Cystic fibrosis (CF) airway epithelia are characterized by enhanced Na+ absorption probably due to a lack of downregulation of epithelial Na+ channels by mutant CF transmembrane conductance regulator. Extracellular nucleotides adenosine 5 ′ -triphosphate (ATP) and uridine 5 ′ -triphosphate (UTP) have been shown to activate alternative Ca2 +-dependent Cl− channels in normal and CF respiratory epithelia. Recent studies suggest additional modulation of Na+ absorption by extracellular nucleotides. In this study we examined the role of mucosal ATP and UTP in regulating Na+ transport in native human upper airway tissues from patients with 16 patients with CF and 32 non-CF control subjects. To that end, transepithelial voltage and equivalent short-circuit current (I sc ) were assessed by means of a perfused micro-Ussing chamber. Mucosal ATP and UTP caused an initial increase in lumen-negative I sc that was followed by a sustained decrease of Isc in both non-CF and CF tissues. The amiloride-sensitive portion of I sc was inhibited significantly in normal and CF tissues in the presence of either ATP or UTP. Both basal Na+ transport and nucleotide-dependent inhibition of amiloride-sensitive I sc were significantly enhanced in CF airways compared with non-CF. Nucleotide-mediated inhibition of Na+ absorption was attenuated by pretreatment with the Ca2 +–adenosine triphosphatase inhibitor cyclopiazonic acid but not by inhibition of protein kinase C with bisindolylmaleimide. These data demonstrate sustained inhibition of Na+ transport in non-CF and CF airways by mucosal ATP and UTP and suggest that this effect is mediated by an increase of intracellular Ca2 +. Because ATP and UTP inhibit Na+ absorption and stimulate Cl− secretion simultaneously, extracellular nucleotides could have a dual therapeutic effect, counteracting the ion transport defect in CF lung disease.
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 (6-9), 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 (10-14). 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 (15-17). 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 (6-9). 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 (21-23). Similar results were obtained in airways of different species and cultured human bronchial epithelial cells expressing wild-type or ΔF508 CFTR. In these tissues, inhibition of Na+ absorption was elicited by extracellular nucleotides and other Ca2+-mediated agonists (24-27). 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 (Vte) and equivalent short-circuit current (I sc ) were determined. The effects of ATP and UTP on native normal and CF upper airways were compared.
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 (Rte) was determined by applying short (1 s) current pulses (ΔI = 0.5 μA) and the corresponding changes in Vte (ΔVte) and basal Vte were recorded continuously. Values for Vte were referred to the serosal side of the epithelium. Rte was calculated according to Ohm's law. The Isc was determined from Vte and Rte, i.e., I sc = Vte/Rte.
After mounting the tissues in the Ussing chamber, an equilibration period of 60 min was allowed for stabilization of basal Vte and Rte. 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.
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 (ΔIAmil–Con) and the amiloride-sensitive I sc in the presence of ATP or UTP (ΔIAmil–ATP/UTP) as follows: inhibition (%) = 1 − (ΔIAmil–ATP/UTP)/(ΔIAmil–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.
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 (Vte = −1.2 ± 0.1 mV; Rte = 22.1 ± 2.4 Ωcm2; n = 21). In CF tissues (n = 12), basal I sc and Vte were significantly increased (−245.6 ± 42.9 μA/cm2 and −3.8 ± 0.8 mV) and Rte was reduced (15.2 ± 1.6 Ωcm2) 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 (ΔI sc = 222.1 ± 37.7 μA/cm2, n = 12) compared with non-CF (ΔI 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: ΔIscAmil = −119.2 ± 22.8 μA/cm2, n = 12 versus non-CF: ΔIscAmil = −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 sc Amil is inhibited in CF airways.
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 (15-17, 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 (ΔI sc Amil = −73.8 ± 12.7 μA/cm2, n = 15) compared with non-CF tissues (ΔI sc Amil = −19.7 ± 3.9 μA/cm2, n = 15) (Figures 3 and 4B).
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.
CF airways demonstrate enhanced Na+ absorption, which is caused by a lack of ENaC downregulation when CFTR is defective (4-9). 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 (24-27). 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 Vte and Rte 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; Vte and Rte 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 Isc 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.
The authors gratefully thank Professor Laszig, ENT Clinic, University Hospital Freiburg, for his cooperation. They further acknowledge the expert technical assistance by Mrs. S. Hirtz and Mrs. C. Hodler. This study was supported by the Deutsche Forschungs Gemeinschaft grant DFG KU1228/ 1-1 and Zentrum Klinische Forschung 1 (ZKF1, A2), University of Freiburg.
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