Pancreatic dysfunction in patients with cystic fibrosis (CF) is felt to result primarily from impairment of ductal secretion. We provide molecular evidence for the expression of NBC-1, an electrogenic Na+- cotransporter (NBC) in cultured human pancreatic duct cells exhibiting physiological features prototypical of CF duct fragments (CFPAC-1 cells) or normal duct fragments [CAPAN-1 cells and CFPAC-1 cells transfected with wild-type CF transmembrane conductance regulator (CFTR)]. We further demonstrate that1) uptake across the basolateral membranes of pancreatic duct cells is mediated via NBC and2) cAMP potentiates NBC activity through activation of CFTR-mediated Cl− secretion. We propose that the defect in agonist-stimulated ductal secretion in patients with CF is predominantly due to decreased NBC-driven entry at the basolateral membrane, secondary to the lack of sufficient electrogenic driving force in the absence of functional CFTR.
- cystic fibrosis
- cystic fibrosis transmembrane conductance regulator
- electrogenic sodium-bicarbonate cotransporter
- pancreatic dysfunction
cystic fibrosis (CF) remains a major healthcare problem worldwide (29). An autosomal recessive disease, CF results from mutational inactivation of a cAMP-sensitive Cl− channel [CF transmembrane conductance regulator (CFTR)] with resultant impairments in the respiratory, pancreatic, hepatobiliary, and genitourinary systems (29). With regard to the pancreas, pancreatic dysfunction is felt to result primarily from impairment of secretin-stimulated ductal Cl− and secretion (17, 22,23). Experimental and histopathological evidence suggests that the reduction in secretin-stimulated secretion from pancreatic duct epithelial cells alters intraductal pH sufficiently to precipitate proteins secreted from acinar cells, resulting in protein plugs, and to disrupt vesicular trafficking in the apical domain of the acinar cell (16, 31). Together, these alterations lead to pancreatic fibrosis and insufficiency in 80% of cases (31).
Although gene therapy has provided the scientific and medical communities with an appealing potential therapeutic option in the treatment of the CF defect, important obstacles have yet to be overcome (2, 21, 42). An alternative hope is that a better understanding of the physiological role(s) of CFTR in various organs will lead to novel strategies capable of correcting the functional defect in the absence of functional CFTR. The currently accepted model of pancreatic ductal secretion suggests that1) intracellular accumulates due to basolateral diffusion of CO2 and subsequent action of carbonic anhydrase, 2) G protein-coupled receptors (e.g., secretin and vasoactive intestinal peptide) activate cAMP-sensitive CFTR, and3) resultant increases in luminal Cl− drive a Cl−/ exchanger (reviewed in Refs. 11, 18, 34). On the basis of the belief that accumulates intracellularly in a passive, essentially unregulated fashion, recent physiological studies have focused on alternate channels capable of conducting Cl− into the pancreatic duct lumen (43, 44).
However, a recent report indicated that uptake at the basolateral membrane of pancreatic duct cells in guinea pig is Na+ dependent (19). We have recently reported the molecular cloning, cellular distribution, and functional expression of NBC-1, an electrogenically driven Na+- cotransporter (NBC) found in human renal epithelial cells (9). Northern blot hybridization utilizing an NBC-1 cDNA probe detected a similarly sized transcript in normal human kidney and pancreas (9). Taken together, these findings raise the possibility that either NBC-1 or a highly homologous electrogenically driven Na+- cotransporter may mediate uptake in human pancreatic duct cells (wheren represents an unknown number ≥2). If this is in fact the case, an important testable hypothesis is that cAMP-mediated activation of CFTR creates an electrogenic potential that drives entry into pancreatic duct epithelium via a Na+- cotransporter.
In an attempt to identify a model system(s) that would allow us to gain initial insights into the functional role of Na+- cotransport in both normal and CF pancreatic duct epithelium, duct cells prototypical of normal or CF duct fragments (CAPAN-1 or CFPAC-1 cells, respectively) were utilized. Interestingly, CAPAN-1 cells, derived from a well-differentiated human ductal pancreatic adenocarcinoma with ductal morphology, represent the only human tumor-derived cell line known to express the cAMP-sensitive CFTR in a confluence-dependent manner and exhibit other functional characteristics prototypical of normal pancreatic ducts (7, 12, 24). CFPAC-1 cells were derived from a well-differentiated human ductal pancreatic cancer resected from a patient with CF due to the F508 mutation in CFTR (32). These cells have been extensively studied and documented to have normal pancreatic ductal physiology, excepting the lack of cAMP-sensitive Cl−secretion (15, 32). Furthermore, a recent report from our group indicated the development of stably transfected CFPAC-1 cell lines bearing functional wild-type CFTR (in this paper referred to as CFPAC-WT), and confirmed the recovery of cAMP-sensitive Cl− secretion in those cells (15).
In this work, we report the results of studies assessing1) the molecular identity and functional expression of this pancreatic Na+- cotransporter in CAPAN-1, CFPAC-1, and CFPAC-WT cells,2) the effect of membrane depolarization on Na+-dependent, cotransport following acid loading of these cells, and 3) the ability of cAMP to influence intracellular pH (pHi) recovery in the same cells either bearing or lacking functional CFTR. Our findings suggest that uptake in these prototypical pancreatic duct cell models is electrogenically regulated by cAMP-sensitive CFTR. These findings have important ramifications regarding our understanding of the physiological role of CFTR in these cells and provide important novel insights into the pancreatic pathophysiology of CF.
MATERIALS AND METHODS
CAPAN-1 and CFPAC-1 cells were obtained from the American Type Culture Collection and cultured as previously described by our group (15, 32). Stably transfected CFPAC-1 cells bearing functional CFTR (CFPAC-WT) were cultured in a similar fashion, excepting the addition of G418 (1 mg/ml) to the medium (15).
RNA isolation and Northern blot hybridization.
Total cellular RNA was extracted from CAPAN-1 and CFPAC-1 cells according to the established methods (13), quantitated spectrophotometrically, and stored at −80°C. Total RNA samples (30 μg/lane) were fractionated on a 1.2% agarose-formaldehyde gel, transferred to Magna NT nylon membranes, cross-linked by ultraviolet light, and baked. Hybridization was performed according to Church and Gilbert (14). The membranes were washed, blotted dry, and exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA). A32P-labeled cDNA fragment corresponding to nucleotides 509–3237 of the mRNA encoding human NBC-1 was used as a specific probe.
Functional localization of Na+- cotransport.
CFPACWT cells were grown in collagen-coated 30-mm Millicell HA culture dish inserts (porosity 0.45 μm) for 10 days and assayed for the presence of -dependent22Na+uptake from the luminal or basolateral surface. The integrity of confluent cell monolayers was assessed by a lack of significant transport of22Na+from the upper compartment to the lower compartment under control conditions (>1% of the22Na+appeared in the lower compartment after 5 min, which is a good indicator of the integrity of the monolayer grown on a filter). Acid loading was accomplished by NH4Cl pulse at each side of the filter (38). The NH4Cl-containing solution consisted of (in mM) 75N-methyl-d-glucamine (NMDG) chloride, 25 KHCO3, and 40 NH4Cl. The uptake medium consisted of (in mM) 10 22NaCl, 105 NMDG chloride, and 25 KHCO3; 0.5 mM ouabain was added to the basolateral uptake medium (to inhibit Na+-K+-ATPase), and 2 mM amiloride was added to the apical or basolateral uptake medium (to inhibit Na+/H+exchange). A Na+-free solution (Na+ was replaced by equimolar NMDG as chloride salt) that in addition contained 2 mM amiloride was used to bathe the side opposite the uptake medium. All solutions contained (in mM) 4 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES (pH 7.4).
Cell pH measurement.
Changes in pHi were monitored using the pH-sensitive fluorescent dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM as described (3, 38, 39). Cells were grown to confluence on glass coverslips and incubated in the presence of 5 μM BCECF in a solution consisting of (in mM) 115 tetramethylammonium (TMA) chloride, 25 KHCO3, and 10 HEPES (pH 7.4) and gassed with 5% CO2-95% O2.1The monolayer was then perfused with the appropriate solutions in a thermostatically controlled holding chamber (37°C) in a Delta Scan dual-excitation spectrofluorometer (PTI, South Brunswick, NJ). The fluorescence ratio at excitation wavelengths of 500 and 450 nm was utilized to determine pHi values. Calibration curves were established daily by the KCl-nigericin technique. -free or -containing solutions were used to determine the dependence of the transporter. The Na+ and dependence of the transporter was examined by monitoring pHirecovery (dpHi/dt, in pH unit/min) from an acid load following NH3/ withdrawal, as employed for Refs. 4 and 9. The -containing solution consisted of (in mM) 75 TMA (or NMDG) chloride, 40 NH4Cl, and 25 KHCO3. The Na+-containing solution consisted of 115 mM NaCl and 25 mM KHCO3. For voltage-clamp studies of cAMP effect, the Na+-containing solution consisted of (in mM) 45 NaCl, 25 NaHCO3, and either 70 TMA chloride or 70 KCl. All solutions contained (in mM) 0.8 K2HPO4, 0.2 KH2PO4, 1 CaCl2, 1 MgCl2, and 10 HEPES (pH 7.4).
Values are expressed as means ± SE. The significance of differences between mean values was examined using ANOVA.P < 0.05 was considered statistically significant.
Molecular expression of NBC-1 in pancreatic duct cells.
On the basis of the high level of expression of NBC-1 mRNA in whole normal human pancreas (9), we first examined whether this transporter is expressed in cultured human duct cells, CAPAN-1 and CFPAC-1 cells. Northern hybridizations utilizing a32P-labeled probe corresponding to nucleotides 509–3237 of human NBC-1 identified an 8.1-kb transcript in both CAPAN-1 cells and CFPAC-1 cells (Fig.1). The expression of this pancreatic NBC was compared with rat kidney cotransporter (Fig. 1). NBC-1 mRNA levels in the duct cells (Fig. 1) were lower than in the whole pancreas (9).
Functional expression of NBC in pancreatic duct cells.
Cells were grown to confluence on glass coverslips and incubated with BCECF. In the presence of 1 mM amiloride (an inhibitor of Na+/H+exchange), switching to a Na+-containing solution resulted in rapid pHi recovery from acidosis in the presence of (Fig.2, A andB). The recovery from cell acidosis was inhibited in both cell lines by 200 μM DIDS (Fig. 2). The results of four separate experiments showed DIDS-sensitive, Na+-dependent -mediated pHi recovery rates of 0.11 ± 0.03 and 0.16 ± 0.03 pH unit/min in CAPAN-1 and CFPAC-1 cells, respectively (Fig. 2, C andD). NBC activity in CFPAC-WT cells showed rates very similar to CFPAC-1 cells (0.15 ± 0.03 pH unit/min, n = 4). In the absence of but in the presence of Na+ and amiloride, no pHi recovery from acidosis was detected, confirming the dependence of pHi recovery in Fig.2. In the absence of Na+ and , there was no pHi recovery from acidosis, indicating functional absence of H+-ATPase in both cell lines (Fig.2, E andF).
Basolateral localization of Na+- cotransport in pancreatic duct cells.
To examine the localization of the NBC in pancreatic duct cells, CFPAC-WT were grown to confluence in 30-mm Millicell HA culture dish inserts (porosity 0.45 μm) for 8–10 days and assayed for the presence of acid-stimulated, -dependent22Na+uptake from the upper compartment (luminal surface) or lower compartment (basolateral surface) (see Functional localization of Na+- cotransport). As illustrated in Fig.3 A, -dependent, acid-stimulated22Na+influx was observed predominantly at the basolateral surface (there is a minimal degree of22Na+influx at the luminal surface that could be due to overgrowth of cells on the filters, which can disturb complete polarization of the cells). Taken together, these experiments indicate that the electrogenic NBC mediates influx into the duct cells across the basolateral membrane.
Electrogenicity of Na+- cotransport in pancreatic duct cells.
The kidney NBC is highly electrogenic and transports three for each Na+ (35, 37, 45), indicating that it carries a net negative charge. As such, this transporter is very sensitive to alterations in the membrane potential (6, 8, 28, 36). Indeed, in the kidney, the Na+- cotransporter that is located on the basolateral membrane of the proximal tubule and carries from the cell to the blood is only driven by inside-negative membrane potential (6, 8, 28, 36). To determine whether alterations in the membrane potential can affect the rate of Na+- cotransport in pancreatic duct cells, CFPAC-1 cells were acid loaded by the pulse method and switched to a Na+-containing solution (70 mM NaCl) that had either low (1.8 mM) or high (72 mM) K+. As indicated in Fig.3 B, in the presence of valinomycin (which increases permeability of the membrane to K+ and causes the flow of K+ down its gradient), the rate of Na+-dependent influx decreased significantly in low-K+ solution (summary of 4 separate experiments showed that NBC activity decreased by 54% in low-K+ solution vs. high-K+ solution,P < 0.02,n = 4). Taken together, these results indicate that changing the membrane potential regulates Na+- cotransport activity.
Effects of cAMP on Na+- cotransport in pancreatic duct cells.
It has been shown that stimulation of CFTR by secretin, which works via cAMP, increases secretion (5, 11,34). In view of the fact that Na+- cotransport is highly electrogenic and is regulated by membrane potential (Fig. 3 B), we entertained the possibility that stimulation of ductal secretion by CFTR activation could be due to enhanced entry at the basolateral membrane via stimulation of the NBC (resulting from depolarization of the membrane potential secondary to Cl− secretion by CFTR). We therefore examined the effect of 8-bromoadenosine 3′5′-cyclic monophosphate (8-BrcAMP) on Na+- cotransport in cells with functional or mutant CFTR. Cells were exposed to cAMP during the withdrawal step. As indicated, addition of 500 μM 8-BrcAMP significantly enhanced the NBC activity in CAPAN-1 cells, increasing it by ∼2.8-fold (Fig.4, A andC). The rate of pHi recovery increased from 0.10 ± 0.02 pH unit/min in the absence of cAMP to 0.28 ± 0.04 pH unit/min in the presence of cAMP (P< 0.001, n = 4). These experiments were performed in the presence of an outward Cl− gradient (extracellular Cl− was replaced with gluconate). However, potentiation of Na+- cotransport activity by cAMP was significantly attenuated when external Cl− was present (apparently due to the lack of a favorable chemical gradient for the outward movement of Cl−), strongly suggesting that the stimulatory effect of cAMP on the NBC is indirect and is mediated via activation of CFTR (data not shown).
Potentiation of Na+- cotransport by cAMP is only observed in duct cells expressing functional CFTR.
To determine whether the presence of a functional CFTR is indeed necessary for enhancement of the NBC by cAMP (Fig. 4), CFPAC-1 cells that express the defective CFTR were examined. As shown in Fig. 4,B andD, in the presence of an outward Cl− gradient, addition of cAMP had no significant effect on the NBC activity. To determine whether stimulation of Na+- cotransport by cAMP is indeed due to functional CFTR and is not a cell-specific property, CFPAC-1 cells stably transfected with wild-type CFTR (CFPAC-WT) were examined. Addition of cAMP resulted in an approximately twofold increase in the rate of Na+- cotransport activity in CFPAC-WT cells (Fig.5, Aand C). The rate of pHi recovery increased from 0.15 ± 0.02 pH unit/min in the absence of cAMP to 0.29 ± 0.04 pH unit/min in the presence of cAMP (P< 0.005, n = 4). The stimulatory effect of cAMP on Na+- cotransport was prevented when extracellular Cl− was present (data not shown).
Potentiation of Na+- cotransport by cAMP in pancreatic duct cells is via membrane depolarization.
The above results indicate that cAMP stimulates the NBC in cells expressing only functional CFTR. They further indicate that stimulation of the NBC by cAMP is due to activation of CFTR. Activation of CFTR by cAMP in vitro or in vivo leads to Cl− secretion and subsequent cell membrane depolarization (reviewed in Refs. 11, 18, 34). To determine whether alteration in cell membrane potential was responsible for stimulation of Na+- cotransport, Na+-dependent transport in CFPAC-WT cells was examined in response to cAMP under voltage-clamped conditions. Cells were acidified and exposed to cAMP in the presence of the K+ ionophore valinomycin and high external K+ to clamp the membrane potential (35, 37). As shown in Fig. 5,B andD, the stimulatory effect of cAMP on the NBC was blocked by voltage clamping in high-K+ solution.
CFTR does not carry in pancreatic duct cells.
It has been shown that stimulation of CFTR by secretin, which works via cAMP, increases secretion (5, 11). One possibility with respect to the underlying stimulatory mechanism of ductal secretion by cAMP was that CFTR had the capability to carry , analogous to findings in tracheal epithelial cells (27, 33). To determine whether CFTR mediates efflux, duct cells were loaded with and monitored for pHi in the presence of but the absence of Na+ in the media (to keep the NBC inactive). As indicated in Fig. 5 E, addition of cAMP had no significant effect on pHi in the presence of an outward Cl− gradient (0 mM external Cl−). Similarly, cAMP had no effect on pHi in the absence of an outward Cl− gradient. These results indicate that CFTR has minimal affinity for in the pancreatic duct cells.
RT-PCR analysis of pancreatic NBC-1 cDNA.
To determine whether the pancreas NBC-1 expresses the protein kinase A (PKA) binding site, a region around the PKA phosphorylation consensus site of the kidney NBC-1 was amplified using RT-PCR, cloned, and sequenced. Accordingly, total RNA from human pancreas was subjected to reverse transcription using an oligo(dT) primer. After the reverse transcription and digestion with RNase H, the pancreatic cDNA was purified on a Microcon-30 ultrafilter (Amicon) to remove the oligo(dT) primers and digested RNA. PCR was then performed using the primers 5′-CAA AGA GTC ACT GGA ACC CTT G and 5′-AAG AGA GAA GCA GAG AGA GCG. Analysis by gel electrophoresis revealed a single band, indicating a single amplification product. The band was excised from the agarose gel, purified, and ligated into a pGEM-T plasmid (Promega). After transformation by electroporation and antibiotic selection, two independent clones were selected. The two clones were grown up, and plasmids were isolated from each for sequencing. Because of the central location of the PKA consensus site within each of the cDNA inserts, it was possible to get high-quality sequence of this region by sequencing from each end of the two plasmids. Thus the sequence of the PKA consensus phosphorylation site was determined by sequencing in both directions from two independent clones. Because bidirectional sequencing was not performed on the flanking regions, some sequence ambiguities remain in those areas. However, all four overlapping sequences showed an intact PKA consensus sequence.
Cultured pancreatic duct cells (CAPAN-1 and CFPAC-1) express the NBC-1 mRNA (Fig. 1) and NBC activity (Fig. 2). The NBC is located on the basolateral membrane domain and dictates uptakes in duct cells (Fig.3 A). This transporter is highly electrogenic and is regulated by alterations in membrane potential (Fig. 3 B). cAMP potentiates uptake via NBC only in duct cells expressing functional CFTR (Figs. 4 and5 A), by depolarizing the cell membrane (Fig. 5 B).
According to the currently accepted model of secretion in the pancreatic duct cells, basolateral transport is via diffusion of CO2 and subsequent action of cytosolic carbonic anhydrase (5, 11, 34). The resultant carbonic acid dissociates into H+ and , with H+ transported back to the blood via a basolateral H+-ATPase and/or a Na+/H+exchanger (5, 11, 34). Our data indicate that intracellular accumulation of occurs as a result of direct transport of via a basolateral electrogenic NBC rather than by diffusion of CO2. This is consistent with the expression of NBC-1 in the pancreatic duct cells (Fig. 1) and is in agreement with recent reports indicating functional expression of NBC in basolateral membranes of pancreatic duct cells in guinea pig and rat (19).
NBC-1 is expressed in both renal proximal tubule cells (10) and pancreatic duct cells (Figs. 1-4). The pancreatic NBC-1 is apparently a spliced variant of kidney NBC-1 (1). In the kidney proximal tubule cells, the Na+- cotransporter mediates the transport of Na+ and from cell to blood against chemical gradients for both ions, as cellular concentrations of Na+ and are less than those of blood (6,8, 28, 36). As such, the only driving force for outward movement of Na+- cotransport is the favorable membrane potential (inside-negative potential). Indeed, with basolateral membrane potentials at −65 to −70 mV in the kidney proximal tubule cells (8, 28, 36, 45), the stoichiometry of three per Na+ allows for the efflux of the Na+- cotransporter from cell to blood against chemical gradients for both Na+ and . Membrane potentials of −60 mV or lower (i.e., −50 or −40 mV) will not be able to overcome the uphill chemical gradients for Na+ and (45). In the pancreatic duct cells, basolateral membrane potentials of −35 mV or lower (which has been shown to be the case in the stimulated state) (26) guarantee that the direction of the NBC is from the blood to the cell. In other words, this transporter mediates the influx of to duct cells, a phenomenon completely opposite to that in the kidney proximal tubule cells. This change in the direction of the transport results from the activity of the apical CFTR that is absent in the kidney proximal tubule cells.
We have examined the effect of cAMP on NBC-1 in kidney HEK-293 cells (transiently transfected with the NBC-1 cDNA) and found that its activity is decreased in response to cAMP (dpHi/dt decreased from 0.24 to 0.16 pH unit/min in response to cAMP,P < 0.04,n = 4 for each group). Inhibition of Na+- cotransport in the kidney by cAMP is consistent with published reports on regulation of this transporter in kidney proximal tubule cells (30). Kidney NBC-1 has only a single PKA phosphorylation site (at the 3′ end of the coding region at amino acid 979), which likely mediates the inhibitory effect of cAMP (9). One possibility with respect to the opposite effects of cAMP on Na+- cotransport activity in the kidney and pancreas (inhibition vs. stimulation, respectively) was that pancreatic NBC lacked the PKA binding site (resulting from either deletion or alternate splicing), leaving the stimulatory effect of membrane depolarization unopposed. Sequence analysis of the 3′ end of the pancreatic NBC-1 cDNA, however, showed an intact PKA consensus sequence in pancreatic NBC-1 (Fig.6).2The stimulatory effect of cAMP on the pancreas cotransporter strongly suggests that the inhibitory phosphorylation effect would have to be superseded by the depolarizing effect of CFTR activation. Alternatively, it is plausible that the inhibitory effect of PKA in the kidney proximal tubule cells is mediated via an intermediary protein (41), and that protein could be absent in the pancreatic duct cells, leaving the stimulatory effect of membrane depolarization unopposed.
Stimulation of NBC by cAMP (which is also observed at baseline pHi, data not shown) in the pancreatic duct cells expressing functional CFTR is intriguing and highlights the coordinated regulation of apical and basolateral transporters in the duct cells. In the currently accepted model of secretion, the defect in secretion presumably lies at the apical membrane of the duct cells, where decreased secretion of Cl− via CFTR inhibits the apical Cl−/ exchange. It is worth mentioning that, although an apical Cl−/ exchanger is expressed in the duct cells (20, 46), its role in mediating secretin-stimulated secretion remains controversial (20). Indeed, a recent report demonstrated that removal of luminal Cl− or addition of DIDS only partially inhibited secretin-stimulated secretion (<25%) in the guinea pig pancreatic duct cells (20), strongly suggesting that the apical Cl−/ exchanger does not play a major role in secretin-stimulated secretion. In addition, CFTR also did not show any significant affinity for in the duct cells (Fig.7), which would have otherwise explained the ductal secretion defect in CF. Taken together, these results suggest that the ductal secretion defect in CF has an alternative molecular basis than the currently accepted one.
Our results indicating lack of stimulation of NBC in cells expressing the mutant CFTR are consistent with a decreased basolateral transport into the duct cells in patients with CF. Although we acknowledge the need for confirmatory studies in primary cultures of both normal and CF human pancreatic duct cells, our findings in these prototypical cells suggest that the limiting step in ductal secretion in CF patients lies at the basolateral membrane of these cells, where decreased electrogenic driving force (resulting from lack of depolarization of the membrane) prevents the entry of via the NBC. Accordingly, we propose the following transport model for the pancreatic duct cells (Fig. 7). According to this model, secretin increases intracellular cAMP, which then results in the activation of CFTR and secretion of Cl−, leading to depolarization of both luminal and basolateral membranes. The depolarization of the basolateral membrane increases the driving force for NBC and, as a result, enhances entry into the duct cells of , which is then secreted at the apical membrane (mostly via a transporter that is poorly defined at the present and partially via an apical Cl−/ exchanger).
Because the identity of the apical transporter mediating the majority of the ductal secretion remains poorly understood (20), and because all acid-base transporters seem to be expressed in both normal and CF cells, it is logical to conclude that an alternative strategy to gene therapy for correcting the secretion defect in CF patients could be via activation of NBC (either directly or indirectly). According to this hypothesis, stimulation of NBC either directly (i.e., via protein kinase C analogs or angiotensin II) or indirectly (i.e., via cell membrane depolarization) should increase entry into the duct cells of , which can be secreted into the lumen via the apical transporter(s). Depolarizing agents such as basolateral K+ channel blockers are excellent candidates to indirectly stimulate NBC.
In summary, a basolateral electrogenic NBC dictates uptake in the pancreatic duct cells. This cotransporter is stimulated by cAMP only in cells expressing functional CFTR. The stimulation of the NBC by cAMP is mediated via membrane depolarization. We propose that the defect in ductal secretion in patients with CF is predominantly due to decreased entry at the basolateral membrane as a result of decreased electrogenic driving force and subsequent inhibition of NBC. Future studies should test the possibility that stimulating the NBC (either directly or indirectly) can increase entry into the duct cells and therefore alleviate pancreatic secretion defect in patients with CF.
We acknowledge the excellent contributions of Elizabeth Kopras, Charles Burnham, and Zhaohui Wang.
Address for reprint requests: M. Soleimani, Division of Nephrology and Hypertension, University of Cincinnati Medical Center, 231 Bethesda Ave., MSB 5502, Cincinnati, Ohio 45267-0585.
These studies were supported by National Institutes of Health Grants DK-46789, DK-52821, and DK-54430 (to M. Soleimani) and CA-74456 (to C. D. Ulrich) and a grant from Dialysis Clinic Inc. (to M. Soleimani).
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. §1734 solely to indicate this fact.
↵1 To use in Na+-free solutions for loading and withdrawal steps, we initially attempted to avoid choline bicarbonate, as choline has been shown to have nonspecific effects on pHi. We therefore used KHCO3. However, subsequent studies indicated similar experimental results when either 25 mM choline bicarbonate (and more physiological concentrations of K+) or 25 mM KHCO3 was used.
↵2 Pancreatic NBC-1 has two consensus PKA binding sites (1). However, the additional phosphorylation site does not play an important role in mediating the stimulatory effect of cAMP on NBC-1, as addition of cAMP had no effect on Na+-dependent cotransport in cells expressing the mutant CFTR.
- Copyright © 1999 the American Physiological Society