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CFTR upregulates the expression of the basolateral Na+-K+-2Clminus cotransporter in cultured pancreatic duct cells

Holli Shumaker and Manoocher Soleimani

Department of Medicine, University of Cincinnati, and Veterans Affairs Medical Center at Cincinnati, Cincinnati, Ohio 45267


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The purpose of the current experiments was 1) to assess basolateral Na+-K+-2Cl- cotransporter (NKCC1) expression and 2) to ascertain the role of cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of this transporter in a prototypical pancreatic duct epithelial cell line. Previously validated human pancreatic duct cell lines (CFPAC-1), which exhibit physiological features prototypical of cystic fibrosis, and normal pancreatic duct epithelia (stable recombinant CFTR-bearing CFPAC-1 cells, termed CFPAC-WT) were grown to confluence before molecular and functional studies. High-stringency Northern blot hybridization, utilizing specific cDNA probes, confirmed that NKCC1 was expressed in both cell lines and its mRNA levels were twofold higher in CFPAC-WT cells than in CFPAC-1 cells (P < 0.01, n = 3). Na+-K+-2Cl- cotransporter activity, assayed as the bumetanide-sensitive, Na+- and Cl--dependent NH+4 entry into the cell (with NH+4 acting as a substitute for K+), increased by ~115% in CFPAC-WT cells compared with CFPAC-1 cells (P < 0.01, n = 6). Reducing the intracellular Cl- by incubating the cells in a Cl--free medium increased Na+-K+-2Cl- cotransporter activity by twofold (P < 0.01, n = 4) only in CFPAC-WT cells. We concluded that NKCC1 is expressed in pancreatic duct cells and mediates the entry of Cl-. NKCC1 activity is enhanced in the presence of an inward Cl- gradient. The results further indicate that the presence of functional CFTR enhances the expression of NKCC1. We speculate that CFTR regulates this process in a Cl--dependent manner.

cystic fibrosis transmembrane conductance regulator; HCO-3 secretion; cystic fibrosis


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ACTIVATION OF cystic fibrosis transmembrane conductance regulator (CFTR) by secretin enhances Cl- secretion into pancreatic duct lumen and, as a result, depolarizes the luminal and basolateral membranes of the duct cells (3, 6, 26, 33). In parallel with enhanced Cl- secretion, secretin-stimulated CFTR activation also increases HCO-3 secretion in the pancreatic duct cells (3, 6, 16, 17, 19, 33). Several studies have demonstrated that the Cl- concentration in the final pancreatic ductal secretion is very low, indicating reabsorption of Cl- along the ductal system (3, 6). This has led to the postulation that the resulting increase in luminal Cl- drives an apical Cl-/HCO-3 exchanger (reviewed in Refs. 3, 6, 33). According to this model, the indirect coupling of CFTR and the apical Cl-/HCO-3 exchanger results in the recycling of Cl- and sustains the driving force for Cl- and HCO-3 secretion in the agonist-stimulated state (3, 6, 33).

Clearly, the degree of Cl- efflux via activated CFTR depends on the availability of intracellular Cl- (3, 6). Recent studies demonstrate that removal of luminal Cl- or addition of DIDS only partially inhibited secretin-stimulated HCO-3 secretion (<25%) in the guinea pig pancreatic duct cells (16, 17), strongly suggesting that the apical Cl-/HCO-3 exchanger does not play a major role in secretin-stimulated HCO-3 secretion. These findings by inference demonstrate that activation of CFTR by secretin and the resultant HCO-3 secretion persist despite lack of availability of luminal Cl-. Together, these studies indicate that a mechanism distinct from the apical Cl-/HCO-3 exchanger is responsible for Cl- entry into the duct cells.

A number of secretory epithelial cells express a Na+-K+-2Cl- cotransporter on their basolateral membrane, which mediates the entry of Cl- into the cell for eventual secretion via apical Cl- channels (i.e., CFTR or a Ca+-sensitive Cl- channel) (4, 10, 22, 24). The best example is the small intestine, in which cAMP-activated Cl- secretion (via CFTR) is dependent on the basolateral Na+-K+-2Cl- cotransporter (23-25, 27). Cloning experiments have identified the cDNA encoding the Na+-K+-2Cl- cotransporter (called NKCC1) (12, 37). NKCC1 is expressed in a variety of epithelial and nonepithelial cells (4, 10, 12, 15, 22-25, 37). In nonepithelial cells such as vascular smooth muscle cells, NKCC1 is predominantly responsible for volume regulation by the transportation of Na+ and Cl- into the cells (12, 15, 37). In epithelial cells such as small intestine or kidney tubules, NKCC1 is localized on the basolateral membrane domain (12, 22-25, 34, 36, 37) and is responsible for the transport of Cl- and Na+ from blood to the cell. Cl- is likely secreted into the lumen via Cl- channels (CFTR or Ca2+-sensitive isoform), whereas Na+ is transported back to the blood via the Na+-K+-ATPase.

In addition to the basolateral cotransporter (NKCC1), an apical Na+-K+-2Cl- cotransporter (NKCC2) has also been cloned (14). NKCC2 is exclusively expressed on the apical membranes of kidney thick limb of Henle and is responsible for the reabsorption of Na+ and Cl- in this nephron segment (14, 29). Both NKCC1 and NKCC2 are electroneutral transporters and are sensitive to inhibition by furosemide and bumetanide (12, 14, 24, 29, 37). In addition to Na+ and Cl-, both the apical and basolateral Na+-K+-2Cl- cotransporters can transport NH+4, with NH+4 substituting for K+ (1, 2, 28, 35).

The purpose of the current experiments was to determine whether NKCC1 is expressed in cultured pancreatic duct cells. In addition, we were interested in testing whether CFTR plays any role in the regulation of this transporter. Accordingly, cultured pancreatic duct cells with or without functional CFTR were utilized. The results demonstrated that NKCC1, but not NKCC2, is expressed in pancreatic duct cells and its expression is regulated by CFTR.


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Cell lines. CFPAC-1 cells were obtained from American Type Culture Collection (ATCC) and cultured as previously described (30, 31). Additional CFPAC-1 clones (1a and 1b) were developed in our laboratory. Stably transfected CFPAC-1 cells bearing functional CFTR (termed CFPAC-WT) were cultured in a similar fashion, except for the addition of G418 (1 mg/ml) to the medium (13, 31). CFPAC-WT clones (CFPAC-WT, CFPAC-WTa, and CFPAC-WTb) were a generous gift from Dr. R. Frizzell, University of Pittsburgh. Capan-1 cells were obtained from ATCC and cultured as previously described (31).

RNA isolation. Total cellular RNA was extracted from CFPAC-1 and CFPAC-WT cells by the method of Chomczynski and Sacchi (7). In brief, cells from three separate flasks were homogenized at room temperature in 10 ml of Tri reagent (Molecular Research Center, Cincinnati, OH). RNA was extracted by phenol-chloroform, precipitated by isopropanol (7), quantitated by spectrophotometry, and stored at -80°C.

Northern hybridization. Total RNA samples (30 µg/lane) were fractionated on a 1.2% agarose-formaldehyde gel and transferred to Magna NT nylon membranes (MSI) using 10× sodium chloride-sodium phosphate-EDTA as transfer buffer. Membranes were cross-linked by ultraviolet light and baked for 1 h. Hybridization was performed according to Church and Gilbert (8). The cDNA probes were labeled with [32P]deoxynucleotides using the RadPrime DNA labeling kit (GIBCO BRL). The membranes were washed twice in 40 mM sodium phosphate buffer, pH 7.2, 5% SDS, 0.5% BSA, and 1 mM EDTA for 10 min at 65°C, washed four times in 40 mM sodium phosphate buffer, pH 7.2, 1% SDS, and 1 mM EDTA for 10 min at 65°C. Thereafter, the membranes were blotted dry and exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA) at room temperature for 24-72 h. Densitometric scanning of the blots was performed by the PhosphorImager. The following rat PCR product fragments were used as specific probes in the Northern blot analyses: for NTCC2, nucleotides 509-3237; for NTCC1, nucleotides 906-1238 and 1631-2300. These PCR fragments have been used as specific probes (2) and under high-stringency hybridization conditions do not cross-react with one another (2).

Intracellular pH measurements. Intracellular pH (pHi) in cultured pancreatic cells was measured with the use of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) as previously described (2, 5, 31). In brief, cells were grown to confluence on coverslips, loaded with BCECF, and monitored for pHi recording with the use of a Delta Scan dual-excitation spectrofluorometer (double-beam fluorometer, Photon Technology International, Brunswick, NJ) that was equipped with a water-jacketed, temperature-controlled system. Fluorescence intensity was recorded at emission wavelength at 525 nm and two excitations wavelengths at 500 and 450 nm. The fluorescence ratios (F500/F450) were converted into pHi values with use of calibration curves that were established by the KCl-nigericin method.

Measurement of Na+-K+-2Cl- cotransporter activity. Na+-K+-2Cl- cotransporter activity was assessed as previously described (2). Briefly, the Na+-K+-2Cl- cotransporter activity was measured by determining the rate of intracellular acidification caused by NH+4 entry into the cells via this transport mechanism on abrupt application of 40 mM NH4Cl (1, 2, 28, 35). NH+4 entry into cultured cells can occur via several transport pathways, including Na+-dependent and Na+-independent pathways (1, 2, 28, 35). The Na+ dependence of NH+4 transport was determined to distinguish this transporter from Na+-independent pathways. Furthermore, bumetanide, which inhibits Na+-K+(NH+4)-2Cl- cotransport, was used to distinguish this transporter from other Na+-dependent pathways (1, 2). NKCC1 activity was therefore determined as the bumetanide-sensitive, Na+-dependent NH+4 entry into the cells.

For the experiments, cultured CFPAC-WT (or CFPAC-1) cells were incubated in a Cl--containing, Na+-free and CO2-free HEPES-Tris-buffered medium (solution A, Table 1). Baseline pHi was 7.28 ± 0.07 for CFPAC-WT cells (and 7.23 ± 0.06 for CFPAC-1 cells, P > 0.05 between the two cell lines). When cells were switched to a NH+4-containing solution (that, in addition, contained Na+ and Cl-, solution B, Table 1), a very rapid initial cell alkalinization occurred due to immediate NH3 entry, which stopped when intracellular and extracellular NH3 concentrations became equal due to NH3 equilibrium (see Fig. 1A). The initial alkalinization was then followed by a relatively rapid drop in pHi (Fig. 1B). The initial rate (dpHi/dt) of this acidification is exclusively due to NH+4 entry (see RESULTS). Furthermore, the NH+4-induced acidification is completely blocked in the presence of 60 mM K+ (high K+) vs. 1.8 mM K+ (low K+) (solutions C and D, Table 1), indicating competition between K+ and NH+4 (Fig. 1C). The inhibitory effect of K+ on NH+4-induced cell acidification was concentration dependent, with 30 mM K+ causing ~50% inhibition vs. 60 mM K+ (data not shown). The experiments were performed at least four separate times for each condition. In the absence of Na+ or presence of bumetanide, NH+4-induced cell acidification was almost completely blocked in cultured cells, indicating that all of the NH+4 entry into the pancreatic duct cells is due to Na+-K+(NH+4)-2Cl- activity (see RESULTS).

                              
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Table 1.   Composition of solutions





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Fig. 1.   NH+4 transport via the Na+-K+-2Cl- cotransporter in wild-type cystic fibrosis transmembrane conductance regulator (CFTR)-bearing pancreatic duct (CFPAC-WT) cells. A and B: schematic diagrams [combined with intracellular pH (pHi) tracings] demonstrating NH+4 transport in wild-type CFTR-bearing pancreatic duct cells. C: NH+4-induced acidification was completely blocked in the presence of high K+ concentration in the medium, indicating competition between K+ and NH+4. [Cl-]o, [Na+]o, and [NH+4]o: extracellular Cl-, Na+, and NH+4 concentration, respectively.

Materials. [32P]dCTP was purchased from NEN (Boston, MA). Nitrocellulose filters and other chemicals were purchased from Sigma Chemical (St. Louis, MO). RadPrime DNA labeling kit was purchased from GIBCO BRL. BCECF was from Molecular Probes (Eugene, OR).

Statistical analysis. The data are expressed as means ± SE where appropriate. For statistical analysis of mRNA expression experiments, the PhosphorImager readings of three separate Northern hybridizations were obtained and analyzed. Statistical analysis was determined using one-way ANOVA. P < 0.05 was considered statistically significant.


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RESULTS
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Molecular expression of NKCC1 in wild type CFTR-bearing pancreatic duct cells. In the first series of experiments, we examined whether NKCC1 is expressed in wild-type CFTR-bearing cultured pancreatic duct cells (CFPAC-WT cells). Northern hybridizations utilizing a 32P-labeled probe corresponding to nucleotides 906-1238 and 1631-2300 of rat NKCC1 identified a 6.5-kb transcript in CFPAC-WT cells (Fig. 2), consistent with the expression of NKCC1 in the pancreatic duct cells.


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Fig. 2.   Molecular expression of the basolateral Na+-K+-2Cl- cotransporter (NKCC1) in CFPAC-WT cells. Top: NKCC1 Northern hybridization. Bottom: 28S rRNA Northern hybridization. Thirty micrograms of RNA were loaded in each lane.

We also assessed the expression of NKCC2 in pancreatic duct cells. As shown in Fig. 3, Northern hybridization blots did not detect the mRNA expression of NKCC2 in cultured human duct cells. The expression of NKCC2 in kidney medulla is shown for comparison (Fig. 3).


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Fig. 3.   Northern hybridizations of the apical Na+-K+-2Cl- cotransporter (NKCC2). Top: NKCC2 Northern hybridization. Bottom: 28S rRNA Northern hybridization. Thirty micrograms of RNA were loaded in each lane. NKCC2 mRNA was not detected in CFPAC-WT cells. Expression of NKCC2 in the kidney medulla is shown for control.

Functional expression of the Na+-K+-2Cl- cotransporter in wild-type CFTR-bearing pancreatic duct cells. CFPAC-WT cells were grown to confluence on glass coverslips, loaded with BCECF, and assayed for Na+-K+-2Cl- cotransporter activity as described in MATERIALS AND METHODS. Figure 4A is a representative pHi tracing that demonstrated that switching the CFPAC-WT cells to an NH+4-containing solution that also contained Na+ and Cl- (solution B, Table 1) resulted in a rapid initial cell alkalinization, likely due to NH3 diffusion (Fig. 4A). After this initial alkalinization, pHi acidified back to baseline in ~10 min (Fig. 4A). This acidification represented NH+4 entry, as induction of equivalent cell alkalinization by acetate withdrawal (solution E, Table 1) did not result in a significant acidification (Fig. 4A, representative tracings). The rate of recovery from acetate withdrawal-induced alkalinization was not different from zero (n = 3). Furthermore, the presence of DIDS did not inhibit NH+4-induced cell acidification, indicating that Cl-/OH- exchange did not play a role in this process (data not shown). To determine whether NH+4 transport in pancreatic duct cells occurs via a Na+-dependent pathway, NaCl was replaced with tetramethylammonium chloride (solution F, Table 1) and the experiments were repeated. Figure 4B indicates that NH+4-induced cell acidification was almost completely inhibited in the absence of Na+. These results are consistent with the transport of NH+4 via a Na+-dependent pathway. To test whether NH+4 transport in pancreatic duct cells is mediated via NKCC1, the experiments were performed in the presence of 500 µM bumetanide, a strong inhibitor of the Na+-K+-2Cl- cotransporter (Na+, Cl-, and NH+4 were present, solution B, Table 1). Figure 4C is a representative pHi tracing that indicates that the Na+-dependent NH+4 entry was almost abolished in the presence of 500 µM bumetanide. These results indicate that NKCC1 is expressed in pancreatic duct cells and is functionally active. The results of several separate experiments on NH+4 transport in the presence or absence of Na+ or bumetanide is shown in Fig. 4D. Cl- was present in all solutions.





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Fig. 4.   Functional expression of NKCC1 in wild-type CFTR-bearing pancreatic duct cells. A: NH+4 transport in the presence of Na+ and Cl- in the medium. Induction of equivalent cell alkalinization by acetate withdrawal did not result in any acidification. B: NH+4 transport in CFPAC-WT cells is Na+ dependent. C: bumetanide (500 µM) completely inhibited the Na+- and Cl--dependent NH+4 transport. D: summary of separate experiments demonstrating that NH+4 entry in CFPAC-WT cells is Na+ dependent and is sensitive to inhibition by bumetanide (n = 8 for NH+4 transport in the presence of Na+, n = 4 for NH+4 transport in the absence of Na+, and n = 4 for NH+4 transport in the presence of bumetanide and Na+). NKCC, Na+-K+-2Cl- cotransporter; TMA, tetramethylammonium.

NKCC1 mRNA expression and activity is enhanced in wild-type CFTR-bearing pancreatic duct cells. In the next series of experiments, we examined whether the mRNA expression of NKCC1 is regulated by the CFTR. Accordingly, the expression of NKCC1 was compared in CFPAC-1 and CFPAC-WT cells. As indicated in Fig. 5A, Northern hybridizations indicated that mRNA levels for the NKCC1 were increased by approximately twofold in CFPAC-WT cells over CFPAC-1 cells (P < 0.02, n = 3).


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Fig. 5.   A: Northern hybridization of NKCC1 in pancreatic duct cells with mutant CFTR (CFPAC-1 cells) or cells transfected with wild-type CFTR (CFPAC-WT cells). Top: NKCC1 Northern hybridization. Bottom: 28S rRNA Northern hybridization. Thirty micrograms of RNA were loaded in each lane. B: Northern hybridization of NKCC1 in independent CFPAC-1 and CFPAC-WT clones. Top: NKCC1 Northern hybridization. Bottom: 28S rRNA Northern hybridization. Thirty micrograms of RNA were loaded in each lane. CFPAC-1a and CFPAC-1b refer to new clones.

Figure 5A demonstrated enhanced expression of NKCC1 in functional CFTR-bearing CFPAC-WT cells. To determine whether enhanced expression of NKCC1 in CFPAC-WT cells was due to the presence of functional CFTR, RNA from two independent CFPAC-WT and CFPAC-1 clones was isolated and analyzed. Northern hybridizations (Fig. 5B) indicated that mRNA levels for NKCC1 were increased by ~90 and 55% in two independent CFPAC-WT clones vs. CFPAC-1 clones. Together with the experiments in Fig. 5A, these results indicate that the presence of a functional CFTR (and not clonal selection or number of passages) is responsible for enhanced expression of NKCC1 in pancreatic duct cells.

To determine whether increased mRNA expression of NKCC1 in CAPAC-WT cells is associated with increased activity, bumetanide-sensitive Na+ and Cl--dependent NH+4 entry in cultured pancreatic duct cells was measured in a manner similar to Fig. 4. As demonstrated in Fig. 6, A and B, Na+-K+-2Cl- cotransporter activity was enhanced by ~115% in wild-type CFTR-bearing duct cells over cells expressing the mutant CFTR (Na+-K+-2Cl- cotransporter activity was 0.066 pH units/min in CFPAC-WT cells vs. 0.031 in CFPAC-1 cells, P < 0.01, n = 6).





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Fig. 6.   Activity of NKCC1 is increased in CFPAC-WT cells. A: representative pHi tracings in CFPAC-WT and CFPAC-1 cells. B: summary of results of A. As indicated, NKCC activity is increased in cells transfected with wild-type CFTR. C and D: representative tracings indicating Na+ dependence (C) and bumetanide sensitivity (D) of NKCC in CFPAC-1 cells (see RESULTS for details).

Similar to CFPAC-WT cells, Na+-K+-2Cl- cotransporter activity in CFPAC-1 cells was Na+ dependent and significantly inhibited by 500 µM bumetanide (Fig. 6, C and D, representative tracings). A summary of multiple experiments indicated that the Na+-K+-2Cl- cotransporter activity decreased by 95% in the absence of Na+ and by 79% in the presence of bumetanide (the rates were 0.031 pH units/min in the presence of Na+, 0.0016 pH units/min in the absence of Na+, and 0.0065 pH units/min in the presence of bumetanide) (n = 4, P < 0.01 for each group vs. Na+).

Intracellular Cl- depletion potentiates NKCC1 activity only in wild-type CFTR-bearing pancreatic duct cells. NKCC1 is responsible for the transport of Cl- from blood to the cell for eventual secretion via the apical Cl- channels. We aimed to determine whether decreased intracellular Cl- concentration can enhance NKCC1. Intracellular Cl- depletion was induced by incubating the CFPAC-WT cells in a Cl--free solution (solution G, Table 1) for 15 min. As shown in Fig. 7A, decreasing the intracellular Cl- concentration significantly enhanced the rate of Na+- and Cl--dependent NH+4 entry. Figure 7B summarizes the results of four separate experiments. Na+-K+-2Cl- cotransporter activity was 0.051 pH units/min in normal condition in CFPAC-WT cells and increased to 0.099 pH units/min in the Cl--depleted state (P < 0.01, n = 4). These results are consistent with activation of Na+-K+-2Cl- cotransporter activity by intracellular Cl- depletion in CFPAC-WT cells. Interestingly, incubation of CFPAC-1 cells in Cl--free medium did not enhance the Na+-K+-2Cl- cotransporter activity (Fig. 7, C and D). Na+-K+-2Cl- cotransporter activity was 0.036 and 0.040 pH units/min in normal condition and in the Cl--depleted state, respectively (P > 0.05, n = 4).





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Fig. 7.   Intracellular Cl- depletion potentiates NKCC1 activity only in CFPAC-WT cells. CFPAC-WT (A) or CFPAC-1 (C) cells were incubated in a Cl--free medium for 15 min before switching to a Na+- and Cl--containing solution for NKCC activity measurement. B and D: summaries of 4 separate experiments (B shows CFPAC-WT cells and D shows CFPAC-1 cells) demonstrating the effect of intracellular Cl- depletion on NKCC activity.

In the last series of experiments, the effect of cAMP on the Na+-K+-2Cl- cotransporter activity in CFPAC-WT and CFPAC-1 cells was examined. Addition of cAMP did not have a consistent stimulatory effect on NKCC1 in either of the two pancreatic duct cell lines (data not shown). To determine whether the absence of an effect by 8-bromo-cAMP on the Na+-K+-2Cl- cotransporter activity was due to poor permeability of cells, forskolin was added. Figure 8, A and B, shows representative pHi tracings and summarizes four separate experiments, respectively, and demonstrate that forskolin had no stimulatory effect on Na+-K+-2Cl- cotransporter activity in CFPAC-WT cells. The effect of forskolin on Na+-K+-2Cl- cotransporter activity was also examined in Capan-1 cells, which represent the only human tumor-derived cell line known to express the cAMP-sensitive CFTR. Capan-1 cells express Na+-dependent, bumetanide-sensitive NH+4 transport, consistent with Na+-K+-2Cl- cotransport (the Na+-K+-2Cl- cotransporter activities were 0.06 ± 0.004 and 0.01 ± 0.001 pH units/min in the absence or presence of bumetanide, respectively, n = 4 for each group, P < 0.01). Addition of forskolin had no stimulatory effect on Na+-K+-2Cl- cotransporter activity in Capan-1 cells (the activities were 94% ± 6 in the presence of forskolin vs. 100% in control, P > 0.05, n = 4). Similarly, CFPAC-1 cells also did not demonstrate any stimulation of Na+-K+-2Cl- cotransporter activity by forskolin (data not shown).



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Fig. 8.   Forskolin does not stimulate NKCC1 in CFPAC-WT cells. A: representative pHi tracings in CFPAC-WT cells with or without forskolin. B: summary of results from A. As indicated, NKCC was not activated by forskolin in cells transfected with wild-type CFTR.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cultured pancreatic duct cells (CFPAC-1 and CFPAC-WT) express NKCC1 mRNA (Fig. 1) and activity (Fig. 4). NKCC1 is responsible for the transport of Cl- from blood to the duct cells. The mRNA expression and activity of NKCC1 is upregulated in functional CFTR-bearing pancreatic duct cells (Figs. 5 and 6). Intracellular Cl- depletion increased the activity of NKCC1 in cells expressing functional CFTR (CFPAC-WT cells) (Fig. 7). Neither cell line expressed NKCC2 (Fig. 3).

The process of agonist stimulation of HCO-3 secretion at the apical membrane of the pancreatic duct cells is entirely dependent on the activation of CFTR (3, 6, 9, 19, 21, 33). This by inference indicates that the transport of Cl- into the pancreatic duct cells is essential for activation of CFTR in the agonist-stimulated state. The currently accepted model of HCO-3 transport in the pancreatic duct cells attributes the restoration of intracellular Cl- to the apical Cl-/HCO-3 exchanger, which presumably recycles the luminal Cl- back to the cell in exchange for HCO-3 (3, 6, 33). This model, however, is inconsistent with recent studies in which HCO-3 efflux across the apical membrane persisted even in the absence of luminal Cl-, indicating that Cl- entrance into the duct cells occurs via a mechanism distinct from the apical Cl-/HCO-3 exchanger (17).

One possible candidate for the transport of Cl- into the duct cells is NKCC1. This possibility was entertained based on the fact that NKCC1 is expressed in a number of secretory epithelial cells (4, 11, 12, 22-25). Recent studies have demonstrated that a bumetanide-sensitive Na+-K+-2Cl- cotransporter is located on the basolateral membranes of intestinal epithelial cells and is responsible for the transport of Cl- from blood to the cell for secretion via CFTR at the luminal membrane (22-25).

The present studies are the first to demonstrate the expression of NKCC1 mRNA in the pancreatic duct cells. Our results further demonstrated the functional presence of NKCC1, indicating that this membrane protein mediates the transport of Cl- from blood to the duct cells. Cl- that enters the duct cells will eventually be secreted at the apical membrane via CFTR and to a lesser extent via other anion channels. The expression of NKCC2, which is responsible for the reabsorption of luminal Cl- in kidney cells (14, 29), was not detected in the pancreatic duct cells. This latter transporter is exclusively expressed in the ascending limb of Henle in the kidney and mediates the reabsorption of Cl- and Na+ from the luminal fluid (14, 29).

Various transporters, including the Na+-nHCO-3 cotransporter (5, 31) and Cl-/HCO-3 exchanger (20), have been found to be upregulated, whereas the Na+ channel is downregulated by CFTR (18). The Na+-nHCO-3 cotransporter activation is likely through membrane depolarization in response to Cl- secretion at the apical membrane secondary to CFTR activation (31). Decreased activity of the Na+ channel (18) and activation of the Cl-/HCO-3 exchanger (20) on the other hand have been postulated to be direct and independent of the Cl- secreting ability of CFTR.

Enhanced expression of NKCC1 in the functional CFTR-bearing pancreatic duct cells is intriguing. It has been proposed that Cl- entry across the basolateral membrane may be the rate-limiting step in regulating the activity of the apical Cl- channels in secretory epithelial cells (22). The current study, however, indicates that CFTR plays an important role in regulating the activity of NKCC1. It is likely that upregulation of NKCC1 by functional CFTR is mediated in a Cl--dependent manner. According to this scheme, the presence of the functional CFTR could increase the secretion of Cl- and result in decreased intracellular Cl- concentration in pancreatic duct cells. This in turn could increase the driving force for NKCC1, leading to its enhanced activity. This is consistent with the speculative model for the indirect regulation of NKCC1 in intestinal epithelial cells (25). The exact mechanism by which CFTR enhances the expression of NKCC1, of course, is unclear. Whether it is the Cl- depletion or an exaggerated Cl- gradient or membrane depolarization that triggers enhanced expression of NKCC1 remains speculative. It would be unlikely that CFTR directly upregulates the expression of the basolateral cotransporter, as these two transporters are located on two separate membrane domains, making the possibility of direct interaction between them unlikely.

Increased activity of NKCC1 in functional CFTR-bearing duct cells (CFPAC-WT) that are depleted of Cl- strongly suggests that an increased inward Cl- gradient across the basolateral membrane increases the driving for this transporter. This is consistent with published reports in intestinal cells in which intracellular Cl- depletion increased the activity of NKCC1 (37). It should be mentioned that the exact mechanism by which Cl- depletion increases Na+-K+-2Cl- cotransporter activity remains speculative. Intracellular Cl- can regulate Na+-K+-2Cl- cotransporter activity by either changing the phosphorylation state of NKCC1 protein or by altering the driving force for ion uptake. Activation of NKCC1 under a Cl--depleted state should result in enhanced Cl- entry into the cells across the basolateral membrane for eventual secretion at the apical membrane. Interestingly, incubation of the mutant CFTR-bearing duct cells (CFPAC-1) in Cl--free medium did not enhance Na+-K+-2Cl- cotransporter activity. Whether this was due to decreased cell volume (shrinkage) or a lack of reduction in intracellular Cl- in CFPAC-1 cells remains speculative. Future studies aimed at measuring the intracellular Cl- concentration in these two cell lines should shed light on this question.

Our results indicating enhanced expression and activity of NKCC1 in wild-type CFTR-bearing duct cells and its potentiation in the Cl--depleted state strongly suggest that activation of CFTR and the subsequent reduction in intracellular Cl- increase NKCC1 activity. This should result in enhanced entry of Cl- into the cells and maintain activation of CFTR and HCO-3 secretion under agonist-stimulated state. Accordingly, we propose a model for Cl- and HCO-3 transport in the pancreatic duct cells, which is shown in Fig. 9. According to this schematic diagram, secretin increases intracellular cAMP, which then results in the activation of CFTR and secretion of Cl-, leading to decreased intracellular Cl- concentration. This will result in depolarization of both the luminal and basolateral membranes. The reduction in cell Cl- concentration activates NKCC1, which restores the intracellular Cl- for secretion via CFTR. The depolarization of the basolateral membrane increases the driving force for Na+-HCO-3 cotransporter and as a result enhances HCO-3 entry into the duct cells for eventual secretion at the apical membrane. The identity of the transporter that mediates the bulk HCO-3 secretion at the apical membrane remains speculative. With Cl-/HCO-3 exchanger playing only a minor role and the CFTR not being able to mediate HCO-3 secretion (32), it is very plausible that HCO-3 secretion at the apical membrane of the pancreatic duct cells occurs via a HCO-3 (anion) channel. Further studies are needed to clarify this issue.


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Fig. 9.   Schematic diagram demonstrating the interaction of CFTR, NKCC1, and the Na+-HCO-3 cotransporter (NBC) in human pancreatic duct cells. A "+" inside the cell shows membrane depolarization due to the loss of anions secondary to CFTR stimulation. AE, anion exchanger (Cl-/HCO-3 exchanger).

In conclusion, NKCC1 is expressed in pancreatic duct cells and mediates the entry of Cl-. The presence of functional CFTR enhances the expression of NKCC1. The results further indicate that NKCC1 activity is enhanced in the presence of an inward Cl- gradient. We speculate that CFTR regulates this process in a Cl--dependent manner.


    ACKNOWLEDGEMENTS

We acknowledge the excellent contributions of Hassane Amlal and Zhaohui Wang.


    FOOTNOTES

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46789, DK-52821, and DK-54430 and a grant from Dialysis Clinic Incorporated.

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.

Address for reprint requests and other correspondence: M. Soleimani, Dept. of Internal Medicine, Univ. of Cincinnati, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585 (E-mail: Manoocher.soleimani{at}uc.edu).

Received 17 March 1999; accepted in final form 20 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 277(6):C1100-C1110
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