Introduction

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Two prevailing alterations of ion transport have been well characterized in cystic fibrosis (CF) lung disease: a defect in cyclic adenosine monophosphate-dependent Cl secretion, and enhanced amiloride-sensitive Na⁺ absorption (1- 3). There is a large body of evidence that the epithelial Na⁺ channel (ENaC) is downregulated by the CF transmembrane conductance regulator (CFTR) (4, 5). Thus, increased ENaC activity in CF respiratory and colonic tissues is caused by a lack of regulation of ENaC by mutant CFTR (6), causing hyperabsorption of electrolytes, thereby leading to increased mucous viscosity and reduced mucociliary clearance in the airways of patients with CF. Therapeutic strategies aim at restoring defective Cl secretion and reducing enhanced Na⁺ absorption. The 5'-nucleotides adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) have been shown to activate alternative Ca²⁺-dependent Cl channels in murine and human normal and CF respiratory epithelia (10). The pharmacologic profile observed in these studies suggests that ATP and UTP act via binding to the P2Y₂ receptor expressed on the luminal membrane of polarized respiratory epithelial cells (15). This was confirmed recently in studies on P2Y₂ receptor (/) mice in which ATP- and UTP-mediated Ca²⁺ signaling and Cl secretory responses were largely abolished (18, 19). These results triggered clinical trials in which both UTP and amiloride were applied simultaneously to counteract the ion transport defect in CF (20).

CFTR plays an important role in regulating ENaC in airway and intestinal epithelia (6). In kidney epithelia, Na⁺ absorption is modulated by changes in intracellular Ca²⁺, and Ca²⁺-mediated agonists have been demonstrated to inhibit ENaC located in the apical membrane of the cortical collecting duct (21). Similar results were obtained in airways of different species and cultured human bronchial epithelial cells expressing wild-type or F508 CFTR. In these tissues, inhibition of Na⁺ absorption was elicited by extracellular nucleotides and other Ca²⁺-mediated agonists (24). Because no data are available from the original human tissue, we examined the effects of extracellular purine and pyrimidine triphosphates on epithelial Na⁺ absorption in native human upper airway epithelium. For this purpose nasal tissues were mounted in a perfused micro-Ussing chamber and the effects of ATP and UTP on transepithelial voltage (V_te) and equivalent short-circuit current (I_SC) were determined. The effects of ATP and UTP on native normal and CF upper airways were compared.

	Materials and Methods

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Ussing Chamber Experiments

Freshly excised nasal tissues were obtained from 32 non-CF individuals (mean age: 37.0 ± 2.8 yr, range 5 to 72 yr; 20 males, 12 females) after surgery for plastic reconstruction or sleep apnea syndrome and from 16 CF patients (13 ± 2.4 yr, range 4 to 38 yr; 7 males, 9 females) after polypectomy. The study was approved by the Ethical Committee at the University Hospital, Albert-Ludwigs-University Freiburg. Nasal tissues were kept in ice-cold buffer solution of the following composition (mmol/liter): NaCl 127, KCl 5, D-glucose 5, MgCl₂ 1, Na-pyruvate 5, N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid 10, CaCl₂ 1.25, and albumin (10 g/liter). A thin layer of nasal epithelium was dissected from the stroma and mounted into a perfused micro-Ussing chamber with a circular aperture of 0.95 mm² as described previously (13). In brief, the luminal and basolateral sides of the epithelium were perfused continuously at a rate of 10 ml/min (chamber volume 1 ml). The bath solution had the following composition (mmol/liter): NaCl 145, KH₂PO₄ 0.4, K₂HPO₄ 1.6, D-glucose 5, MgCl₂ 1, and Ca-gluconate 1.3. The pH was adjusted to 7.4. Bath solutions were heated by a water jacket and all experiments were carried out at 37°C. Experiments were performed under open-circuit conditions. Transepithelial resistance (R_te) was determined by applying short (1 s) current pulses (I = 0.5 µA) and the corresponding changes in V_te (V_te) and basal V_te were recorded continuously. Values for V_te were referred to the serosal side of the epithelium. R_te was calculated according to Ohm's law. The I_sc was determined from V_te and R_te, i.e., I_SC = V_te/R_te.

Experimental Protocols

After mounting the tissues in the Ussing chamber, an equilibration period of 60 min was allowed for stabilization of basal V_te and R_te. Continuous perfusion of the luminal and basolateral sides of the epithelium allowed us to study ATP- and UTP-mediated inhibition of Na⁺ absorption in a strictly paired fashion. First, the effect of amiloride (10 µmol/liter, luminal solution) was determined under control conditions. The effect of amiloride was entirely reversible on washout for 30 min. Next the effect of mucosal ATP (100 µmol/liter, luminal solution) or UTP (100 µmol/liter, luminal solution) was examined. Both ATP and UTP typically induced a biphasic response with a transient initial increase in I_SC (peak) followed by prolonged inhibition (plateau). In the plateau phase, amiloride was added in the presence of ATP or UTP. Inhibition of Na⁺ absorption was determined by comparing the amiloride-sensitive I_SC in the absence and presence of ATP or UTP. Inhibition of Na⁺ transport by extracellular nucleotides was reversible upon 60 min washout. To examine the role of intracellular Ca²⁺ and protein kinase (PK) C as possible second messengers involved in nucleotide-mediated inhibition of amiloride-sensitive I_SC, tissues were incubated for 20 min with either the Ca²⁺-adenosine triphosphatase (ATPase) inhibitor cyclopiazonic acid (CPA) (50 µmol/ lliter) or the PKC inhibitor bisindolylmaleimide (BIM) (200 nmol/ liter) added on both sides of the epithelium.

Compounds and Data Analysis

Amiloride, ATP, UTP, CPA, and BIM I were all obtained from Sigma (Deisenhofen, Germany). All used chemicals were of highest grade of purity available. Data are shown as individual recordings or as means ± standard error of the mean (n = number of tissue samples). The fractional (%) inhibition of Na⁺ transport was determined from the amiloride-sensitive I_SC under control conditions (I_Amil-Con) and the amiloride-sensitive I_SC in the presence of ATP or UTP (I_Amil-ATP/UTP) as follows: inhibition (%) = 1  (I_Amil-ATP/UTP)/(I_Amil-Con). Statistical analysis was performed using paired Student's t test. Data obtained from CF and non-CF tissues were compared by unpaired Student's t test. P values < 0.05 were accepted to indicate statistical significance.

	Results

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Effect of Luminal ATP on Na⁺ Transport in Normal and CF Nasal Epithelia

The effect of extracellular ATP on Na⁺ absorption in native human nasal tissues was studied after mounting small epithelial sheaths in a perfused micro-Ussing chamber. Amiloride-sensitive I_SC was measured in the absence and presence of ATP (100 µmol/liter), applied to the mucosal side of the epithelium. Basal bioelectric properties were determined after a 60-min equilibration period in the Ussing chamber. In non-CF tissues lumen-negative I_SC was 61.0 ± 6.4 µA/cm² (V_te = 1.2 ± 0.1 mV; R_te = 22.1 ± 2.4 cm²; n = 21). In CF tissues (n = 12), basal I_SC and V_te were significantly increased (245.6 ± 42.9 µA/cm² and 3.8 ± 0.8 mV) and R_te was reduced (15.2 ± 1.6 cm²) compared with non-CF (Figures 1 and 2A). The contribution of electrogenic Na⁺ absorption to transepithelial transport was determined by adding amiloride (10 µmol/liter) to the luminal side of the epithelium. As expected from previous studies (8, 28) the amiloride-sensitive I_SC was significantly increased in CF (I_SC = 222.1 ± 37.7 µA/cm², n = 12) compared with non-CF (I_SC = 45.7 ± 4.8 µA/cm², n = 21) (Figures 1 and 2B). The effect of amiloride was entirely reversible on washout for 30 min. Next, the effect of mucosal ATP (100 µmol/liter) was examined. In non-CF tissues, perfusion with luminal ATP caused a transient increase of lumen-negative I_SC that was followed by sustained inhibition. When amiloride was added in the presence of ATP the amiloride-sensitive I_SC was significantly reduced by 50.3 ± 3.9% (n = 21; Figures 1A and 2). In CF tissues, mucosal ATP induced a similar I_SC response with an initial transient increase followed by prolonged inhibition of lumen-negative I_SC. Addition of amiloride in the presence of ATP revealed a reduction of amiloride-sensitive I_SC by 54.8 ± 4.3% (n = 12; Figures 1B and 2). Thus, the fractional (%) inhibition of amiloride-sensitive I_SC by extracellular ATP was similar in normal and CF tissues. However, the absolute magnitude of amiloride-sensitive I_SC inhibited by ATP was significantly increased in CF compared with that in non-CF tissues (CF: I_scAmil = 119.2 ± 22.8 µA/cm², n = 12 versus non-CF: I_scAmil = 22.6 ± 3.1 µA/cm², n = 21). The data demonstrate that Na⁺ absorption is significantly reduced by application of luminal ATP in normal and CF nasal tissue and that a larger amount of I_SCAmil is inhibited in CF airways.

View larger version (104K): [in this window] [in a new window]   Figure 1.   Effect of amiloride (10 µmol/liter) and extracellular ATP (100 µmol/liter) added to the luminal side of human non-CF (A) and CF (B) nasal epithelia. CF tissues showed an increased response toward amiloride. In the absence of amiloride (30-min washout), ATP caused an initial increase of lumen-negative Vte which was followed by sustained inhibition in non-CF (A) and CF (B) tissues. In the presence of luminal ATP the effect of amiloride was markedly attenuated. Rte was determined continuously from the Vte downward deflections obtained by pulsed current injection. Time gaps between recordings were 30 min.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Summary of the effect of ATP (100 µmol/liter) in human non-CF and CF nasal tissues. (A) Luminal ATP caused an initial lumen-negative peak response (measurements taken during the first minute) which was followed by sustained inhibition of transepithelial ISC (measurements taken after 10 min) in non-CF and CF tissues. (B) In the presence of ATP the amiloride-sensitive ISC was significantly decreased in non-CF and CF tissues. ATP-mediated inhibition of amiloride-sensitive ISC was enhanced in CF and entirely reversible on washout for 60 min. *Statistical significance for the effect of ATP and amiloride (paired t test). #Significant effects of ATP on amiloride-sensitive ISC (paired t test). §Statistical significance when comparing the effects of ATP and amiloride in non-CF and CF tissues (unpaired t test). (n) = number of tissue samples.

Effect of Luminal UTP on Na⁺ Transport in Normal and CF Nasal Epithelia

Different subtypes of nucleotide receptors have been described on the luminal surface of epithelia with a different rank order of potency for ATP, UTP, and their diphosphate nucleotides and analogues. P2Y₂ receptors expressed on murine and human airway epithelial cells are similarly activated by ATP or UTP but not by diphosphate nucleotides (15, 19). To determine whether nucleotide-induced inhibition of Na⁺ absorption was mediated by P2Y₂ receptors, we further examined the effect of UTP (100 µmol/liter) on amiloride-sensitive I_SC. In both non-CF and CF tissues, UTP-induced changes of I_SC were comparable with the observations made for ATP. Addition of UTP resulted in a lumen-negative peak response that was followed by marked and sustained inhibition of transepithelial I_SC (Figures 3 and 4). A detailed analysis of the time course of the UTP response was obtained from five CF tissues (Figure 5); the figure shows that the transient lumen-negative UTP response returned to baseline within 2 min. Maximal UTP-induced inhibition of I_SC was observed after approximately 7 min. Compared with the transient initial increase, inhibition of I_SC was sustained in the presence of UTP and gradually reversible on washout (Figure 5). A similar time course was obtained in non-CF tissues. In the presence of UTP, the amiloride-sensitive I_SC was significantly inhibited in non-CF (by 39.5 ± 6.1%, n = 15) and CF tissues (by 45.4 ± 2.6%, n = 15), respectively. As observed for ATP, the absolute inhibitory effect of UTP on amiloride-sensitive I_SC was significantly increased in CF (I_SCAmil = 73.8 ± 12.7 µA/cm², n = 15) compared with non-CF tissues (I_SCAmil = 19.7 ± 3.9 µA/cm², n = 15) (Figures 3 and 4B).

View larger version (80K): [in this window] [in a new window]   Figure 3.   Effect of amiloride (10 µmol/liter) and mucosal UTP (100 µmol/liter) on human non-CF (A) and CF (B) nasal epithelia. CF tissues showed an increased response toward amiloride. In the absence of amiloride (30-min washout), UTP caused an initial increase of lumen-negative Vte which was followed by prolonged inhibition in non-CF (A) and CF (B) tissues. In the presence of luminal UTP the effect of amiloride was markedly attenuated. Rte was determined continuously from the Vte downward deflections obtained by pulsed current injection. Time gaps between recordings were 30 min.

View larger version (17K): [in this window] [in a new window]   Figure 4.   Summary of the effect of UTP (100 µmol/liter) in human non-CF and CF nasal tissues. (A) Luminal UTP caused an initial lumen-negative peak response (measurements taken during the first minute) which was followed by sustained inhibition of transepithelial ISC (measurements taken after 10 min) in non-CF and CF tissues. (B) In the presence of UTP the amiloride-sensitive ISC was significantly decreased in non-CF and CF tissues. UTP-mediated inhibition of amiloride-sensitive ISC was enhanced in CF and entirely reversible on washout for 60 min. *Statistical significance for the effect of UTP and amiloride (paired t test). #Significant effects of UTP on amiloride- sensitive ISC (paired t test). §Statistical significance when comparing the effects of UTP and amiloride in non-CF and CF tissues (unpaired t test). (n) = number of tissue samples.

View larger version (8K): [in this window] [in a new window]   Figure 5.   Time course of the effect of luminal UTP (100 µmol/ liter) on Na+ absorption. In five CF tissues UTP was applied for 15 min and the time course of the UTP response on transepithelial Isc was recorded. After a transient increase in ISC, perfusion with UTP on the mucosal side caused sustained inhibition of ISC, slowly returning to baseline values after 45 min washout. (n) = number of tissue samples.

Signal Transduction Pathways in UTP-Mediated Inhibition of Na⁺ Absorption

Amiloride-sensitive Na⁺ conductance in non-CF and CF upper airway tissues was equally inhibited by ATP and UTP, suggesting that the response was mediated by the P2Y₂ receptor. P2Y₂ receptor activation has been shown to be coupled to phospholipase (PL) C, resulting in an increase in inositol trisphosphate and diacylglycerol (16, 29). Therefore, the inhibitory effects of ATP and UTP could be mediated by either increase of intracellular Ca²⁺ or activation of PKC. We examined the potential role of intracellular Ca²⁺ in inhibition of Na⁺ absorption in CF tissues by adding the Ca²⁺-ATPase inhibitor CPA (50 µmol/liter). Basal I_SC and amiloride-sensitive I_SC were significantly inhibited by CPA (Figure 6B). After a 20-min incubation period with CPA, both the initial lumen-negative UTP peak response and the prolonged UTP plateau response were significantly reduced in CF nasal tissues (Figures 6A and 6C). These results indicate that UTP-dependent inhibition of Na⁺ absorption is mediated by an increase of intracellular Ca²⁺. To investigate a possible role of PKC activation in nucleotide-mediated inhibition of amiloride-sensitive I_SC, CF tissues were incubated with the PKC inhibitor BIM (200 nmol/liter). As shown in Figure 7, PKC inhibition had no effect on basal I_SC, amiloride-sensitive I_SC, or UTP-dependent inhibition of Na⁺ absorption (Figure 7). These data suggest that intracellular Ca²⁺ signaling, but not PKC, is involved in nucleotide-mediated inhibition of Na⁺ transport.

View larger version (53K): [in this window] [in a new window]   Figure 6.   Effect of the Ca2+-ATPase inhibitor CPA (50 µmol/ liter) on UTP-dependent inhibition of Na+ absorption in CF nasal tissues. Original recording of Vte (A) and summary of the effect of CPA on amiloride-sensitive ISC (B) and UTP-induced peak and plateau responses (C). Basal ISC and amiloride-sensitive ISC were significantly inhibited by CPA (B). After a 20-min incubation period with CPA, both the initial lumen-negative UTP peak response and the prolonged UTP plateau response were significantly reduced in the presence of CPA. Time gaps between recordings were 60 min. *Statistical significance for the effects of amiloride, CPA, and UTP (paired t test). #Effect of amiloride and UTP significantly different in the absence and presence of CPA (paired t test). (n) = number of tissue samples.

View larger version (51K): [in this window] [in a new window]   Figure 7.   Effect of the PKC inhibitor BIM (200 nmol/liter) on UTP-dependent inhibition of Na+ absorption in CF nasal tissues. Original recording of Vte (A) and the summary of the effect of BIM on amiloride-sensitive ISC (B) and UTP-induced peak and plateau ISC responses (C). In strictly paired experiments, incubation with the PKC inhibitor BIM for 20 min had no effect on basal ISC, amiloride-sensitive ISC, or UTP-mediated inhibition of Na+ transport. Time gaps between recordings were 60 min. *Statistical significance for the effect of amiloride and UTP (paired t test). (n) = number of tissue samples.

	Discussion

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CF airways demonstrate enhanced Na⁺ absorption, which is caused by a lack of ENaC downregulation when CFTR is defective (4). Apart from CFTR, binding of nucleotides such as ATP or UTP to luminal P2Y₂ receptors has recently been shown to modulate Na⁺ absorption in various epithelia. Inhibition of Na⁺ transport by extracellular nucleotides has been reported for rabbit trachea, rat distal airway cells, porcine bronchi, and cultured human bronchial epithelial cells (24). This effect exists in addition to the well-described activation of Ca²⁺-dependent Cl channels, apparently expressed in the luminal membrane of normal and CF airways (10, 11, 14, 17, 19, 29). These previous reports prompted us to examine whether (1) nucleotide-dependent inhibition of Na⁺ absorption takes place in native human airways; (2) inhibition of Na⁺ transport is different in CF compared with normal airways; and (3) Ca²⁺-dependent intracellular signaling participates in inhibition of Na⁺ absorption. Due to limited accessibility of lower airway tissue from normal and CF individuals we used freshly excised nasal epithelium, which has been shown to have similar ion conduction properties compared with lower airways and has been well characterized for the ion transport defects in CF (2, 28).

Regulation of Na⁺ transport in human proximal airways was studied by assessing the effects of ATP and UTP on amiloride-sensitive I_SC in epithelial sheaths mounted in a modified perfused micro-Ussing chamber. As reported previously (8), the exposed tissue area was reduced to 0.95 mm² to be able to examine very small samples of native human epithelium. Due to edge leak conductance, the measured V_te and R_te values were certainly underestimated compared with in vivo studies (11) or previous measurements on human nasal tissues using Ussing chambers with a larger-size open area (28). However, bioelectric properties of our preparations corresponded well to measurements of upper murine airways mounted in small-size Ussing chambers (7). Despite imperfect edge-sealing, typical CF alterations of ion transport (e.g., enhanced Na⁺ absorption) were well preserved; V_te and R_te were stable during the whole course of the experiments; and robust responses were obtained upon addition of amiloride, ATP, or UTP. We present here data which confirm nucleotide-mediated inhibition of Na⁺ absorption in native human upper airway tissue from normal individuals and patients with CF. We demonstrate that both nucleotides, in strictly paired experiments, induced a transient increase followed by prolonged inhibition of Na⁺ absorption. Similar fractional (%) inhibition of the amiloride-sensitive I_sc in the range of 40 to 55% was observed for both nucleotides in non-CF and CF tissues, respectively. However, due to enhanced basal Na⁺ conductance in CF, the absolute magnitude of nucleotide-mediated inhibition of Na⁺ absorption was significantly increased in CF compared with non-CF tissues. Extracellular ATP and UTP were equally effective in reducing Na⁺ transport, which suggests that the responses are mediated by luminal P2Y₂ receptors (18), coupling to intracellular PLC and G proteins (16, 19). Previous studies on Na⁺ absorption in renal epithelia showed attenuation of Na⁺ transport by an increase in intracellular Ca²⁺ (21, 23). In another report, ATP-induced inhibition of Na⁺ absorption did not depend on Ca²⁺ but was mediated by PKC (22). We show here that an increase in intracellular Ca²⁺ by adding the Ca²⁺-ATPase inhibitor CPA results in inhibition of Na⁺ absorption in the absence of extracellular nucleotides. Further, nucleotide-mediated inhibition of Na⁺ transport is largely reduced in the presence of CPA. The PKC inhibitor BIM had no effect on nucleotide-mediated inhibition of amiloride-sensitive I_SC. These data suggest that a rise of intracellular Ca²⁺, but not activation of PKC, is involved in inhibition of epithelial Na⁺ transport in human upper airway tissues, which confirms previous results obtained from cultured human bronchial epithelial cells (24). However, in the presence of amiloride, CPA also inhibited P2Y₂-mediated activation of Cl secretion (unpublished observation from the authors' laboratory). Therefore, the present data do not allow us to discriminate whether Ca²⁺ acts directly on ENaC or whether activation of P2Y₂-coupled Cl conductance is required for inhibition of Na⁺ absorption. In that respect it is noteworthy that Cl transport was shown to be essential for CFTR-mediated inhibition of ENaC (30). Moreover, at this stage it cannot be excluded that G proteins or direct protein-protein interaction between luminal P2Y₂ receptors and ENaC do contribute to the inhibition of Na⁺ transport.

According to the present and previously published results, autocrine secretion of nucleotides to the luminal surface of airways inhibits Na⁺ absorption and activates Cl secretion. By this mechanism, airway cells could switch from NaCl absorption to NaCl secretion even in the absence of intact CFTR. Extracellular nucleotides may therefore play an important physiologic role in fine-tuning of the airway surface liquid lining the superficial respiratory epithelium. Thus, an increase in luminal nucleotide concentration by inhalation of ATP or UTP is expected to counteract enhanced amiloride-sensitive Na⁺ conductance caused by CFTR mutations.

Earlier studies have shown that extracellular nucleotides activate an alternative Ca²⁺-dependent Cl conductance and increase mucociliary clearance in CF airways, and have proposed the use of ATP and UTP as therapeutic drugs in the treatment of CF lung disease (11, 20). In the present report we demonstrate that extracellular nucleotides induce significant inhibition of Na⁺ absorption in native human non-CF and CF airway tissues. Therefore, topical application of aerosolized ATP or UTP could have a dual therapeutic effect by counteracting increased Na⁺ absorption and reduced Cl secretion in airways from patients with CF.