Pharmacologic characterization of novel adenosine A2A receptor agonists in equine neutrophils

Wan-chun Sun Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602

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James N. Moore Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602
Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602

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David J. Hurley Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602
Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602

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Michel L. Vandenplas Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602

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Joel M. Linden Department of Physiology, School of Medicine, University of Virginia, Charlottesville, VA 22908

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Thomas F. Murray Department of Pharmacology, School of Medicine, Creighton University, Omaha, NE 68178

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Abstract

Objective—To evaluate anti-inflammatory effects of several novel adenosine receptor agonists and to determine their specificity for various adenosine receptor subtypes on neutrophils, cells heterologously expressing equine adenosine receptors, or equine brain membranes.

Sample Population—Neutrophils isolated from 8 healthy horses.

Procedures—Radioligand binding experiments were performed to compare binding affinities of adenosine receptor agonists to equine adenosine A1, A2A, and A3 receptor subtypes. Effects of these agonists on endotoxin-induced production of reactive oxygen species (ROS) by equine neutrophils and roles of specific adenosine receptor subtypes and cAMP production in mediating these effects were determined.

Results—Radioligand binding experiments yielded a ranked order of affinity for the brain equine A2A receptor on the basis of 50% inhibitory concentrations (IC50) of the agonists as follows: ATL307 (IC50 = 1.9nM) and ATL313 > ATL309 and ATL310 > ATL202 > 2-([p-2- carboxyethyl] phenylethylamino)-5′-N-ethylcarboxyamidoadenosine > 5′-N-ethylcarboxamidoadenosine. Furthermore, ATL313 had approximately 100-fold greater selectivity for A2A over A1 and A3 receptors. In functional assays with equine neutrophils, the compounds inhibited endotoxin-induced ROS production and stimulated production of cAMP with the same ranked order of potency. Results of experiments performed with selective adenosine receptor antagonists indicated that functional effects of ATL313 were via stimulation of A2A receptors.

Conclusions and Clinical Relevance—Results indicated that activation of A2A receptors exerted anti-inflammatory effects on equine neutrophils and that stable, highly selective adenosine A2A receptor agonists may be developed for use in management of horses and other domestic animals with septic and nonseptic inflammatory diseases.

Abstract

Objective—To evaluate anti-inflammatory effects of several novel adenosine receptor agonists and to determine their specificity for various adenosine receptor subtypes on neutrophils, cells heterologously expressing equine adenosine receptors, or equine brain membranes.

Sample Population—Neutrophils isolated from 8 healthy horses.

Procedures—Radioligand binding experiments were performed to compare binding affinities of adenosine receptor agonists to equine adenosine A1, A2A, and A3 receptor subtypes. Effects of these agonists on endotoxin-induced production of reactive oxygen species (ROS) by equine neutrophils and roles of specific adenosine receptor subtypes and cAMP production in mediating these effects were determined.

Results—Radioligand binding experiments yielded a ranked order of affinity for the brain equine A2A receptor on the basis of 50% inhibitory concentrations (IC50) of the agonists as follows: ATL307 (IC50 = 1.9nM) and ATL313 > ATL309 and ATL310 > ATL202 > 2-([p-2- carboxyethyl] phenylethylamino)-5′-N-ethylcarboxyamidoadenosine > 5′-N-ethylcarboxamidoadenosine. Furthermore, ATL313 had approximately 100-fold greater selectivity for A2A over A1 and A3 receptors. In functional assays with equine neutrophils, the compounds inhibited endotoxin-induced ROS production and stimulated production of cAMP with the same ranked order of potency. Results of experiments performed with selective adenosine receptor antagonists indicated that functional effects of ATL313 were via stimulation of A2A receptors.

Conclusions and Clinical Relevance—Results indicated that activation of A2A receptors exerted anti-inflammatory effects on equine neutrophils and that stable, highly selective adenosine A2A receptor agonists may be developed for use in management of horses and other domestic animals with septic and nonseptic inflammatory diseases.

Polymorphonuclear neutrophils are the most abundant circulating leukocytes in horses and are the first to migrate to sites of infection, where they participate in the initial phases of the inflammatory response. At those sites, they ingest invading microorganisms and cell debris, generate ROS, and release proteolytic enzymes.1 Although these functions of neutrophils are important in limiting the spread of pathogens, these same inflammatory products also are capable of causing tissue damage at the site of infection and exacerbating inflammation in pathologic conditions such as endotoxemia, arthritis, and myocardial infarction.2

Adenosine, a ubiquitous endogenous purine nucleoside released during ATP metabolism, has anti-inflammatory effects and may be an important part of the natural dampening mechanism for the inflammatory response.3,4 Cellular signaling by adenosine is through 4 subtypes of adenosine receptors (ie, A1, A2A, A2B, and A3), all of which are G-protein–coupled receptors.5,6

Results of studies2,7-9 indicate that activation of the adenosine A2A receptor subtype exerts anti-inflammatory effects by several mechanisms, including suppressing the production of ROS by activated neutrophils. A common problem associated with the administration of adenosine receptor agonists is the lack of selectivity of compounds for the various adenosine receptor subtypes and the concomitant development of important adverse cardiovascular effects, such as heart block mediated by activation of the adenosine A1 receptor.6

The goal of our laboratory group's research in this area is to identify highly selective and potent adenosine analogues for A2A receptors that may be useful in the management of inflammation in horses. In the study reported here, a series of novel adenosine A2A receptor agonists were investigated. We compared their selectivity for binding to equine receptor subtypes, ability to reduce LPS-induced production of ROS by equine neutrophils, and selectivity for exerting this effect via adenosine A2A receptors. Results obtained in this study may serve as a foundation for the development of a new class of pharmaceutical agents that can be used to manage septic and nonseptic inflammatory responses in horses and other domestic animals.

Materials and Methods

Sample population—Blood samples for all experiments were obtained from 8 healthy adult horses. Brain tissue was obtained from a single cadaveric horse. Use of these horses was approved by the Institutional Animal Care and Use Committee of the University of Georgia.

Isolation of neutrophils—Blood samples were obtained from the jugular vein from each horse into syringes that contained EDTA as an anticoagulant, and RBCs were allowed to settle for approximately 20 minutes. Leukocyte-rich plasma was layered onto a solution of polysucrose and sodium diatrizoatea and centrifuged at 400 × g for 30 minutes at 20°C. The RBCs that contaminated the resulting pellet were lysed with distilled water, and tonicity was then restored by the addition of 2X PBS solution.

Granulocytes (> 95% neutrophils) were washed 3 times with PBS solution and then suspended at a final concentration of 3 × 107 cells/mL in RPMI 1640 medium containing 10% fetal bovine serumb (without phenol red), 2mM L-glutamine, 2mM sodium pyruvate, and gentamicin (50 μg/mL). Cells were then incubated in 5% carbon dioxide at 37°C for 90 minutes to reduce signal induced by exposure to the solution of polysucrose and sodium diatrizoate. After that incubation, it was determined by use of trypan blue dye exclusion that viability of the neutrophils was ≥ 98%. Neutrophils were diluted by the addition of the aforementioned medium to achieve a final concentration of 3 × 106 cells/mL.

Radioligand binding assays with equine A1, A2A, and A3 receptors—Specific binding of [3H]DPCPX,c an A1-selective antagonist radioligand, to equine cerebellar membranes was determined by use of a rapid filtration assay. The cerebellum was removed from the calvarium of a euthanized horse and dissected into blocks. These blocks were then immediately frozen on dry ice and stored in sealed containers at −80°C until use (tissues were used within 1 month after collection). On the day of the assay, cerebellar tissue was thawed, weighed, and homogenizedd in 100 volumes of ice-cold 50mM Tris-HCl buffer. The homogenate was centrifuged (48,000 × g for 10 minutes); pellets were then suspended in the same volume of ice-cold buffer and homogenized again. The homogenate was centrifuged, and the pellet was suspended in 100 volumes of 50mM Tris-HCl buffer containing 2 U of adenosine deaminasee/mL. After incubation at 22°C for 30 minutes, the homogenate was cooled on ice and centrifuged; the pellet was then suspended in 100 volumes of ice-cold 50mM Tris-HCl by use of the homogenizer.

Equilibrium binding reactions were performed in triplicate. In equilibrium competition binding assays, aliquots (0.175 mL) of the brain membrane suspension were incubated at 22°C for 90 minutes with 0.2nM [3H]DPCPX and increasing concentrations of adenosine analogues (ATL202,f ATL307,g ATL309,h ATL310,i or ATL313j), NECA,k or CGS21680.l Aliquots of the membrane suspension were incubated with 25 μL of [3H]DPCPX (120 Ci/mmol) and 25 μL of buffer or N6-cyclopentyladenosine (final concentration, 10μM) to define nonspecific binding. Aliquots of the membrane suspension were dissolved in 0.5 N NaOH for protein concentration determination. Total binding minus nonspecific binding with N6-cyclopentyladenosinem was designated as specific binding, which was approximately 95% of the total binding. Binding reactions for all 3 radioligands were terminated by adding 2 mL of ice-cold 50mM Tris-HCl buffer followed by solution filtration with filter stripsn and in a 24-well cell harvester.° Filter strips were soaked in 0.5% polyethyleneamine before use to reduce nonspecific adsorption of ligand. The filter strips were rinsed 4 times with 4 mL of ice-cold 50mM Tris-HCl buffer. For [3H]-labeled ligands, filter strips were placed in 7-mL vials that contained 3.5 mL of scintillation cocktail, which was followed by measurement of radioactivity in a scintillation counter.p

For determination of specific binding to A2A receptors in equine brain tissues, striatal membranes were prepared by Dounce homogenization (20 strokes) of dissected equine striatum in ice-cold 50mM Tris-HCl buffer (pH, 7.7) and centrifuged at 40,000 × g for 20 minutes. Membrane pellets were then resuspended in buffer, centrifuged, and washed 2 additional times prior to performing the binding assays. For the equilibrium competition assays, 100 μg of equine striatal membranes were incubated with approximately 1.0nM of the selective A2A receptor antagonist [3H]ZM241385q and increasing concentrations of adenosine analogues (ATL202, ATL307, ATL309, ATL310, or ATL313), NECA, or CGS21680. Aliquots of membrane suspension were incubated with [3H]ZM241385, with or without the addition of 10μM 6-amino-2-chloropurine riboside,r to define nonspecific binding. Binding reactions were allowed to proceed at 22°C for 45 minutes.

For determination of specific binding to equine adenosine A3 receptors, HEK293 cells that heterologously but stably expressed these receptors were used. Cell membranes were harvested from equine A3–expressing HEK293 cells, as described elsewhere.10 Briefly, 25 μg of the transfected HEK293 cell membranes were incubated with 0.25nM [125I] AB-MECAs and increasing concentrations of adenosine analogues (ATL202, ATL307, ATL309, ATL310, or ATL313), NECA, or CGS21680. The binding reaction was allowed to proceed at 22°C for 45 minutes. Equine A3 receptor–expressing HEK cell membranes were incubated with [125I] AB-MECA alone or with 10μM 4-(3-[cyclopentyloxy]-4-methoxyphenyl)-2-pyrrolidinonet to define nonspecific binding. The binding reaction was terminated via rapid filtration onto filter stripsn by use of a 24-well cell harvester.o Radioactivity was then determined by use of an automatic gamma counter.u

Measurement of ROS production by isolated neutrophils—Production of ROS by the neutrophils (3 × 105 cells/well) was monitored in 96-well flat-bottom tissue culture plates. Selected wells contained specific adenosine A2A receptor agonists with or without adenosine deaminase and with or without Escherichia coli O55:B5 LPS.v Phorbol myristate acetate,w a direct activator of protein kinase C, was used to determine the maximal amount of ROS production. To identify the involvement of specific adenosine receptor subtypes, selected wells also contained ZM241385,x MRS1706,y or MRS1220.z To detect ROS, 10 μL of nonfluorescent dihydrorhodamine dyeaa (final concentration, 10μM) was added to each well. The dye oxidizes to green-fluorescent rhodamine in response to hydrogen peroxide produced by neutrophils. Plates were incubated in a humidified atmosphere of 5% carbon dioxide at 37°C for 2 hours. Fluorescence was measured by use of a fluorescent plate readerbb with a 485-nm excitation filter and 538-nm emission filter. Values were reported as the number of AFUs. All reagents were diluted with RPMI 1640 medium not containing phenol red, but that did contain 10% fetal bovine serum, 2mM L-glutamine, 2mM sodium pyruvate, and gentamicin (50 μg/mL).

To permit comparison of data among experiments, fluorescence in each group of wells was adjusted relative to that obtained for unstimulated cells and thus was standardized for endogenous production of ROS. The degree of inhibition of LPS-induced responses was evaluated by use of the following equation:

article image

where AFU LPS is the value for cells incubated with LPS alone, AFU treatment is the value for cells incubated with the particular treatment being evaluated and with LPS, and AFU cells are the basal value for unstimulated cells.

To provide a quality-control index, a parallel set of wells was included in each experiment in which ROS production was determined for unstimulated cells incubated with polymyxin B.cc If ROS production by those cells was < 90% of ROS production by cells incubated without polymyxin B, we concluded that there was LPS contamination at some point during the experiment and that experiment was excluded from the study.

In preliminary experiments, we did not observe a significant effect of the addition of adenosine deaminase on ROS production. However, adenosine deaminase (1 U/mL) was included in the experiments reported here to counter possible effects of endogenously produced adenosine and minimize variability.

Measurement of total cellular cAMP concentration—Neutrophils (2 × 106 cells/ mL) were suspended in RPMI 1640 medium that contained adenosine deaminase (1 U/mL) and 50μM 4-(3-[cyclopentyloxy]-4-methoxyphenyl)-2-pyrrolidinone,dd a type IV–specific phosphodiesterase inhibitor. Aliquots (160 μL) of the cell suspensions were placed into duplicate wells of 96-well plates. Various concentrations of adenosine analogues (ATL202, ATL307, ATL309, ATL310, or ATL313) were added in 20 μL of RPMI 1640 medium with or without the A2A receptor antagonist ZM241385 or the A2B receptor antagonist MRS1706. After incubation in a humidified atmosphere of 5% carbon dioxide at 37°C for 20 minutes, reactions were terminated by the addition of 20 μL of stop solution to each well. Concentrations of cAMP were then determined by use of a commercially available EIA kitee performed in accordance with the manufacturer's protocol.

Data analysis—All experiments were repeated 3 times, and the ROS production assay was performed in quadruplicate. Concentration-response data were analyzed by use of nonlinear regression analysis with commercially available software.ff The EC50 values were expressed as mean and 95% confidence intervals. Bestfit values for 3 variables (log EC50 and the ROS values at the top and bottom plateaus of the curves) were compared in a pairwise manner by use of the F test to identify significant differences between concentration-response curves generated after incubation with and without each of the receptor antagonists. Significance was defined at values of P < 0.05.

Results

Comparative binding affinities of adenosine A2A receptor agonists—Equilibrium competition experiments performed with the selective adenosine A2A receptor antagonist [3H]ZM241385 yielded a ranked order of agonist affinities of ATL307 > ATL309 and ATL310 and ATL313 > ATL202 > CGS21680 > NECA (Figure 1; Table 1). To determine the selectivity of these compounds for equine adenosine A1, A2A, and A3 receptors, equilibrium competition experiments were also performed with selective adenosine A1 and A3 receptor ligands, [3H]DPCPX and [125I]AB-MECA, respectively. All 5 adenosine analogues were highly selective for equine adenosine A2A receptors, compared with selectivity for equine adenosine A1 and A3 receptors, with ATL313 having approximately 100- and 93-fold greater selectivity for adenosine A2A receptors than for A1 or A3 receptors, respectively.

Figure 1—
Figure 1—

Competitive binding of adenosine receptor agonists incubated with [3H]ZM241385 (A2A receptors; A), [3H]DPCPX (A1 receptors; B), and [125I]AB-MECA (A3 receptors; C). Each point represents the mean ± SEM for 3 to 6 replicates. Results are reported as the percentage of radioligand binding without addition of an agonist. Membranes for binding of [3H]DPCPX were prepared from equine cerebellum, membranes for binding of [3H]ZM241385 were prepared from equine corpus striatum, and membranes for binding of [125I]AB-MECA were prepared from HEK293 cells stably transfected with equine A3 adenosine receptors. Similar results were obtained in 3 additional experiments.

Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.981

Table 1—

Binding of various receptor agonists to adenosine receptors in equine brain membranes.

Receptor agonistIC50 (nM)*  
A1A2AA3  
NECA174.0 (123.0–246.0)149.5 (106.0–211.0158.7 (109.0–232.0)1.21.1
CGS216804,393.0 (3,312.0–5,825.0)103.3 (84.2–127.0)167.5 (100.0–280.0)42.51.6
ATL202156.7 (99.5–246.0)5.3 (4.7–5.9)45.9 (31.6–66.6)29.88.7
ATL30745.1(34.1–59.5)1.9 (1.7–2.2)42.5 (29.3–61.7)23.622.3
ATL309150.5 (97.7–232)2.8 (2.5–3.2)44.6 (34.5–57.6)53.015.7
ATL310164.3 (95.5–286.0)3.4 (2.9–4.0)49.0 (35.9–66.8)48.214.4
ATL313345.4 (214.0–559.0)3.4 (2.8–4.2)318.9 (230.1–442.0)100.492.7

Values are expressed as mean(95% confidence interval).

Functional effects of adenosine A2A receptor agonists—Experiments evaluating the inhibitory effects of the adenosine A2A receptor agonists on LPS-induced production of ROS by equine neutrophils yielded a ranked order of potency of ATL307 > ATL309 and ATL310 and ATL313 > ATL202 > CGS21680 > NECA (Figure 2; Table 2). This ranked order was the same as that obtained for the radioligand binding experiments. Inhibition of LPS-induced ROS production was reduced significantly by a selective adenosine A2A receptor antagonist, ZM241385, which suggested that this effect of the adenosine analogues used in the study was predominantly via activation of adenosine A2A receptors.

Table 2—

Mean (95% confidence interval) values of the LPS-induced ROS production for neutrophils obtained from 8 healthy horses after neutrophils were incubated with various analogues of A2A receptor agonists with and without the A2A receptor antagonist ZM241385.

Receptor agonistIC50 (nM)*
0nM ZM241385100nM ZM241385
NECA111.0 (46.4–265.7)4,885.0 (2,104.0–11,340.0)
CGS2168065.7 (40.9–105.6)4,222.0 (685.1–26,010.0)
ATL20213.9 (6.8–28.4)673.8 (343.5–1,322.0)
ATL3072.5 (1.9–3.2))214.5 (82.9–555.0)
ATL3095.3 (4.1–6.8)690.5 (575.7–828.3)
ATL3105.4 (3.3–8.8)609.6 (381.8–973.4)
ATL3134.9 (3.6–6.7)1,130.0 (733.4–1,740.0)

Values are expressed as mean (95% confidence interval).

Figure 2—
Figure 2—

Inhibition of LPS-induced neutrophil ROS production by various adenosine receptor agonists. Each point represents the mean ± SEM for 6 replicates. Results are reported as the percentage of inhibition of ROS production induced by LPS. Similar results were obtained in 3 additional experiments.

Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.981

To more fully evaluate the involvement of various adenosine receptor subtypes in the inhibition of LPS-induced production of ROS, experiments were performed with different consentrations of ZM241385 as well as the selective adenosine A2B receptor antagonist MRS1706. Inhibition of LPS-induced ROS production by ATL313 was reduced in a concentration-dependent manner by the A2A antagonist ZM241385. A Schild plot of these data yielded a ZM241385 KB value of 3.8nM, which is consistent with the KD value reported elsewhere11 for radioligand binding experiments performed with HEK293 cells expressing equine A2A receptors (Figure 3). Furthermore, incubation of neutrophils with the adenosine A2B receptor antagonist MRS1706 did not significantly alter the calculated IC50 value for inhibition of ROS production by ATL313. Collectively, these findings indicated that ATL313-mediated inhibition of LPS-induced ROS production was via activation of A2A receptors.

Figure 3—
Figure 3—

Results of LPS-induced neutrophil ROS production when incubated with ATL313 and various concentrations of ZM241385 (A), a Schild plot of those results (B), and effects of MRS1706 on LPS-induced ROS production (C). For the Schild plot, the negative logarithm of KD (ie, PA2) was 8.419. Results in panels A and C represent mean ± SEM of 6 replicates and are reported as the percentage of inhibition of LPS-induced ROS production. Similar results were obtained in 3 additional experiments. DR=EC50 with the addition of various concentrations of receptor antagonist)/EC50 without the addition of a receptor antagonist.

Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.981

cAMP production and activation of adenosine A2A receptors—In other species, activation of adenosine A2A receptors is coupled to G-protein–mediated responses that lead to the accumulation of cAMP. Results obtained from experiments in which equine neutrophils were incubated with the adenosine A2A receptor agonists yielded a ranked potency order of ATL307 and ATL313 > ATL309 and ATL310 > ATL202 (Figure 4). This order was nearly identical to that obtained for both A2A receptor binding and inhibition of ROS production.

Figure 4—
Figure 4—

Concentration of cAMP in equine neutrophils induced by 5 adenosine A2A receptor agonists (A) and effects of ZM241385 and MRS1706 on ATL313-induced accumulation of cAMP (B). Each point represents the mean ± SEM of 3 replicates. Values for the control samples were obtained without addition of receptor antagonists.

Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.981

To determine the adenosine receptor subtypes responsible for the increase in cAMP production, experiments were performed in which equine neutrophils were incubated with the A2A receptor antagonist ZM241385 or the A2B receptor antagonist MRS1706. Incubation of neutrophils with ZM241385 (100nM) significantly increased the calculated EC50 value for ATL313-induced cAMP accumulation (Figure 4). In contrast, incubation of neutrophils with MRS1706 (100nM) did not affect the EC50 value for ATL313. These findings indicated that the ATL313-mediated increase in cAMP accumulation was via activation of A2A receptors.

Discussion

Adenosine exerts its biological effects by interacting with the 4 known adenosine receptor subtypes. Results of most of the conducted studies12–15 indicate that activation of A2A receptors is responsible for the anti-inflammatory effects attributed to adenosine. In 1 study,9 our laboratory group determined that NECA inhibits ROS production in LPS-stimulated equine neutrophils via activation of adenosine A2A receptors.

In a previous study reported here, we expanded on the other studies conducted by our laboratory group by characterizing the adenosine receptor signature and functional effects of an array of novel synthetic adenosine analogues. Similar to information reported for other species, NECA had equivalent binding affinities for A1, A2A, and A3 receptors, whereas CGS21680 had greater affinity for A2A and A3 receptors than for A1 receptors. More importantly, however, we found that all 5 adenosine analogues tested in these experiments were highly selective for equine adenosine A2A receptors, relative to their selectivity for equine A1 and A3 receptors. Of the adenosine analogues tested here, ATL313 had approximately 100-fold greater affinity for the equine adenosine A2A receptor than for the A1 or A3 receptors. The other 4 adenosine analogues tested were less selective for A2A receptors than for A3 receptors. These analogues vary only in the constituent at the 2 position of adenine, which suggests that modification in this region influences potency for A2A and A3 receptors.

Although neutrophils express all 4 adenosine receptor subtypes,16–18 analysis of results of the study reported here suggested that adenosine A2A receptors were primarily responsible for the anti-inflammatory effects of ATL313. For example, the inhibitory effects of ATL313 on LPS-induced ROS production by equine neutrophils were prevented by coincubation of the cells with the adenosine A2A receptor antagonist ZM241385. In addition, a Schild plot of the data yielded a KB value of 3.8nM for ZM241385 . This latter finding is consistent with the reported11 affinity of ZM241385 for equine adenosine A2A receptors. Furthermore, the effect of ATL313 on LPS-induced ROS production was not affected by coincubation with an antagonist for the adenosine A2B receptor. These findings are consistent with the effects of stimulation of adenosine A2A receptors on oxidative activity of human neutrophils.8

Considerable evidence exists19 to support the concept that agents that increase intracellular concentrations of cAMP also modulate neutrophil function. Analysis of results of the study reported here revealed that incubation of equine neutrophils with adenosine receptor agonists increased cellular cAMP concentration. In addition, the ranked order of potency of these compounds for initiating this response was the same as the ranked order for both binding affinity to equine A2A receptors and inhibition of LPS-induced ROS production. By use of ATL313 (ie, the compound with the highest binding selectivity for equine A2A receptors), we found a concentration-dependent increase in cellular cAMP that was prevented by coincubation with ZM241385 but not by incubation with MRS1706. The lack of an effect of MRS1706 indicated that the increase in cellular cAMP concentration did not involve activation of the other adenosine receptor subtype that also activates adenylyl cyclase (ie, the adenosine A2B receptor).8

Analysis of results of the study reported here indicated that stimulation of adenosine A2A receptors on equine neutrophils inhibited LPS-induced ROS production by increasing intracellular concentrations of cAMP. These findings suggested that synthetic adenosine receptor agonists that have high selectivity for A2A receptors may prove to be useful as anti-inflammatory agents in horses.

ABBREVIATIONS

ROS

Reactive oxygen species

LPS

Lipopolysaccharide

DPCPX

1,3-dipropyl-8-cyclopentylxanthine

NECA

5′-N-ethylcarboxamidoadenosine

CGS21680

2-([p-2-carboxyethyl]phenylethylamino)-5′-N-ethylcarboxyamidoadenosine

[125I]AB-MECA

4-amino-3-[125I]iodobenzyl-5′-N-methylcarbamoyladenosine

AFU

Arbitrary fluorescence unit

EC50

Effective concentration that induces 50% of the maximum response

KB

Dissociation constant

KD

Equilibrium dissociation constant

IC50

50% inhibitory concentration

a.

Histopaque 1077, Sigma-Aldrich, St Louis, Mo.

b.

Fetal bovine serum, Hyclone, Logan, Utah.

c.

[3H]DPCPX, PerkinElmer Life Sciences, Wellesley, Mass.

d.

Polytron, Brinkmann Instruments Inc, Westbury, NY.

e.

Adenosine deaminase, Roche Diagnostics Corp, Indianapolis, Ind.

f.

ATL202, Adenosine Therapeutics LLC, Charlottesville, Va.

g.

ATL307, Adenosine Therapeutics LLC, Charlottesville, Va.

h.

ATL309, Adenosine Therapeutics LLC, Charlottesville, Va.

i.

ATL310, Adenosine Therapeutics LLC, Charlottesville, Va.

j.

ATL313, Adenosine Therapeutics LLC, Charlottesville, Va.

k.

NECA, Tocris Bioscience, Ellisville, Mo.

l.

CGS21680, Tocris Bioscience, Ellisville, Mo.

m.

N6-Cyclopentyladenosine, Sigma-Aldrich, St Louis, Mo.

n.

Whatman GF/C filter strips, Whatman Inc, Sanford, Me.

o.

Brandell cell harvester, Brandell, Gaithersburg, Md.

p.

Beckman LS 6000 counter, Beckman Coulter Inc, Fullerton, Ca.

q.

[3H]ZM241385, Tocris Bioscience, Ellisville, Mo.

r.

2-CADO, Sigma-Aldrich, St Louis, Mo.

s.

[125I]AB-MECA, PerkinElmer Life Sciences, Wellesley, Mass.

t.

IB-MECA, Sigma-Aldrich, St Louis, Mo.

u.

PerkinElmer 1470 automatic gamma counter, PerkinElmer Life Sciences, Downers Grove, Ill.

v.

Escherichia coli O55:B5 LPS, List Biologics, Campbell, Calif.

w.

Phorbol myristste acetate, Sigma-Aldrich, St Louis, Mo.

x.

ZM241385, Tocris Bioscience, Ellisville, Mo.

y.

MRS1706, Tocris Bioscience, Ellisville, Mo.

z.

MRS1220, Tocris Bioscience, Ellisville, Mo.

aa.

DHR123, Invitrogen-Molecular Probes, Carlsbad, Calif.

bb.

Fluoroskan Ascent FL, Thermo Labsystems, GMI Inc, Albertville, Minn.

cc.

Polymyxin B, Bedford Laboratories, Bedford, Ohio.

dd.

Rolipram, Sigma-Aldrich, St Louis, Mo.

ee.

cAMP Biotrak EIA kit, Amersham Biosciences, Piscataway, NJ.

ff.

Prism software, version 4.0, GraphPad Software Inc, San Diego, Calif.

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  • 9.

    Sun W, Moore JN, Hurley DJ, et al. Effects of stimulation of adenosine A2A receptors on lipopolysaccharide-induced production of reactive oxygen species by equine neutrophils. Am J Vet Res 2007;68:649656.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Brandon CI, Vandenplas ML, Dookwah H, et al. Cloning and pharmacological characterization of the equine adenosine A3 receptor. J Vet Pharmacol Ther 2006;29:255263.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Brandon CI, Vandenplas M, Dookwah H, et al. Cloning and pharmacological characterization of the equine adenosine A2A receptor: a potential therapeutic target for the treatment of equine endotoxemia. J Vet Pharmacol Ther 2006;29:243253.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Al-Ayadhi LY, Al-Tuwajri AS. The synergistic effect of adenosine A2A receptors agonist, type IV phosphodiesterase inhibitor and ATP-sensitive K channels activation on free radicals production and aggregation of human polymorphonuclear leukocytes. Pharmacol Res 2004;50:157163.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Sullivan GW, Carper HT, Mandell GL. The specific type IV phosphodiesterase inhibitor rolipram combined with adenosine reduces tumor necrosis factor-alpha-primed neutrophil oxidative activity. Int J Immunopharmacol 1995;17:793803.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Sullivan GW, Linden J, Buster BL, et al. Neutrophil A2A adenosine receptor inhibits inflammation in a rat model of meningitis: synergy with the type IV phosphodiesterase inhibitor, rolipram. J Infect Dis 1999;180:15501560.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Sullivan GW, Linden J, Hewlett EL, et al. Adenosine and related compounds counteract tumor necrosis factor-alpha inhibition of neutrophil migration: implication of a novel cyclic AMP-independent action on the cell surface. J Immunol 1990;145:15371544.

    • Search Google Scholar
    • Export Citation
  • 16.

    Gessi S, Varani K, Merighi S, et al. A(3) adenosine receptors in human neutrophils and promyelocytic HL60 cells: a pharmacological and biochemical study. Mol Pharmacol 2002;61:415424.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Gessi S, Varani K, Merighi S, et al. Expression, pharmacological profile, and functional coupling of A2B receptors in a recombinant system and in peripheral blood cells using a novel selective antagonist radioligand, [3H]MRE 2029-F20. Mol Pharmacol 2005;67:21372147.

    • Crossref
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  • 18.

    Spicuzza L, DiMaria G, Polosa R. Adenosine in the airways: implications and applications. Eur J Pharmacol 2006;533:7788.

  • 19.

    Lin P, Welch EJ, Gao XP, et al. Lysophosphatidylcholine modulates neutrophil oxidant production through elevation of cyclic AMP. J Immunol 2005;174:29812989.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Figure 1—

    Competitive binding of adenosine receptor agonists incubated with [3H]ZM241385 (A2A receptors; A), [3H]DPCPX (A1 receptors; B), and [125I]AB-MECA (A3 receptors; C). Each point represents the mean ± SEM for 3 to 6 replicates. Results are reported as the percentage of radioligand binding without addition of an agonist. Membranes for binding of [3H]DPCPX were prepared from equine cerebellum, membranes for binding of [3H]ZM241385 were prepared from equine corpus striatum, and membranes for binding of [125I]AB-MECA were prepared from HEK293 cells stably transfected with equine A3 adenosine receptors. Similar results were obtained in 3 additional experiments.

  • Figure 2—

    Inhibition of LPS-induced neutrophil ROS production by various adenosine receptor agonists. Each point represents the mean ± SEM for 6 replicates. Results are reported as the percentage of inhibition of ROS production induced by LPS. Similar results were obtained in 3 additional experiments.

  • Figure 3—

    Results of LPS-induced neutrophil ROS production when incubated with ATL313 and various concentrations of ZM241385 (A), a Schild plot of those results (B), and effects of MRS1706 on LPS-induced ROS production (C). For the Schild plot, the negative logarithm of KD (ie, PA2) was 8.419. Results in panels A and C represent mean ± SEM of 6 replicates and are reported as the percentage of inhibition of LPS-induced ROS production. Similar results were obtained in 3 additional experiments. DR=EC50 with the addition of various concentrations of receptor antagonist)/EC50 without the addition of a receptor antagonist.

  • Figure 4—

    Concentration of cAMP in equine neutrophils induced by 5 adenosine A2A receptor agonists (A) and effects of ZM241385 and MRS1706 on ATL313-induced accumulation of cAMP (B). Each point represents the mean ± SEM of 3 replicates. Values for the control samples were obtained without addition of receptor antagonists.

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  • 9.

    Sun W, Moore JN, Hurley DJ, et al. Effects of stimulation of adenosine A2A receptors on lipopolysaccharide-induced production of reactive oxygen species by equine neutrophils. Am J Vet Res 2007;68:649656.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Brandon CI, Vandenplas ML, Dookwah H, et al. Cloning and pharmacological characterization of the equine adenosine A3 receptor. J Vet Pharmacol Ther 2006;29:255263.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Brandon CI, Vandenplas M, Dookwah H, et al. Cloning and pharmacological characterization of the equine adenosine A2A receptor: a potential therapeutic target for the treatment of equine endotoxemia. J Vet Pharmacol Ther 2006;29:243253.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Al-Ayadhi LY, Al-Tuwajri AS. The synergistic effect of adenosine A2A receptors agonist, type IV phosphodiesterase inhibitor and ATP-sensitive K channels activation on free radicals production and aggregation of human polymorphonuclear leukocytes. Pharmacol Res 2004;50:157163.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Sullivan GW, Carper HT, Mandell GL. The specific type IV phosphodiesterase inhibitor rolipram combined with adenosine reduces tumor necrosis factor-alpha-primed neutrophil oxidative activity. Int J Immunopharmacol 1995;17:793803.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Sullivan GW, Linden J, Buster BL, et al. Neutrophil A2A adenosine receptor inhibits inflammation in a rat model of meningitis: synergy with the type IV phosphodiesterase inhibitor, rolipram. J Infect Dis 1999;180:15501560.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Sullivan GW, Linden J, Hewlett EL, et al. Adenosine and related compounds counteract tumor necrosis factor-alpha inhibition of neutrophil migration: implication of a novel cyclic AMP-independent action on the cell surface. J Immunol 1990;145:15371544.

    • Search Google Scholar
    • Export Citation
  • 16.

    Gessi S, Varani K, Merighi S, et al. A(3) adenosine receptors in human neutrophils and promyelocytic HL60 cells: a pharmacological and biochemical study. Mol Pharmacol 2002;61:415424.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Gessi S, Varani K, Merighi S, et al. Expression, pharmacological profile, and functional coupling of A2B receptors in a recombinant system and in peripheral blood cells using a novel selective antagonist radioligand, [3H]MRE 2029-F20. Mol Pharmacol 2005;67:21372147.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Spicuzza L, DiMaria G, Polosa R. Adenosine in the airways: implications and applications. Eur J Pharmacol 2006;533:7788.

  • 19.

    Lin P, Welch EJ, Gao XP, et al. Lysophosphatidylcholine modulates neutrophil oxidant production through elevation of cyclic AMP. J Immunol 2005;174:29812989.

    • Crossref
    • Search Google Scholar
    • Export Citation

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