Introduction

Top Summary Introduction Procedures Results Discussion References

Adenosine exerts numerous physiological effects that were originally thought to be mediated by three adenosine receptors, A₁, A_2A, and A_2B. In the early 1990s, a new adenosine receptor was cloned from rat tissues, first by Meyerhof et al. (1) and then by Zhou et al. (2), who named it A₃. More recently, human (3), sheep (4), and rabbit (5) A₃ adenosine receptors have been cloned and characterized. Functional expression of A₃ adenosine receptors from various species indicates that A₁ and A₃ receptors bind the radioligands [¹²⁵I]APNEA, [¹²⁵I]ABA, and [¹²⁵I]AB-MECA and are negatively coupled to adenylyl cyclase (2, 4, 6). One unusual property of A₃ adenosine receptors is a major difference among species in the binding affinity of xanthine antagonists. In particular, the rat receptor is resistant to blockade by xanthines, whereas sheep, human, and rabbit receptors bind certain xanthines with high affinity, although with distinct potency orders (3).

The addition of adenosine to rat basophilic leukemic cells (RBL 2H3 cells; a tumor cell line resembling mast cells) causes facilitation of the release of granules, which is mediated by A₃ adenosine receptors (7, 8). A₃ receptor activation also triggers the degranulation of mast cells surrounding hamster cheek pouch arterioles (9). Based on these results, the observation that the inhalation of adenosine produces histamine release and bronchoconstriction in asthmatics but not in nonasthmatics (10, 11) and the discovery of high levels of A₃ adenosine receptor transcript in human and sheep lung, we proposed a role for the A₃ adenosine receptor in the pathophysiology of asthma (12). Because rodents may be poor animal models for the investigation of the role of A₃ receptors in human allergy and asthma, we decided to clone the canine A₃ adenosine receptor as a first step toward characterizing the role of A₃ adenosine receptors in canine models of asthma.

Here, we report the cloning, expression, and pharmacological characterization of an A₃ adenosine receptor cDNA isolated from BR cells [canine mastocytoma cells (13)]. Low levels of both A₁ and A₃ adenosine receptors are found on BR cells, but these are not primarily responsible for stimulating degranulation of this canine mastocytoma cell line. Rather, an A_2B adenosine receptor causes degranulation via a pertussis toxin-insensitive pathway that mobilizes mastocytoma cell Ca²⁺ and can be blocked by the antiasthmatic xanthine enprofylline (14).

	Experimental Procedures

Top Summary Introduction Procedures Results Discussion References

Materials. All chemicals were obtained from Sigma Chemical (St. Louis, MO) with the following exceptions. IB-MECA was from Dr. Saul Kadin (Pfizer, Groton, CT). I-ABA and I-ABOPX (also known as BW-A522) were from Dr. Susan Daluge (Glaxo-Wellcome, Research Triangle Park, NC). WRC-0571 [C⁸-(N-methylisopropyl)-amino-N⁶-(5-endohydroxy)-endonorbornan-2-yl-9-methyladenine] was from Dr. Pauline Martin (Discovery Therapeutics, Richmond, VA). APNEA was from Dr. Ray Olsson (University of South Florida, Tampa, FL). RDC7 (dog A₁ adenosine receptor cDNA) was from Dr. Guy Vassart (Brussels, Belgium). Rat A₃ adenosine receptor cDNA was from Dr. Fereydoun Sajjadi (Gensia, La Jolla, CA). Human A₃ adenosine receptor cDNA was from Dr. Marlene Jacobson (Merck, West Point, PA). Rabbit A₃ adenosine receptor cDNA was from Dr. Scott Kennedy (Pfizer, Groton, CT). HMC-1 mast cells were from Dr. J. H. Butterfiled (Mayo Clinic, Rochester, MN). NECA, CGS 21680 (2-[4-(2-carboxyethyl)phenethylamino]-5-N-ethylcarboxamidoadenosine), (R)-PIA, CPA, CPX, XAC, 8-SPT, theophylline, and enprofylline were purchased from Research Biochemicals (Natick, MA). Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone] was from BIOMOL Research Laboratories (Plymouth Meeting, PA). Adenosine deaminase was from Boehringer-Mannheim Biochemicals (Indianapolis, IN). Fura-2/AM was from Molecular Probes (Eugene, OR). myo-[³H]Inositol was from Amersham Life Sciences (Arlington Heights, IL). Dowex AG 1- X8 was from BioRad (Richmond, CA). [¹²⁵I]ABA was synthesized as described previously (15). Cell culture media and supplies were from GIBCO BRL (Gaithersburg, MD).

Cell culture. COS-7 cells were grown in DMEM with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Canine BR mastocytoma cells were grown in low-glucose DMEM supplemented with 2% calf serum, 25 mM HEPES, 1.5 mM l-histidine, 100 units/ml penicillin G, and 100 µg/ml streptomycin. The medium was changed every 3 days, and the cells were replated weekly.

Molecular cloning. To obtain the full-length sequence of the canine A₃ adenosine receptor cDNA, a library prepared in gt10 from BR cell poly(A)⁺ RNA was screened using a probe generated by RT-PCR of total RNA isolated from dog tissues using the cDNA cycle kit (InVitrogen, La Jolla, CA). Primers for amplification were primer A (sense 132-152), 5-GACCACCACCTTCTATTTCA-3; and primer B (antisense 660-680), 5-GTCTTGAACTCCCGA/TCC-3. The primers correspond to conserved regions within the first and third intracellular loops of the human, sheep, and rat A₃ adenosine receptor cDNAs. Each reaction cycle consisted of incubations at 95° for 1 min, 55° for 2 min, and 72° for 3 min with 0.02 unit/ml of Taq polymerase (Promega, Madison, WI). PCR fragments were subcloned into the TA vector (InVitrogen) and sequenced with Sequenase (United States Biochemical, Cleveland, OH) using modifications for double-stranded sequencing. One fragment from lung RNA was found to be ~90% identical to the human, rat, and sheep A₃ receptor transcripts. This probe was labeled with [-³²P]dCTP (Primit II; Stratagene, La Jolla, CA) and used to screen the BR cell cDNA library. Library screening was carried out by plaque-filter hybridization. Filters were hybridized at 65° overnight in 10% dextran sulfate, 1 M NaCl, 100 µg/ml herring sperm DNA, and 1 × 10⁶ cpm/ml radiolabeled probe and then washed in 0.5× SSC/0.5% SDS at 65°. Recombinants hybridizing to the probe were plaque-purified and reprobed. Recombinant phage DNA isolated by the plaque lysate method were digested with EcoRI and electrophoresed through a 1% agarose gel to determine the insert sizes. Several clones were identified ranging in size from 0.9 to 3 kb. One clone (cA₃13.1), which was 1.6 kb long, was subcloned into the EcoRI site of the plasmid vector pGEM-7z() (Promega). Double-stranded DNA was isolated, and both strands were sequenced in full, first by using T7 and Sp6 primers to get nucleotide sequence information near the 5 termini, and then with a series of synthetic oligonucleotide primers derived from sequences determined previously.

Radioligand binding studies. Membranes were prepared from COS-7 cells expressing the canine A₃ receptor (cA₃13.1) or the canine A₁ receptor (RDC7); HEK 293 cells stably expressing human, rabbit or rat A₃ adenosine receptors; or canine BR cells. The full coding region of the receptor cDNAs were subcloned into the expression vector CLDN10B and transiently expressed (60 hr) in COS cells by the DEAE-dextran method (16) or stably expressed in HEK 293 cells after transfection by the Ca²⁺ phosphate precipitation method (17) and selection in 2 mg/ml G-418. Transfected cells were washed in phosphate-buffered saline; homogenized in 10 mM EDTA, 10 mM Na-HEPES, pH 7.4, and 0.1 mM benzamidine; and centrifuged at 20,000 × g for 20 min. Pellets were resuspended and washed in 10 mM Na-HEPES, 1 mM EDTA, pH 7.4, and 0.1 mM benzamidine (HE buffer) and resuspended in the same buffer with 10% (w/v) sucrose (sucrose buffer) at a membrane protein concentration of 1 mg/ml. Because [¹²⁵I]ABA bound poorly to crude BR cell membranes, plasma membranes were enriched by preparing P2 pellets. Cells were homogenized in sucrose buffer and centrifuged at 500 × g for 10 min. The pellet was resuspended in sucrose buffer and centrifuged again at 500 × g. The pooled supernatants were diluted 3-fold, pelleted, and washed twice by centrifugation at 20,000 × g for 20 min in HE buffer; resuspended; and frozen in sucrose buffer. Protein concentrations were determined using fluorescamine with BSA as standard. Membranes were frozen in aliquots and stored at 80°. For radioligand binding studies, cell membranes were incubated in 0.1 ml for 3 hr at 21° with 5 mM MgCl₂ and 5 units/ml adenosine deaminase. For equilibrium binding assays, 6-8 concentrations of [¹²⁵I]ABA were used in triplicate in tubes, each containing 10-60 µg of membrane protein, and the specific activity of [¹²⁵I]ABA was reduced 10-20-fold with the nonradioactive compound. Nonspecific binding was measured in the presence of 5 µM I-ABA. [¹²⁵I]ABA was found to have a higher ratio of specific to nonspecific binding than an alternative radioligand, [¹²⁵I]AB-MECA. For competition experiments, 0.5-1 nM [¹²⁵I]ABA was added to tubes, and competing ligands were added over a range of concentrations; the tubes contained 10-50 µg of membrane protein in a final volume of 0.1 ml.

Analysis of binding data. Specific [¹²⁵I]ABA binding to A₁ adenosine receptors was optimally fit to a single site binding model using Marquardt's nonlinear least-squares interpolation (18). [¹²⁵I]ABA was found to bind to two affinity states of the recombinant canine, human, and rat A₃ receptors. For two-site Scatchard transformation, the relationship between bound/free and bound can be shown to be described by a quadratic equation: bound/free = A * X * X + B * X + C, where A = bound; X = K_d1/K_d2; B = K_d1 * X + K_d2 * X  B_max1 * K_d2  B_max2 * K_d1; C = X * X  B_max1 * X  B_max2 * X. Optimal parameters for two-site Scatchard plots were generated by using the binomial theorem to solve this equation within each iteration of nonlinear least-squares analysis.

Northern blots. Northern analysis and RT-PCR were used to determine the tissue distribution of A₃ adenosine receptor transcript and to identify A_2B, A₁, and A₃ receptor transcripts in BR cells. Total RNA was extracted and poly(A)⁺ RNA was selected using oligo(dt) cellulose. Five micrograms of poly(A)⁺ RNA was electrophoresed through 1% agarose gels containing 1% formaldehyde and then transferred to nylon membranes (Genescreen Plus; DuPont). The membranes were hybridized in 10% dextran sulfate, 1 M NaCl, and 100 µg/ml herring sperm DNA with 1 × 10⁶ cpm/ml random-labeled probe at 65° overnight. Filters were washed with 0.5× SSC/0.5% SDS at 65° and then exposed to Amersham Hyperfilm MP for 24-48 hr. The A₃ receptor probe consisted of a 600-bp PCR fragment of cA₃13.1, corresponding to approximately half of the carboxyl-terminal sequence and the 3 noncoding region. The A_2B receptor probe consisted of a 500-bp PCR fragment generated by RT-PCR from BR cell RNA corresponding to transmembrane regions I-IV.

Mastocytoma cell degranulation. As an indicator of degranulation of BR cells, we measured the release of -hexosaminidase (a granule-associated protein that parallels histamine release) using a modification of the method of Schwartz et al. (20). BR cells grown in suspension were washed twice in Ca²⁺/Mg²⁺-free Tyrode's buffer and then resuspended in complete Tyrode's at a density of 1.2 × 10⁶ cells/ml. Cells were then transferred to a 96-well plate in 250-µl aliquots and prewarmed to 37° for 15 min. Cells were stimulated with agonists added in 50-µl aliquots for 20 min at 37° with shaking. The reactions were stopped by placing the plate on ice for 10 min and then pelleting the cells by centrifugation at 200 × g for 10 min (4°). Two hundred microliters of the supernatant was removed and added to 50 µl of 5 mM p-nitrophenyl-N-acetyl-D-glucosaminide, and 100 mM citric acid, pH 3.8, and incubated at 37° for 2 hr with shaking before the addition of 50 µl of 0.4 M NaCO₃. Total cellular -hexosaminidase was determined by adding 50 µl of lysis buffer (complete Tyrode's buffer plus 0.6% Triton A-100) to 250-µl aliquots of cells, and 20 µl was removed and assayed. Absorbance was read at 405 nm using a Titertech Multiskan II plate reader. Experiments were performed in triplicate, and release of -hexosaminidase is expressed as percentage of the total content of unstimulated cells.

cAMP. BR cells were washed twice and resuspended in serum-free low-glucose DMEM containing 25 mM HEPES, 1 unit/ml adenosine deaminase, and 20 µM Ro 20-1724 and then transferred to polypropylene test tubes (1 × 10⁶ cells/0.2 ml, 21°). Drugs were added in 50-µl aliquots, and the tubes were placed in a 37° shaking water bath for 20 min. Assays were terminated by the addition of 500 µl of 0.15 N HCl. cAMP in the acid extract (500 µl) was acetylated and quantified by automated radioimmunoassay.

Intracellular Ca²⁺. BR cells were loaded with 1 µM Fura-2/AM in buffer containing 100 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 1 mM KH₂PO₄, 25 mM NaHCO₃, 0.5 mM CaCl₂, 2.7 g/liter D-glucose, 20 mM Na-HEPES, pH 7.4, and 0.25% BSA for 45 min. Cells were washed and resuspended in the same buffer without BSA, plus 1 unit/ml adenosine deaminase to a density of 1 × 10⁶ cells/ml. Fluorescence was measured with an SLM spectrofluorimeter in a thermostable cuvette (37°).

InsP₃. BR cells were preincubated for 24 hr with 2.5 µCi/ml myo-[³H]inositol in inositol-free low-glucose DMEM supplemented with 2% dialyzed fetal calf serum. The labeled cells were washed and resuspended in low-glucose DMEM with 25 mM HEPES, 1 unit/ml adenosine deaminase, and 100 mM LiCl and then transferred to polypropylene test tubes (4 × 10⁵ cells/0.2 ml) at 37° in a shaking water bath and stimulated by 5× agonists added in 50-µl aliquots for 10 min. Assays were terminated by the addition of 400 µl stop solution (0.5 M HCLO₄, 5 mM EDTA, and 1 mM diethylenetriaminpentacetic acid) plus 1 mg/ml phytic acid and placed on ice for 30 min before the addition of 5 M K₂CO₃ to raise the pH to 8-9. After centrifugation, the supernatants were passed through a 0.2-µm filter, applied to 1-ml Dowex AG 1-X8 columns (200-400 mesh), and washed with 5 ml of H₂O and 5 ml of 40 mM HCl; then, InsP₃ was eluted with 5 ml of 170 mM HCl.

	Results

Top Summary Introduction Procedures Results Discussion References

Molecular cloning of the canine A₃ adenosine receptor. The screening of a canine mastocytoma cDNA library with an A₃ adenosine receptor probe generated by RT-PCR resulted in the identification of several positively hybridizing clones. A clone designated cA₃13.1 contains a 1.6-kb insert with an open reading frame corresponding to 314 amino acids and 181 and 480 bp of 5 and 3 untranslated sequence, respectively (Fig. 1). A hydrophilicity plot of the deduced amino acid sequence predicts seven transmembrane domains, which are indicated in Fig. 2A. Sites found to be conserved within all species of A₃ adenosine receptors cloned to date include a putative palmitoylation site at Cys305 of the consensus sequence and two putative N-linked glycosylation sites at Asn4 and Asn162. Several putative phosphorylation sites are conserved among the A₃ adenosine receptors, including four potential sites for phosphorylation by protein kinase C (Thr124, Thr125, Ser/Thr215, and Thr230); one potential site for phosphorylation by tyrosine kinases (Tyr120); and one potential site for phosphorylation by cAMP/cGMP-dependent protein kinases (Thr294). The carboxyl tail distal to the palmitoylation site contains several serine/threonine residues that are surrounded by acidic groups that may be sites for phosphorylation by G protein receptor kinases.

View larger version (56K): [in this window] [in a new window]   Fig. 1.   Nucleotide and amino acid sequences of cA313.1, the canine A3 adenosine receptor. The sequence has been deposited in GenBank (accession no. U54792).

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Deduced amino acid sequence of cA313.1, the canine A3 adenosine receptor. A, Alignment with human, sheep, rat, and rabbit A3 adenosine receptor sequences. Solid lines, putative transmembrane (TM) domains with numbered designations (I-VII). Dashes, sequence gaps. Sites conserved among all species for possible N-linked glycosylation (glyc); phosphorylation by protein kinase C (PKC), cAMP-dependent protein kinase (cAMP), tyrosine kinases (tyr), and palmitoylation (palm) are designated. B, Dendogram illustrating species and subtype sequence differences among adenosine receptors. Distance along the horizontal axis is proportional to divergence of the amino acid sequences.

Tissue distribution of A₃ mRNA. Northern blots probing for cA₃13.1 transcripts in several different canine tissues revealed two hybridizing bands of 1.9 and 2.7 kb (Fig. 3). Transcripts were most abundantly expressed in spleen, but high levels also were detected in lung and liver. Two major hybridizing bands were also observed in testes, but the sizes were 1.3 and 2.4 kb. Transcripts were not detected in heart or kidney by Northern analysis. Using the sensitive technique of RT-PCR, trace transcripts were observed in all six tissues studied (data not show). Transcripts for A₁, A₃, and A_2B adenosine receptors were detected by Northern blotting of BR cell poly(A)⁺ mRNA (data not shown). The size of the A₃ transcripts, 1.9 and 2.7 kb, is the same as in dog spleen, lung, and liver. The A_2B transcript sizes are 1.6 and 1.8 kb and correspond to transcript sizes noted previously for A_2B receptor transcripts in mouse bone marrow-derived mast cells (21).

View larger version (48K): [in this window] [in a new window]   Fig. 3.   Tissue localization of the canine A3 adenosine receptor transcript. Northern blots of poly(A)+ RNA (5 µg/lane) from six different dog tissues using a probe corresponding to the carboxyl-terminal tail and 3 untranslated region of cA313.1. The same blot was stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase transcript (G3PDH). RNA size markers are indicated.

Pharmacological characterization of canine A₃ adenosine receptors. Binding of [¹²⁵I]ABA was measured to membranes prepared from COS-7 cells transfected with cA₃13.1 (Fig. 4). Specific binding was absent in untransfected cells and was abolished by 1 µM nonradioactive I-ABA. N-Ethylmaleimide has been reported to alkylate G proteins in the G_i/o family and cause them to become uncoupled from receptors. GTPS and N-ethylmaleimide both reduced specific binding of [¹²⁵I]ABA to canine A₃ adenosine receptors by ~60%, indicating that the radioligand is an agonist and that G_i/G_o proteins couple to the A₃ receptor (Fig. 4A). In equilibrium binding studies, [¹²⁵I]ABA specific binding was consistently found to fit significantly (p < 0.01) better to a two-site than to a one-site model (22). The respective high and low affinity K_d values of [¹²⁵I]ABA are 0.53 ± 0.13 and 16.4 ± 0.8 nM, and B_max values are 250 ± 9 and 768 ± 123 fmol/mg of membrane protein. In the presence of 50 µM GTPS, [¹²⁵I]ABA binds only to the low affinity site, with a K_d value of 17.4 ± 0.1 nM and a B_max value of 768 ± 123.0 fmol/mg of total protein. The conversion of receptors from two affinity states to a single low affinity state on the addition of GTPS is most clearly illustrated by Scatchard analysis (Fig. 4C). These results suggest that the high affinity site reflects binding to G protein-coupled receptors and the low affinity site reflects binding to uncoupled receptors. A similar analysis indicates that [¹²⁵I]ABA also binds to two affinity states of recombinant rabbit A₃ adenosine receptors with K_D values of 1.2 and 34 nM (not shown). In contrast, in filtration assays, [¹²⁵I]ABA detects only the high affinity state of canine A₁ receptors transiently expressed in COS-7 cells (K_d = 2.67 ± 0.50 nM, B_max = 1275 ± 52 fmol/mg protein; Fig. 5), and specific binding is almost completely abolished by the addition of GTPS (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Radioligand binding to recombinant canine A3 adenosine receptors. A, Inhibition of [125I]ABA (1.4 nM) binding to COS-7 cell membranes expressing cA313.1 by unradiolabeled I-ABA, GTPS, and N-ethylmaleimide. B, Equilibrium specific and nonspecific (NS) binding of [125I]ABA to transfected COS-7 cell membranes in the presence and absence of GTPS. C, Scatchard transformation of the specific binding data shown in B. For B and C, control data were fit optimally to two-site equations as described in Experimental Procedures. Values are mean ± standard error of triplicate determinations (25 µg of membrane protein/tube); where omitted, standard error bars are smaller than the symbols. The results are typical of three experiments. Binding parameters are summarized in Table 1.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Radioligand binding to recombinant canine A1 adenosine receptors (RDC7). Equilibrium specific () and nonspecific binding () binding of [125I]ABA to transfected COS-7 cell membranes is plotted. Inset, Scatchard transformation of the specific binding. Values are mean ± standard error of triplicate determinations (10 µg of membrane protein/tube); where omitted, standard error bars are smaller than the symbols. The results are typical of three experiments. Binding parameters are summarized in Table 1.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Competition for [125I]ABA binding to COS-7 cell membranes derived from cells transfected with canine A3 or A1 adenosine receptors. Binding is plotted as the fraction of control specific binding (>90% of total binding). Values are mean ± standard error of triplicate determinations from three experiments. Protein, 25 µg/tube (A3) or 10 µg/tube (A1), [125I]ABA, 150,000-300,000 cpm/tube (0.4-0.8 nM). The binding of [125I]ABA and competing ligands was fit to one or two binding sites as described in Experimental Procedures.

[¹²⁵I]ABA binding to canine BR mastocytoma cell membranes. Because both A₁ and A₃ adenosine receptor transcripts were detected in canine BR cells, we next determined whether [¹²⁵I]ABA binding to A₁ and/or A₃ receptors could be detected in membranes prepared from these cells. Little specific binding could be detected to crude membranes, but the binding of 0.4 nM radioligand to an enriched P2 membrane preparation was 80% specific (Fig. 7). Of the [¹²⁵I]ABA binding site on BR cell membranes, 24 ± 3% (three experiments) bind WRC-0571 with low affinity (IC₅₀ = 22 ± 9 µM) characteristic of A₃ receptors; the remainder bind WRC-0571 with high affinity (IC₅₀ = 114 ± 38 nM) characteristic of A₁ receptors (Fig. 7, Table 2). When added at 0.4 nM, [¹²⁵I]ABA labeled only 2.4 fmol/mg of protein of A₃ receptors in the P2 membranes of BR cells, suggesting the density of A₃ receptors on BR cells is low.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Competition by WRC-0571 for [125I]ABA binding to enriched plasma membranes derived from BR cells. P2 membranes were prepared as described in Radioligand Binding Studies. Binding was determined in triplicate to membranes containing 156,000 cpm (0.4 nM) [125I]ABA, 60 µg of P2 membrane protein, and various concentrations of WRC-0571. Nonspecific binding (10 µM I-ABA) is 846 ± 18 cpm (dashed line). Standard error bars are smaller than the symbols. The data were fit to a two-site model probably reflecting WRC-0571 binding to A1 and A3 adenosine receptors. Binding parameters from triplicate experiments are summarized in the text.

Characterization of the adenosine receptor that causes degranulation of canine mastocytoma cells. Adenosine agonists have been shown to enhance A23187 (calcimycin)-stimulated degranulation of several types of mast cells, including murine bone marrow-derived mast cells, human lung mast cells, RBL-2H3 cells, and rat peritoneal mast cells (23, 24). Fig. 8 shows the effect of increasing concentrations of Ca²⁺ ionophore to elicit -hexosaminidase release from BR cells when administered alone or in combination with the nonselective adenosine receptor agonist NECA (10 µM). When administered alone, A23187 evokes a maximal release of ~15.6% of the total cellular content after 20 min of stimulation. NECA (10 µM) alone also stimulates -hexosaminidase release (5.81 ± 0.59%), and costimulation with NECA and A23187 increases -hexosaminidase release to 28.2 ± 1.8%. NECA decreases the EC₅₀ for A23187 from 0.32 ± 0.06 to 0.13 ± 0.08 µM.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Stimulation of -hexosaminidase release from BR cells by A23187 and NECA. A, Cells (300,000/tube) were stimulated with increasing concentrations of A23187 in the presence or absence of 10 µM NECA for 20 min. -Hexosaminidase released into the supernatant was measured as described in Experimental Procedures. B, Effects on NECA-stimulated -hexosaminidase release of pretreatment of cells for 15 min with NBTI (1 µM) or XAC (1 µM). Data are pooled from three separate experiments, each assayed in triplicate. *, Different from control (p < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Effect of pretreatment of BR cells with pertussis toxin (PTX) on NECA-stimulated degranulation. Cells were pretreated for 24 hr with 0.3 µg/ml (A) or 1 µg/ml (B) pertussis toxin and then stimulated for 20 min with 1 µM A23187 and various concentrations of NECA. Data are pooled from three separate experiments, each assayed in triplicate.

View larger version (19K): [in this window] [in a new window]   Fig. 10.   -Hexosaminidase release from BR cells in response to adenosine receptor agonists. A, Cells were stimulated with adenosine agonists and 1 µM A23187 for 20 min. B, Cells were stimulated with adenosine agonists alone. Values are mean ± standard error of data pooled from three separate experiments, each assayed in triplicate.

View this table: [in this window] [in a new window]   TABLE 4 Potency of adenosine analogs to stimulate -hexosaminidase release from canine BR mastocytoma cells mean ± SEM, n = 3

View larger version (25K): [in this window] [in a new window]   Fig. 11.   Effects of enprofylline and Ro 20-1724 on -hexosaminidase release from BR cells. A, Antagonistic effect of enprofylline on NECA-induced -hexosaminidase release from BR cells costimulated with 1 µM A23187 and various concentrations of NECA. Inset, Schild plot; for enprofylline, pA2 = 5.1. B, Cells were pretreated with or without 20 µM Ro 20-1724 for 15 min and then stimulated with NECA. Data are mean ± standard error of three determinations; similar results were observed in a replicate experiment.

Second messenger responses evoked by A_2B receptor activation in canine mastocytoma cells. We next measured second messenger responses to adenosine receptor activation in BR cells. Unlike A₃ adenosine receptors, which are inhibitory to adenylyl cyclase, A_2B adenosine receptors stimulate adenylyl cyclase (26). We measured changes in intracellular levels of cAMP, Ca²⁺, and InsP₃ in response to NECA, CGS 21680, and IB-MECA. As shown in Fig. 12, NECA, but not CGS 21680 or IB-MECA, produces a concentration-dependent increase in intracellular levels of cAMP. The absence of cAMP accumulation in response to CGS-21680 indicates that accumulation of the cyclic nucleotide in response to NECA is not mediated by A_2A adenosine receptors. The calculated EC₅₀ for NECA to increase cAMP in BR cells is 0.9 ± 0.2 µM, which is similar to the EC₅₀ value for NECA to stimulate -hexosaminidase release (0.6 ± 0.3 µM), suggesting that both responses are mediated by the same receptor subtype. NECA (1 µM) also increased intracellular levels of Ca²⁺ and InsP₃, whereas 1 µM CGS -21680 or 1 µM IB-MECA had little effect (Fig. 12). Small responses to CGS 21680 and IB-MECA to influence cAMP, Ca²⁺, or InsP₃ suggest that selective activation of A_2A or A₃ receptors has little effect on cAMP or Ca²⁺ signaling in canine BR mastocytoma cells.

View larger version (16K): [in this window] [in a new window]   Fig. 12.   Changes in intracellular levels of cAMP, Ca2+, and InsP3 in BR cells during stimulation with adenosine agonists. A, cAMP levels were measured in suspended BR cells incubated for 10 min with 20 µM Ro 20-1724. B, Ca2+ levels were measured in suspended cells loaded with FURA-2/AM. C, InsP3 levels were measured in cells prelabeled with myo-[3H]inositol. All incubations included 1 unit/ml adenosine deaminase. Data are mean ± standard error of three experiments. *, Greater than control and IB-MECA (p < 0.01).

View larger version (25K): [in this window] [in a new window]   Fig. 13.   Antagonistic effects of enprofylline on second messenger responses in canine BR cells. A, cAMP responses to NECA and enprofylline. Inset, Schild plot; for enprofylline, pA2 = 5.3. B, Concentration-dependence of Ca2 responses to NECA with and without enprofylline (100 µM); the EC50 values for NECA are 760 and 5725 nM in the absence and presence of enprofylline, respectively. The pA2 value for enprofylline is estimated to be 4.8. Results are typical of three experiments.

	Discussion

Top Summary Introduction Procedures Results Discussion References

We cloned and characterized a canine A₃ adenosine receptor cDNA designated cA₃13.1 from canine BR mastocytoma cells. The clone is homologous to A₃ receptors cloned from other species. Transcripts for A₁ and A_2B adenosine receptors also were detected in BR cells, and evidence of A₁, A_2B, and A₃ receptor expression was found on the basis of radioligand binding or functional assays. The A_2B receptor predominates in the regulation of BR cell degranulation, as discussed in detail below.

The tissue distribution of A₃ adenosine receptor transcript in dog is similar to the human distribution (3), with highest levels expressed in spleen, followed by lung and liver. The tissue distribution is different from rat, in which transcript is much more abundant in testes than in other tissues (1). In terms of sequence homology and pharmacology, the canine A₃ adenosine receptor is more similar to the human than to the rat A₃ receptor.

The results of radioligand binding assays with the A₁/A₃ agonist [¹²⁵I]ABA indicate that the canine A₃ adenosine receptor binds to both G protein-coupled and -uncoupled receptors with K_D values that differ by ~30-fold. The potency order of agonists for the A₃ receptor, IB-MECA > R-PIA  NECA > CPA, is consistent among species. In all species, IB-MECA and CPA are A₃ and A₁ selective, respectively. Specific binding of [¹²⁵I]ABA to the uncoupled conformation of the canine A₃ adenosine receptor distinguishes the canine A₃ receptor from the uncoupled canine A₁ receptor, which has too low affinity for [¹²⁵I]ABA binding to be detected in filtration assays. However, [¹²⁵I]ABA binds with 10 times higher affinity to bovine than to canine A₁ receptors, and the radioligand can detect two affinity states of the bovine A₁ adenosine receptor [K_d = 0.09 and 10.4 nM (29)]. The detection in filtration assays of two affinity states of A₃ receptors complicates the analysis of competition binding assays because the radioligand binds with two affinities and competing agonists and antagonists bind with two or one affinities, respectively. To calculate the K_i values of competing compounds required the derivation of nonstandard analytical procedures (see Analysis of Binding Data). As summarized in Table 1, I-ABA and IB-MECA both bind with high affinity (K_D < 1 nM) to the G protein-coupled conformation of canine A₃ receptors. Failure to analytically resolve the two agonist affinity states in radioligand binding assays will result in underestimation of high affinity agonist dissociation constants as well as errors in the calculation of the dissociation constants of competing compounds based on the Cheng and Prusoff formula (30). It is notable that in the range of 0.1-1 µM, compounds that often are used as selective agonists of A₁ receptors (CPA) or A_2A receptors (CGS-21680) also will bind to canine A₃ receptors. Hence, caution must be taken in attributing functional responses of these compounds to particular adenosine receptor subtypes.

This study confirms and extends the observation that there are substantial species differences in the binding of xanthines to A₃ adenosine receptors. The rat A₃ adenosine receptor, the first A₃ adenosine receptor to be cloned, was originally reported not to bind xanthine antagonists (2). Subsequent studies have shown that xanthines bind weakly to the rat receptor. The most potent xanthine antagonist, I-ABOPX, binds to the rat A₃ receptor with a K_I value of 1.5 µM. In contrast, sheep, human, and canine A₃ receptors bind I-ABOPX with 80-500 times higher affinity (3, 4).

CPX is widely regarded as a selective antagonist of A₁ adenosine receptors. Although CPX is >250-fold selective for human A₁ over A₃ receptors (27), this selectivity drops to only 10-fold in the case of canine receptors. This is partly due to the fact that compared with human and sheep A₃ receptors, canine A₃ receptors bind CPX with relatively high affinity. In addition, canine A₁ adenosine receptors have lower affinity than other species for CPX. Consequently, CPX is not particularly useful for discriminating between A₁ and A₃ receptor-mediated responses in the dog. A preferable compound for this purpose is WRC-0571, an A₁-selective nonxanthine antagonist that is >4000-fold selective for human A₁ over A₃ receptors (31). Although WRC-0571 binds with much lower affinity to canine A₁ receptors (K_I = 484 nM) than to human A₁ receptors (K_I = 3 nM), it still is 35-fold selective as an antagonist of canine A₁ over A₃ receptors. Species differences in binding affinity also are significant for BW-A1433 [8-(4-carboxyethenylphenyl)-1,3-dipropylxanthine], which is sometimes used as a A₃ receptor antagonist on the basis of its moderate affinity for sheep and human A₃ receptors (3, 4). BW-A1433 is a relatively weak and nonselective antagonist of canine A₃ receptors, binding with 10-fold lower affinity to canine than to human A₃ receptors.

Enprofylline, an antiasthmatic agent that has moderate affinity for human A₃ receptors [K_I = 156 ± 110 µM (27)], binds poorly to the canine A₁ and A₃ receptors (K_I > 100 µM). Because enprofylline binds to the human A_2B adenosine receptors with a K_I value of 7 µM (27), the compound was evaluated in this study to discriminate between canine A_2B and A₁ or A₃ adenosine receptor-mediated responses. Inasmuch as the canine A₃ receptor clone was isolated from a canine mastocytoma cDNA library, we anticipated that the A₃ receptor subtype would be responsible for stimulating the release of granule-associated mediators. However, A_2B and A₁ as well as A₃ transcript were found in BR cells, and low levels of A₁ and A₃ receptor binding sites could be detected on enriched plasma membranes prepared from the canine mastocytoma cells. Nevertheless, several lines of evidence indicate that BR cell degranulation requires activation of the A_2B but not the A₁ or A₃ adenosine receptor: (i) degranulation of BR cells is not prevented by pretreatment of cells with pertussis toxin; (ii) the response is blocked by enprofylline with a pA₂ value near 5, an affinity similar to that of human A_2B receptors and higher that the affinity of enprofylline for canine A₁ or A₃ receptors; (iii) the potency order of agonists to stimulate degranulation, NECA > PIA > CGS-21680 > IB-MECA, differs from the potency order of these compounds to bind to recombinant canine A₃ adenosine receptors; and (iv) NECA, but not CGS-21680, elevates cAMP, which is consistent with the existence of functional A_2B receptors on BR cells.

It was somewhat unexpected that A_2B adenosine receptors seem to couple to Ca²⁺ mobilization in canine mastocytoma cells inasmuch as A_2B adenosine receptors have been shown to couple to stimulation of cAMP accumulation (26). Apparent dual coupling to cAMP and Ca²⁺ has also been noted in HEK 293 cells stably transfected with recombinant human A_2B receptors,² and it is significant in this regard that the expression of recombinant rat A_2B receptors in Xenopus laevis oocytes results in the appearance of adenosine-mediated Ca²⁺-dependent Cl current (32). Dual coupling of G protein coupled receptors to G_s and G_q/11 is not unprecedented. For example, the human prostacyclin receptor also displays such dual coupling (33). Coupling of A_2B adenosine receptors to a Ca²⁺-mobilizing G protein resistant to pertussis toxin (G_q/11) may be essential for triggering BR cell degranulation because agents that elevate cAMP in various kinds of mast cells, including agonists of A_2A adenosine receptors, either have no effect or are inhibitory to degranulation (34, 35).

The conclusions of previous studies have been inconsistent regarding the adenosine receptor subtype that mediates mast cell degranulation. Recent DNA antisense experiments suggest that activation of A₁ adenosine receptors may contribute to bronchoconstriction in a rabbit model of asthma (36). However, the low potency of various A₁-selective xanthines to block histamine release from asthmatic human lung fragments (10) and the low potency of enprofylline to block human A₁ adenosine receptors (27) are consistent with the participation of A_2B and/or A₃ receptors in human disease. The A₃ adenosine receptor has been implicated in the degranulation of RBL 2H3 rat mast cells and in triggering vascular responses³ secondary to degranulation of mast cells in the hamster cheek pouch (9, 37) and the pithed rat (25). A_2B adenosine receptors seem to mediate the degranulation of murine bone marrow-derived mast cells (21), and although pretreatment of RBL 2H3 rat mast cells with pertussis toxin abolishes NECA-mediated degranulation, Ca²⁺ mobilization in these cells requires activation of G_i3 or G_q (38). Moreover, activation of phosphoinositide breakdown in RBL 2H3 cells is not well correlated with the affinity of adenosine analogs for A₃ adenosine receptors (39). The treatment of murine bone marrow mast cells with pertussis toxin produces a decrease in the potency of adenosine to enhance degranulation in response to A23187, similar to the result in the current study with canine mastocytoma cells. Pretreatment of murine mast cells with pertussis toxin fails to reduce adenosine-mediated Ca²⁺ mobilization, which is consistent with an A_2B-mediated, but not an A₃-mediated, response (40). In the human HMC-1 mast cell leukemia line, the ability of 300 µM enprofylline to block NECA-stimulated interleukin-8 release was taken as evidence that this response also is mediated by A_2B adenosine receptors (41). The current study tends to substantiate previously published reports that suggest the receptor subtype primarily responsible for adenosine-mediated mast cell degranulation is variable. It is not yet clear whether this variability depends on species, tissue source, or environmental factors that affect signaling cascades and/or the phenotype of various mast cells. The results of this study indicate that A_2B receptors play a major role in the regulation of mast cell degranulation but are consistent with possible participation of multiple adenosine receptor subtypes and multiple G proteins.

The canine A₃ adenosine receptor described in this study is structurally and pharmacologically more similar to human A₃ receptors than are A₃ receptors from rodent species. This finding, along with the fact that it seems that murine bone marrow mast cells, canine mastocytoma, and human HMC-1 leukemic mast cells are regulated by adenosine, primarily via A_2B receptors, raises the possibility that canine models of asthma may be better predictors of human disease than rodent models. It will be important to determine which adenosine receptor subtype or subtypes are responsible for facilitating mast cell degranulation in the asthmatic human lung. Once the predominant receptor or receptors are identified, novel antagonists superior to theophylline and enprofylline for the treatment of asthma may be developed.