Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, North Carolina 27858
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ABSTRACT |
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We recently reported that adenosine caused
bronchoconstriction and enhanced airway inflammation in an allergic
mouse model. In this study, we further report the characterization of
the subtype of adenosine receptor(s) involved in bronchoconstriction.
5'-(N-ethylcarboxamido)adenosine (NECA), a nonselective
adenosine agonist, elicited bronchoconstriction in a dose-dependent
manner. Little effects of
N6-cyclopentyladenosine
(A1-selective agonist) and
2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (A2A-selective agonist) compared with NECA were observed in
this model.
2-Chloro-N6-(3-iodobenzyl)-9-[5-(methylcarbamoyl)--D-ribofuranosyl]adenosine, an A3-selective receptor agonist, produced a
dose-dependent bronchoconstrictor response, which was blocked by
selective A3 antagonist
2,3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS1523). However, MRS1523 only partially inhibited NECA-induced bronchoconstriction. Neither selective A1 nor
A2A antagonists affected NECA-induced bronchoconstriction.
Enprofylline, a relatively selective A2B receptor
antagonist, blocked partly NECA-induced bronchoconstriction.
Furthermore, a combination of enprofylline and MRS1523 completely
abolished NECA-induced bronchoconstrictor response. Using RT-PCR, we
found that all four adenosine receptor subtypes are expressed in
control lungs. Allergen sensitization and challenge significantly
increased transcript levels of the A2B and A3
receptors, whereas the A1 receptor message decreased. No
change in transcript levels of A2A receptors was observed
after allergen sensitization and challenge. These findings suggest that A2B and A3 adenosine receptors play an
important role in adenosine-induced bronchoconstriction in our allergic
mouse model. Finally, whether the airway effects of the receptor
agonists/antagonists are direct or indirect needs further investigations.
adenosine agonists; adenosine antagonists; mouse lung; asthma
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INTRODUCTION |
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AMONG THE MANY ACTIONS of adenosine, several lines of evidence suggest a contribution of adenosine to the pathophysiology of asthma. Adenosine is present in high concentrations in the bronchoalveolar lavage fluid of asthmatics (8). Airway preparations from allergic asthmatic subjects are more sensitive to the contractile responses of adenosine and related analogs (4). Inhaled adenosine causes bronchoconstriction in asthmatics and allergic nonasthmatics compared with normal subjects (24).
The response of the asthmatic airways to adenosine involves the activation of extracellular adenosine receptor(s). Theophylline, a nonselective adenosine receptor antagonist, selectively (relative to histamine) inhibited adenosine-induced bronchoconstriction at doses generating plasma concentrations that are insufficient to inhibit phosphodiesterases but well above those needed to block adenosine receptors (22). Moreover, both rebound hyperresponsiveness to adenosine and exacerbation of symptoms have been reported when chronic theophylline therapy was withdrawn (36).
Adenosine receptors are seven-transmembrane-spanning receptors that are coupled to the effector system through heterotrimeric G proteins. Four subtypes of adenosine receptors, A1, A2A, A2B, and A3, have been identified based on the order of agonist potency and antagonist affinity, G protein coupling, cellular responses, and receptor cloning studies (11). Each of these receptor subtypes is expressed in lung and may have a role in asthma.
Most evidence indicates that activation of the A1 receptors, and to some extent the A3 receptors, induces adenylyl cyclase inhibition with subsequent decrease in adenosine 3',5'-cyclic monophosphate (cAMP) level. This intracellular signal induces contraction of the airway smooth muscle. In contrast, when adenosine activates the A2A receptors, cAMP level increases, which causes airway relaxation (29). Adenosine A2B receptors have long been known to be coupled to Gs protein, which mediates the activation of adenylyl cyclase (12, 33). Recent studies indicate that A2B receptors are also coupled to Gq and result in Ca2+ mobilization and mitogen-activated protein kinase activation (20, 21). Also, the A3 receptors have been suggested to mediate the facilitation of antigen-induced release of bronchoconstrictor mediators through inositol triphosphate and the Ca2+ pathway (34). Despite the pivotal role that adenosine receptors play in bronchoconstriction and inflammatory reactions under allergic conditions, the subtype(s) of adenosine receptors mediating bronchoconstriction remain poorly defined. The accumulated evidence shows that the receptor subtype varies widely among species, for example, the involvement of A3 receptors in the guinea pig (18), A1 receptors on airway smooth muscle cell in the rabbit (28), and a combination of A1, A2B, and A3 receptors in the BDE rat (31). In mice, our data (9) and those of others (6) have shown that exogenous and endogenous adenosine mediates bronchoconstriction and airway hyperresponsiveness through specific adenosine receptors, but the relative contribution of each adenosine receptor subtype mediating bronchoconstriction in mice is still unresolved.
The present study was initiated following our previous study demonstrating that ragweed sensitization and challenge of mice induced a profound increase in bronchoconstrictor response to adenosine and lung inflammation (9). Here, we investigated the involvement of the subtype of adenosine receptor(s) mediating bronchoconstriction to adenosine using selective adenosine receptor agonists and antagonists and studied a profile of adenosine receptor expression in allergic mouse lung using RT-PCR.
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MATERIALS AND METHODS |
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Mice and Sensitization
Male BALB/c mice, 6-8 wk of age and free of specific pathogens, were obtained from Harlan Laboratories (Indianapolis, IN). The animals were maintained on a ragweed-free diet. All experimental animals used in this study were under a protocol approved by the Institutional Animal Care and Use Committee of East Carolina University.Sensitization was performed according to a method described previously (9). In brief, mice were sensitized with two intraperitoneal injections of ragweed allergen (Greer Laboratories, Lenoir, NC), 200 µg per dose with 200 µl of Imject alum (Pierce Laboratories, Rockford, IL) on days 1 and 6. Nonsensitized control animals received only the Imject alum with the same volumes. After sensitization, the mice were placed in a Plexiglas chamber and challenged with 0.5% aerosolized ragweed or with 0.9% saline as a control, via an ultrasonic nebulizer (DeVilbiss, Somerset, PA) for 20 min both in the morning and afternoon on days 11, 12, and 13.
Experimental Protocol
Airway responsiveness to adenosine agonists.
Airway responsiveness was assessed with whole body plethysmograph (Max
II; Buxco, Troy, NY). This system estimates total pulmonary air flow
using a dimensionless parameter known as enhanced pause (Penh).
Pressure differences were used to extrapolate Penh value, which is a
function of the sum of the air flows in the upper and lower respiratory
tracts during a respiratory cycle. Penh has been shown to correlate
with direct measurement of airway obstruction (lung resistance and
dynamic compliance) (17). Twenty-four hours after
the last challenge with aerosolized ragweed or saline, mice were placed
in the chamber and exposed to adenosine agonists
5'-(N-ethylcarboxamido)adenosine (NECA, a nonselective
adenosine receptor agonist),
N6-cyclopentyladenosine (CPA, a selective
A1 adenosine receptor agonist) in 10% ethanol,
2-p-(2-carboxyethl)
phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS21680, a
selective A2A adenosine receptor agonist) in 0.9% saline,
2-chloro-N6-(3-iodobenzyl)-9-[5-(methylcarbamoyl)--D-ribofuranosyl]adenosine (CL-IB-MECA, a selective A3 adenosine receptor agonist) in
20% DMSO via Buxco Aerosol Delivery System (version 1.5;
Buxco, Sharon, CT) at 2.5 l/min of the dilution flow and 0.15 l/min of the trickle flow for 2 min at increasing concentrations
(11.72-375 µg/ml) for dose responsiveness. The next dose was not
given until the animal returned to baseline Penh. Airway responsiveness
was expressed as percentage increase in Penh compared with vehicle.
Readings were taken for 5 min following each nebulization. For the
quantification of the dose responsiveness to adenosine agonists, the
linear regression of Penh was calculated for individual mice. The dose
corresponding to an increase in Penh of 100% (EC100) was
determined, and average dose in different groups was compared by
analysis of variance.
Adenosine antagonist challenges.
Adenosine antagonists 1,3,-dipropyl-8-cyclopentylxanthine (DPCPX,
A1 selective, 104 M),
7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidine (SCH58261, A2A selective, 10
4 M),
3-n-propylxanthine (enprofylline, a relative A2B
antagonist, 10
4 M) in 10% ethanol, and
2,3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS1523, A3 selective, 10
4 M) in 20% DMSO
were aerosolized with the Buxco Aerosol Delivery System at 2.5 l/min of
the dilution flow and 0.15 l/min of the trickle flow for 5 min. After
15 min, dose responsiveness to a corresponding adenosine agonist and
NECA was performed in the same manner as mentioned above. Pulmonary
measurements were recorded before and after pretreatment with an
adenosine antagonist to determine its effect on basal bronchial tone.
Also, the effect of vehicle per se on Penh was examined.
RT-PCR.
Mice were immunized with the protocol described above. Twenty-four
hours after the last challenge with aerosolized allergen or 0.9%
saline, mice were killed by injection (0.1 ml pentobarbitone sodium 200 mg/ml ip). Lungs were taken and immediately frozen in liquid nitrogen.
Total RNA was isolated from the mouse lung of both ragweed-sensitized
and -challenged mice and control animals with TRIzol reagent (Roche
Diagnostics, Indianapolis, IN) according to the manufacturer's
instructions, and RT-PCR was conducted as described earlier
(5). Briefly, for the RT reaction, 5 µg of purified
total RNA were reverse transcribed in the presence of an anchored
oligo-p (dT)15 primer, by use of avian myeloblastosis virus
RT (Roche Diagnostics) based on the manufacturer's
recommendations. Sets of specific primers for adenosine
receptors for A1 (37), A2A
(15), A2B (5), and A3
(3) are presented in Table
1.
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Chemicals
CPA, CGS21680, NECA, DPCPX, enprofylline, MRS1523, ethanol, and DMSO were purchased from Sigma Chemical (St. Louis, MO). SCH58261 was a gift from Dr. A. Monopoli (Shearing Plough, Milan, Italy). CL-IB-MECA was obtained by SRI International from the National Institute of Mental Health Chemical Synthesis and Drug Supply Program.Statistical Analysis
Experimental values are presented as means ± SE. EC100 was calculated. Analysis of variance was used to determine the level of significance among all groups. Pairs of groups were compared with unpaired two-tailed Student's t-test. The P value for significance was set at 0.05. ![]() |
RESULTS |
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Dose Responsiveness to Adenosine Receptor Agonists and the Effect of Corresponding Adenosine Receptor Antagonists
Aerosol challenge with NECA, a nonselective adenosine receptor agonist, elicited a dose-dependent increase in Penh in both ragweed-sensitized and -challenged and control mice. Bronchoconstrictor responsiveness to NECA was significantly enhanced in allergen-sensitized and -challenged mice compared with the controls. The maximum responsiveness to NECA (375 µg/ml) was 405.9 ± 35.6% in sensitized and challenged mice and 181.8 ± 7.7% in controls, respectively (P < 0.001). The EC100 was 30.66 µg/ml (95% confidence limits: 24.75-36.58) in ragweed-sensitized and -challenged mice and 142.03 µg/ml (95% confidence limits: 124.47-159.60) in control animals, respectively (P < 0.001, Fig. 1). The baseline Penh values between control (0.72 ± 0.06) and ragweed-sensitized and -challenged mice (0.78 ± 0.05) were not different. Vehicle (10% ethanol aerosolized for 2 min) used in experiment with agonists had no significant effect on Penh in both control and immunized mice. Also, 10% ethanol aerosolized for 5 min (vehicle for antagonists) was without an effect on basal bronchial tone.
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Because NECA, like adenosine, has affinity for each of the four
adenosine receptor subtypes (19), the question arises as to which adenosine receptor subtype is being activated in NECA-induced bronchoconstriction. To distinguish the subtype of adenosine receptors involved in NECA-induced bronchoconstrictor responsiveness, we employed
selective adenosine agonists and antagonists. CPA is a potent and
selective agonist at A1 receptors with reported inhibitor constant (Ki) values of 0.59, 460, and 240 nM at
rat A1, A2A, and A3 receptors,
respectively (11). Aerosol challenge of mice with CPA
elicited little change in Penh in both control and immunized mice (Fig.
2A). Further experimentation
was done to examine the bronchoconstrictor responsiveness to NECA with
a selective A1 receptor antagonist, DPCPX, for which the
Ki values at rat A1, A2A, and A3 receptors are 0.3 nM, 340 nM, and
>10 µM, respectively (11). There was no effect of DPCPX
on dose responsiveness to NECA observed after mice were pretreated with
DPCPX (Fig. 2B). DPCPX per se had no effect on baseline Penh
in both groups. Penh values before and after DPCPX under baseline
conditions were 0.68 ± 0.03 and 0.62 ± 0.05 in control
animals and 0.73 ± 0.06 and 0.67 ± 0.08 in sensitized and
challenged mice, respectively (P > 0.05).
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CGS21680 is the most potent, highly selective agonist at
A2A receptors and is the ligand of choice for the
characterization of A2A receptors. Aerosol challenge with
CGS21680 did not affect the Penh (Fig.
3A). Pretreatment with an
A2A receptor antagonist, SCH58261, a potent and highly
selective A2A receptor antagonist both in vivo and in vitro
(30), was unable to alter the bronchoconstrictor dose-response curve to NECA in either control or sensitized and challenged groups (Fig. 3B). There was no difference in
baseline Penh value before and after SCH58261 in both groups of
animals. The values were 0.64 ± 0.04 and 0.66 ± 0.05 (before vs. after SCH58261) in the control group and 0.74 ± 0.04 and 0.79 ± 0.05 (before vs. after SCH58261) in the sensitized and
challenged groups, respectively.
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CL-IB-MECA is a highly potent and selective A3 receptor
agonist with Ki values of 820, 470, and 0.33 nM
for rat A1, A2A, and A3 receptors,
respectively (11). CL-IB-MECA challenge produced marked
bronchoconstrictor responsiveness in a dose-dependent manner (Fig.
4A). The maximum
responsiveness to CL-IB-MECA (375 µg/ml) was 334.2 ± 34.6% in
sensitized and challenged mice and 169 ± 19.4% in controls,
respectively (P < 0.001). The EC100 dose
of CL-IB-MECA was 62.81 µg/ml (95% confidence limits:
52.52-73.10) in ragweed-sensitized and -challenged mice and 158.55 µg/ml (95% confidence limits: 121.75-195.35) in control
animals, respectively. CL-IB-MECA-induced bronchoconstrictor
responsiveness was completely inhibited by an A3-selective
antagonist, MRS1523, that is effective in various species
(11) (Fig. 4A). MRS1523 itself did not affect basal bronchial tone. The basal Penh values before and after
pretreatment with MRS1523 were 0.61 ± 0.03 and 0.72 ± 0.09 in control mice (P > 0.05) and in sensitized and
challenged animals were 0.71 ± 0.05 and 0.64 ± 0.07 (P > 0.05), respectively. Vehicle alone (20% DMSO
aerosolized for 2 or 5 min) had no effect on Penh in both control and
allergen-sensitized and -challenged mice.
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Furthermore, the effect of MRS1523 on a dose-response curve for NECA was also tested. It was found that NECA-induced bronchoconstriction was partially inhibited by MRS1523 (Fig. 4B), especially at the highest concentration (375 µg/ml of NECA). This suggests the involvement of other adenosine receptors, possibly the A2B receptors, in addition to A3 receptors.
Because there is a lack of availability of A2B-selective
antagonists commercially, enprofylline, which is somewhat selective for
A2B adenosine receptors with Ki
values of 156, 32, 7, and 65 µM at human A1,
A2A, A2B, and A3 receptors,
respectively (10), was used to examine the effect of
A2B receptors in NECA-induced bronchoconstrictor
responsiveness. As expected, enprofylline did block NECA-induced
bronchoconstriction as reflected by rightward shift of the
dose-response curve (Fig. 5).
EC100 changed from 30.66 µg/ml (95% confidence limits:
24.75-36.58) in sensitized and challenged mice to 157.40 µg/ml
(95% confidence limits: 114.59-200.21) after pretreatment with
enprofylline in sensitized and challenged mice (P < 0.01). Also, a combination of enprofylline and MRS1523 completely
inhibited the bronchoconstrictor responsiveness to NECA in both groups
(Fig. 6), which was different from the
effect of MRS1523 alone on a dose-response curve for NECA (Fig.
4B), suggesting the involvement of A2B and
A3 receptors in NECA-induced bronchoconstriction.
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Adenosine Receptor Transcript Levels in Mouse Lungs
Adenosine receptor transcript levels were quantified in total RNA extracts isolated from whole lungs of both ragweed-sensitized and -challenged mice and control animals. RT-PCR revealed expression of four subtypes of adenosine receptors in the control lungs, with relatively high levels of A2B receptor message, intermediate levels of A1 and A3 receptor message, and low levels of A2A receptors (Fig. 7A). Mice sensitized and challenged with ragweed showed changes in the message for adenosine receptors in lungs. There were significant elevations in transcript levels of A2B and A3 receptors after ragweed sensitization and challenge, which increased by 42.58% for A2B receptors (P < 0.01) and 29.05% for A3 receptors (P < 0.05), respectively, and there was a decrease in the expression of A1 adenosine receptors by 22.85% (P < 0.05). Little change in the expression of A2A receptors was observed (
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DISCUSSION |
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Consistent with our previous study (9), our present results show that NECA, a nonselective adenosine receptor agonist, causes bronchoconstriction, which is significantly greater in systemically allergen-sensitized, airway-challenged mice compared with control animals. Furthermore, the A1 agonist CPA and A2A agonist CGS21680 produced little increase in Penh in this allergic mouse model. The selective A1 or A2A antagonist did not affect bronchoconstrictor responsiveness to NECA.
However, the A3 agonist CL-IB-MECA induced dose-dependent bronchoconstriction, which was completely inhibited by the selective A3 antagonist MRS1523 (Fig. 4A). At the same time, NECA-induced bronchoconstriction was partially inhibited by MRS1523, especially at the highest concentration of NECA, indicating the involvement of other adenosine receptors, possibly A2B receptors. To confirm our observations further, we employed a relatively selective A2B receptor antagonist, enprofylline, to study the effect of A2B receptors on NECA-induced bronchoconstriction. Enprofylline showed an attenuation of NECA-induced bronchoconstriction (Fig. 5). Because enprofylline is a relatively selective A2B antagonist with Ki values of 7 and 65 µM for A2B and A3 receptors, respectively (10), it was still difficult to make conclusions about the role of A2B receptors from this experiment. Further supporting data were obtained using a combination of enprofylline with MRS1523, which showed that the partial inhibition of NECA-induced bronchoconstriction was completely blocked by a combination of enprofylline and MRS1523. These results were supported by the transcript levels of both A2B and A3 receptors being higher in the sensitized and challenged mice as opposed to control mice. Together, these results suggest that A2B and A3 adenosine receptors are possibly involved in NECA-induced bronchoconstriction in an allergic mouse model.
Adenosine and its agonist-induced bronchoconstriction have been studied in several animal models. A series of studies carried out by Pauwels and colleagues (31, 32) showed that BDE rats respond to adenosine agonists with the following order of potency: NECA = CPA > N6-2-(4-aminophenyl)ethyladenosine > N6-cyclohexyladenosine > R-N6-(2-phenylisopropyl)adenosine > CGS21680. In the same study, A3-selective agonist produced a dose-dependent bronchoconstriction, and selective A1 receptor antagonists KF15372 and KW3902 inhibited bronchoconstrictor responsiveness to NECA (31, 32). These observations led the investigators to conclude that adenosine-induced bronchoconstriction in the BDE rat was most likely through A1, A2B, and A3 receptors (31, 32). In Brown Norway rats, Hannon and coworkers (13) suggested the involvement of A2B receptors in bronchoconstrictor responsiveness to adenosine. The same investigators, in a more recent study, questioned the role of A2B receptors in the bronchoconstrictor responsiveness to adenosine in Brown Norway rats (14). Studies using guinea pigs suggest the existence of a mechanism involving adenosine A3 receptors (18, 38). In another animal model, the animals immunized neonatally with ragweed or house dust mite developed bronchoconstrictor responsiveness to adenosine agonists (1). In this model, the order of agonist potency was CPA >> NECA >> CGS21680, and CPA-induced bronchoconstrictor responsiveness was blocked by a selective A1 antagonist, DPCPX (2). This pharmacological profile is typical of an adenosine A1 receptor subtype. In an extension of these studies, Nyce and Metzger (28) confirmed that an aerosolized phosphorothiorate antisense oligodeoxynucleotide directed against the A1 receptors (but not the appropriate sense control) attenuated bronchoconstrictor responsiveness of immunized rabbits to either adenosine or adenosine agonists or dust mite allergen. To the best of our knowledge, the present study demonstrates in mice for the first time that adenosine- and its agonist-induced bronchoconstriction was most likely mediated by the activation of A2B and A3 adenosine receptors. Furthermore, an expression of all four subtypes of adenosine receptors was found in normal mouse lung using RT-PCR (Fig. 7A). Allergen sensitization and airway challenge produced a significant increase in the transcript levels of A2B and A3 adenosine receptors in mouse lung (Fig. 7B).
Whether the effect of receptor activation in this model is a direct effect on airway smooth muscle or secondary to an effect on the activation of other cells (e.g., mast cells, eosinophils, neutrophils, lymphocytes, and macrophages) cannot be determined from this study. However, there is evidence that A2B adenosine receptors are expressed in human airway smooth muscle cells (26). Adenosine could directly affect airway smooth muscle cell contractility by modulating the intracellular [Ca2+] through the activation of adenosine A3 receptors (25). Mice lacking the adenosine A3 receptors showed attenuation of adenosine-induced bronchoconstriction (39). The A1 receptors located directly on the smooth muscle in monkey and rabbit (27, 28) may play a role, since the selective depletion of the airway smooth muscle A1 receptors by antisense reduced the bronchoconstrictor response to adenosine (28). However, our data showed a decrease in transcript level for A1 receptors after allergen sensitization and challenge of mice, as well as the absence of CPA-induced bronchoconstriction in both control and sensitized and challenged mice. This discrepancy may be attributed to the use of a different animal model with varying pathophysiological conditions. A decrease in the expression of adenosine A1 receptors has been reported in chronic inflammatory disease (16). Diminished A1 receptor mRNA in mouse lung in our study may reflect a compensatory mechanism for the lung inflammation, since proinflammatory actions of adenosine A1 receptor agonists on inflammatory cells have been suggested (7).
A variety of evidence points to the bronchoconstrictor responsiveness to adenosine being a consequence of activation of inflammatory cells in airways, especially mast cells and eosinophils (40, 41). It is well documented that A2A, A2B, and A3 receptor mRNA is expressed in mouse bone marrow-derived mast cells (23, 35). In a functional study, Zhong et al. reported that enprofylline was able to protect ~30% of the adenosine-dependent mast cell degranulation in adenosine deaminase (ADA)-deficient mice (41). These authors further demonstrated with ADA-deficient and A3 receptor double-knockout mice that A3 receptors also played an important role in adenosine-mediated murine lung mast cell degranulation (42). Enhanced expression of adenosine A3 receptors on eosinophils has also been reported in the asthmatic lung (40). Our findings of increased expression of A2B and A3 adenosine receptors in ragweed-sensitized and -challenged mouse lung was also confirmed by Chunn and colleagues in partially ADA-deficient mouse lung (6).
In summary, our data show an increased expression of A2B and A3 receptor transcripts and no change in A2A receptor transcript with a decrease in A1 message in sensitized and challenged mice. Although A1/A2A receptors were expressed in mouse lung, on the basis of Penh they seemed to play a little role in adenosine-mediated bronchoconstriction in our studies. Taking these data together, we conclude that adenosine A2B and A3 receptors play an important role in adenosine- and its agonist-induced bronchoconstriction in our allergic mouse model.
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ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge the generous gifts of CL-IB-MECA from the National Institute of Mental Health's Chemical Synthesis and Drug Supply Program (SRI International, Menlo Park, CA) and of SCH58261 from Dr. A. Monopoli (Shearing Plough, Milan, Italy).
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-50049. Some of the data presented here are a part of U. S. patent no. 6,387,913 B1.
Address for reprint requests and other correspondence: S. J. Mustafa, Dept. of Pharmacology and Toxicology, Brody School of Medicine, E. Carolina Univ., Greenville, NC 27858 (E-mail: mustafas{at}mail.ecu.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.
First published January 17, 2003;10.1152/ajplung.00353.2002
Received 22 October 2002; accepted in final form 14 January 2003.
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