UTP inhibits Na+ absorption in wild-type and Delta F508 CFTR-expressing human bronchial epithelia

Daniel C. Devor1 and Joseph M. Pilewski1,2

Departments of 1 Physiology and Cell Biology and 2 Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ca2+-mediated agonists, including UTP, are being developed for therapeutic use in cystic fibrosis (CF) based on their ability to modulate alternative Cl- conductances. As CF is also characterized by hyperabsorption of Na+, we determined the effect of mucosal UTP on transepithelial Na+ transport in primary cultures of human bronchial epithelia (HBE). In symmetrical NaCl, UTP induced an initial increase in short-circuit current (Isc) followed by a sustained inhibition. To differentiate between effects on Na+ absorption and Cl- secretion, Isc was measured in the absence of mucosal and serosal Cl- (INa). Again, mucosal UTP induced an initial increase and then a sustained decrease that reduced amiloride-sensitive INa by 73%. The Ca2+-dependent agonists histamine, bradykinin, serosal UTP, and thapsigargin similarly induced sustained inhibition (62-84%) of INa. Mucosal UTP induced similar sustained inhibition (half-maximal inhibitory concentration 296 nM) of INa in primary cultures of human CF airway homozygous for the Delta F508 mutation. BAPTA-AM blunted UTP-dependent inhibition of INa, but inhibitors of protein kinase C (PKC) and phospholipase A2 had no effect. Indeed, direct activation of PKC by phorbol 12-myristate 13-acetate failed to inhibit Na+ absorption. Apyrase, a tri- and diphosphatase, did not reverse inhibitory effects of UTP on INa, suggesting a long-term inhibitory effect of UTP that is independent of receptor occupancy. After establishment of a mucosa-to-serosa K+ concentration gradient and permeabilization of the mucosal membrane with nystatin, mucosal UTP induced an initial increase in K+ current followed by a sustained inhibition. We conclude that increasing cellular Ca2+ induces a long-term inhibition of transepithelial Na+ transport across normal and CF HBE at least partly due to downregulation of a basolateral membrane K+ conductance. Thus UTP may have a dual therapeutic effect in CF airway: 1) stimulation of a Cl- secretory response and 2) inhibition of Na+ transport.

cystic fibrosis; cystic fibrosis transmembrane conductance regulator; epithelial sodium channel; uridine 5'-triphosphate; human airway


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CYSTIC FIBROSIS (CF) is characterized by a defect in ion transport consisting of both a reduced or absent Cl- secretory response to cAMP-mediated agonists and a hyperabsorption of Na+ (2). Recent evidence has suggested that, in addition to functioning as a Cl- channel, the CF transmembrane conductance regulator (CFTR) regulates additional ion conductive pathways, including the amiloride-sensitive epithelial Na+ channel (ENaC; Ref. 35). CFTR appears to act as a negative modulator of ENaC activity (35), which likely explains the Na+ hyperabsorption observed in CF patients.

The human airway surface epithelium is a Na+-absorbing tissue under basal conditions. Cl- is at or near its electrochemical equilibrium across the apical membrane in both non-CF and CF epithelia (5, 41). Thus Cl- secretory agonists fail to stimulate a response in this basal, Na+ absorptive, state due to the lack of driving force for Cl- exit across the apical membrane. Therefore, current therapeutic protocols for CF consist of initially inhibiting the apical membrane Na+ conductance with amiloride. This serves to hyperpolarize the apical membrane, thereby increasing the electrochemical driving force for Cl- exit across the apical membrane. In contrast to cAMP-dependent agonists, increased intracellular Ca2+ has been shown to stimulate Cl- secretion across CF airway (40). On the basis of this information, Ca2+-dependent agonists have been proposed as therapeutically useful agents in CF therapy. In support of this approach, Mason et al. (26) demonstrated that purine and pyrimidine nucleotide triphosphates (ATP, UTP), acting at P2U (P2Y2) receptors, are capable of stimulating a Cl- secretory response in CF tracheal epithelium via an increase in intracellular Ca2+. Knowles et al. (19, 20) have demonstrated that UTP induces a hyperpolarization of nasal potential difference in both normal and CF patients that is consistent with stimulation of Cl- secretion.

It is well known that increasing intracellular Ca2+ in kidney epithelia inhibits Na+ absorption via an inhibition of apical membrane Na+ channels (ENaC; Refs. 4, 25, 31). Indeed, such an inhibition of Na+ transport was recently reported in the cortical collecting duct of rabbit in response to luminal UTP (22). Therefore, we determined whether, in addition to stimulation of Cl- secretion, a second effect of UTP in the human airway might be the inhibition of transepithelial Na+ absorption. We demonstrate that UTP, as well as bradykinin and histamine, inhibits transepithelial Na+ absorption across primary cultures of human bronchial epithelium (HBE) expressing both wild-type (wt) and Delta F508 CFTR. These results suggest that amiloride pretreatment may not be a prerequisite for mucosal UTP to have therapeutic benefit in CF.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Primary cultures of HBE. HBE was obtained from excess pathological tissue remaining after lung transplant under a protocol approved by the University of Pittsburgh Investigational Review Board. Tissue expressing wt CFTR was obtained following lung transplant for a variety of pathological conditions including emphysema, primary pulmonary hypertension, pulmonary fibrosis, and alpha 1-antitrypsin deficiency. All CF tissue employed in this study was shown to be homozygous for the Delta F508 CFTR mutation by allele-specific hybridization (performed at Genzyme, Framingham, MA). Bronchi of the second to sixth generation were dissected, rinsed thoroughly, and incubated overnight at 4°C in MEM containing 0.1% protease (type XIV; Sigma Chemical, St. Louis, MO). The epithelial cells were isolated by centrifugation and washed in MEM containing 5% fetal bovine serum (FBS). After centrifugation, the cells were resuspended in serum-free bronchial epithelial growth medium (Clonetics, San Diego, CA) and plated into type VI human placental collagen (HPC; Sigma)-coated t-25 tissue culture flasks. When the cells reached 80-90% confluence, they were trypsinized, resuspended in MEM plus 5% FBS, and seeded onto HPC-coated Costar Transwell filters (0.33 cm2) at a density of ~2 × 106 cells/cm2. After 24 h, the medium was changed to DMEM/F-12 (1:1) plus 2% Ultroser G (BioSepra, Villeneuve-la-Gavenne, France) and an air interface at the apical membrane established. The medium bathing the basolateral surface was changed every 48 h. Measurements of short-circuit current (Isc) were performed after ~10-20 additional days in culture.

Isc measurements. Costar Transwell cell culture inserts were mounted in an Ussing chamber (Jim's Instruments, Iowa City, IA), and the monolayers were continuously short-circuited via an automatic voltage clamp (Dept. of Bioengineering, University of Iowa, Iowa City, IA). Transepithelial resistance was measured by periodically applying a 5-mV pulse, and the resistance was calculated using Ohm's law. The bath solution contained (in mM) 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose. The pH of this solution was 7.4 when gassed with a mixture of 95% O2-5% CO2 at 37°C. In Cl--free solutions, all Cl- was replaced with gluconate. The effects of UTP on basolateral membrane K+ currents (IK) were assessed following permeabilization of the apical membrane with nystatin (180 µg/ml) for 15-30 min and establishment of a mucosa-to-serosa K+ concentration gradient (11). For measurements of IK, mucosal NaCl was replaced with equimolar potassium gluconate, and serosal NaCl was replaced with equimolar sodium gluconate. Cl- was removed from these solutions to prevent cell swelling that may be associated with the limited Cl- permeability of the nystatin pore. In all gluconate solutions, the CaCl2 was increased to 4 mM to compensate for the Ca2+-buffering capacity of the gluconate anion. UTP was added to the indicated side of the monolayer. Bumetanide, histamine, and bradykinin were added only to the serosal bathing solution, whereas amiloride was added only to the mucosal bathing solution. Changes in Isc were calculated as the difference in current between either the peak or sustained phase of the response and the respective baseline value.

Chemicals. Nystatin was a generous gift from Dr. S. Lucania (Bristol Meyers-Squibb). RG-80267, a generous gift of Dr. Kim Barrett (University of California, San Diego, CA), was made as a 1,000-fold stock solution in DMSO. UTP, histamine, bradykinin, bisindolylmaleimide I, calphostin C, staurosporine, thapsigargin, forskolin, phorbol 12-myristate 13-acetate (PMA), and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM were obtained from Calbiochem (La Jolla, CA). Bumetanide and apyrase were obtained from Sigma. Arachidonyl trifluoromethyl ketone (AACOCF3), palmitoyl trifluoromethyl ketone (PACOCF3), and E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (HELSS) were obtained from Biomol (Plymouth Meeting, PA) and made up as 1,000-fold stock solutions in DMSO. Charybdotoxin (CTX) was obtained from Accurate Chemical and Scientific (Westbury, NY). Forskolin was made up as a 1,000-fold stock solution in ethanol. Nystatin was made up as a 180 mg/ml stock solution in DMSO and sonicated for 30 s just before use. Cell culture media were obtained from GIBCO except as noted above.

Data analysis. All data are presented as means ± SE; n indicates the number of experiments. The percent inhibition of Na+ current was calculated for each experiment under the assumption that all of the current in the absence of Cl- (wt CFTR HBE) or in Delta F508 HBE was due to Na+ absorption, such that %inhibition = (Ibaseline - IUTP)/(Ibaseline - Iamiloride), where Ibaseline is the baseline current and IUTP and Iamiloride are the currents in presence of UTP and amiloride, respectively. Statistical analysis was performed on the raw data using Student's t-tests. A value of P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Ca2+-mediated agonists on HBE expressing wt CFTR. The hyperabsorption of Na+ across human CF airway may contribute to dehydration of airway secretions and impairment of mucociliary clearance. This has led to clinical trials designed to determine whether pharmacological inhibition of Na+ transport would be therapeutically beneficial in CF patients (14, 20, 39). Ca2+-mediated agonists are known negative modulators of Na+ absorption in both kidney (4, 25, 31) and colonic (38, 39) epithelia. Therefore, we determined the effect of Ca2+-mediated agonists on Na+ absorption in primary cultures of HBE. In a total of 25 filters expressing wt CFTR that were studied in a symmetrical NaCl bath solution, the baseline Isc averaged 26.0 ± 1.6 µA/cm2, with a transepithelial potential difference of -19.5 ± 1.5 mV and a transepithelial resistance of 888 ± 68 Omega  · cm2. The effect of mucosal UTP (100 µM) on the basal Isc response of one representative HBE filter expressing wt CFTR is shown in Fig. 1, left. We chose to use 100 µM UTP in these experiments because this concentration has been shown to be maximally effective at modulating ion transport in human nasal epithelium in vivo (19, 20). Addition of UTP to the mucosal compartment resulted in a transient increase in Isc followed by a decrease to below baseline levels. Subsequent addition of the Na+ channel blocker amiloride (10 µM) and the Na+-K+-2Cl- cotransport inhibitor bumetanide (20 µM) reduced Isc further. In six experiments, UTP induced an initial increase in Isc from 27.5 ± 4.1 to 36.2 ± 4.3 µA/cm2 (P < 0.001) followed by a sustained inhibition to 21.2 ± 2.3 µA/cm2 (P < 0.01). The subsequent addition of amiloride and bumetanide further reduced Isc to 14.0 ± 1.9 and 7.3 ± 0.9 µA/cm2, respectively. The initial increase in Isc is likely due to the stimulation of Na+ absorption, since Cl- is at or below electrochemical equilibrium across the apical membrane in human airway (41). Boucher and colleagues (5-7) have demonstrated the existence of similar driving forces for Cl- across the apical membrane of human airway in primary culture. Also, Clarke et al. (7) previously demonstrated, using microelectrode techniques, that the Ca2+-mediated agonist bradykinin induces an initial transient increase in Na+ absorption across human airway, consistent with our results. However, as we have not directly measured the electrochemical driving force for Cl- across the apical membrane of our cultures, we cannot rule out the possibility that a portion of this initial increase is due to Cl- secretion. Our results suggest that, following the transient activation of Na+ absorption, the effect of UTP is inhibitory in nature. This was confirmed by comparing the amiloride-sensitive Na+ current (INa-amil) in the absence or presence of mucosal UTP (Fig. 1, right) on monolayers cultured from a single patient and run on the same day. In 19 filters, amiloride reduced Isc by 12.5 ± 1.4 µA/cm2. In contrast, following mucosal UTP, amiloride reduced Isc by only 6.2 ± 0.5 µA/cm2 (n = 6; P < 0.01). This result is consistent with an inhibition of active Na+ absorption by mucosal UTP.


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Fig. 1.   Effect of mucosal UTP (100 µM) on amiloride-blockable Na+ current (INa-amil) in human bronchial epithelia (HBE) expressing wild-type (wt) cystic fibrosis transmembrane conductance regulator (CFTR). Left: UTP induced a transient increase in short-circuit current (Isc) followed by a sustained decrease. Subsequent addition of amiloride (10 µM) and bumetanide (20 µM; serosal addition) further reduced Isc. Dashed line, zero-current level. Right: average INa-amil in absence [control (Cont)] or presence of UTP. INa-amil was determined in separate experiments before addition of UTP (control) or after stimulation by mucosal UTP. No. of experiments is indicated in parentheses. * P < 0.01. All experiments were carried out in presence of mucosal and serosal NaCl (see METHODS).

Although the above results suggest that UTP inhibits Na+ transport across airway epithelia, the interpretation of Isc changes in symmetrical NaCl is not straightforward. Any inhibition of apical Na+ channels would be expected to hyperpolarize the apical membrane, resulting in the establishment of a favorable electrochemical gradient for Cl- secretion. Because UTP is known to activate a Ca2+-dependent Cl- channel, resulting in a stimulation of Cl- secretion (26), this will confound attempts to isolate the effects of UTP on Na+ absorption. To overcome this, we performed similar experiments in the absence of apical and basolateral Cl- to study the Na+ absorptive process in isolation. As shown in Fig. 2, left, addition of mucosal UTP (100 µM) produced a response similar to that seen in the presence of Cl-, i.e., a transient increase in Na+ absorption and then a dramatic inhibition. Amiloride caused a further reduction in the Isc measured in the absence of mucosal and serosal Cl- (INa). To confirm that the reduction in Isc was truly a reduction in INa-amil, we inhibited the basal Isc with amiloride in the absence of UTP (Fig. 2, right). As is apparent, the postamiloride current level is similar in the two monolayers, indicating that changes in Isc reflect changes in INa-amil. In 15 HBE monolayers, the basal INa averaged 13.9 ± 1.2 µA/cm2. Mucosal UTP (100 µM) induced a transient increase in INa to 16.9 ± 1.1 µA/cm2 (P < 0.001) that was followed by a decline to 5.0 ± 0.5 µA/cm2. The subsequent addition of amiloride (10 µM) further reduced INa to 2.7 ± 0.3 µA/cm2. In seven monolayers from the same HBE culture, the baseline INa averaged 11.8 ± 1.0 µA/cm2, and this was reduced to 3.0 ± 0.5 µA/cm2 by amiloride (10 µM). These results demonstrate that mucosal UTP inhibits 76 ± 4% of the INa-amil. Also, these results confirm that UTP initially increases electrogenic Na+ transport.


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Fig. 2.   Effect of mucosal UTP (100 µM) on Isc in absence of mucosal and serosal Cl- (see METHODS) in HBE expressing wt CFTR. This current is referred to as INa. Left: mucosal UTP induced a transient increase in INa followed by a sustained inhibition. Subsequent addition of amiloride (10 µM) further reduced INa. Right: effect of amiloride (10 µM) on INa. Dashed line, zero-current level.

The above results demonstrate that mucosal UTP inhibits transepithelial Na+ transport in human airway. We next determined whether this effect was unique to mucosal UTP or whether UTP added to the serosal membrane would have a similar effect. The results of one representative experiment are shown in Fig. 3, top left. In the absence of mucosal and serosal Cl-, addition of UTP to the serosal membrane induced a response that was qualitatively similar to that seen with mucosal UTP, i.e., a transient increase in Na+ absorption followed by a dramatic inhibition. In six experiments, the baseline INa averaged 23.2 ± 3.7 µA/cm2, and this increased to a peak of 31.8 ± 3.9 µA/cm2 (P < 0.001) following stimulation with serosal UTP. This increase was followed by a decrease in INa to 6.6 ± 0.7 µA/cm2. After administration of serosal UTP, amiloride further reduced INa to 1.5 ± 0.2 µA/cm2. Thus serosal UTP inhibited INa by an average of 74 ± 5%. These results indicate that the inhibition of Na+ transport observed is not restricted to mucosal UTP.


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Fig. 3.   Effect of Ca2+-mediated agonists on INa in HBE expressing wt CFTR. Addition of UTP (100 µM; top left), histamine (30 µM; top right), or bradykinin (100 nM; bottom left) to serosal membrane of HBE expressing wt CFTR resulted in a transient stimulation of Na+ transport followed by a sustained inhibition. Ca2+-ATPase inhibitor thapsigargin similarly transiently increased INa, followed by a sustained inhibition (bottom right). In each case, subsequent addition of amiloride (10 µM) further reduced INa. Dashed line, zero-current level. All experiments were carried out in absence of mucosal and serosal Cl-.

We next determined whether other Ca2+-dependent agonists would induce a similar inhibition of Na+ transport. The effects of two inflammatory mediators, histamine and bradykinin, were evaluated. Both of these agonists have previously been shown to induce a Ca2+-dependent Cl- secretory response in human airway following inhibition of Na+ transport with amiloride (6, 7, 43-45). Both histamine (30 µM; Fig. 3, top right) and bradykinin (100 nM; Fig. 3, bottom left) induced an increase in INa followed by an inhibition, similar to what is observed following stimulation with UTP. In three filters, histamine increased INa from 19.1 ± 1.5 to 29.7 ± 1.6 µA/cm2 (P < 0.01) before a sustained inhibition to 8.2 ± 1.1 µA/cm2. The subsequent addition of amiloride further reduced INa to 1.5 ± 0.4 µA/cm2, demonstrating that histamine inhibited 62 ± 4% of the INa-amil. Similarly, bradykinin increased INa from 30.9 ± 1.5 to 43.2 ± 1.8 µA/cm2 (P < 0.01) before a sustained inhibition to 12.0 ± 1.5 µA/cm2 (n = 3). Amiloride subsequently reduced INa to 2.2 ± 0.5 µA/cm2, demonstrating that bradykinin inhibits the INa-amil by 65 ± 3%. These results indicate that the inhibition of Na+ transport is not unique to purinergic agonists. Rather, they suggest that any increase in intracellular Ca2+ will inhibit Na+ transport. This was confirmed by utilizing the Ca2+-ATPase inhibitor thapsigargin (1 µM) to elevate intracellular Ca2+. Thapsigargin initially increased INa and then caused a marked inhibition (Fig. 3, bottom right). In six experiments, thapsigargin increased INa from 25.2 ± 5.1 to 26.0 ± 5.1 µA/cm2 (P < 0.05) followed by a sustained inhibition to 3.1 ± 1.3 µA/cm2. Amiloride further reduced INa to 0.0 ± 1.3 µA/cm2, indicating an 84 ± 3% inhibition of the INa-amil.

Effect of UTP on HBE homozygous for the Delta F508 CFTR mutation. The above results demonstrate that UTP inhibits Na+ transport in human airway expressing wt CFTR. We next determined whether UTP would similarly inhibit Na+ transport in HBE expressing the Delta F508 CFTR mutation. We evaluated a total of 60 filters using symmetrical NaCl bath solutions. The basal Isc, transepithelial potential difference, and transepithelial resistance averaged 30.1 ± 1.4 µA/cm2, -15.2 ± 0.9 mV, and 539 ± 24 Omega  · cm2, respectively. In contrast to wt CFTR-expressing HBE, for which ~50% of this basal Isc is sensitive to block by amiloride, nearly 90% of the basal current of Delta F508 CFTR-expressing HBE is amiloride sensitive (unpublished observations). Thus our primary HBE cultures exhibit the characteristic hyperabsorption of Na+ associated with CF. The effect of mucosal UTP (100 µM) on Isc in one representative monolayer expressing Delta F508 CFTR is shown in Fig. 4, left. UTP induced a response qualitatively identical to that observed in wt CFTR-expressing HBE. That is, UTP induced an initial increase in Isc followed by a sustained inhibition. Addition of amiloride (10 µM) further reduced Isc. In eight experiments, UTP increased Isc from 38.2 ± 5.5 to 46.4 ± 3.7 µA/cm2 (P < 0.01) followed by a decline to 14.0 ± 3.0 µA/cm2. The subsequent addition of amiloride further reduced Isc to 5.2 ± 1.3 µA/cm2. Addition of bumetanide had no effect on the small remaining Isc, suggesting that the inhibition of Isc is unrelated to any Cl- secretory current evoked by UTP. If it is assumed that Isc is a purely Na+ absorptive current, mucosal UTP inhibits 75 ± 2% of the amiloride-blockable current. This is similar to the results obtained in wt CFTR-expressing HBE. These results suggest that mucosal UTP inhibits transepithelial Na+ absorption in human CF airway. To confirm this observation, we evaluated the effect of mucosal UTP (100 µM) on Na+ transport across CF airway in the absence of mucosal and serosal Cl-. The results of one representative experiment are shown in Fig. 4, right. Similar to our previous results, UTP induced an initial increase in INa that was followed by a sustained inhibition. This initial transient response to UTP appears to have a shorter duration in CF HBE than in wt CFTR-expressing HBE (compare Figs. 1 and 2 with Figs. 4, 7, and 8), although this was not further analyzed. The subsequent addition of amiloride further reduced INa. In 12 experiments, UTP increased INa from 12.0 ± 0.9 to 15.2 ± 1.2 µA/cm2 (P < 0.001) followed by a sustained decrease to 3.4 ± 0.8 µA/cm2. Amiloride further reduced INa to 1.1 ± 0.3 µA/cm2, indicating that UTP inhibited 80 ± 4% of the amiloride-blockable current.


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Fig. 4.   Effect of mucosal UTP (100 µM) on Na+ transport in HBE homozygous for Delta F508 mutation either in presence (left) or absence (right) of mucosal and serosal NaCl. Under both conditions, mucosal UTP induced a transient increase in Na+ transport followed by a sustained inhibition. Subsequent addition of amiloride (10 µM) further reduced Na+ current. Dashed line, zero-current level.

The demonstration of similar changes in INa in the absence of mucosal and serosal Cl- indicates that our primary CF airway cultures can be studied using a standard NaCl bath solution on both the serosal and mucosal membranes. Because this provides more stable baseline currents, the remainder of our studies on CF epithelia were carried out using standard bath solutions (120 NaCl). To determine the half-maximal inhibitory concentration (Ki) for UTP on Na+ transport in CF airway, each filter was challenged with one concentration of UTP and then with amiloride, to avoid difficulties associated with downregulation of the purinergic receptor. The data were fitted to a Michaelis-Menten function with an apparent Ki of 296 nM (Fig. 5).


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Fig. 5.   Concentration-response curve for UTP-dependent inhibition of Na+ transport in Delta F508/Delta F508 HBE. Na+ current after UTP stimulation (INa-UTP) minus Na+ current before UTP stimulation (INa-Cont) was normalized to INa-Cont minus INa-amil (10 µM amiloride). Data (means ± SE of 4 or 5 individual experiments) were fitted to a Michaelis-Menten function with an apparent half-maximal inhibitory concentration of 296 nM. Experiments were carried out in presence of mucosal and serosal NaCl. C, control (0 UTP).

We also determined whether serosal UTP and other Ca2+-mediated agonists would inhibit Na+ absorption across CF airway in a manner similar to that of mucosal UTP. In four experiments, serosal UTP (100 µM) increased INa from 33.4 ± 2.1 to 38.6 ± 2.2 µA/cm2 (P < 0.001) before a sustained inhibition to 11.3 ± 0.9 µA/cm2. The subsequent addition of amiloride further reduced INa to 2.5 ± 0.2 µA/cm2, indicating that serosal UTP inhibits 72 ± 1% of the Na+ current across human CF airway. We next determined whether an additional Ca2+-mediated agonist, bradykinin (100 nM), would similarly inhibit Na+ transport in Delta F508 CFTR HBE. In three filters, bradykinin increased INa from 66.3 ± 14.4 to 72.1 ± 13.9 µA/cm2 (P < 0.01) before a sustained inhibition to 25.6 ± 8.1 µA/cm2. Amiloride further reduced INa to 5.5 ± 1.0 µA/cm2, indicating that bradykinin inhibits 69 ± 4% of the Na+ current across Delta F508 CFTR-expressing HBE. These results indicate that, similar to the situation in wt CFTR-expressing airway, the inhibition of Na+ transport is not restricted to mucosal UTP. They suggest instead that Ca2+-mediated agonists in general are capable of inhibiting Na+ transport across human CF airway.

In summary, our results indicate that an increase in intracellular Ca2+ underlies the inhibition of Na+ current observed. To evaluate this possibility further, we incubated the HBE monolayers in the cell-permeant Ca2+ chelator BAPTA-AM (50 µM for 1 h) before determining the effect of mucosal UTP on Na+ transport. In 11 monolayers, the baseline Isc averaged 35.2 ± 1.6 µA/cm2. Mucosal UTP (100 µM) induced only a small increase in Isc to 36.5 ± 1.8 µA/cm2 (P < 0.02) followed by a sustained inhibition to 24.8 ± 1.4 µA/cm2. The subsequent addition of amiloride further reduced Isc to 2.4 ± 0.3 µA/cm2. Thus mucosal UTP inhibited only 31 ± 3% of the amiloride-blockable current in the presence of BAPTA-AM. Because the total amiloride-sensitive Isc was not different in the absence (33.0 ± 4.3 µA/cm2, n = 8) or presence (32.8 ± 1.5 µA/cm2, n = 11) of BAPTA-AM, we directly compared the UTP-dependent portion of these currents (control, 24.3 ± 2.6 µA/cm2; BAPTA-AM, 10.3 ± 1.3 µA/cm2). These results demonstrate that incubating the HBE monolayers in BAPTA-AM significantly attenuated the UTP-dependent inhibition of Na+ absorption (P < 0.0001). It should be noted that BAPTA-AM also significantly attenuated the initial increase in Isc induced by mucosal UTP (P < 0.01), consistent with this being due to stimulation of Na+ transport via a Ca2+-mediated process.

Effects of second messenger modulators on the UTP-dependent inhibition of Na+ transport. Our results with thapsigargin suggest that an increase in Ca2+ alone is sufficient to inhibit Na+ transport. However, a second possibility is that the increase in intracellular Ca2+ by UTP increases additional second messengers that may be important in the observed inhibition of Na+ transport. Boucher and colleagues previously demonstrated that UTP induces an increase in both protein kinase C (PKC) (3) and arachidonic acid (23) in human airway epithelia, and these second messengers are known to modulate Na+ transport in kidney epithelia (12, 14). Therefore, we evaluated the effects of inhibitors of PKC and phospholipase A2 (PLA2) on the UTP-dependent inhibition of Na+ transport in wt CFTR-expressing HBE in the absence of Cl- and Delta F508 CFTR-expressing HBE in standard NaCl bath solutions. In the presence of the cytosolic PLA2 inhibitor AACOCF3 (100 µM; 15-min pretreatment), mucosal UTP inhibited 76 ± 2% (n = 4) of the INa-amil in Delta F508 CFTR-expressing HBE. Similarly, in the presence of a different cytosolic PLA2 inhibitor, PACOCF3 (100 µM; 15-min pretreatment), and a secreted PLA2 inhibitor, HELSS (2 µM; 15-min pretreatment), UTP inhibited 77 (n = 2) and 90 ± 1% (n = 3) of INa, respectively. An alternate means of generating arachidonic acid is via the conversion of diacylglycerol (DAG) via DAG lipase. Mucosal UTP inhibited 74% of the INa-amil in the presence of the DAG lipase inhibitor RG-80267 (50 µM; 15-min pretreatment; n = 2). These results suggest that arachidonic acid does not mediate the UTP-dependent inhibition of Na+ transport observed.

The effects of the PKC inhibitors staurosporine (100 nM), bisindolylmaleimide I (200 nM), and myristolated PKC (amino acids 19-27 fragment; 10 µM) were evaluated in wt CFTR-expressing HBE. After incubation of the monolayers in staurosporine (15 min), bisindolylmaleimide I (15 min), or myristolated PKC (25 min), mucosal UTP inhibited 84 ± 3 (n = 3), 79 ± 4 (n = 3), and 71 ± 6% (n = 4) of INa, respectively. The effects of the PKC inhibitors bisindolylmaleimide I (1 µM) and calphostin C (2 µM) were also evaluated in Delta F508 CFTR-expressing HBE. After incubation of the monolayers in bisindolylmaleimide I (15 min) or calphostin C (15 min), mucosal UTP inhibited 72 ± 1 (n = 3) and 77 ± 1% (n = 3) of the INa-amil, respectively. These results suggest that PKC is not involved in the UTP-dependent inhibition of Na+ transport across HBE.

Our results with the PKC inhibitors were somewhat surprising, since Eaton and colleagues (21, 25) previously demonstrated that the Ca2+-dependent inhibition of Na+ transport in kidney epithelia is dependent on PKC. Therefore, we directly evaluated the effects of the PKC agonist PMA on Na+ transport across HBE expressing Delta F508 CFTR. The results of one experiment are shown in Fig. 6, left. PMA (100 nM) had no effect on Isc, whereas the subsequent addition of amiloride inhibited Isc as expected. In six monolayers, the baseline Isc averaged 29.0 ± 2.6 µA/cm2; this was unaffected by PMA, whereas amiloride reduced Isc to 7.8 ± 1.6 µA/cm2. As a positive control, we evaluated the effect of PMA on Cl- secretion after amiloride in wt CFTR-expressing HBE (Fig. 6, right). Hanrahan and colleagues (36) previously demonstrated that PKC activates CFTR in excised patches, and we have demonstrated that PMA stimulates Cl- secretion across T84 cells (9). After inhibition of Na+ absorption by amiloride, PMA (100 nM) stimulated a bumetanide-sensitive Cl- secretory response, as expected. In six experiments, amiloride reduced Isc from 38.4 ± 3.0 to 11.9 ± 2.1 µA/cm2. Addition of PMA increased Isc an average of 3.8 ± 0.7 µA/cm2 (P < 0.01). These results demonstrate that direct activation of PKC has no effect on Na+ transport across HBE and confirm our PKC inhibitor studies described above.


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Fig. 6.   Effect of protein kinase C activator phorbol 12-myristate 13-acetate (PMA; 100 nM) on Na+ absorption across Delta F508 CFTR-expressing HBE (left) and Cl- secretion across wt CFTR-expressing HBE (right). Addition of PMA had no effect on Na+ absorption across Delta F508 CFTR HBE, whereas subsequent addition of amiloride (10 µM) inhibited Isc as expected (left). After inhibition of Na+ absorption with amiloride (10 µM), PMA stimulated a bumetanide (Bumet)-sensitive increase in Cl- secretion across HBE expressing wt CFTR (right).

Because Ca2+-dependent agonists often modulate tyrosine kinase activity, we determined whether the protein tyrosine kinase inhibitors genistein (50 µM) and lavendustin A (5 µM) would modulate the effect of mucosal UTP on Na+ transport in HBE expressing Delta F508 CFTR. Genistein induced a sustained increase in Na+ transport, as shown in Fig. 7. After genistein, mucosal UTP increased Na+ transport before a sustained inhibition. Addition of amiloride further reduced Na+ absorption. In seven filters, genistein increased Isc from 32.9 ± 4.6 to 43.0 ± 5.8 µA/cm2 (P < 0.001). The subsequent addition of mucosal UTP decreased Isc to 16.4 ± 1.7 µA/cm2. Amiloride further reduced Isc to 3.4 ± 1.0 µA/cm2, demonstrating that UTP inhibited 56 ± 5% of the amiloride-dependent Na+ transport. This large increase in Isc is unlikely to be due to a genistein-induced Cl- secretory response, since we have demonstrated that genistein increases Cl- current by only ~2 µA/cm2 in Delta F508 CFTR-expressing HBE after amiloride (10). The presence of amiloride would enhance the electrochemical driving force for Cl- secretion in the experiments of Ref. 10 relative to those reported here. In four additional experiments, the effect of lavendustin A on Na+ transport was evaluated. Lavendustin A had no effect on Isc, although the subsequent addition of genistein further increased Isc from 26.6 ± 2.9 to 37.0 ± 4.8 µA/cm2 (P < 0.01). These results suggest that the effect of genistein on Na+ absorption is independent of tyrosine kinase inhibition and further indicate that the UTP-dependent inhibition of Na+ transport is not due to the activation of a tyrosine kinase.


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Fig. 7.   Effect of genistein on Na+ current in Delta F508/Delta F508 HBE. Addition of genistein (50 µM; mucosal and serosal addition) induced a sustained increase in Na+ current. Subsequent addition of mucosal UTP (100 µM) induced a transient increase in Na+ current followed by a sustained inhibition. Amiloride (10 µM) further reduced Na+ current. Dashed line, zero-current level.

An additional possibility is that the UTP-dependent increase in intracellular Ca2+ inhibits adenylyl cyclase in the cell. Increased cAMP is well known to activate Na+ transport, and increased Ca2+ potently downregulates adenylyl cyclase (8). Therefore, we determined whether exogenous cAMP could either prevent or reverse the effect of mucosal UTP on Na+ transport in Delta F508 CFTR-expressing HBE. We utilized the cell-permeant cAMP analog 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) to bypass the adenylyl cyclase. The results of one representative experiment are shown in Fig. 8. Addition of CPT-cAMP (500 µM) stimulated a small increase in Na+ transport. The subsequent addition of mucosal UTP (100 µM) resulted in an increase in Na+ transport followed by a sustained inhibition. Addition of amiloride further reduced Isc. In six filters, CPT-cAMP increased Isc from 32.5 ± 6.7 to 38.8 ± 6.8 µA/cm2 (P < 0.001). Mucosal UTP increased Isc to 44.6 ± 8.2 µA/cm2 (P < 0.05) before a sustained inhibition to 19.5 ± 3.6 µA/cm2. The addition of amiloride further reduced Isc to 4.4 ± 0.4 µA/cm2; thus UTP inhibited 48 ± 6% of the amiloride-sensitive current. This demonstrates that cAMP stimulates Na+ transport in CF airway and suggests that exogenous cAMP partially prevents the effects of UTP on Na+ transport.


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Fig. 8.   Effect of CPT-cAMP and apyrase on Na+ current in Delta F508/Delta F508 HBE. CPT-cAMP (500 µM) induced a sustained increase in Na+ current. Addition of mucosal UTP (100 µM) induced a further transient increase followed by a sustained decrease in Na+ current. Addition of tri- and diphosphatase (apyrase; 5 U/ml; mucosal addition) failed to induce a recovery of Na+ current. Amiloride (10 µM) further reduced Na+ current. Dashed line, zero-current level.

To determine whether cAMP could reverse the effect of UTP on Na+ transport, the monolayer was first challenged with mucosal UTP, and then CPT-cAMP was added during the sustained inhibitory phase. UTP increased Isc from 20.7 ± 0.8 to 30.4 ± 1.2 µA/cm2 (P < 0.001) before a sustained inhibition to 6.7 ± 0.4 µA/cm2 (n = 4). Addition of CPT-cAMP increased Isc to 11.0 ± 1.1 µA/cm2 (P < 0.05), and this was inhibited by amiloride (4.1 ± 0.9 µA/cm2). Bumetanide reduced Isc to only 3.2 ± 1.0 µA/cm2, confirming that CPT-cAMP was not increasing Cl- secretion across the CF airway. These results demonstrate that cAMP partially reverses the inhibitory effect of mucosal UTP.

Effect of apyrase on the UTP-dependent inhibition of Na+ transport. We next determined whether the inhibitory effect of UTP was dependent on the continued presence of the agonist. To accomplish this, apyrase, which cleaves both tri- and diphosphate nucleotides, thus removing UTP from the mucosal solution, was added during the sustained inhibitory phase of the UTP response. As shown in Fig. 8, apyrase failed to induce a recovery of current; it increased Isc by only 1.1 ± 0.5 µA/cm2 (n = 4). In the absence of CPT-cAMP, mucosal UTP induced a sustained inhibition of Na+ transport from 31.8 ± 6.8 to 9.2 ± 2.6 µA/cm2, and subsequent addition of apyrase increased Isc slightly to 13.0 ± 3.3 µA/cm2 (n = 7; P < 0.05). Amiloride reduced Isc to 2.5 ± 0.9 µA/cm2. To ensure that apyrase was removing UTP from the mucosal solution, apyrase was added before UTP in parallel filters. In the presence of apyrase, mucosal UTP had no effect on Na+ transport, but the subsequent addition of serosal UTP inhibited Na+ transport as described above (data not shown). Collectively, these results indicate either that mucosal UTP generates an inhibitory second messenger that remains active despite the removal of the agonist or that Na+ channels have been removed from the membrane and are no longer available for activation despite the removal of UTP.

Effect of mucosal UTP on IK. Modulation of Na+ transport across epithelia is typically associated with parallel changes in apical and basolateral membrane conductances. Therefore, we determined the effect of mucosal UTP on the IK after establishment of a mucosa-to-serosa K+ concentration gradient and permeabilization of the mucosal membrane with nystatin (see Isc measurements). As shown in Fig. 9, after inhibition of apical Na+ channels with amiloride, permeabilization of the mucosal membrane with nystatin revealed a K+ current (IK) across the basolateral membrane. The subsequent addition of mucosal UTP induced an increase in IK followed by a sustained inhibition. Addition of Ba2+ (10 mM) further inhibited IK. In a total of 17 filters, nystatin increased IK from 2.8 ± 0.9 to 58.6 ± 5.8 µA/cm2. In 13 of these filters, the subsequent addition of mucosal UTP increased IK to 79.8 ± 8.9 µA/cm2 (P < 0.001) before a sustained decrease to 40.0 ± 3.9 µA/cm2 (P < 0.001). As expected for a K+ conductance, in nine of these filters, addition of Ba2+ further reduced IK to 18.6 ± 3.4 µA/cm2 (P < 0.001); quinine similarly inhibited IK (data not shown). These results suggest that the basal IK represents the K+ conductance associated with Na+ absorption across human airway epithelia. In support of this, both Ba2+ and quinine inhibit Na+ absorption across CF HBE (unpublished observations). To determine whether the increase in IK is due to activation of a Ca2+-dependent K+ channel, we evaluated the effect of CTX (50 nM) on the initial increase in IK induced by mucosal UTP in the additional four filters. CTX completely inhibited the transient increase in IK (n = 4), indicating that this increase is due to the activation of a Ca2+-dependent K+ channel.


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Fig. 9.   Effect of mucosal UTP on basolateral membrane K+ current (IK) in Delta F508/Delta F508 HBE. After establishment of a mucosa-to-serosa K+ gradient, inhibition of Na+ transport with amiloride (10 µM), and permeabilization of mucosal membrane with nystatin (Nyst; see METHODS), mucosal UTP (100 µM) induced a transient increase in IK followed by an inhibition. Subsequent addition of Ba2+ (10 mM; serosal addition) further inhibited IK. Dashed line, zero-current level.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Both a defective cAMP-dependent Cl- secretion and a hyperabsorption of Na+ across airway epithelia characterize CF. However, Ca2+-dependent Cl- secretion remains intact across CF airway epithelia (40); this has led to the evaluation of Ca2+-dependent agonists as potential therapeutics in CF (19, 20). Because Cl- has been shown to be at electrochemical equilibrium across the apical membrane of airway epithelia (41), clinical trials have been conducted in the presence of the Na+ channel blocker amiloride to hyperpolarize the apical membrane and thereby increase the driving force for Cl- secretion. However, Ca2+-dependent agonists have previously been shown to inhibit Na+ absorption in other epithelia, including kidney (4, 21, 22, 24, 25, 31) and colon (39). We therefore speculated that Ca2+-mediated agonists would similarly inhibit transepithelial Na+ absorption across human airway epithelia, obviating the requirement for exogenous Na+ channel inhibition. Indeed, if Ca2+-mediated agonists are in fact Cl- secretagogues, then inhibition of Na+ absorption, and the resultant hyperpolarization of the apical membrane, would be a prerequisite to inducing Cl- secretion by these agonists. We demonstrate that Ca2+-dependent agonists are capable of dramatically inhibiting Na+ transport across HBE and that this occurs in both wt and Delta F508 CFTR-expressing monolayers.

The role of basolateral membrane K+ channels in Na+ transport. We demonstrate that Ca2+-mediated agonists initially stimulate Na+ absorption across human airway before a sustained inhibition. Although we have been unable to electrically isolate the apical membrane Na+ conductance, we have succeeded in obtaining information with regard to the regulation of the basolateral membrane K+ conductances involved in modulating Na+ absorption. We hypothesize that the initial increase in Na+ absorption is due to the activation of a Ca2+-dependent basolateral membrane K+ channel that results in a hyperpolarization and increased driving force for Na+ entry across the apical membrane. Incubation of our cultures in the cell-permeant Ca2+ chelator BAPTA-AM resulted in an attenuation of this initial increase in Na+ absorption, consistent with our hypothesis. Also, addition of agonists (UTP, histamine, or bradykinin) to the serosal membrane resulted in a greater increase in Isc than did mucosal UTP. Clarke et al. (5-7) demonstrated, using microelectrode techniques, that Ca2+-dependent agonists activated a basolateral K+ conductance that results in a hyperpolarization of human nasal epithelium. Indeed, activation of this basolateral K+ conductance was greater when agonist was added to the serosal membrane than when it was added to the mucosal side (5). Finally, in permeabilized monolayers, we demonstrate that mucosal UTP activates a basolateral membrane, CTX-sensitive K+ conductance (Fig. 9). We (10) and others (28) previously characterized this CTX-sensitive K+ channel in human airway epithelia. We conclude that activation of this basolateral membrane K+ conductance results in the initial stimulation of Na+ absorption across human airway.

Apical Na+ entry is considered the rate-limiting step in Na+ absorption. Abundant experimental data employing kidney and colonic epithelia demonstrate that increasing cellular Ca2+ results in an inhibition of Na+ conductance (4, 14, 21, 22, 24, 25, 31, 39). Thus, although there is little doubt that inhibition of Na+ conductance is important in the observed UTP-dependent inhibition of Isc, we demonstrate that mucosal UTP also inhibits a basolateral IK (Fig. 9). Indeed, inhibition of this K+ conductance is sufficient to account for a large portion of the observed inhibition of Na+ absorption. Unfortunately, the nature of this inhibited basolateral K+ channel is not known at the single-channel level. Dawson and colleagues (38, 39) demonstrated previously that the Ca2+-dependent agonist carbachol inhibited Na+ absorption across the turtle colon. Similar to our results, these authors (38) demonstrated that carbachol induced a profound inhibition of the basolateral IK in turtle colon. In addition to this effect on the K+ conductance, it was also demonstrated that carbachol inhibited the apical membrane Na+ conductance in these cells (39), consistent with the concept of cross talk between apical and basolateral membranes. Also, Harvey (14) demonstrated that the basolateral ATP-sensitive K+ channel of frog principal cells is inhibited by Ca2+ with an affinity similar to inhibition of the Na+ channel, suggesting a parallel downregulation of both membranes. Thus it is impossible to separate the relative contributions of these two membranes to the overall inhibitory response observed because both are regulated in parallel to maintain cellular homeostasis. A complete understanding of the Ca2+-dependent inhibition of Na+ transport across human airway will require an analysis of this basolateral membrane K+ conductance at the single-channel level, including its regulation by Ca2+.

Second messenger regulation of Na+ transport across human airway. Although Ca2+-dependent agonists are well-known negative modulators of Na+ absorption in the kidney, this inhibition appears to be indirect, involving an activation of PKC (21, 25). In addition, other second messengers associated with Ca2+-dependent agonists (e.g., oxidative metabolites of arachidonic acid) have been shown to modulate ENaC activity during patch-clamp recordings (12). Boucher and colleagues (3, 23) previously demonstrated that luminal purinergic agonists increase intracellular Ca2+, PKC, and arachidonic acid in human airway epithelia. In contrast to previous reports from kidney epithelia (12, 21, 25), we demonstrate that inhibitors of PKC and PLA2 have no effect on the UTP-dependent inhibition of Na+ transport across HBE. Indeed, we demonstrate that the direct activation of PKC by PMA has no effect on Na+ transport (Fig. 6). Together, these results argue against a role for PKC in the observed inhibition of Na+ transport by UTP. We also demonstrate that the Ca2+-ATPase inhibitor thapsigargin similarly inhibits Na+ transport across HBE. Thapsigargin fails to induce the accumulation of inositol phosphates in both human airway (23) and colonic (18) epithelia, further suggesting that PKC accumulation is not involved in the observed inhibitory response. Finally, we demonstrate that incubation of HBE in the cell-permeant Ca2+ chelator BAPTA-AM results in a significant attenuation of the UTP-dependent inhibition of Na+ transport. Similar to our results, Yamaya et al. (45, 46) were unable to completely suppress the response in HBE to Ca2+-mediated agonists in the presence of BAPTA-AM. On the basis of our results with both thapsigargin and BAPTA-AM, we speculate that Ca2+ itself may act as a negative modulator of Na+ transport across human airway. In this regard, Rotin and colleagues (17) recently demonstrated that ENaC, heterologously expressed in MDCK cells, could be directly inhibited by Ca2+ in excised, inside-out patches. However, previous reports demonstrated that increasing Ca2+ at the cytoplasmic face of ENaC excised from rat and rabbit cortical collecting tubule cells does not affect the channel open probability (14, 25). Thus the mechanism for Ca2+ regulation of ENaC remains to be determined.

Our results demonstrate that UTP induces a long-term inhibition of Na+ transport that is independent of the continued presence of agonist (Fig. 8). One explanation for these results is that UTP generates a second messenger that has long-term inhibitory effects on Na+ transport. Barrett and colleagues (37) recently demonstrated, in the T84 cell line, that the generation of inositol 3,4,5,6-tetrakisphosphate by muscarinic agonists produces a long-term inhibitory effect on Cl- secretion. An additional possibility is that increased cellular Ca2+ causes the retrieval of Na+ channels from the apical membrane and thus long-term inhibition. Wilkinson and Dawson (39) concluded, employing fluctuation analysis, that increased Ca2+ resulted in a decrease in apical Na+ channel density in turtle colon. Recently, Rotin and colleagues (32-34) characterized the ubiquitin-ligase Nedd4 as an important negative modulator of ENaC. Nedd4 was shown to translocate to the apical membrane of MDCK cells following an increase in cellular Ca2+. These authors speculated that Nedd4 may be important in the endocytosis of ENaC from the apical membrane of epithelia. Interestingly, an increase in cAMP was able to partially reverse the effect of UTP on Na+ absorption (Fig. 8). cAMP is known to increase apical membrane Na+ channel density (12). Further studies are required to directly determine whether UTP modulates Na+ channel density, open probability, or both in HBE and the potential role of Nedd4 in this process.

We also evaluated the effect of the tyrosine kinase inhibitor genistein on the UTP-dependent inhibition of Na+ transport. Surprisingly, genistein alone stimulated Na+ transport across human CF airway (Fig. 7). In contrast, the structurally unrelated tyrosine kinase inhibitor lavendustin A failed to modulate Na+ transport, suggesting that the effect of genistein is unrelated to its known tyrosine kinase inhibitory activity. Despite this stimulation of Na+ absorption, genistein failed to modulate the inhibitory effect of UTP. Our results with genistein are opposite to what has been reported in A6 kidney epithelia. Matsumoto et al. (27) demonstrated that genistein inhibited Na+ transport across A6 cells, suggesting that a tonic tyrosine kinase activity modulated Na+ transport.

In conclusion, we demonstrate that Ca2+-mediated agonists dramatically inhibit Na+ transport across human airway epithelia expressing both wt and Delta F508 CFTR. Indeed, the Ki for UTP-dependent inhibition of Na+ absorption is similar to the half-maximal stimulatory concentration previously reported for the stimulation of Cl- secretion in primary HBE cultures (46). This inhibition of Na+ transport is directly dependent on a rise in intracellular Ca2+, but it is independent of activation of either the PKC or PLA2 second messenger cascades. These results may help explain the observation that UTP increased mucociliary clearance in normal volunteers and that this effect was not potentiated by amiloride (30). However, it should be noted that in CF patients a combination of amiloride plus UTP was required to increase mucociliary clearance (1), although, as indicated by the authors of that study, a greater number of subjects need to be studied to rule out effects of either agent alone. We also demonstrate that genistein stimulates Na+ absorption across CF epithelia. Because genistein and its analogs have been shown to stimulate Cl- secretion across epithelia (15, 29) and are being evaluated as Cl- secretory agonists in human nasal epithelia (13, 16), this potentially confounding stimulation of Na+ absorption warrants further investigation. On the basis of these results, we predict that amiloride may not be required in combination with Ca2+-mediated agonists to stimulate Cl- secretion across human CF airway. Ca2+-mediated agonists have a dual therapeutic role in CF: 1) inhibition of Na+ absorption and 2) stimulation of Cl- secretion.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the excellent secretarial skills of Michele Dobransky, the technical assistance of Cheng Zhang Shi and Joe Latoche in both tissue culture and Ussing chamber experiments, the assistance of Drs. Jan Manzetti and Robert Keenan (University of Pittsburgh Medical Center lung transplant program) in obtaining human lung tissue, and the help of Mark Gerardi (Genzyme) in facilitating genotype analysis.


    FOOTNOTES

This work was supported by Cystic Fibrosis Foundation Grants DEVOR960 and Q933 and a Cystic Fibrosis Research Development Program Center grant.

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: D. C. Devor, Dept. of Cell Biology and Physiology, S312 BST, 3500 Terrace St., University of Pittsburgh, Pittsburgh, PA 15261 (E-mail: dd2+{at}pitt.edu).

Received 13 October 1998; accepted in final form 13 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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