40 mM HCO3- up-regulated sAC protein expression in Calu-3 cells by 93%. On the other hand, sAC expression in BCECs and Calu-3 cells was unaffected by changes in bath pH or osmolarity. Interestingly, BCECs pre-treated with10 μM adenosine or 10 μM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively. Intracellular cAMP production by sAC paralleled the time and [HCO3-]-dependent expression of sAC. Bicarbonate-induced apical Cl- permeability increased by 78% (P < 0.01) in BCECs cultured in HCO3-. However for cells cultured in the absence of HCO3-, apical Cl- permeability increased by only 10.3% (P > 0.05).
HCO3- not only directly activates sAC, but also up-regulates the expression of sAC. These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.
Soluble adenylyl cyclase (sAC) has recently been characterized as a unique means to generate the ubiquitous signaling molecule, cyclic adenosine 3', 5'-monophosphate (cAMP) [1–3]. Elucidation of the properties of sAC indicate that this enzyme is biochemically and chromatographically different from the transmembrane adenylyl cyclases (tmACs) [4, 5]. Unlike the tmACs, sAC activity depends on the divalent cation Mn2+ [1, 3, 6], is insensitive to G protein regulation and forskolin [2, 4], and displays approximately 10-fold lower affinity for ATP (Km ≈ 1 mM)  than the tmACs (Km ≈ 100 μM) . HCO3- directly binds to and activates sAC in a pH-independent manner . HCO3- is the primary physiological activator of sAC and a recent study has shown that this can be modulated by Ca2+ .
Immunocytochemistry studies have demonstrated that sAC is distributed in specific subcellular compartments: mitochondria, centrioles, mitotic spindles, and nuclei, all of which contain cAMP-dependent targets . Distribution at these intracellular sites indicates that sACs are in close proximity to all cAMP effectors, suggesting a model in which local concentrations of cAMP are regulated by individual adenylyl cyclases . Therefore, sAC can activate many potential targets. For many years, models describing cAMP signaling in mammalian cells relied on tmAC-dependent generation of cAMP. Activation of PKA, which is anchored at various places in the cell by the scaffolding protein A kinase-anchoring protein (AKAP) [10–12], required cAMP to diffuse through the cytoplasm to propagate its signal. The elucidation of sAC removes the membrane-proximal limitation on cAMP generation and reveals new aspects of what was previously thought to be a very well characterized signaling pathway.
sAC is the predominant form of adenylyl cyclase in mammalian sperm [1–3]. The direct activation of sAC by bicarbonate in sperm provides a mechanism for generating the cAMP required for fertilization, including hyperactivated motility, capacitation, and the acrosome reaction [13–15]. sAC is also widely expressed in various tissues at low level . Since bicarbonate is almost always present in vivo, sAC may function as a general bicarbonate/CO2 sensor throughout the body . Furthermore, regulation of basal levels of cAMP will be influenced by active bicarbonate transport systems in those cells that express sAC. More recently, we have shown that BCECs, which express robust HCO3- transport activity [16, 17] due to the presence of the Na+/2HCO3- cotransporter (NBC1) , also express sAC [18, 19]. The presence of bicarbonate increases the steady-state [cAMP], leading to phosphorylation of the apical CFTR and increased apical Cl- permeability , which is an important component of the secretory function of corneal endothelial cells.
In the current study, we asked whether sAC expression can be influenced by its primary ligand, HCO3-. We used BCECs to show that HCO3- not only directly activates sAC, but also up-regulates the expression of sAC, generating a higher level of cAMP. We also examined the airway surface epithelial cell line, Calu-3, which we have also found to express sAC. These results could have relevance to many other tissues where sAC is expressed.
[HCO3-]- and time-dependent expression of sAC
The effects of extracellular pH and intracellular [cAMP] on sAC expression
Gene transcription regulated by cAMP, is mainly by PKA-dependent phosphorylation of the cyclic AMP response element binding (CREB) family of transcription factors (CREB, CREM, and ATF-1) [23, 24]. CREB is expressed in corneal endothelium , so it is possible that the addition of HCO3- induces basal levels of sAC to produce cAMP, which in turn upregulates further sAC expression. Therefore, we asked whether changes in intracellular cAMP can regulate the sAC expression in BCECs. In BCECs, 10 μM adenosine can increase [cAMP] by ~50%, while forskolin produces a 6–8 fold increase in cAMP . The increase in cAMP induced by adenosine is similar to the increase in cAMP induced by HCO3-. BCECs were treated with 10 μM forskolin or 10 μM adenosine for 24 hours in the absence of bicarbonate. As shown in figure 3C, the expression of sAC mRNA was decreased in adenosine and forskolin treated cells relative to the control. The ratios of band densities (sAC/GAPDH) are summarized in Figure 3D. These results suggest that the increase in [cAMP] in cells bathed in HCO3- rich solutions is not responsible for increasing sAC expression.
The effect of bath osmolarity on sAC expression
The effect of HCO3- induced sAC expression on intracellular [cAMP]
The effect of HCO3--induced sAC expression on apical Cl permeability in BCECs
This study is the first demonstration that bicarbonate up-regulates sAC expression in a dose- and time-dependent manner. In BCECs, RT-competitive PCR identified that sAC mRNA expression is significantly increased with increasing [HCO3-] or incubation time in HCO3-. Western blot gave a major band with BCECs at the expected size, which was confirmed using the human calu-3 cells. Furthermore, sAC protein expression is increased by incubation in HCO3- in both BCECs and Calu-3 cells.
Since bath pH is changed at different [HCO3-], sAC expression could be induced by pH rather than HCO3-. Previous studies have shown that pH changes can be a regulatory factor for stabilization of some mRNA . Direct regulation of mRNA stability by pH requires that the 3'-untranslated region of the regulated mRNA contain a direct repeat of an eight-base AU sequence that functions as a pH-response element . This sequence increases the binding of ζ-crystallin/NADPH:quinine reductase, which may initiate the pH-responsive stabilization of the regulated mRNA . Therefore, in Fig. 3A and 3B, we tested whether varying bath pH may induce sAC mRNA and protein expression in BCECs and Calu-3 cells. Bath pH from 7.0 to 8.0 had no effect on sAC expression. This finding is consistent with the fact that sAC does not contain an eight-base AU repeat in the 3'-untranslated region [2, 4].
sAC activity is not only modulated by bicarbonate, but also by calcium . Therefore cytosolic [Ca2+] could also be a possible regulatory factor of sAC expression. In some cell types, changes in external pH can alter cytosolic [Ca2+]. However, the pH-independence of sAC expression shown here (figure 3A and 3B) excludes this possibility. Furthermore, we have previously shown that changing perfusion of cultured BCEC between bicarbonate-free to bicarbonate-rich conditions has no effect on [Ca2+]i . However, this does not exclude the possibility that altering [Ca2+]i by some other means could affect sAC expression.
Changing [NaHCO3] between 0 and 80 mM significantly affects medium osmolarity, which could be responsible for the change in sAC expression. However, decreasing or increasing osmolarity or increasing [NaCl] had no effect. Moreover, from figure 1A, the biggest change in sAC expression is between 20 mM and 40 mM HCO3-. However, the osmolarity change caused by this increase in NaHCO3 is only 18 mmol/kg (from 299–317 mmol/kg). Much larger changes in osmolarity (e.g., 40 to 80 mM NaHCO3) had no effect on sAC expression.
Bicarbonate-regulated sAC represents an alternate source for cAMP [1–3, 18]. sAC is not solely a soluble protein but is specifically targeted to well-defined intracellular compartments – mitochondria, centrioles, mitotic spindles and nuclei – in close proximity to intracellular effectors of cAMP signaling . As shown in Fig. 3C, either forskolin or adenosine can significantly decrease sAC expression compared to bicarbonate-free control, suggesting that higher [cAMP]i down-regulates the expression of sAC. These observations indicate that HCO3--induced up-regulation of sAC is not caused by increasing [cAMP]. Conversely, these results are consistent with the notion that sAC contributes to regulating basal levels of cAMP. Possibly, when cAMP levels increase above some basal set-point, sAC is down-regulated. This raises the following questions: 1) Is cAMP-induced sAC down-regulation caused by phosphorylated CREB?2) Would activated CREB decrease the transcription of sAC mRNA?3) How does HCO3- increase sAC mRNA level? sAC can be found in the nucleus , thus possibly sAC itself is associated with a transcription factor that is activated upon binding by HCO3-.
As we demonstrated previously , activated sAC in BCECs can stimulate CFTR via up-regulating the intracellular [cAMP] and increasing PKA activity, leading to increased phosphorylation of CFTR and thus increased apical Cl- permeability. In the current study, we used HCO3--starved BCECs to test the effect of down-regulated sAC on the apical Cl- permeability. Due to the basal levels of sAC expression in HCO3--starved BCECs (as shown in figure 2A and 2B), HCO3- produced a small (10.3%), but not significant, increase in apical Cl- permeability compared to the control (fig 6B). It is possible that some cAMP is likely being produced, but it is also being quickly degraded by the cAMP specific phosphodiesterase, PDE4 . Thus the basal amount of sAC is not sufficient to raise the steady-state level of [cAMP] such that CFTR permeability is affected. However, the HCO3- stimulated apical Cl- permeability in BCECs cultured in HCO3- was significantly increased relative to the control. This finding not only is consistent with our previous report , but also confirms that HCO3--regulated sAC expression affects the basal level of apical Cl- permeability in BCECs.
In the current study, we identified that HCO3- can up-regulate functional sAC. Coupled with our recent study that HCO3--activated sAC in BCECs can generate cAMP leading to phosphorylation of apical CFTR, which significantly enhances apical chloride permeability, this novel finding has important implications for mammalian cells such as corneal endothelium and Calu-3 cells. In corneal endothelial cells, chloride and bicarbonate are essential for transendothelial fluid transport to maintain corneal transparency [30–33]. Chloride and bicarbonate secretion at the apical membrane (aqueous humor side) is stimulated by increasing cytosolic [cAMP] [34–36]. Furthermore, cAMP activates chloride transport in cultured corneal endothelial cells [37, 38] and cAMP-dependent CFTR significantly contributes to apical Cl- and HCO3- efflux in corneal endothelium . Therefore, HCO3--regulated sAC expression may play a pivotal role in maintaining steady-state anion transport and fluid secretion by the corneal endothelium. In addition, Calu-3 cells share many of the same anion transport mechanisms as corneal endothelium. Thus the presence of HCO3- and sAC need to be considered when evaluating basal cAMP dependent anion transport in Calu-3 cells.
Fresh cow eyes were obtained from a local slaughterhouse and kept on ice until use, ~4 hours after death. Primary cultures of bovine corneal endothelial cells (BCEC) were grown to confluence as previously described [39, 40]. Calu-3 cells were purchased from ATCC and cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum.
Analysis of sAC mRNA expression by competitive RT-PCR
A pair of human sAC primers was constructed on the basis of the published cDNA sequence . The sAC sense primer was 5'-CCTGGAATAACCTGTTCAAG-3' and the sAC antisense primer was 5'-TCTGGTCCTTGAGCCACAG-3'. The expected length of PCR products for sAC was 544 bp. Another pair of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers was constructed on the basis of the published bovine cDNA sequence (GenBank access no. U85042). The GAPDH sense primer was 5'-TGACCCCTTCATTGACCTTC-3' and the antisense primer was 5'-GGTCATAAGTCCCTCCAGGA-3'. The expected length of PCR products for GAPDH was 460 bp.
Confluent cultured BCECs in 35 mm Petri dishes were starved for bicarbonate in HCO3--free DMEM for 48 hours at 37°C. Cells were then incubated either for different times in 40 mM HCO3- DMEM or for 24 hours in varying concentrations of HCO3-. HCO3--free DMEM was purchased from GIBCO (Carlsbad, CA). Different concentrations of HCO3- DMEM were prepared by adding varying amounts of NaHCO3. Different osmolarity DMEM solutions were adjusted using sucrose to be equivalent to the osmolarities of the different [HCO3-] DMEM solutions. The method to prepare different [NaCl] solution is the same as preparing different [HCO3-] DMEM solutions except for replacing NaHCO3- with NaCl. In addition, BCECs without bicarbonate starvation were incubated in HCO3--free DMEM either at different bath pH or with10 μM adenosine or 10 μM forskolin for 24 hours at 37°C.
Total RNA was extracted from BCECs using TRIzol reagent (Invitrogen) as previously described [17, 41]. The RNA was DNase-treated using an RNase-free DNase set (Qiagen). Reverse transcription was performed using Superscript™ cDNA synthesis system (Invitrogen) and oligo (dT) primers as previously described [17, 41]. Competitive PCR amplifications were carried out in a thermocycler using the high fidelity TaKaRa Ex Taq PCR system kit (TaKaRa Shuzo) under the following conditions: denaturation at 94°C for 3 min for one cycle, 30 cycles of denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 45 seconds, and a final extension for one cycle at 72°C for 10 min. The PCR products were loaded onto 1% agarose gels, electrophoresed, and stained with 0.5 μg/ml ethidium bromide. The density of each band was digitized using Un-Scan-It software (Silk Scientific). The ratio of sAC band density to parallel GAPDH band density was compared within each experiment.
Competitive RT-PCR was used throughout this study. Thus, as sAC expression increases, the amount of GAPDH PCR product formed drops. The ratio of the two sets of primers (sAC and GAPDH) in the starting amplification reaction was varied for each type of experiment in order to assure that bands for both sAC and GAPDH could be detected. Thus comparisons can be made within each type of experiment (e.g., within figure 1 or figure 3), but not between experiments (i.e., figure 1 cannot be compared with figure 3).
PCR products were purified using a 1% low-melting point agarose gel. Freshly purified products were inserted into pCR 4-TOPO vector (Invitrogen, San Diego, CA) and sequenced as previously described [17, 41]. Sequences were assembled and compared using Vector NTI version 5.2 software (InforMax, North Bethesda, MD).
Confluent cultured BCECs and Calu-3 cells were starved for bicarbonate, followed by a 24-hour incubation in different concentrations of bicarbonate in DMEM at 37°C. Calu-3 cells were also incubated in HCO3--free DMEM with different pH for 24 hours. Cells were then lysed in chilled lysis buffer (150 mM NaCl, 0.5% sodium deoxycholate, 1% SDS, 50 mM Tris, pH 8.0, 1 mM EDTA, 0.1 mM PMSF, 10 μg/ml leupeptin, 1 μg/ml pepstatin) and cleared by centrifugation. Cell lysates (60 ug/lane) were separated by 8% SDS-PAGE, and transferred to a polyvinylidene fluoride (PVDF) membrane, as previously described . The membrane was blocked and probed with mouse anti-human sAC primary antibody (1:1000, a kind gift from Dr. J Buck and L Levin, Medical College of Cornell University)  and goat anti-mouse secondary antibody coupled to horseradish peroxidase (1:5000, Sigma). The same transferred membrane was rinsed and reprobed using mouse anti-human β-actin to test the loading of total protein. Exposed films were scanned and the density of equal areas of the developed bands was estimated using Un-Scan-It software (Silk Scientific).
Determination of Intracellular cAMP Accumulation
Culture medium was removed from confluent cultured BCECs and replaced with HCO3--free DMEM for 48 hours at 37°C. Cells were then incubated either for different time in 40 mM HCO3- or for 24 hours in different concentrations of HCO3- at 37°C. At the end of the incubation, cells were washed with PBS and lysed in 0.1 N HCl and cleared of cellular debris by centrifugation. [cAMP] was measured by an enzyme immunoassay kit (R&D Systems) and an ELISA plate reader (Fluostar galaxy, BMG Labtechnologies).
Apical Cl- permeability
Relative changes in apical Cl- permeability were assessed with the halide-sensitive fluorescent dye MEQ. Confluent BCEC, grown on permeable Anodiscs and starved for HCO3- for 24 hours, were loaded with MEQ by exposure to diH-MEQ for 10 min [37, 38] 42. Anodiscs were placed in a double-sided microscope perfusion chamber and apical and basolateral compartments were independently perfused at 37°C. MEQ fluorescence (excitation: 365 ± 10 nm; emission: 420–450 nm) was measured as previously described [37, 38] 42. Anodiscs were initially perfused with a Cl- and HCO3--free solution (in mM: 150 Na+, 4 K+, 0.6 Mg2+, 1.4 Ca2+, 148.5 NO3-, 2 HPO42-, 10 HEPES, 2 gluconate-, and 5 glucose, pH 7.5) and the apical side briefly pulsed with Cl--rich Ringer's (equimolar replacement of 118 NaNO3- with NaCl). HCO3--Rich solutions were prepared by equimolar substitution of 28.5 NaNO3- with NaHCO3, gassed with 5% CO2, pH 7.5. Relative changes in apical Cl- permeability between control and experimental conditions in the same cells were determined by comparing the percentage change in MEQ fluorescence (F/F0) after addition of Cl-, where F0 is the fluorescence in the absence of Cl-. The maximum slope of fluorescence change was determined by calculating the first derivative using Felix software (Photon Technology International).
All data were expressed as means ± SE and Student's paired t-test was used for statistical analysis at p < 0.05.
We would like to thank Dr. J. Buck and L. Levin, from Medical College of Cornell University, for the kind gift of mouse anti-human sAC monoclonal antibody. This study was supported by a grant from the NIH EY08834 (JAB).
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