Adenosine A3 receptor-mediated potentiation
of mucociliary transport and epithelial ciliary motility
Manako
Taira,
Jun
Tamaoki,
Kazuyuki
Nishimura,
Junko
Nakata,
Mitsuko
Kondo,
Hisashi
Takemura, and
Atsushi
Nagai
First Department of Medicine, Tokyo Women's Medical University
School of Medicine, Tokyo 162-8666, Japan
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ABSTRACT |
To examine the effect of adenosine
A3 receptor stimulation on airway mucociliary clearance, we
measured transport of Evans blue dye in rabbit trachea in vivo and
ciliary motility of epithelium by the photoelectric method in vitro.
Mucociliary transport was enhanced dose dependently by the selective
A3 agonist
N6-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine
(IB-MECA) and to a lesser extent by the less-selective
N6-2-(4-amino-3-iodophenyl)ethyladenosine,
whereas the A1 agonist N-cyclopentyladenosine (CPA) and the A2 agonist
CGS-21680 had no effect. The effect of IB-MECA was abolished by
pretreatment with the selective A3 antagonist MRS-1220
but not by the A1 antagonist 1,3-dipropyly-8-cyclopentylxanthine or the A2 antagonist
3,7-dimethyl-L-propargylxanthine. Epithelial ciliary beat
frequency was increased by IB-MECA in a concentration-dependent manner,
the maximal increase being 33%, and this effect was inhibited by
MRS-1220. The IB-MECA-induced ciliary stimulation was not altered by
the Rp diastereomer of cAMP but was greatly inhibited by
Ca2+-free medium containing BAPTA-AM. Incubation with
IB-MECA increased intracellular Ca2+ contents. Therefore,
A3 agonist enhances airway mucociliary clearance probably
through Ca2+-mediated stimulation of ciliary motility of
airway epithelium.
airway epithelium; adenine nucleotide; ciliary beat frequency; calcium
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INTRODUCTION |
MUCOCILIARY
CLEARANCE is one of the most important nonspecific host defense
mechanisms in the lungs whereby locally produced biological debris and
trapped inhaled particles and bacteria are removed from the conducting
airways of the respiratory tract (35). The rate of mucus
transport toward the oropharynx depends on ciliary motion of airway
epithelium, physicochemical properties of periciliary fluid, and mucus
secretion (27, 30). Because the impairment of mucociliary
transport is a well-documented feature of chronic airway diseases, such
as chronic bronchitis, bronchiectasis, cystic fibrosis, and asthma
(2, 31), stimulation of ciliary motility may be desirable
in the treatment of such conditions.
Adenine nucleotides and nucleosides are present in the airways and
induce a variety of cellular responses via cell surface receptors.
Previous studies have shown that triphosphate nucleotides, such as ATP
and UTP, stimulate P2Y2 receptors and promote
transepithelial chloride transfer and mucociliary transport (13,
16, 20). On the other hand, nucleosides exert their effects
through stimulation of adenosine receptors, which have been classified
into three main subtypes based on both functional characterization and
gene cloning [A1, A2 (A2a and
A2b), and A3 (7, 19)]. Adenosine A3 receptors are richly expressed in human lungs
(25), and recent studies have reported that A3
receptor agonists may modulate allergic reactions by acting on
eosinophils, i.e., stimulation of the receptors potentiates or
attenuates migration of eosinophils (13, 32) and inhibits
degranulation and superoxide release (4, 5). Regarding the
role of adenosine receptors in airway mucociliary transport, we have
previously shown that A1 agonist inhibits ciliary motility
of airway epithelium (34), but the effect of
A3 agonist remains unknown. Therefore, in the present
study, to elucidate whether stimulation of adenosine A3
receptors affects mucociliary transport and, if so, whether the
alteration of epithelial ciliary motility is involved and what the
mechanism of action is, we measured mucociliary transport in the rabbit
tracheal mucosa by the Evans blue method in vivo and ciliary beat
frequency (CBF) of the tracheal epithelium in vitro.
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METHODS |
Agents.
The following drugs were purchased from Research Biochemicals (Natick,
MA):
N6-2-(4-amino-3-iodophenyl)ethyladenosine
(APNEA), N-cyclopentyladenosine (CPA),
3,7-dimethyl-L-propargylxanthine (DMPX), and MRS-1220. N6-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine
(IB-MECA), CGS-21680, and 1,3-dipropyly-8-cyclopentylxanthine
(DPCPX) were purchased from Tocris Cookson (Bristol, UK). The Rp
diastereomer of cAMP (Rp-cAMPS) was supplied by BIOLOG Life Science
Institute (Bremen, Germany). Evans blue, ionomycin, and EGTA were
purchased from Sigma-Aldrich (St. Louis, MO).
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM) and fura 2-AM were purchased from Dojin
(Kumamoto, Japan).
Stock solutions of all adenosine receptor agonists and antagonists were
made in dimethyl sulfoxide (DMSO) to concentrations in the range of
1-4 × 10
1 M and then diluted in sterile
saline. The final concentration of DMSO present at the highest drug
concentrations did not exceed 0.05%, a concentration that had no
effect on mucociliary transport rate or ciliary beat frequency in our
experimental system.
Measurement of mucociliary transport.
The experiments were approved by the Committee on Animal Research,
Tokyo Women's Medical University. Specific pathogen-free male Japanese
white rabbits (2.0-2.4 kg) were anesthetized with intraperitoneal
-chloralose (50 mg/kg) and urethan (500 mg/kg), and the trachea was
explored and covered with a Lucite moist chamber maintained at 37°C.
A polyethylene tube was cannulated 2 mm above the carina, and
mechanical ventilation (tidal volume 10 ml/kg, respiratory rate 60/min)
was performed (model SN-480-7; Shinano, Tokyo, Japan). The
cartilage rings of upper trachea were incised transaxially, and the
surface of the membranous portion was exposed; 1 µl of 0.5% Evans
blue dye in sterile saline warmed at 37°C was gently placed on the
membranous portion by a microsyringe 1.5 cm above the carina (Fig.
1). Immediately after this procedure, either the selective A1 agonist CPA, the selective
A2 agonist CGS-21680, or the selective A3
agonist IB-MECA (1) at a dose of 30 µg/kg was given from
the left femoral vein as a bolus injection. Next, 2.5, 5, 10, and 20 min after the injection, the trachea was removed and cut into four
1.0-cm-long transverse sections sequentially from the carina toward the
larynx (section 1 to section 4). Because Evans
blue dye placed on section 2 can be transported toward
section 3 and then section 4, higher levels of
Evans blue contents in sections 3 and 4 were
assumed to represent increased tracheal mucociliary clearance. In our
preliminary experiment, during a 20-min observation, Evans blue
transported to a section more cephalad than section 4 was
<5% of the total, even in the animals treated with 100 µg/kg
IB-MECA. For all sections, Evans blue dye was extracted in 2 ml of
formamide, kept in water at 40°C for 24 h, and measured by a
spectrophotometer (V-550; Nihon Bunko, Tokyo, Japan) at 620 nm. The
Evans blue level in each tracheal section was expressed as a percentage
of the total amount of the dye in sections 1-4.

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Fig. 1.
Time-dependent changes in Evans blue (EB) dye contents in
rabbit tracheal sections after application of the dye to the mucosal
surface 1.5 cm above the carina. The EB level in each tracheal section
was expressed as a percentage of the total amount of the dye in
sections 1-4. Values are means ± SE;
n = 10 experiments for each point. *P < 0.05 and **P < 0.01, significantly different from
the values at time 0.
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For the assessment of dose-dependent effects of adenosine receptor
agonists, various doses (1-100 µg/kg) of IB-MECA, the
less-selective A3 agonist APNEA (6), CPA, and
CGS-21680 were given, and, 10 min later, the contents of Evans blue in
section 4 were measured. To confirm whether the stimulation
of mucociliary transport by IB-MECA is mediated by A3
receptors, the effects of pretreatment with the following drugs were
tested: the selective A1 antagonist DPCPX (1 mg/kg), the
selective A2 antagonist DMPX (1 mg/kg), and the selective
A3 antagonist MRS-1220 (100 µg/kg; see Ref.
11). After 15 min of the intravenous injection of an
antagonist, IB-MECA (1-100 µg/kg) was given, and the Evans blue
contents in section 4 were determined 10 min later. These
adenosine receptor antagonists were determined in pilot studies to have
no effect per se on the transport of Evans blue dye.
Measurement of CBF.
The method for the measurement of CBF of rabbit tracheal epithelium has
been described in detail previously (15). Briefly, the
mucosa of excised rabbit trachea was cut into small pieces (1-2
mm2) and rinsed several times with Hanks' balanced salt
solution (HBSS). Next, the tissues were placed on a coverglass (18 × 24 mm) coated with human placental collagen (5.8 µg/cm2; Sigma-Aldrich) in a petri dish and incubated in
Ham's nutrient F-12 medium containing 10 µg/ml insulin, 5 µg/ml
transferrin, 25 ng/ml epidermal growth factor, 7.5 µg/ml endothelial
cell growth supplement, 50 U/ml penicillin, 50 µg/ml streptomycin,
and 50 µg/ml gentamicin at 37°C in a CO2 incubator
(95% air-5% CO2). On the seventh day of incubation, the
cover glass on which the cultured explant was adhered was mounted in a
Rose chamber (Sasaki Medical, Tokyo, Japan) that was then placed on the
temperature-controlled stage (37°C) of the microscope equipped with a
phase-contrast condenser and an on-base type of halogen illuminator
(Optiphoto-XF; Nikon, Tokyo, Japan). The photometer (NFX-II; Hamamatsu
Photonics, Hamamatsu, Japan) with a built-in periplanatic eyepiece, a
limiting aperture, and a lateral focusing telescope was attached to the head of the microscope. Because of the beating action of cilia, light
from the illuminator passed through the preparation in varying intensities. These variations of light intensity were detected by the
photometer and transduced to voltage impulses, which were recorded by a
pen recorder (VP-6213A; Panasonic, Osaka, Japan). Measurements of CBF
were averaged from clumps of two or more cells with free borders devoid
of debris. In our separate experiment, variation of CBF among
preparations was <0.6 Hz (< 7%), and there were no significant
differences in the variations between experimental groups. In addition
to CBF, we assessed ciliary coordination by the image of the beating
pattern recorded on a video camera (VO-5800; Sony, Tokyo, Japan) with a
videocassette recorder capable of freeze-frame replay. Ciliary
discoordination was defined as the loss of a metachronal wave on the
free border of the cell clump (26, 33).
Before the measurement of CBF, the preparation was allowed to stabilize
for 30 min in Krebs-Henseleit (KH) solution of the following
composition (in mM): 118 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 25.5 NaHCO3, and 5.6 D-glucose, with pH adjusted to
7.4 and warming at 37°C. After determination of the baseline CBF,
medium was drained off the chamber and replaced with KH solution containing CPA, CGS-21680, IB-MECA, or APNEA at a concentration of
10
8 M, and CBF was continuously recorded for 20 min. To
examine the effects of adenosine receptor antagonists, the epithelial
cells were incubated for 15 min with DPCPX, DMPX, or MRS-1220 at
10
7 M, and the maximal response of CBF to the subsequent
addition of 10
8 M IB-MECA was determined. To study the
dose-response relationship, IB-MECA (10
10 to
10
6 M) was applied to the chamber, and the highest
recorded value in response to each concentration was determined. In
this experiment, only one dose of IB-MECA was given per preparation,
because tachyphylaxis is a characteristic of adenosine A3
receptor activation (21).
Because both intracellular cAMP and Ca2+ play a major role
in the regulation of airway epithelial ciliary motility (15,
24), we assessed the contributions of these to the action of
IB-MECA. To do so, the cells were treated for 15 min with the cAMP
antagonist Rp-cAMPS (10
4 M) or with Ca2+-free
KH solution containing both EGTA (5 × 10
3 M) and
the intracellular Ca2+-chelating agent BAPTA-AM (5 × 10
5 M), and the concentration-response curves for IB-MECA
were generated. In our preliminary experiment, Rp-cAMPS
(10
4 M) per se had no effect on the baseline value of CBF
but abolished the increase in CBF produced by 8-bromo-cAMP
(10
6 M). In addition, Ca2+-free medium
containing EGTA and BAPTA-AM decreased baseline CBF by only 7.3 ± 1.1% (P < 0.05, n = 9).
Measurement of intracellular Ca2+.
To confirm whether the effects of adenosine receptor agonists on
ciliary motility were associated with Ca2+ mobilization, we
measured intracellular levels of Ca2+
([Ca2+]i) in response to 10
8 M
CPA, CGS-21680, and IB-MECA. We also determined the effect of
pretreatment for 15 min with MRS-1220 (10
7 M) on
IB-MECA-induced [Ca2+]i responses. The cells
grown on a cover glass were washed with HBSS that contained 10 mM HEPES
(pH 7.4) and loaded with fura 2-AM for 20 min at 37°C. The cover
glass was then washed again and held with a rigid holder in a
continuously stirred cuvette containing HEPES-buffered HBSS maintained
at 37°C, and the fluorescence intensity was measured with a
spectrophotometer (CAF-110; Japan Spectroscopic, Tokyo, Japan). For
excitation of fura 2 fluorescence, ultraviolet lights of 340- and
380-nm wave lengths were automatically exchanged at a rate of 50 Hz,
the emitted light from cells [fluorescence at 340 (F340)
and 380 nm (F380)] was detected with a photomultiplier tube through a 510 ± 10-nm band-pass filter, and the fluorescence intensity ratio, F340/F380, was automatically
calculated. Maximal and minimal values for the ratio were determined in
the presence of ionomycin (10
5 M) and EGTA (5 × 10
3 M), respectively, and
[Ca2+]i was calculated using the external
calibration standards and formula previously described
(9).
Statistics.
All values were expressed as means ± SE. The drug concentrations
producing a half-maximal response of CBF (EC50 values) were calculated using the concentration-effect curves by nonlinear regression analysis. Statistical analysis was performed by ANOVA using
Scheffé's F-test (Unistat 3.0 statistical
software; Megalon, Novato, CA), and a P value <0.05 was
considered statistically significant.
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RESULTS |
Mucociliary transport.
Spontaneous mucociliary transport in the rabbit tracheal mucosa is
shown in Fig. 1. The contents of Evans blue dye in section 2, where the dye had been placed at time 0 on the
mucosal surface, gradually decreased. During the first 10 min, the
decrease in Evans blue contents in section 2 was accompanied
by corresponding increases in the dye contents in sections 3 and 4. Next, the contents in section 3 decreased,
whereas those in section 4 increased. These findings
indicate that Evans blue was transported from the lower trachea toward
the larynx in the control condition.
The effects of intravenous administration of adenosine A1,
A2, and A3 receptor agonists (30 µg/kg) on
mucociliary transport are demonstrated in Fig.
2. When CPA was given, Evans blue
contents at 10 min tended to be greater in section 2 and
were significantly smaller in section 4 compared with
controls (P < 0.05, n = 10), suggesting that mucociliary transport was retarded. Administration of
CGS-21680 was without effect, and IB-MECA caused a decrease in Evans
blue contents in section 2 (P < 0.01, n = 10) and increases in the dye contents in
sections 3 (P < 0.05, n = 10) and 4 (P < 0.01, n = 10), indicating a potentiation of mucociliary transport. As shown in
Fig. 3, IB-MECA enhanced tracheal
mucociliary transport in a dose-dependent manner, where the maximal
increase in Evans blue contents in section 4 was observed at
a dose of 30 µg/kg (9.7 ± 0.9 to 24.0 ± 1.5%,
P < 0.01, n = 8) and the effect of a
higher dose (100 µg/kg) was less potent. Administration of APNEA likewise caused a dose-dependent but weaker stimulation of Evans blue
transport, CGS-21680 had no significant effect, and CPA at 30 and 100 µg/kg caused an inhibition of the transport (P < 0.05 for each dose). The IB-MECA-induced increase in Evans blue
transport was not altered by pretreatment with DMPX but was almost
completely abolished by MRS-1220 (Fig.
4). Pretreatment with DPCPX only enhanced the response to 100 µg/kg IB-MECA (20.9 ± 1.0 to 29.8 ± 1.3%. P < 0.05, n = 8).

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Fig. 2.
Effects of adenosine receptor agonists on mucociliary
transport in the rabbit trachea. EB contents in the tracheal sections
were determined 10 min after iv administration of
N-cyclopentyladenosine (CPA), CGS-21680, or
N6-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine
(IB-MECA; 30 µg/kg for each). In the control experiment, no drug was
given. The EB level in each tracheal section was expressed as a
percentage of the total amount of dye in sections 1-4.
Values are means ± SE; n = 10 for each column.
*P < 0.05 and **P < 0.01, significantly different from control values.
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Fig. 3.
Dose-dependent effect of adenosine receptor agonists on
mucociliary transport. EB contents in tracheal section 4 were determined 10 min after iv administration of CPA, CGS-21680,
N6-2-(4-amino-3-iodophenyl)ethyladenosine
(APNEA), or IB-MECA (1-100 µg/kg for each). In the control
experiment (C), no drug was given. The EB level was expressed as a
percentage of the total amount of dye in sections 1-4.
Values are means ± SE; n = 8 for each point.
*P < 0.05 and **P < 0.01, significantly different from control values.
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Fig. 4.
Effects of adenosine receptor antagonists on
IB-MECA-induced stimulation of mucociliary transport. Rabbits received
iv 1,3-dipropyly-8-cyclopentylxanthine (DPCPX; 1 mg/kg),
3,7-dimethyl-L-propargylxanthine (DMPX; 1 mg/kg), or
MRS-1220 (100 µg/kg), and 15 min later IB-MECA (1-100 µg/kg).
EB contents in tracheal section 4 were determined 10 min
after administration of IB-MECA. The EB level was expressed as a
percentage of the total amount of dye in sections 1-4.
Values are means ± SE; n = 8 for each point.
*P < 0.05 and **P < 0.01, significantly different from values for IB-MECA alone.
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Ciliary motility.
Addition of IB-MECA (10
8 M) to the chamber elicited a
rapid increase in CBF of rabbit tracheal epithelium from the baseline value of 11.2 ± 0.3 to 14.4 ± 0.6 Hz (P < 0.01, n = 11) within 1 min, which was followed by a
small decrease and the subsequent stable response (Fig.
5). The CBF value 20 min after the
addition was still significantly greater than the baseline value
(P < 0.01). Addition of APNEA produced a smaller
increase in CBF at 1 and 3 min, CGS-21680 was without effect, and CPA
caused a small but significant decrease in CBF at 3 and 5 min (maximal
decrease: 11.0 ± 0.3 to 9.5 ± 0.3 Hz, P < 0.05, n = 11). The increase in CBF produced by IB-MECA
was not altered by DPCPX or DMPX but was abolished by MRS-1220
(P < 0.01, n = 10; Fig.
6). Discoordination of ciliary beating
was not observed in the recorded video film throughout the experiments.

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Fig. 5.
Time course of effects of adenosine receptor agonists on
ciliary beat frequency (CBF) of rabbit tracheal epithelium. IB-MECA,
APNEA, CGS-21680, or CPA at a concentration of 10 8 M was
added to the chamber at time 0 (arrow). Values are
means ± SE; n = 11 for each point.
*P < 0.05 and **P < 0.01, significantly different from baseline values.
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Fig. 6.
Effects of adenosine receptor antagonists on
IB-MECA-induced increase in CBF. Epithelial cells were incubated with
10 7 M DPCPX, DMPX, or MRS-1220, and 15 min later IB-MECA
(10 8 M) was added. Responses are expressed as the maximal
increase from baseline CBF. Data are means ± SE;
n = 10 for each column. **P < 0.01, significantly different from values for IB-MECA alone.
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As shown in Fig. 7, IB-MECA
(10
10 to 10
7 M) increased CBF in a
concentration-dependent fashion: the maximal increase from the baseline
value was 33.2 ± 4.6% (P < 0.01, n = 8), and the EC50 value was 3.1 ± 0.5 × 10
9 M (n = 8). The CBF value
declined at the higher concentration of 10
6 M IB-MECA.
Incubation of cells with Ca2+-free medium containing EGTA
and BAPTA-AM greatly attenuated the IB-MECA-induced increase in CBF,
but Rp-cAMPS did not alter the effect of IB-MECA.

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Fig. 7.
Effects of the Rp diastereomer of cAMP (Rp-cAMPS;
10 4 M) and Ca2+-free medium containing
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA)-AM (5 × 10 5 M) on CBF in response to
IB-MECA. Cells were incubated for 15 min with each blocker, and then
the concentration-response curves for IB-MECA were generated. Responses
are expressed as the maximal increase from baseline CBF. Brackets
denote concentration. Data are means ± SE; n = 8 for each point. **P < 0.01, significantly different
from corresponding response to IB-MECA alone.
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Intracellular Ca2+ levels.
The baseline [Ca2+]i in the rabbit tracheal
epithelium was 147 ± 11 nM (n = 16). Exposure of
cells to CPA or CGS-21680 at 10
8 M had no effect on
[Ca2+]i. However, IB-MECA (10
8
M) caused a rapid increase in F340/F380 (Fig.
8). This
[Ca2+]i response was biphasic, consisting of
an initial transient rise that peaked within 15 s and a following
sustained response. In this phase, periodic increases in
[Ca2+]i (Ca2+ oscillations) were
not observed. The peak value of [Ca2+]i was
566 ± 39 nM, indicating an increase in
[Ca2+]i by 419 ± 32 nM
(P < 0.001, n = 16). Pretreatment with
MRS-1220 (10
7 M) did not affect the baseline
[Ca2+]i levels but reduced the subsequent
IB-MECA-induced [Ca2+]i rise by 84 ± 9% (P < 0.001, n = 16).

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Fig. 8.
Representative recordings of adenosine receptor
antagonist (10 8 M)-induced Ca2+ responses in
fura 2-loaded rabbit tracheal epithelial cells. IB-MECA induced a
transient rapid increase in intracellular Ca2+
concentration ([Ca2+]i) followed by a
sustained response, whereas CPA and CGS-21680 had no effect. The
IB-MECA-induced rise in [Ca2+]i was inhibited
by pretreatment of cells for 15 min with MRS-1220 (10 7
M). F340, fluorescence at 340 nm; F380,
fluorescence at 380 nm.
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DISCUSSION |
In the present experiment, we developed an in vivo method to
evaluate airway mucociliary clearance by determining the transport rate
of Evans blue dye that had been placed on the tracheal mucosal surface
above the carina in rabbits and demonstrated for the first time that
stimulation of adenosine A3 receptors enhances mucociliary transport. This conclusion is based on the following findings. First,
the rate of propulsion of the dye toward the larynx was increased by
intravenous administration of IB-MECA, a potent and selective
A3 receptor agonist (1), and to a lesser
extent by the relatively less selective A3 agonist APNEA
(6), but not with the selective A1 and
A2 agonists CPA and CGS-21680, respectively. Second,
pretreatment with the selective A3 receptor antagonist MRS-1220 (11) blocked the IB-MECA-induced stimulation of
Evans blue transport, whereas DPCPX and DMPX, selective A1
and A2 antagonists, respectively, did not.
It has been generally accepted that mucociliary transport is governed
by ciliary activity and the depth and rheological properties of
periciliary fluid (27, 30), and we hypothesized that the A3 receptor-mediated transport of Evans blue dye may be
accounted for by the stimulation of ciliary activity. Furthermore,
because adenosine A3 receptors are present on mast cells
and the receptor activation results in the release of a variety of
mediators (22), the observed effect of IB-MECA could be
attributed to mast cell-derived mediators. To test these possibilities,
we conducted in vitro experiments using cultured tracheal epithelium,
and the results were consistent with our in vivo findings. Addition of
IB-MECA rapidly increased CBF, indicating a direct action on airway
epithelial cells. The ciliary stimulatory effect is likely mediated by
the A3 receptor, since IB-MECA was effective at nanomolar
concentrations, consistent with A3 selectivity
(12), and since it was specifically blocked by MRS-1220.
To date, A3 receptors have been cloned from rats
(17), rabbits (10), and humans
(25), and mRNA for these receptors are expressed in
particularly high abundance within the human lung (25),
but localization of the receptors to airway epithelial cells warrants
further studies. Additionally, the effectiveness of ciliary action in
transporting Evans blue dye may depend on several characteristics of
ciliary beating, of which the CBF is but one. The coordination of the
beating pattern, for instance, also plays a role in ciliary performance
(27). In the present study, no ciliary discoordination was
noted among adjacent cilia on the same cell or several bordering cells
in association with the increased CBF in response to IB-MECA. Thus it
seems reasonable to speculate that the observed increase in CBF can be
translated into the enhanced mucociliary transport, as predicted by
theoretical models of mucociliary pumping (23). However,
although there is no evidence that selective stimulation of adenosine
A3 receptors causes bronchoconstriction, possible influence
of the contraction of trachealis musculature on the IB-MECA-induced
mucociliary transport cannot be ruled out.
In both in vivo and in vitro experiments, the A1 agonist
CPA caused small but significant decreases in Evans blue transport and
CBF. This is consistent with our previous finding that the A1 agonist CPA inhibited rabbit tracheal ciliary motility
(34). In addition, we noted that, although IB-MECA dose
dependently increased mucociliary transport and ciliary motility at
relatively low doses (up to 30 µg/kg and 10
7 M,
respectively), the responses to high doses (100 µg/kg and 10
6 M) tended to decline. The reason for this is
uncertain, but desensitization of A3 receptors is unlikely
because only one dose of IB-MECA was given for each experiment. Another
possible reason would be that the high dose of IB-MECA is not
A3 receptor selective. In fact, it has been shown that
IB-MECA bind to cloned adenosine A3 receptors at
concentrations of as low as 10 nM (8, 10) and also bind to
cloned rabbit adenosine A1 receptors at high concentrations (10). This hypothesis seems likely, because the decline in
Evans blue transport in response to 100 µg/kg IB-MECA was reversed by the selective A1 receptor antagonist DPCPX.
Ciliary motility of airway epithelium is regulated mainly by cAMP and
Ca2+ (15, 24). Intracellular cAMP activates
glycogenolysis and subsequently stimulates the production of ATP, an
energy source of ciliary beating, via the tricarboxylic acid cycle, and
the mobilization of intracellular Ca2+ apparently acts on
the ciliary axoneme via formation of Ca2+-calmodulin
complexes. In our experiment, the increase in CBF produced by IB-MECA
was not altered by pretreatment of cells with the cAMP antagonist
Rp-cAMPS, but it was greatly attenuated by Ca2+-free
solution containing the intracellular Ca2+-chelating agent
BAPTA-AM, which inhibits both Ca2+ influx and
Ca2+ release from intracellular stores. These results
suggest that the ciliary stimulatory action of IB-MECA may be mediated
by Ca2+ mobilization. We confirmed this by measuring
[Ca2+]i and found that IB-MECA but not CPA or
CGS-21680 increased [Ca2+]i and that the
IB-MECA-induced response was greatly attenuated in the presence of
MRS-1220. In support of this, recent studies have shown that
stimulation of A3 receptors elevate intracellular Ca2+ in human nonpigmented ciliary epithelial cells
(18), rat cardiomyocytes (29), and the rat
mast cell line (28) probably via generation of inositol
phosphate through phosphoinositide breakdown. It has been demonstrated
that stimulation of nucleotide receptors with ATP and UTP produces an
initial transient increase in [Ca2+]i and the
following Ca2+ oscillations in airway epithelium
(3). In contrast, such Ca2+ oscillations were
not noted in the response to IB-MECA. The reason for the difference is
uncertain, but this could be because of the difference in receptors
and/or experimental condition.
In conclusion, our present studies show that stimulation of adenosine
A3 receptors increases ciliary motility of airway
epithelium and mucociliary transport in the respiratory tract.
Therefore, selective A3 receptor agonists might be useful
in the treatment of impaired mucociliary clearance.
 |
ACKNOWLEDGEMENTS |
We thank Masayuki Shino and Yoshimi Sugimura for technical assistance.
 |
FOOTNOTES |
This work was supported in part by Grant no. 06670243 from the Ministry
of Education, Science and Culture, Japan.
Address for reprint requests and other correspondence: J. Tamaoki, First Dept. of Medicine, Tokyo Women's Medical Univ. School of Medicine, 8-1 Kawada-Cho, Shinjuku, Tokyo 162-8666, Japan (E-mail: jtamaoki{at}chi.twmu.ac.jp).
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.
10.1152/ajplung.00360.2001
Received 10 September 2001; accepted in final form 26 October 2001.
 |
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