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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 ![]() |
RESULTS |
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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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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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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) (ATPS, 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 ATP
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/ATP
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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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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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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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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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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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DISCUSSION |
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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 > ATPS; 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 ATP
S (Fig. 2). Given that ATP, UTP, and ATP
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:
ATPS > 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, ATP
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 ClMixed 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.
|
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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.
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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.
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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.
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