Differential effects of UTP, ATP, and adenosine on ciliary activity of human nasal epithelial cells

Diane M. Morse1, Jennifer L. Smullen1, and C. William Davis1,2

1 Cystic Fibrosis/Pulmonary Research and Treatment Center, 2 Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, North Carolina 27599-7248


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The purinergic regulation of ciliary activity was studied using small, continuously superfused explants of human nasal epithelium. The P2Y2 purinoceptor (P2Y2-R) was identified as the major purinoceptor regulating ciliary beat frequency (CBF); UTP (EC50 = 4.7 µM), ATP, and adenosine-5'-O-(3-thiotriphosphate) elicited similar maximal responses, approximately twofold over baseline. ATP, however, elicited a post-peak sustained plateau in CBF (1.83 ± 0.1-fold), whereas the post-peak CBF response to UTP declined over 15 min to a low-level plateau (1.36 ± 0.16-fold). UDP also stimulated ciliary beating, probably via P2Y6-R, with a maximal effect approximately one-half that elicited by P2Y2-R stimulation. Not indicated were P2Y1-R-, P2Y4-R-, or P2Y11-R-mediated effects. A2B-receptor agonists elicited sustained responses in CBF approximately equal to those from UTP/ATP [5'-(N-ethylcarboxamido)adenosine, EC50 = 0.09 µM; adenosine, EC50 = 0.7 µM]. Surprisingly, ADP elicited a sustained stimulation in CBF. The ADP effect and the post-peak sustained portion of the ATP response in CBF were inhibited by the A2-R antagonist 8-(p-sulfophenyl)theophylline. Hence, ATP affects ciliary activity through P2Y2-R and, after an apparent ectohydrolysis to adenosine, through A2BAR.

cilia; ciliated cells; purinergic agonists; regulation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OVER THE LAST FEW YEARS chloride and fluid transport (7, 8, 38, 39) and mucin secretion (18, 35) in the airways have been shown to be regulated in fundamentally important ways by ATP and UTP acting from the lumen via apical membrane P2Y2 purinoceptors (P2Y2-R; Ref. 61). These purinergic agonists also stimulate mucociliary clearance (4, 63), and ATP has been shown to stimulate ciliary activity directly (25, 68). In rabbit trachea epithelial cells, there is a concentration-dependent relationship between ATP and ciliary beat frequency (CBF) with an EC50 of ~10 µM (40), and there is a good correlation between the ATP-induced responses in intercellular Ca2+ and CBF (40). These results are consistent with the direct effects of ATP on ciliary activity being mediated by P2Y2-R, but they are not definitive. Whereas the effects of UTP would help greatly in determining the participation of P2Y2-R in this response, surprisingly there are no reports regarding the effects of the agonist on ciliary activity. The identity and characteristics of the receptors and other components of the nucleotide signaling system regulating ciliary activity may have great clinical impact on the treatment of cystic fibrosis and other airway diseases (21, 50).

Five P2Y G protein-coupled receptors are known to mediate the effects of purinergic agonists on cellular functions: P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 (13, 28, 61). All of these receptors couple to phospholipase C (PLC) through Gq/11, but some may couple also to adenylyl cyclase either through Gi (P2Y2-R, P2Y4-R) or Gs (P2Y11-R). ATP is an effective agonist at P2Y2-R and P2Y11-R, whereas UTP activates P2Y2-R and P2Y4-R. The other P2Y receptors are activated principally by nucleoside diphosphates, P2Y1-R by ADP and P2Y6-R by UDP. Purinoceptors figure broadly in autocrine and paracrine responses in the body, and the number of physiological systems they influence or control is increasing rapidly (28, 61). In the airways, ATP and UTP are approximately equipotent in their stimulatory effects on chloride and, potentially, fluid secretion (17, 47), and the agonists are equipotent and cross-desensitize in their activation of PLC (10, 56). That ATP and UTP effect these responses selectively through P2Y2-R was indicated in a recent study showing that >85% of the chloride secretory response to each agonist was lost in the trachea of the P2Y2-R-/- mouse (17). In addition to its actions at P2Y receptors, ATP also acts to effect responses through a family of ligand-gated P2X purinoceptors (61). At the present time, however, there is no direct evidence for P2X receptor involvement in the regulation of airway ion transport activities. Adenosine also stimulates chloride secretion in airways (60, 65), a response mediated by P1 purinoceptor A2BAR (42, 61).

The initial goal of the present experiments was to test the responsiveness of human airway epithelial cell ciliary activity to purinergic stimulation. In pursuit of that goal we discovered a major difference between the effects of ATP and UTP on ciliary activity, a difference that appears to result from a dynamic interplay involving ATP, nucleotide ectohydrolysis, and P2Y2 and A2B purinoceptors. These elements may comprise the core components of a purinergic signaling system operative on the luminal surface of the airways.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

Tissue culture media and supplements were purchased from Collaborative Research or Sigma Chemical; nucleotides and adenosine were purchased from Boehringer-Mannheim Biochemicals, and adenylyl cyclase inhibitors, adenosine receptor agonists, and antagonists were from RBI. All other reagents were purchased from Sigma Chemical. To remove nucleotide triphosphate contamination from solutions containing nucleotide diphosphates, 1 mM stock solutions were pretreated with 10 U/ml hexokinase, plus 5 mM glucose, for 30 min at 37°C (43). If necessary, hexokinase was adjusted to 1 U/ml after the nucleotide diphosphate stock was diluted to its respective working concentration.

ATP was judged to be essentially free of adenosine by high-pressure liquid chromatography; the material was 98.97% pure and contained a maximum of 0.03% adenosine. Thus 100 µM ATP contained no more than 30 nM adenosine, a concentration ~24-fold below the apparent EC50 measured for adenosine in the experiment of Fig. 4.

Epithelial Cell Culture and Superfusion

Human nasal epithelial (HNE) cells were removed by protease XIV digestion from turbinates obtained from patients undergoing elective surgeries. All such procedures were approved by the University of North Carolina Committee for the Rights of Human Subjects. The cellular products of the protease digestion were seeded onto 12-mm Transwell-Col inserts (TCol; Costar) at a density of 3 × 105 cells/TCol, and the cultures were maintained as previously described (26, 48). Over the first 24 h, small clumps of intact epithelium settled out and attached to the TCol; ciliated cells within these small explants of native epithelium were used to study the regulation of CBF over the next 1-5 days. Within this period of time, many cultures became confluent through the multiplication of basal-like cells, but in no case did ciliogenesis occur de novo because this process requires ~2 wk of air:liquid culture after confluence.

TCols bearing epithelial explants were mounted with the TCol serosal surface positioned within 0.5 mm of a coverslip forming the bottom of a simple chamber on the stage of a Zeiss IM 35 inverted microscope. Serosal bath volume was ~1 ml. Culture lumina were superfused using delivery and uptake stainless steel needles positioned within 1 mm of the TCol substratum; the needles were held in a custom-fabricated collar, which interlocked with the flange at the upper end of the TCol. Solutions were delivered to the chamber at 250 µl/min using a peristaltic pump and were removed by suction; luminal bath volume was ~250 µl. The control bathing solution was Krebs-bicarbonate-Ringer (KBR) with the following composition (in mM): 125 NaCl, 5.2 KCl, 1.2 MgCl2, 1.2 CaCl2, 25 NaHCO3, 10 TES, and 5 glucose (pH 7.4 when gassed with 5% CO2). Solutions were changed by moving the intake of the inlet tubing quickly between holding vessels; the small bubble of air introduced into the TCol lumen by this action floated off and burst without interfering with data collection. The chamber and solutions were maintained at 35-36°C by a temperature-controlled box (Digi-Sense proportional controller, Cole-Parmer Instruments, and a 1,200-W resistive heater), which enclosed the microscope stage and perfusion system.

Ciliary Activity Measurements

Ciliated cells were viewed by phase-contrast microscopy using a Zeiss ×32 objective, and the image was monitored with a Dage 72 monochrome charge-coupled device video camera. CBF was determined as previously described (25). Briefly, a photodarlington detector was positioned on the video monitor over a ciliated cell, and its amplified (2-20×), low-pass filtered (5 kHz) output voltage was digitized at 40 kHz by an analog-to-digital converter in a personal computer under the control of a custom software program. The program monitored this signal in real time for the spike in the 60-Hz video signal, which corresponded to the electron beam of the monitor passing beneath the sensor. The peak amplitude of this spike, which varies sinusoidally as a result of the beating cilia in the video image, was determined by the software program and stored. During experiments, ciliary activity was so sampled for 10 s every minute, and after the experiment the data collected were analyzed by a fast Fourier transform (FFT) for CBF. The power spectral density of each FFT analysis was inspected visually to ensure that a single dominate frequency was reported by the analysis software program. In the rare case (<5% of all experiments) when multiple frequencies were apparent, the entire data set was rejected. The temporal limit of the measurement system, given its dependence on the video field rate (60 Hz), is 30 Hz.

Experimental Protocols and Data Analysis

In all experiments, after being mounted in the experimental chamber, HNE cultures were superfused for 1.5 h in KBR before experimentation. Beginning with a 10-min baseline period and through the remainder of the experiment, data were recorded every minute for the determination of CBF. After the FFT analyses for each experiment, the resulting CBF data were normalized to the mean CBF recorded during the baseline period. Responses in ciliary activity to experimental maneuvers are reported as the means ± SE of the response ratio, relative to baseline, for n cultures. For each experimental protocol, cultures derived from the tissues of three or more patients were used. Differences between means were tested for significance with a t-test, using paired or grouped data as appropriate to the analysis. From such comparisons, differences yielding P <=  0.05 were judged as being significant. Results found not to be statistically significant are indicated as NS or P > 0.05.


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MATERIALS AND METHODS
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Effects of P2Y2-R Agonists on CBF

P2Y2-R-based regulation of ciliary activity of HNE cells was assessed initially by examining UTP concentration-effect relationships. After equilibration and the determination of baseline CBF, the cells were exposed to 100 µM UTP as an internal control for 10 min, agonist was washed out for 30 min, and then on the same cell a second baseline CBF frequency and response to a variable concentration of UTP were recorded. The first baseline CBF was 9.35 ± 0.38 Hz, and the second was 9.05 ± 0.42 Hz [n = 33, not significant (NS), Fig. 1A]. UTP stimulated ciliary activity over its basal level in a concentration-dependent manner. At each concentration, CBF rose to a peak 2-3 min after the change in solution, generally followed by a monotonic decline toward baseline values. The relatively slow onset of agonist effects on ciliary activity observed was consistent with the low rate of superfusion used in these experiments. Note that the peak responses in CBF elicited by the control, 100 µM UTP challenges were similar at ~1.6-fold over baseline. Although other experiments (below) yielded lower and higher values for peak UTP responses, the similarity of control values in this experiment allowed a simple evaluation of cell responsiveness to the variable concentrations of UTP (Fig. 1B). The CBF response to UTP saturated at 100 µM, and the EC50 was 4.7 µM, consistent with other whole cell responses mediated by P2Y2 receptors (see DISCUSSION).


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Fig. 1.   Effects of UTP on ciliary activity in human nasal epithelial (HNE) cells. A: responsiveness of ciliary activity to increasing concentrations of UTP. After the determination of basal ciliary beat frequency (CBF) in each experiment, cells were exposed to 100 µM UTP as an internal control. After a 30-min washout and a second basal CBF determination, they were then exposed to a variable concentration of UTP as indicated. B: concentration-effect relationship for UTP on CBF. The peak responses in CBF from the experiments in A are plotted against the concentration of UTP. In both panels, the data are expressed as the means ± SE CBF relative to baseline, n>= 6.

In the control responses to 100 µM UTP, a variability in the post-peak behavior in CBF was noted during the 10-min exposure to agonist (Fig. 1). In the majority of cases CBF declined to some degree from its peak, but in a few cases the signal appeared more stable. In preliminary experiments using ATP as an agonist, the post-peak behavior in CBF appeared to differ from that observed with UTP, but again the variability in the response prevented a definitive conclusion. When, however, the effects of 100 µM UTP and ATP were compared over a longer, 20-min time course, definitive differences were revealed. Namely, from similar peak responses (UTP peak = 2.20 ± 0.11-, ATP peak = 2.13 ± 0.11-fold over baseline), ciliary activity declined at a more rapid rate to a significantly lower plateau with UTP than with ATP (Fig. 2A). The rates of post-peak decline in CBF observed during exposure to 100 µM UTP and ATP, determined between 6 and 14 min post-agonist, were -0.068 ± 0.035 and -0.027 ± 0.029-fold/min (P < 0.05). The plateau phase of the responses, measured at 20 min post-agonist, were 1.36 ± 0.16- and 1.83 ± 0.10-fold over baseline, respectively (P < 0.05, n = 6 each).


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Fig. 2.   Time courses of CBF responses by HNE cells to purinoceptor agonists. A: responses to ATP and UTP. After determination of basal CBF, the superfusate bathing HNE cells was switched to one containing 100 µM ATP or UTP. After 20 min of exposure, the dose of agonist was doubled (n = 6 each). B: response to adenosine-5'-O-(3-thiotriphosphate) (ATPgamma S). Experiment as in A, except the cells were exposed to 100 µM ATPgamma S (n = 6). For comparison, dotted line reproduces the UTP data from A.

The more sustained ATP response indicates that it may have effects on CBF that are independent of P2Y2-R. Two simple possibilities are that ATP could also interact directly with a different receptor, e.g., the P2X receptor recently suggested to regulate rabbit tracheal ciliated cells (46), or ATP could have indirect effects on CBF through the interaction of an ectometabolite with another receptor. As an initial test of the possibility that ATP is ectometabolized to another ciliostimulatory agonist, we challenged HNE cells with the poorly hydrolyzable adenosine-5'-O-(3-thiotriphosphate) (ATPgamma S, Fig. 2B). This P2Y2-R agonist elicited a peak response in ciliary activity 2.07 ± 0.14-fold over baseline CBF that was not different from those elicited by UTP and ATP. Subsequently, CBF declined in a manner similar to the responses elicited by UTP; the post-peak rate of decline in ATPgamma S-stimulated CBF was -0.054 ± 0.040-fold/min (n = 6).

Effects of Nucleoside Diphosphates on CBF

If the differences in the ciliary activity response patterns elicited by UTP/ATPgamma S and ATP reflect ecto-nucleotide hydrolysis of ATP, then one or more products of that hydrolysis should possess stimulatory effects on HNE cells. This possibility was tested initially using ADP. ADP concentrations of 3 and 100 µM elicited strong responses in ciliary activity, with the peak response to the higher concentration being similar (1.99 ± 0.09-fold over baseline) to that elicited by P2Y2 agonists (Fig. 3A). At both concentrations, the response in CBF elicited by ADP was sustained, similar to the effects of ATP. After 10 min, the ADP solutions were exchanged for one containing 100 µM UTP alone. UTP (100 µM) had little additional effect on ciliary activity when switching from the same concentration of ADP, and from 3 µM ADP the switch to UTP caused an elevation in CBF to levels close to those achieved by 100 µM ADP (Fig. 3A). In each case, however, the CBF response pattern shifted from the sustained elevation elicited by ADP to a declining pattern similar to that observed with UTP stimulation. The post-ADP rates of decline were -0.012 ± 0.003- and -0.13 ± 0.016-fold/min, determined between 3 and 10 min post-agonist for 3 and 100 µM ADP (Fig. 3A, NS, P > 0.05). In contrast, the post-UTP rates of decline for these cultures, determined between 6 and 10 min post-agonist, were -0.049 ± 0.034- and -0.041 ± 0.039-fold/min, for the cultures exposed initially to 3 and 100 µM ADP, respectively.


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Fig. 3.   Effects of nucleoside diphosphates on ciliary activity of HNE cells. After determination of basal CBF, the superfusate bathing HNE cells was switched to one containing 3 or 100 µM ADP (A, n = 4 or 6, respectively), 100 µM 2-methylthio-ADP (2-MeS-ADP, B, n = 6), or 100 µM UDP (C, n = 6) for 10 min. At that time, in each case, the superfusate was switched to one containing 100 µM UTP as a control.

A P2Y1 archetypal agonist, 2-methylthio-ADP (2-MeS- ADP) (54, 61), was used to test whether the P2Y1 purinoceptor mediates ADP effects on CBF. 2-MeS-ADP had no effect on ciliary activity, whereas the same cells responded normally to a subsequent exposure to UTP (Fig. 3B). Additionally, the P2Y1-R-selective antagonist adenosine-2'-phosphate-5'-phosphate (PAP; Ref. 9) had no effect on the response of HNE cells to ADP (data not shown). Hence, it is unlikely that the P2Y1 purinoceptor mediates apical membrane effects on CBF in these cells.

UDP, the principal agonist for P2Y6-R, stimulated HNE cell ciliary activity (Fig. 3C) to a peak response of 1.42 ± 0.06 over baseline, a level approximately one-half that expected from a full P2Y2-R-mediated response. When UTP was substituted for UDP in this experiment CBF increased again to 1.75 ± 0.09 over baseline, a level similar to UTP controls (Figs. 1 and 2).

Effects of Adenosine Receptor Agonists on CBF

Adenosine and its nonmetabolizable analog 5'-(N-ethylcarboxamido)-adenosine (NECA) were next tested for effects on CBF. HNE cell ciliary activity was stimulated by NECA in a concentration-dependent manner (Fig. 4); the response saturated above 1 µM NECA and the EC50 was 0.09 µM. Most notably, the effects of NECA on HNE cells were generally sustained over the period tested. The effects of adenosine on CBF in HNE cells were similar to those of NECA, with the primary exception being a rightward shift in the concentration-response curve such that the apparent EC50 was 8.5-fold higher at 0.72 µM (Fig. 4).


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Fig. 4.   Effects of adenosine and 5'-N-ethylcarboxamido-adenosine (NECA) on HNE ciliary activity. Left: time courses of responses to NECA. After determination of basal CBF, HNE cells were exposed to a superfusate containing a variable concentration of NECA as indicated. Right: concentration-response curves. Curves were constructed from the maximum responses to NECA (left) or from similar experiments for adenosine. For each dose of NECA and adenosine, n = 5 or 6.

The A2B receptor mediates other adenosine-regulated responses of airway epithelial cells (42), and the following experiments tested whether this receptor participates in the regulation of ciliary beating. First, the adenylyl cyclase activator forskolin was used to test whether ciliary activity in HNE cells is responsive to cAMP. As expected, CBF was stimulated over baseline by 1 and 10 µM forskolin, by a maximum of about 50% at the higher concentration (Fig. 5A). Second, the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ-22536) was used to test whether stimulation by NECA leads to cAMP production (23), as reflected by changes in ciliary beating. When SQ-22536 was added to the superfusate bathing HNE cells 5 min after a submaximal stimulation by NECA, it inhibited CBF by 57 ± 2% from a mean NECA-stimulated response of 1.60 ± 0.10-fold above baseline (Fig. 5B, P < 0.05, n = 3). Third, to distinguish between adenosine acting via A2AAR vs. A2BAR to affect ciliary activity, we tested the effects of the A2AAR-specific agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxyamino-adenosine (CGS-21680). As shown in Fig. 5C, this agonist had no effect on CBF, whereas the same HNE cells responded robustly to a subsequent addition of NECA. Collectively, these data are consistent with the effects of adenosine and NECA on ciliary activity being mediated through A2BAR.


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Fig. 5.   Identification of adenosine receptor subtype mediating NECA and adenosine effects on HNE ciliary activity. A: effects of forskolin. After determination of basal CBF, HNE cells were exposed to superfusates containing forskolin at the indicated concentrations (n = 4 each). B: effects of adenylyl cyclase inhibition. After determination of basal CBF and stimulation with 0.3 µM NECA, HNE cells were exposed to NECA plus 100 µM 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ-22536, n = 3). C: effect of an A2A-selective agonist. After determination of basal CBF, HNE cells were exposed first to a superfusate containing 10 µM 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxyamino-adenosine (CGS-21680) for 10 min and then to one containing 3 µM NECA as a control (n = 4).

Effects of Adenosine Receptor Blockade on CBF Responses to Adenosine, ADP, and ATP

To test whether the products of adenine nucleotide ectohydrolysis affect ciliary beating via A2BAR, we used the antagonist 8-(p-sulfophenyl)theophylline (8-SPT; see Refs. 23, 52, 59). As a control, 3 µM adenosine, 100 µM 8-SPT, and 100 µM UTP were added sequentially, at 10-min intervals, to the superfusate bathing HNE cells (Fig. 6A). Significantly, the stimulatory effects of adenosine on ciliary activity were reversed almost completely by 8-SPT, whereas the cells were still fully capable of responding to UTP in the presence of the inhibitor. In a second control experiment, 100 µM 8-SPT added to the superfusate alone had no effect on CBF, and the cells responded subsequently to the addition of UTP in the continued presence of blocker (data not shown).


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Fig. 6.   Effects of 8-(p-sulfophenyl)theophylline (8-SPT) on adenosine nucleoside and nucleotide stimulation of CBF. A: stimulation with adenosine. After determination of basal CBF and stimulation with 3 µM adenosine, the superfusate bathing HNE cells was switched to one also containing 8-SPT. After 10 min, 100 µM UTP was added as a control (n = 6). B and C: stimulation with ADP and ATP. After the determination of basal CBF, HNE cells were exposed to a superfusate containing 100 µM 8-SPT plus, either 3 µM ADP for 10 min or 100 µM ATP for 20 min (n = 7, each).

8-SPT inhibited the effects of ADP on CBF (Fig. 6B). Ciliary activity rose slightly to a peak of 1.17 ± 0.06-fold over baseline following the addition of ADP + 8-SPT, which was significantly lower than the peak response elicited by 3 µM ADP alone (Fig. 3A, peak = 1.71 ± 0.18, P < 0.05). Furthermore, the CBF response to ADP in the presence of the antagonist was not only slow to develop but it collapsed back to baseline after a few minutes.

The A2BAR blocker had more subtle but significant effects on the stimulation of ciliary activity by ATP (Fig. 6C). In the presence of 8-SPT, 100 µM ATP-stimulated HNE CBF to a peak response of 1.72 ± 0.10-fold over baseline, after which it declined over the next 15 min to a plateau at 17-20 min post-agonist of 1.23 ± 0.03-fold over baseline. This plateau was significantly lower than that elicited by ATP alone (Fig. 2, P < 0.05) but not from the UTP-elicited CBF plateau (Fig. 2). Together, these results with 8-SPT suggest that adenine nucleotides are ectometabolized to adenosine on the surface of HNE cells and that all (ADP) or some (ATP) of their effects on ciliary activity are mediated by adenosine interacting with A2BAR.

ATP Stimulation of Ciliary Activity During the UTP Plateau

8-SPT was useful in revealing those actions of ATP mediated by P2Y2-R. To similarly isolate the A2BAR portion of the ATP response as another means of testing the independence of ATP effects mediated by the two receptors, HNE cultures were treated first with a saturating concentration of UTP to minimize cellular responsiveness to P2Y2-R activation (Fig. 7A). When 100 µM ATP was added to the superfusate, concurrent with UTP during the UTP-induced CBF plateau, there was rapid increase in ciliary activity from 1.23 ± 0.04- to 1.60 ± 0.03-fold over baseline (P < 0.05, n = 3). That this increase in CBF was due to adenosine generation was indicated by the decrease in CBF back to the UTP plateau level of 1.19 ± 0.03-fold over baseline following the inclusion of 8-SPT in the superfusate. As controls for the effective doubling of the concentration of P2Y2-R agonists in this experiment, note the lack of additional responses in CBF when the concentrations of ATP and UTP were doubled in the experiment of Fig. 2.


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Fig. 7.   Effect of ATP on ciliary activity following prolonged UTP exposure in HNE cells. A: time course of effects. After determination of basal CBF, HNE cells were exposed to a superfusate containing 100 µM each of UTP for 20 min, ATP for 15 min, and then 8-SPT for 10 min; the additions were cumulative (n = 3). B: concentration-dependence of the ATP effect. Two additional experiments were conducted with protocols identical to that in A except that the ATP concentration was either 10 (n = 4) or 1 (n = 5) µM. This plot depicts the increment in CBF (Delta CBF) elicited by ATP applied during the plateau phase of the UTP response. *P < 0.05 relative to the UTP plateau (see text).

Because the HNE preparations were superfused in these studies, the sustained responses of ciliary activity to ATP (Figs. 2 and 7A) most likely indicate that the exogenously added nucleotide is hydrolyzed continuously to adenosine, which stimulates CBF through A2BAR. To test the sensitivity of this dynamic system, the protocol of Fig. 7A was repeated with superfusate ATP concentrations of 10 and 1 µM. Significant increases in CBF over the UTP-elicited plateau were elicited by 10 and 100 µM ATP under these conditions (Fig. 7B).

Interactions of cAMP and UTP

One of the surprising observations in this study was the sustained effects of ATP, ADP, and adenosine on ciliary activity. To test whether these sustained actions are due to a depression of events distal to receptor activation along the cellular messenger pathway, we studied the relative effectiveness of the permeant cAMP analog chlorophenylthio-cAMP (cpt-cAMP) and UTP in promoting ciliary activity. Addition of 0.5 mM cpt-cAMP to the superfusate bathing HNE cells increased ciliary activity to a sustained level of about 1.6-fold over baseline for at least 5 min (Fig. 8A). The subsequent addition of 100 µM UTP to the superfusate caused a small, additional increase to 1.8- to 1.9-fold over baseline; importantly, the combined effects of cpt-cAMP and UTP were sustained for the duration of the 10-min combined exposure (compare with UTP alone, Figs. 2, 7, and 8B). When UTP was added first to the superfusate and cpt-cAMP was added 10 min later, during the UTP post-peak decline in CBF, ciliary activity was stimulated substantially from 1.39 ± 0.15- to 1.64 ± 0.11-fold over baseline (Fig. 8B, P < 0.05, n = 6). Again, UTP and cpt-cAMP together elicited a sustained elevation in ciliary activity, relative to UTP alone; CBF at the end of the UTP + cpt-cAMP exposure was 1.60 ± 0.13-fold over baseline, a value that is indistinguishable from the UTP peak response.


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Fig. 8.   Effects of chlorophenylthio-cAMP (cpt-cAMP) and UTP on ciliary activity. After determination of basal CBF, HNE cells were exposed to either 500 µM cpt-cAMP followed by the addition of 100 µM UTP (A, n = 6) or the reversed order (B, n = 4).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The biological actions of extracellular nucleotides, first revealed during World War II for their role in traumatic shock (27), have come into focus recently with the pharmacological characterization and cloning of the nine G protein-coupled (P1 and P2Y; Refs. 28, 59, 61) and eight ligand-gated (P2X; Refs. 51, 61) purinergic receptors presently known. These receptors mediate a broad spectrum of signaling events, such as sensory perception (11, 20), cell growth (22), allergic responses in asthma (24), and the regulation of insulin secretion (30, 58) and microvascular tone (45). Purinoceptors also figure broadly in pathophysiology (1). In the airways, ATP and UTP have been implicated in the stimulation of the components comprising the mucociliary clearance system, Cl- and fluid secretion (37, 47), ciliary activity (25, 40, 68), and mucin secretion (18, 35), as well as of mucociliary clearance per se (4, 63). Recent evidence that nucleotide triphosphates are secreted from nonneuronal cells in culture (e.g., see Refs. 19, 29, 30, 53, 64), including secretion into the luminal compartment of cultured airway epithelial cells (41, 67), indicates that ATP and UTP may act as autocrine and paracrine mediators. Interestingly, ATP and/or UTP secreted across both the apical and basolateral membranes of airway epithelial cells may mediate cell-cell communication elicited by the mechanical stimulation of individual cells (31); previously, inositol triphosphate diffusion via gap junctions was implicated in this role (e.g., see Ref. 6).

Of the three principal components of the mucociliary clearance system, the purinergic regulation of ciliary activity is the least well characterized. We tested the participation of P2Y2 receptors in the regulation of ciliary activity of airway columnar epithelial cells by challenging cells with suitable agonists. As detailed below, the study successfully identified, pharmacologically, P2Y2-R as an important component in the regulation of CBF; however, it also revealed that P2Y2-R is coupled with a dissimilar receptor system, A2BAR, through the ectometabolism of ATP to adenosine. In the following, we treat each purinergic receptor individually in terms of its likely participation in ciliary regulation and then discuss the apparent coupling between the dominant P2Y2 and A2B receptors.

P2Y Receptor-Mediated Effects on Ciliary Activity

P2Y1-R. The P2Y1-R rank order potency (human) is as follows: 2-MeS-ADP > ADP > 2-MeS-ATP > ATP; UTP ineffective (54, 61). The physiologically most relevant active agonist at P2Y1-R is ADP, and although ADP does stimulate ciliary beating in HNE cells (Fig. 3A), this action appears to be indirect and independent of P2Y1-R. The most important piece of evidence favoring a non-P2Y1-R action of ADP is the lack of effect of the P2Y1-R hallmark agonist 2-MeS-ADP (28, 54) on CBF (Fig. 3B). Also consistent with this notion was the lack of an effect against ADP stimulation of CBF by the P2Y1 antagonist PAP (data not shown). Interestingly, rather than eliciting a peak and plateau response, the response in ciliary activity after ADP addition was sustained (Fig. 3A). Sustained CBF responses were similarly elicited by NECA and adenosine (Fig. 4). Finally, the effects of ADP were blocked by the adenosine receptor antagonist 8-SPT (Fig. 6), suggesting that the actions of ADP on ciliary activity are in fact due to its ectohydrolysis to adenosine (see below). The apparent absence of P2Y1-R in the regulation of ciliary activity is consistent with ion transport studies in the airways, which show a general lack of responsiveness of transepithelial Cl- secretion to ADP or 2-MeS-ADP (17, 33).

P2Y2-R. The P2Y2-R rank order potency (human) is as follows: ATP = UTP > Ap4p > ATPgamma S; 2-MeS-ATP and 2-MeS-ADP ineffective (44, 61). The data resulting from this study suggest strongly that P2Y2-R participates directly in the regulation of ciliary activity in human airway epithelial cells. The strongest evidence favoring this role is the concentration-dependent stimulation of ciliary activity on HNE cells by UTP (Fig. 1). The EC50 of this response for UTP was in the low range (4.7 µM), consistent with the affinity of P2Y2-R for agonist (10) and with the EC50 found for ATP in rabbit ciliated cells (40). Also indicating an approximate equipotency between ATP and UTP was the finding that the peak response in CBF to a saturating, 100 µM concentration of UTP was indistinguishable from that elicited by the same concentrations of ATP and ATPgamma S (Fig. 2). Given that ATP, UTP, and ATPgamma S are full agonists for P2Y2-R (28) and that ATP and UTP are equipotent at both the effector PLC (10, 56) and whole cell levels (2, 17, 47), the data from this study strongly support a direct role for this receptor in mediating the effects of nucleotide triphosphates on ciliary activity. Given the additional role of this receptor in the regulation of transepithelial chloride and fluid transport across airway ciliated cells (7, 8, 17), P2Y2-R is a major apical membrane signaling system, which regulates the two physiological modalities contributed by ciliated cells to the mucociliary clearance system. The third modality of the system, mucin secretion from goblet cells, is also regulated positively by ATP and UTP acting via P2Y2-R (2, 36). Hence, P2Y2-R signaling appears to play a central role in stimulating and possibly coordinating the individual elements comprising the mucociliary clearance system.

P2Y4-R. The P2Y4-R rank order potency (human) is as follows: UTP > GTP = ITP; ATP antagonizes (34, 61). Although UTP is an effective agonist at P2Y4-R, in addition to P2Y2-R, it is highly unlikely that the agonist acts at both receptors in airway ciliated cells. From the data presented (Fig. 2), ATP and UTP elicited equal responses in ciliary activity that, judging from rank order potency data, is more consistent with agonist interactions with P2Y2-R (34, 44). Furthermore, ATP, which is an agonist for the rat isoform of P2Y4-R (5), has been shown recently to be an antagonist against the human isoform (34), a feature of P2Y4-R inconsistent with our results. In the airways of the P2Y2-R knockout mouse, there was very little response in either intercellular Ca2+ mobilization or Cl- secretion to UTP, suggesting that this alternate receptor for UTP is not expressed in the airways (17, 32).

P2Y6-R. The P2Y6-R rank order potency (human) is as follows: UDP > UTP > ADP > ATP (15, 61). UDP elicited an increase in ciliary activity that was approximately one-half that elicited by UTP and ATP (Fig. 3C). UDP is also known to cause intercellular Ca2+ mobilization in cultured HNE cells (43), and of the adenine and uridine nucleotides tested, it elicited the largest purinergic response in Cl- secretion across the trachea of the P2Y2-R-/- mouse (17). Human P2Y6-R mRNA has been detected in lung (15), in cell lines derived from human airway epithelial cells (14), and in primary cultures of HNE cells (Ref. 43 and E. R. Lazarowski, personal communication). Hence, P2Y6-R appears to be the most likely receptor underlying the ciliostimulatory effects of UDP observed in this study (Fig. 3). Interpretation of this result as indicating UDP stimulation of a physiological activity through P2Y6-R, however, needs to be weighed carefully against other possibilities. For instance, the solution bathing the tissue could be contaminated with UTP, either directly, as received from the manufacturer, or in solution during the experiment as a result of the conversion of UDP to UTP by the action of ectonucleoside diphosphokinases. To minimize potential problems from contaminating UTP, our UDP stock and working solutions were treated with hexokinase/glucose (43, 49). Another possibility is that instead of acting at P2Y6-R, UDP is a partial agonist at P2Y2-R, as was reported originally (10, 44). This possibility was discounted, however, by studies in which the receptors were expressed independently in a null cell line. UDP, in the presence of hexokinase/glucose, had potent effects at P2Y6-R and was without demonstrable effects at P2Y2-R; in the absence of hexokinase, UDP-stimulated cells selectively express P2Y2-R (49). With these considerations, and given that the receptor is expressed in HNE cells (43), the ciliostimulatory effects of UDP observed in this study are most likely due to direct effects of the agonist at P2Y6-R.

P2Y11-R. The P2Y11-R rank order potency (human) is as follows: ATPgamma S > ATP > 2-MeS-ATP; UTP, ADP, and UDP ineffective (12, 16, 61). The P2Y11 receptor does not appear to be expressed in lung (12). Furthermore, the rank order potency of ATP, ATPgamma S, and UTP for this receptor conflicts substantially with the essentially equipotent effects of these agonists on ciliary activity (Fig. 2). Hence, it is highly unlikely that P2Y11-R regulates CBF in the airways.

P1 Receptor-Mediated Effects on Ciliary Activity

A2BAR. The A2BAR rank order potency (human) is as follows: NECA > adenosine > CGS-21680 (59, 61). Previous studies yielded a mixed view of adenosine effects on ciliary activity. With rabbit trachea in vitro adenosine was reported to inhibit CBF (66). The IC50 for this inhibition was ~50 µM, and NECA was ineffective. In contrast, with canine trachea in vivo adenosine delivered as a 10 µM aerosol-stimulated ciliary activity (68). Our data showing that adenosine and NECA are stimulatory in their actions on ciliary activity in HNE cells with low EC50s (Fig. 4) are consistent with the findings in canine trachea. With respect to the inhibitory effects of adenosine suggested for rabbit trachea, our results consequently suggest a re-evaluation to determine whether the regulation of ciliary activity in the mammalian airways is species specific. The results of this study do differ in an important way, however, with those from canine trachea. Namely, pretreatment with adenosine in the latter effort effectively blocked subsequent responses to aerosolized ATP (68). Although our experimental protocols did not duplicate those of the in vivo study, they do suggest that the mixed stimulation through A2BAR and P2Y2-R results in a sustained stimulation (Figs. 2, 4, and 7, and see below), not an inhibition. This difference may be due to the in vitro, rather than an in vivo, design of this study; delivery of bulk adenosine to the tracheal lumen of an intact animal (68) could affect ciliary activity indirectly in many ways by acting through neuronal and/or paracrine and autocrine pathways.

The effects of adenosine and NECA on HNE cell ciliary activity appear to be mediated through A2BAR, which couples to adenylyl cyclase through Gs (23, 52, 59). Consistent with this coupling, direct stimulation of adenylyl cyclase with forskolin enhances, and its inhibition by SQ-22536 after application of agonist suppresses, ciliary activity (Fig. 5, A and B). These two experiments, along with the stimulatory effects of adenosine and NECA with EC50s of ~1 and 0.1 µM (Fig. 4), respectively, indicate an A2 receptor (59). A2BAR was indicated as the specific adenoceptor mediating adenosine effects by the failure of the A2AAR-specific agonist CGS-21680 to affect ciliary activity (Fig. 5C). Consistent with its identification as a ciliostimulatory receptor, A2BAR has been implicated also in the effects of adenosine on Cl- secretion in the airways (42, 60, 65).

The actions of A2BAR agonists on ciliary activity were sustained for the duration of exposure (Fig. 4); by comparison, P2Y2-R activation by UTP elicited a peak and plateau response during a 15- to 20-min exposure to a saturating agonist concentration (Fig. 2). A sustained action of adenosine was also observed for the stimulation of Cl- secretion across canine trachea; short-circuit currents elevated in response to 2-chloroadenosine were undiminished for 1 h (60). Because the A2BAR antagonist 8-SPT (Fig. 6A) and the adenylyl cyclase inhibitor SQ-22536 (Fig. 5B) both inhibited the effects of agonist-stimulated ciliary beating in this study and because a permeant analog of cAMP also caused a sustained increase in CBF (Fig. 8), it is likely that the sustained actions of adenosine and NECA reflect a prolonged time course for receptor desensitization (52).

Mixed effects of ATP on ciliary activity. Since their discovery as potent stimulators of Cl- secretion (37, 47) and mucociliary clearance (4, 63) in the airways and the pharmacological identification (10) and cloning of the receptor (56) from airway epithelial cells, ATP and UTP have been viewed as effecting their actions through P2Y2-R. Consequently, we were surprised to find in this study that ATP elicited a sustained response in HNE cell ciliary activity relative to that elicited by UTP (Fig. 2). The decline in ciliary activity during a 20-min challenge with UTP could be due to P2Y2-R desensitization or to a decline in cellular messenger availability. Although intercellular Ca2+, which appears to underlie the ciliostimulatory effects of P2Y2-R activation in airway ciliated cells (40), has been observed to hold a stable plateau for at least 8-10 min after UTP activation of nasal epithelial cells (47, 55), it is conceivable that this signal declines with longer exposures. Hence, we cannot differentiate formally between these two possibilities. If the more sustained post-peak effects of ATP on ciliary activity are due to activation of an independent receptor/cellular messenger system, however, the actual mechanism of the post-peak decline elicited by UTP is irrelevant. As discussed immediately below, the weight of the evidence presented herein favors a scenario in which ATP elicits its effects through P2Y2-R and, after ectohydrolysis to adenosine, through A2BAR which appears to couple to adenylyl cyclase.

Three considerations support the notion of a dynamic coupling of these two receptors through ecto-nucleotide metabolism. First, the finding that ADP was also ciliostimulatory (Fig. 3A), despite the fact that the P2Y1-R hallmark agonist, 2-MeS-ADP, was ineffective (Fig. 3B), indicated that these unexpected actions were most likely indirect, and possibly due to the ectohydrolysis of the adenine nucleotides to adenosine. Second, that ADP and ATP were, indeed, hydrolyzed to adenosine was indicated by the inhibition of their putative adenosine-related effects by an A2 receptor antagonist; in the presence of 8-SPT, the effects of ADP on ciliary activity were abolished (Fig. 6B) and those of ATP were indistinguishable from the effects of UTP and ATPgamma S (compare Figs. 6C and 2). Lastly, the component of ATP's actions mediated by adenosine via A2BAR appeared to be independent of P2Y2-R. After minimization of the P2Y2-R contribution to CBF stimulation with a prolonged exposure to UTP, the addition of ATP caused a significant, concentration-dependent, 8-SPT-sensitive increment in ciliary activity (Fig. 7).

Hence, the effects of ATP on apical membrane P2Y2-R appears to be coupled indirectly to those of adenosine acting via A2BAR; the coupling appears to be effected by ectoenzymes which hydrolyze nucleotides (Fig. 9). Although nucleotide- and nucleoside-hydrolyzing ectoenzymes are best characterized in the nervous system (69, 70), they also occur on the surface of the airways (57) and most likely wherever P2 and P1 signaling systems are resident. The results of this work indicate that the ectohydrolysis of ATP to adenosine is remarkably rapid. For instance, ATP added to a 350-µl volume in the lumen of a fully ciliated culture disappears with a t0.5 of 5 min; if this volume is extrapolated to the ~50 µl in the film on such a culture under air:liquid conditions, the t0.5 would be closer to 30 s (57). As indicated in Fig. 9, ATP was supplied by continuous superfusion in our experiments. As ATP is degraded in this system, the bulk of the hydrolytic products, including adenosine, are most likely lost to the superfusion. Additionally, adenosine is most likely removed from the luminal surface across the apical membrane of the epithelial cells by a specific transporter (3). Nonetheless, the results indicate that adenosine concentrations in the near-membrane environment are sufficiently high as to activate A2BAR and stimulate ciliary beating. These concentrations of adenosine may be estimated from the data presented in Figs. 4 and 7. Comparing the 100 µM ATP-induced Delta CBF with the concentration-response data for adenosine (Fig. 4), we estimate the concentration of adenosine in the vicinity of A2BAR to be near its EC50 for agonist at ~0.4 µM. The adenosine concentration corresponding to the increase in CBF elicited by 10 µM ATP was too low to estimate from the data in Fig. 4.


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Fig. 9.   Airway lumen near membrane purinergic signaling system. The system is proposed to consist of the P2Y2 and A2B purinergic receptors coupled by ectoenzymes responsible for hydrolyzing adenine nucleotides to adenosine (AD0). The system depicted also uses ATP secretion (mechanism presently unknown) as the in vivo source of ATP and an adenosine uptake system. In the experimental system used in this study, the superfusion system supplied ATP, ex vivo, and removed all nucleotide hydrolysis products at constant rates.

Similar to adenosine acting via A2BAR after its ectohydrolysis from ATP, UDP could also stimulate ciliary activity via P2Y6-R after ectohydrolysis from UTP. Given an EC50 for this receptor of ~0.2 µM (15, 49) and the prolonged time for P2Y6-R desensitization reported in other systems (62), some portion of the plateau phase of the UTP/P2Y2-R response in our experiments (Fig. 2) could be due to stimulation of P2Y6-R by UDP. Assessment of this possibility is made difficult, however, by the lack of an effective antagonist.

In vivo, of course, nucleotide triphosphates are available only from cellular sources and, as noted above, ATP and UTP secretion have been demonstrated in vitro (19, 29, 41, 53, 64, 67). Given that airway surface liquid in vivo is relatively static and its volume minimal, we speculate that ATP secretion in the near-membrane environment could result in concentrations that activate P2Y2-R in an autocrine manner. Furthermore, because the agonist affinity of A2BAR is effectively 10-fold higher than that of P2Y2-R, ectohydrolysis of this secreted ATP to adenosine conceivably could result in A2BAR activation. Lastly, we speculate that P2Y2-R and A2BAR may be clustered with nucleotide degrading ectoenzymes to form signaling complexes that inform the epithelial cells about important physical aspects of the airway surface liquid in vivo.

In summary, we have pharmacologically identified P2Y2-R as the major nucleotide triphosphate purinoceptor regulating ciliary activity in airway epithelial cells. ATP and UTP appear to be equipotent at this receptor in terms of their effects on ciliary activity. UDP also stimulates ciliary beating, most likely via the P2Y6 purinoceptor, though its apparent maximal effect is approximately one-half that of the stimulation associated with ATP and UTP and mediated via P2Y2-R. A third purinoceptor, the adenosine A2B receptor, is also present on ciliated cells, and through it adenosine is capable of exerting a maximal effect on activity that is of the same order of magnitude as for ATP and UTP. Last, ATP and ADP appear to be hydrolyzed to adenosine, and, at least under the conditions of our experiments, this breakdown appears to occur at rates sufficiently high that the nucleotides stimulate ciliary activity indirectly through the A2B receptor.


    NOTE ADDED IN PROOF

As this manuscript was being submitted, a report appeared showing stimulation of ciliary activity in oviduct epithelial cells by ATP and UTP and by adenosine (Morales B, Barrera N, Uribe P, Mora C, and Villalon M. Functional cross talk after activation of P2 and P1 receptors in oviductal ciliated cells. Am J Physiol Cell Physiol 279: C658-C669, 2000). Whereas adenosine acts via A2BAR to stimulate ciliary activity in airway epithelial cells, in oviduct epithelial cells adenosine acts via A2AAR.


    ACKNOWLEDGEMENTS

We express our gratitude to Drs. Jackson Stutts, Eduardo Lazarowski, and Richard Boucher for advice during the course of this study and to Dr. Sam Shaver of Inspire Pharmaceuticals for the high-performance liquid chromatography analysis of the ATP used in these studies.


    FOOTNOTES

Financial support for this work was received under the elective portion of a research contract with Inspire Pharmaceuticals, Inc.

Address for reprint requests and other correspondence: C. W. Davis, 6009 Thurston-Bowles, CB 7248, Univ. of North Carolina, Chapel Hill, NC 27599-7248 (E-mail: cwdavis{at}med.unc.edu).

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. Section 1734 solely to indicate this fact.

Received 21 August 2000; accepted in final form 5 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abbracchio, MP, and Burnstock G. Purinergic signalling: pathophysiological roles. Jpn J Pharmacol 78: 113-145, 1998[ISI][Medline].

2.   Abdullah, LH, Davis SW, Burch L, Yamauchi M, Randell SH, Nettesheim P, and Davis CW. P2u purinoceptor regulation of mucin secretion in SPOC1 cells, a goblet cell line from the airways. Biochem J 316: 943-951, 1996[ISI][Medline].

3.   Baldwin, SA, Mackey JR, Cass CE, and Young JD. Nucleoside transporters: molecular biology and implications for therapeutic development. Mol Med Today 5: 216-224, 1999[ISI][Medline].

4.   Bennett, WD, Olivier KN, Zeman KL, Hohneker KW, Boucher RC, and Knowles MR. Effect of uridine 5'-triphosphate plus amiloride on mucociliary clearance in adult cystic fibrosis. Am J Respir Crit Care Med 153: 1796-1801, 1996[Abstract].

5.   Bogdanov, YD, Wildman SS, Clements MP, King BF, and Burnstock G. Molecular cloning and characterization of rat P2Y4 nucleotide receptor. Br J Pharmacol 124: 428-430, 1998[Abstract].

6.   Boitano, S, Dirksen ER, and Sanderson MJ. Intercellular propagation of calcium waves mediated by inositol trisphosphate. Science 258: 292-295, 1992[ISI][Medline].

7.   Boucher, RC. Human airway ion transport. Part one. Am J Respir Crit Care Med 150: 271-281, 1994[ISI][Medline].

8.   Boucher, RC. Human airway ion transport. Part two. Am J Respir Crit Care Med 150: 581-593, 1994[ISI][Medline].

9.   Boyer, JL, Romero-Avila T, Schachter JB, and Harden TK. Identification of competitive antagonists of the P2Y1 receptor. Mol Pharmacol 50: 1323-1329, 1996[Abstract].

10.   Brown, HA, Lazarowski ER, Boucher RC, and Harden TK. Evidence that UTP and ATP regulate phospholipase C through a common extracellular 5'-nucleotide receptor in human airway epithelial cells. Mol Pharmacol 40: 648-655, 1991[Abstract].

11.   Burnstock, G. P2X receptors in sensory neurones. Br J Anaesth 84: 476-488, 2000[Abstract].

12.   Communi, D, Govaerts C, Parmentier M, and Boeynaems JM. Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase. J Biol Chem 272: 31969-31973, 1997[Abstract/Free Full Text].

13.   Communi, D, Janssens R, Suarez-Huerta N, Robaye B, and Boeynaems J. Advances in signalling by extracellular nucleotides. The role and transduction mechanisms of P2Y receptors. Cell Signal 12: 351-360, 2000[ISI][Medline].

14.   Communi, D, Paindavoine P, Place GA, Parmentier M, and Boeynaems JM. Expression of P2Y receptors in cell lines derived from the human lung. Br J Pharmacol 127: 562-568, 1999[Abstract/Free Full Text].

15.   Communi, D, Parmentier M, and Boeynaems JM. Cloning, functional expression and tissue distribution of the human P2Y6 receptor. Biochem Biophys Res Commun 222: 303-308, 1996[ISI][Medline].

16.   Communi, D, Robaye B, and Boeynaems JM. Pharmacological characterization of the human P2Y11 receptor. Br J Pharmacol 128: 1199-1206, 1999[Abstract/Free Full Text].

17.   Cressman, VL, Lazarowski E, Homolya L, Boucher RC, Koller BH, and Grubb BR. Effect of loss of P2Y2 receptor gene expression on nucleotide regulation of murine epithelial Cl- transport. J Biol Chem 274: 26461-26468, 1999[Abstract/Free Full Text].

18.   Davis, CW. Goblet cells: physiology and pharmacology. In: Airway Mucus: Basic Mechanisms and Clinical Perspectives, edited by Rogers DF, and Lethem MI.. Basel: Berkhauser, 1997, p. 150-177.

19.   Dezaki, K, Tsumura T, Maeno E, and Okada Y. Receptor-mediated facilitation of cell volume regulation by swelling-induced ATP release in human epithelial cells. Jpn J Physiol 50: 235-241, 2000[ISI][Medline].

20.   Ding, Y, Cesare P, Drew L, Nikitaki D, and Wood JN. ATP, P2X receptors and pain pathways. J Auton Nerv Syst 81: 289-294, 2000[ISI][Medline].

21.   Donaldson, SH, and Boucher RC. Therapeutic applications for nucleotides in lung disease. In: The P2 Nucleotide Receptors, edited by Turner JT, Weisman GA, and Fedan JS.. Totowa, NJ: Humana, 1998, p. 413-424.

22.   Erlinge, D. Extracellular ATP: a growth factor for vascular smooth muscle cells. Gen Pharmacol 31: 1-8, 1998[Medline].

23.   Feoktistov, I, and Biaggioni I. Adenosine A2B receptors. Pharmacol Rev 49: 381-402, 1997[Abstract/Free Full Text].

24.   Forsythe, P, and Ennis M. Adenosine, mast cells and asthma. Inflamm Res 48: 301-307, 1999[ISI][Medline].

25.   Geary, CA, Davis CW, Paradiso AM, and Boucher RC. Role of CNP in human airways: cGMP-mediated stimulation of ciliary beat frequency. Am J Physiol Lung Cell Mol Physiol 268: L1021-L1028, 1995[Abstract/Free Full Text].

26.   Gray, TE, Guzman K, Davis CW, Abdullah LH, and Nettesheim P. Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am J Respir Cell Mol Biol 14: 104-112, 1996[Abstract].

27.   Green, HN, and Stoner HB. Biological actions of the adenine nucleotides. London: H. K. Lewis, 1950, p. 1-221.

28.   Harden, TK, Boyer JL, and Nicholas RA. P2-purinergic receptors: subtype-associated signaling responses and structure. Annu Rev Pharmacol Toxicol 35: 541-579, 1995[ISI][Medline].

29.   Harden, TK, and Lazarowski ER. Release of ATP and UTP from astrocytoma cells. Prog Brain Res 120: 135-143, 1999[ISI][Medline].

30.   Hazama, A, Hayashi S, and Okada Y. Cell surface measurements of ATP release from single pancreatic beta cells using a novel biosensor technique. Pflügers Arch 437: 31-35, 1998[ISI][Medline].

31.   Homolya, L, Steinberg TH, and Boucher RC. Cell to cell communication in response to mechanical stress via bilateral release of ATP and UTP in polarized epithelia. J Cell Biol 150: 1349-1360, 2000[Abstract/Free Full Text].

32.   Homolya, L, Watt WC, Lazarowski ER, Koller BH, and Boucher RC. Nucleotide-regulated calcium signaling in lung fibroblasts and epithelial cells from normal and P2Y2 receptor (-/-) mice. J Biol Chem 274: 26454-26460, 1999[Abstract/Free Full Text].

33.   Inglis, SK, Collett A, McAlroy HL, Wilson SM, and Olver RE. Effect of luminal nucleotides on Cl- secretion and Na+ absorption in distal bronchi. Pflügers Arch 438: 621-627, 1999[ISI][Medline].

34.   Kennedy, C, Qi AD, Herold CL, Harden TK, and Nicholas RA. ATP, an agonist at the rat P2Y4 receptor, is an antagonist at the human P2Y4 receptor. Mol Pharmacol 57: 926-931, 2000[Abstract/Free Full Text].

35.   Kim, KC, McCracken K, Lee BC, Shin CY, Jo MJ, Lee CJ, and Ko KH. Airway goblet cell mucin: its structure and regulation of secretion. Eur Respir J 10: 2644-2649, 1997[Abstract/Free Full Text].

36.   Kim, KC, Park HR, Shin CY, Akiyama T, and Ko KH. Nucleotide-induced mucin release from primary hamster tracheal surface epithelial cells involves the P2U purinoceptor. Eur J Respir Dis 9: 542-548, 1996.

37.   Knowles, MR, Clarke LL, and Boucher RC. Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis. N Engl J Med 325: 533-538, 1991[Abstract].

38.   Knowles, MR, Olivier K, Noone P, and Boucher RC. Pharmacologic modulation of salt and water in the airway epithelium in cystic fibrosis. Am J Respir Crit Care Med 151: S65-S69, 1995[ISI][Medline].

39.   Knowles, MR, Olivier KN, Hohneker KW, Robinson J, Bennett WD, and Boucher RC. Pharmacologic treatment of abnormal ion transport in the airway epithelium in cystic fibrosis. Chest 107: 71S-76S, 1995[Abstract/Free Full Text].

40.   Korngreen, A, and Priel Z. Purinergic stimulation of rabbit ciliated airway epithelia: control by multiple calcium sources. J Physiol (Lond) 497: 53-66, 1996[Abstract].

41.   Lazarowski, ER, Boucher RC, and Harden TK. Constitutive release of ATP and evidence for major contribution of ecto-nucleotide pyrophosphatase and nucleoside diphosphokinase to extracellular nucleotide concentrations. J Biol Chem 275: 31061-31068, 2000[Abstract/Free Full Text].

42.   Lazarowski, ER, Mason SJ, Clarke L, Harden TK, and Boucher RC. Adenosine receptors on human airway epithelia and their relationship to chloride secretion. Br J Pharmacol 106: 774-782, 1992[Abstract].

43.   Lazarowski, ER, Paradiso AM, Watt WC, Harden TK, and Boucher RC. UDP activates a mucosal-restricted receptor on human nasal epithelial cells that is distinct from the P2Y2 receptor. Proc Natl Acad Sci USA 94: 2599-2603, 1997[Abstract/Free Full Text].

44.   Lazarowski, ER, Watt WC, Stutts MJ, Boucher RC, and Harden TK. Pharmacological selectivity of the cloned human P2U-purinoceptor: potent activation by diadenosine tetraphosphate. Br J Pharmacol 116: 1619-1627, 1995[Abstract].

45.   Lewis, CJ, Ennion SJ, and Evans RJ. P2 purinoceptor-mediated control of rat cerebral (pial) microvasculature; contribution of P2X and P2Y receptors. J Physiol (Lond) 527: 315-324, 2000[Abstract/Free Full Text].

46.   Ma, W, Korngreen A, Uzlaner N, Priel Z, and Silberberg SD. Extracellular sodium regulates airway ciliary motility by inhibiting a P2X receptor. Nature 400: 894-897, 1999[ISI][Medline].

47.   Mason, SJ, Paradiso AM, and Boucher RC. Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium. Br J Pharmacol 103: 1649-1656, 1991[Abstract].

48.   Matsui, H, Randell SH, Peretti SW, Davis CW, and Boucher RC. Coordinated clearance of periciliary liquid and mucus from airway surfaces. J Clin Invest 102: 1125-1131, 1998[Abstract/Free Full Text].

49.   Nicholas, RA, Watt WC, Lazarowski ER, Li Q, and Harden K. Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor. Mol Pharmacol 50: 224-229, 1996[Abstract].

50.   Noone, PG, Bennett WD, Regnis JA, Zeman KL, Carson JL, King M, Boucher RC, and Knowles MR. Effect of aerosolized uridine-5'-triphosphate on airway clearance with cough in patients with primary ciliary dyskinesia. Am J Respir Crit Care Med 160: 144-149, 1999[Abstract/Free Full Text].

51.   North, RA, and Surprenant A. Pharmacology of cloned P2X receptors. Annu Rev Pharmacol Toxicol 40: 563-580, 2000[ISI][Medline].

52.   Olah, ME, and Stiles GL. The role of receptor structure in determining adenosine receptor activity. Pharmacol Ther 85: 55-75, 2000[ISI][Medline].

53.   Ostrom, RS, Gregorian C, and Insel PA. Cellular release of and response to ATP as key determinants of the set-point of signal transduction pathways. J Biol Chem 275: 11735-11739, 2000[Abstract/Free Full Text].

54.   Palmer, RK, Boyer JL, Schachter JB, Nicholas RA, and Harden TK. Agonist action of adenosine triphosphates at the human P2Y1 receptor. Mol Pharmacol 54: 1118-1123, 1998[Abstract/Free Full Text].

55.   Paradiso, AM, Brown HA, Ye H, Harden TK, and Boucher RC. Heterogeneous responses of cell Ca2+ in human airway epithelium. Exp Lung Res 25: 277-290, 1999[ISI][Medline].

56.   Parr, CE, Sullivan DM, Paradiso AM, Lazarowski ER, Burch LH, Olsen JC, Erb L, Weisman GA, Boucher RC, and Turner JT. Cloning and expression of a human P2U nucleotide receptor, a target for cystic fibrosis pharmacotherapy. Proc Natl Acad Sci USA 91: 3275-3279, 1994[Abstract].

57.  Picher M and Boucher RC. Metabolism of extracellular nucleotides in human airways by a multi-enzyme system. Drug Dev Res. In press.

58.   Poulsen, CR, Bokvist K, Olsen HL, Hoy M, Capito K, Gilon P, and Gromada J. Multiple sites of purinergic control of insulin secretion in mouse pancreatic beta-cells. Diabetes 48: 2171-2181, 1999[Abstract].

59.   Poulsen, SA, and Quinn RJ. Adenosine receptors: new opportunities for future drugs. Bioorg Med Chem 6: 619-641, 1998[ISI][Medline].

60.   Pratt, AD, Clancy G, and Welsh MJ. Mucosal adenosine stimulates chloride secretion in canine tracheal epithelium. Am J Physiol Cell Physiol 251: C167-C174, 1986[Abstract/Free Full Text].

61.   Ralevic, V, and Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 50: 413-492, 1998[Abstract/Free Full Text].

62.   Robaye, B, Boeynaems JM, and Communi D. Slow desensitization of the human P2Y6 receptor. Eur J Pharmacol 329: 231-236, 1997[ISI][Medline].

63.   Sabater, JR, Mao YM, Shaffer C, James MK, O'Riordan TG, and Abraham WM. Aerosolization of P2Y2-receptor agonists enhances mucociliary clearance in sheep. J Appl Physiol 87: 2191-2196, 1999[Abstract/Free Full Text].

64.   Sauer, H, Hescheler J, and Wartenberg M. Mechanical strain-induced Ca2+ waves are propagated via ATP release and purinergic receptor activation. Am J Physiol Cell Physiol 279: C295-C307, 2000[Abstract/Free Full Text].

65.   Stutts, MJ, Fitz JG, Paradiso AM, and Boucher RC. Multiple modes of regulation of airway epithelial chloride secretion by extracellular ATP. Am J Physiol Cell Physiol 267: C1442-C1451, 1994[Abstract/Free Full Text].

66.   Tamaoki, J, Kondo M, and Takizawa T. Adenosine-mediated cyclic AMP-dependent inhibition of ciliary activity in rabbit tracheal epithelium. Am Rev Respir Dis 139: 441-445, 1989[ISI][Medline].

67.   Watt, WC, Lazarowski ER, and Boucher RC. Cystic fibrosis transmembrane regulator-independent release of ATP. Its implications for the regulation of P2Y2 receptors in airway epithelia. J Biol Chem 273: 14053-14058, 1998[Abstract/Free Full Text].

68.   Wong, LB, and Yeates DB. Luminal purinergic regulatory mechanisms of tracheal ciliary beat frequency. Am J Respir Cell Mol Biol 7: 447-454, 1992[ISI][Medline].

69.   Zimmermann, H, and Braun N. Extracellular metabolism of nucleotides in the nervous system. J Auton Pharmacol 16: 397-400, 1996[ISI][Medline].

70.   Zimmermann, H, and Braun N. Ecto-nucleotidases-molecular structures, catalytic properties, and functional roles in the nervous system. Prog Brain Res 120: 371-385, 1999[ISI][Medline].


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