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Cloning and pharmacological characterization of the monkey histamine H 3 receptor

Cloning and pharmacological characterization of the monkey histamine H 3 receptor
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  Cloning and pharmacological characterization of the equine adenosine A 3 receptor C. I. BRANDON*M. VANDENPLAS  H. DOOKWAH  &T. F. MURRAY* * Department of Physiology and Pharmacology;   Large Animal Medicine;and    Anatomy and Radiology, College of Veterinary Medicine, University of Georgia,Athens, GA, USA Brandon CI, Vandenplas M, Dookwah H, Murray TF. Cloning and pharmaco-logical characterization of the equine adenosine A 3  receptor.  J. vet. Pharmacol.Therap.  29,  255–263.The aim of this study was to establish a heterologous expression system for theequine adenosine A 3  receptor (eA 3 -R) in an effort to ascertain its pharmacologicprofile. Initially, radioligand binding assays identified clones expressing the eA 3 -R in human embryonic kidney cells (HEK) based on the specific binding of [ 125 I]AB-MECA. Subsequently, adenylate cyclase assays were utilized todemonstrate functional coupling of the eA 3 -R to the G-protein/adenylatecyclase system. Equilibrium competition binding assays were then performedusing selective and non-selective A 3  agonists and antagonists. Results fromthese experiments revealed a rank order of agonist potency to beIB-MECA > NECA > CGS21680, and an antagonist potency of MRS1220 > ZM241385 > 8-  p -sulphophenyltheophylline; these rank orderswere in agreement with that of other mammalian A 3 -R’s. Lastly, NF- j Breporter gene assays revealed an IB-MECA concentration-dependent inhibitionof TNF a -stimulated NF- j B activity. These results indicate that the heterolo-gously expressed eA 3 -R is functional, has a pharmacological profile similar tothat of other mammalian A 3  receptors, and its activation has an inhibitoryeffect on a key regulatory pathway in the inflammatory response. Thus, theeA 3 -R may serve as a pharmacological target in the treatment of equineinflammatory disease.(Paper received 29 March 2006; accepted for publication 7 April 2006) Thomas F. Murray, PhD, Editor, Critical Reviews in Neurobiology, Distinguished Research Professor and Head, Department of Physiology and Pharmacology, Collegeof Veterinary Medicine, University of Georgia Athens, GA 30602, USA. INTRODUCTIONAdenosine is an endogenous purine nucleoside that modulatesmany physiologic processes, and initiates signal transductionpathways through four known adenosine receptor subtypes (A 1 ,A 2A , A 2B , and A 3 ); all of which are seven transmembranespanning G-protein coupled receptors (Hettinger  et al. , 1998;Olah & Stiles, 2000). These receptors have been classified furtherbased on their ability to either stimulate or inhibit adenylatecyclase activity; specifically, the A 2A  and A 2B  receptors couple toG a s  and stimulate adenylate cyclase, whereas the A 1  and A 3 adenosine receptors couple intracellularly to G a i , which inhibitsadenylate cyclase (Cronstein, 1994; Linden, 2001). Additionally, A 1  receptors couple to G a o , which mediates adenosine inhibitionof Ca 2+ conductance (Gao  et al. , 2001), while A 2B  and A 3 receptors also couple to G a q  and stimulate phospholipase activity(Schulte & Fredholm, 2003).Adenosine has long been known to play a protective role incells undergoing ischmic or hypoxic stress. For example,extracellular adenosine concentrations from normal cells areapproximately 300 n M  (Hirschhorn  et al. , 1981); however, inresponse to cellular damage (e.g. in inflammatory or ischemictissue), adenosine concentrations rapidly exceed 1  l M  (Cronstein et al. , 1995). Thus, in regards to stress or injury, the function of adenosine is primarily that of cytoprotection by preventing tissuedamage during instances of hypoxia, ischemia, or seizure activity(Ibayashi  et al. , 1988). Because adenosine has an extremelyshort half-life of approximately 2 sec, and its administration is Abbreviations: [ 125 I]AB-MECA, [ 125 I] N6-4-amino-3-iodobenzyladeno-sine-5- N  -methyluronamide; CGS21680, 2-p-(2-Carboxyethyl)pheneth-ylamino-5 ¢ - N  -ethylcarboxamidoadenosine; eA 3 -R, equine adenosine A 3 receptor; IB-MECA, N6-(3-iodobenzyl)adenosine-5 ¢ - N  -methyluronamide;MRS1220, 9-Chloro-2-(2-furanyl)-5-((phenylacetyl)amino)-[1,2,4]triaz-olo[1,5- c]quinazoline; NECA, 5 ¢ - N  -ethylcarboxamido-adenosine;ZM241385, 4-(2-[7-Amino-2-(2-furyl)-[1,2,4]-triazolo-[2,3-a]-[1,3,5]-triazin-5-ylamino]ethyl)phenol.  J. vet. Pharmacol. Therap.  29,  255–263, 2006.   2006 The Authors. Journal compilation    2006 Blackwell Publishing Ltd 255  associated with adverse side effects (e.g. hypotension, bradycar-dia, and hypothermia), its therapeutic potential is limited. Toovercome these problems, research into the activation of adenosine receptor subtypes has primarily focused on the useof adenosine analogs that are highly specific as well as efficaciousfor individual adenosine receptor subtypes.One of the more important effects of adenosine receptoractivation is the reported anti-inflammatory properties of thenucleoside. One of the central mediators of the inflammatoryresponse is nuclear factor- j B (NF- j B), with >100 genes beingactivated by this transcription factor; most of which areproinflammatory in nature (Ghosh  et al. , 1998; May & Ghosh,1998). NF- j B is primarily composed of two proteins havingmolecular masses of 50 kDa (p50) and 65 kDa (p65), and isretained in the cytoplasm in an inactive form by an inhibitorysubunit I j B (Majumdar & Aggarwal, 2003). NF- j B can beactivated by an array of inflammatory stimuli including TNF a ,IL-1, H 2 O 2 , as well as endotoxin; these stimuli induce phos-phorylation-dependent degradation of I j B isoforms allowingnuclear translocation of NF- j B where it then regulates thetranscription of a host of inflammatory genes. Activation of adenosine receptors selectively suppresses NF- j B activation,which may contribute to its anti-inflammatory effects (Majum-dar & Aggarwal, 2003). Thus, adenosine receptors are likelycandidates as therapeutic targets for the treatment of inflamma-tory and immune disorders.The goal of this study was to characterize the equineadenosine A 3  receptor [equine adenosine A 3  receptor (eA 3 -R)]to determine if it might serve as a therapeutic target for thetreatment of equine endotoxemia. Thus, the objectives of thisstudy were (1) to heterologously express the equine A 3  receptorsin Human Embryonic Kidney 293 (HEK) cells; (2) to characterizethe pharmacological signature of this receptor utilizing radiolig-and-binding assays; and (3) to determine the effect of eA 3 -Ractivation on TNF a -stimulated NF- j B activity.MATERIALS AND METHODS Cloning and sequencing of the equine A 3  receptor cDNA An equine mesenteric lymph node cDNA library was constructed,and subsequently screened for the equine A 3  receptor cDNA byhybridization, utilizing a  32 P end-labeled rat A 3  oligonucleotideas a probe. A single hybridization positive plasmid was isolated byalkali lysis over Qiagen columns (Valencia, CA, USA), and foundto contain an insert of the estimated size for the full length equineA 3  receptor; this plasmid was subsequently selected for sequen-cing. The plasmid was sequenced in either direction with an ABIPrism BigDye TM Terminator Cycle Sequencing Ready ReactionKit (versions 3.0; Applied Biosystems, Foster City, CA, USA) inboth 96- and 384-well format. Sequencing reactions included4  l l of plasmid DNA (100 ng), 44 pmol of primer, 0.70  l l of BigDye TM , 0.58  l l ddH 2 O, 0.34  l l DMSO, 1.4  l l 5 ·  reactionbuffer (5x  ¼  10 mM MgCl 2 , 400 mM Tris-Cl pH 9.0), resulting ina total reaction volume of 7.2  l l. Thermal cycling reactions wereperformed on a 96-well GeneAmp  PCR System (AppliedBiosystems, Foster City, CA, USA) and an Autolid Dual 384-wellGeneAmp  PCR System 9700 (Applied Biosystems). The purifiedextension products were separated on an ABI Prism  3700 DNAAnalyzer (Applied Biosystems).The eA 3 -R-containing plasmid was then amplified by trans-formation into bacterial cells (Top 10 cells; Invitrogen, Carlsbad,CA, USA), which were then grown overnight at 37   C on LB agarplates containing 50  l g/ml ampicillin (Sigma, St Louis, MO,USA). Ampicillin-resistant colonies were then inoculated in300 mL of LB broth (Sigma) containing 50  l g/mL ampicillinand shaken overnight at 37   C, 5% CO 2 . Plasmid purification of the resulting culture was achieved using an endotoxin-freeplasmid purification kit (Endo-Free Maxi Kit; Qiagen, Valencia,CA, USA) as described previously (Meyer, 1990). An  EcoRI  restriction fragment of the eA 3 -R cDNA was then non-direction-ally subcloned into an  EcoRI   restriction site of the pcDNA3.1expression vector (Invitrogen) using a Takara DNA ligation kit(Fisher Scientific, Suwannee, GA, USA). Specifically, the eA 3 -RcDNA and pcDNA3.1 expression vector were digested in separatetubes with  EcoRI   at 37   C for 3 h. Before ligation, the pcDNA3.1vector was treated further with calf intestinal alkaline phospha-tase to prevent re-circularization of the plasmid DNA; after whichboth the eA 3 -R and pcDNA3.1 were extracted in phenol– chloroform to remove extraneous salts and proteins. Theresulting eA 3 -R  EcoRI   restriction fragment and  EcoRI   digestedpcDNA3.1 were resolved on an agarose gel, purified and ligatedin a reaction using T4 DNA ligase at 16   C, 30 min. As the eA 3 -RcDNA was nondirectionally cloned, a restriction enzyme mappingexperiment was first performed to identify clones in which theeA 3 -R cDNA was in its properly oriented 5 ¢  to 3 ¢  direction. Briefly,2  l l of the eA 3 -R cDNA was incubated with the restrictionenzymes  Not I and  Pst I, and the digestion reaction was allowed toproceed at 37   C for 3 h. Subsequent agarose gel analysisrevealed clones having the properly oriented eA 3 -R cDNA. Fromthis subgroup, the eA 3 .03 clone was selected for sequencing.The eA 3 .03-R cDNA was then sequenced fully from either endusing a forward primer constructed for the T7 promoter region of the pcDNA3.1 vector (5 ¢ -TAATACGACTCACTATAGCG-3 ¢ ) andan internal forward primer 550 bp into the eA 3 .03-R cDNA (5 ¢ -CCTGAGCATCACAATCCAC-3 ¢ ). Sequencing from the 3 ¢  endwas achieved using a pcDNA3.1 reverse primer (5 ¢ -TAG-AAGGCACAGTCGAGG-3 ¢ ) for the bovine growth hormonereverse priming site of pcDNA3.1, and an eA 3 .03-R cDNAinternal reverse primer (5 ¢ -GCTAAATCTGAAGGTCAATGGC-3 ¢ ).Sequencing reactions were performed at the University of Georgia Molecular Genetics Instrumentation Facility (Athens,GA, USA) on an ABI 3700 automated sequencer (AmershamBiosciences, Piscataway, NJ, USA) as previously described.Sequence fragments were then assembled into a contig usingAssemblyLIGN software (Accelrys, Norwalk, CT, USA), andtranslation start and stop codons identified via sequence analysisutilizing MacVector 6.5.3 software (Accelrys). The resultingcoding region of the eA 3 -R cDNA was then translated into itscorresponding amino acid sequence and aligned with othermammalian A 3 -R proteins (MacVector). Conserved amino acid 256  C. I. Brandon  et al.   2006 The Authors. Journal compilation    2006 Blackwell Publishing Ltd  residues were then divided by the total alignment score todetermine receptor sequence homology. Heterologous expression system For stable transfections, human HEK293 were seeded in six-wellmicrotiter plates (Becton-Dickinson, Bedford, MA, USA) at adensity of 6  ·  10 4 cells/well, and cultured in complete mediumcontaining Minimal Essential Media (MEM; Sigma), 10% FCS,and 1% penicillin–streptomycin to approximately 75% conflu-ence. Transfection of the eA 3 -R cDNA was performed usingPolyfect transfection reagent (Qiagen, Valencia, CA, USA).Initially, 2  l g of the eA 3 -pcDNA plasmid were added toEppendorf tubes, and brought to a final volume of 100  l l withTE buffer (Tris-EDTA), pH 7.5. Twenty microliters of Polyfecttransfection reagent (40  l g) was then added and allowed toincubate at room temperature for 10 min. HEK cell growthmedia was removed and replaced with 1.5 mL complete media inthe absence of antibiotics, followed by the addition of the eA 3 -RcDNA/Polyfect reaction complex. The transfection reaction wasincubated for 6 h at 37   C, 5% CO 2 , and 95% relative humidity,after which the transfection media was replaced with MEMsupplemented with 10% FCS and 1% penicillin–streptomycin. At24 h post-transfection, the selection media, comprised of com-plete media that included 0.25 mg/mL of the antibiotic geneticin(G418; Invitrogen) was added. To isolate individual eA 3 -Rclones, plaques were isolated using 6.4  ·  8 mm cloning cylin-ders (Fisher), trypsinized, and transferred to separate cultureflasks. Individual G418-resistant eA 3 -R/HEK cell clones werethen maintained at 37   C, 5% CO 2 , 95% relative humidity untilpharmacological characterization experiments were performed. [  125 I]AB-MECA binding assay To screen individual clones for A 3  receptor expression, radiolig-and binding assays were performed. Initially, eA 3 -R expressingHEK cells were grown to confluence in 150  ·  15 mm Nuncculture plates (Fisher), after which cells were harvested in icecold 25 m M  Tris, pH 7.5. Equine A 3 /HEK cell membranepreparations were then collected by Dounce homogenization(20 strokes), and the resulting membranes centrifuged at40 000  ·  g  for 20 min. The membranes were then resuspendedin the Tris buffer and washed two additional times before thebinding assays. [ 125 I] N6-4-amino-3-iodobenzyladenosine-5- N  -methyluronamide ([ 125 I]AB-MECA) binding assays were per-formed using 25  l g of eA 3 /HEK cell membrane and incubated inthe presence of 0.25 n M  of the selective A 3  agonist [ 125 I]AB-MECA (Amersham) alone or in the presence of 10  l M  N6-(3-iodobenzyl)adenosine-5 ¢ - N  -methyluronamide (IB-MECA) to de-fine non-specific binding. The binding reaction proceeded for45 min at room temperature, after which the membranes wereharvested via rapid filtration onto Brandell GF/B filters using a24-well cell harvester (Brandell, Gaithersburg, MD, USA). Radio-activity was then determined on a Perkin Elmer 1470 automaticgamma counter (Perkin Elmer, Downers Grove, IL, USA), andspecific binding was determined by subtracting non-specific fromtotal binding. Data were analyzed using GraphPad Prism Soft-ware (Prism 4.0; Graphpad Software Inc., San Diego, CA, USA). Adenylate cyclase assays To determine that selected clones functionally coupled tointracellular G-proteins, adenylate cyclase assays were per-formed. Briefly, eA 3 -HEK cells were plated in six-well microtiterplates at a density of 7.5  ·  10 4 cells/well and cultured for 24 h.The cells were then washed three times in serum-free media, andpreloaded for 5 h with [ 3 H]-adenine at a final concentration of 1.2  l Ci/mL. Cells were then washed three times in serum-freemedia followed by the addition of 10  l L of the selective A 3 agonist IB-MECA in a buffer containing 50  l M  of the phospho-diesterase inhibitor rolipram (Sigma), 50  l M  forskolin – a directactivator of adenylate cyclase (Sigma), 2.0 U/mL adenosinedeaminase, and incubated for 40 min at 37   C, and 5% CO 2 . Thecells were then lysed, and the supernatant transferred to 1.5 mLmicrocentrifuge tubes and centrifuged at 10 000  ·  g  for 15 minat 4   C. The resulting supernatant was washed over Dowex(50W-X4 Resin; Bio-Rad, Hercules, CA, USA) followed byalumina columns, and [ 3 H]cAMP eluted from the aluminacolumns by the addition of 0.1  M  imidazole buffer. Radioactivitywas determined using a Beckman LS6000 scintillation counter(Beckman Coulter, Fullerton, CA, USA). Concentration–responsedata were analyzed utilizing nonlinear regression analysis withGraphPad Prism Software (Prism 4.0). Equilibrium competition binding Todeterminethepharmacologicalsignatureoftheheterologouslyexpressed e-A 3 -R’s, equilibrium competition experiments wereperformed to determine the rank order of agonist and antagonistpotencies at the eA 3 -R. Briefly, 25  l g of e-A 3 /HEK cell membranewere incubated with 0.25 nM [ 125 I]AB-MECA and increasingconcentrations of either the selective A 3  agonist IB-MECA, thenonselective agonist 5 ¢ - N  -ethylcarboxamido-adenosine (NECA),and the A 2A  agonist 2-p-(2-Carboxyethyl)phenethylamino-5 ¢ - N  -ethylcarboxamidoadenosine (CGS21680). To determine the rankorder of antagonist potency at the eA 3 -R cell membranes wereincubated with [ 125 I]AB-MECA in the presence of either the A 3 selective antagonist 9-Chloro-2-(2-furanyl)-5-((phenylace-tyl)amino)-[1,2,4]triazolo[1,5- c]quinazoline (MRS1220), theA 2A  selective 4-(2-[7-Amino-2-(2-furyl)-[1,2,4]-triazolo-[2,3-a]-[1,3,5]-triazin-5-ylamino]ethyl)phenol (ZM241385), or the non-selective 8-para-sulfophenyltheophylline. The binding reactionwas allowed to proceed for 45 min at room temperature.Competition data were analyzed utilizing nonlinear regressionanalysis with GraphPad Prism Software (Prism 4.0). Reporter gene and electrophoretic mobility shift assays To determine the effect of receptor activation on signaltransduction, NF- j B reporter gene assays were performed. TheeA 3  2.0 B receptor clone was seeded in a 96-well microtiter plate(Becton-Dickinson) at a density of 2.5  ·  10 4 cells and incubated Equine adenosine A 3  receptor   257   2006 The Authors. Journal compilation    2006 Blackwell Publishing Ltd  until approximately 75–80% confluence was achieved. The cellswere then transiently transfected with 50 ng/well of theendothelial leukocyte adhesion molecule (ELAM) NF- j B-depend-ent firefly luciferase reporter construct (a generous gift from DrDouglas Golenbock, University of Massachusetts Medical School,Worcester, MA, USA), and 5 ng/well of the synthetic  Renilla luciferase reporter plasmid (Promega, Madison, WI, USA) usingPolyfect transfection reagent (Qiagen). Initially, 100 ng totalDNA (50 ng ELAM, 5 ng  Renilla , 45 ng empty vector [pcDNA])was added to individual tubes, followed by the addition of serum-free media to achieve a final volume of 20  l L DNA/well. Three l g of Polyfect transfection reagent was then added per well, andincubated at room temperature for 10 min, after whichMEM + 10% FCS was added to achieve a final volume of 100  l L/well. After the Media was then removed and replacedwith 100  l L complete media containing 10% FCS, 1% penicil-lin–streptomcyin, followed by the addition of 100  l L/well of theELAM/ Renilla /pcDNA-Polyfect reaction mixture. The transfec-tion reaction was allowed to proceed for 6 h, after which thecells were washed in serum-free media. The media was thenreplaced with MEM containing of 10% FCS, 1% penicillin– streptomycin, and 0.25 mg/mL G418. Following a 48 h recov-ery period, the cells were then stimulated with human recom-binant TNF a  at a final concentration of 10 ng/mL for 4 h in thepresence of the selective A 3  agonist IB-MECA at a concentrationrange from 100 n M  to 10  l M . After the stimulation reaction,cells were lysed with 50  l L passive lysis buffer and assayed forfirefly and  Renilla  luciferase activities, respectively using theDual–Luciferase Reporter Assay kit (Promega). Luciferase activ-ity was normalized relative to the  Renilla  luciferase activity tocontrol for differences in transfection efficiencies.To determine the effect of eA 3  receptor activation on NF- j Bnuclear translocation, electrophoretic mobility shift assays wereperformed. Briefly, cells were plated in 15 mm culture plates(Becton-Dickinson) and allowed to reach 75–80% confluency.The cells were then stimulated as in the reporter gene assays for50 min. Growth media used were either MEM + 10% FCS, orMEM + 10% FCS including 50  l M  of the phosphodiesteraseinhibitor rolipram. After stimulation, cells were trypsinized,washed 2X in ice cold PBS, and the nuclear extracts collected byincubation in a hypotonic HEPES buffer, followed by the additionof 10% NP-40. Cells were then centrifuged, supernatantsdiscarded, and the nuclear pellet suspended in a hypertonicHEPES buffer, extracted at 4   C for 30 min with mixing, followedby centrifugation at 14 000  ·  g , 4   C for 5 min. Equivalentamounts of nuclear extract were then incubated with a [ c 32 P]end-labeled NF- j B oligonucleotide for 20 min at room tempera-ture. A 1/10 volume of 10X gel loading buffer was then added toeach reaction tube, and the protein/oligonucleotide complexeswere resolved on a 4% non-denaturing polyacrylamide gel. Afterprotein migration, the gel was dried and opposed to aphosphoimager for at least 24 h; the resulting bands were thenanalyzed on a 70–7856 Molecular FX-Pro imager (Bio-Rad).RESULTS Equine A 3 -R cloning and sequencing Cloning and sequencing of the eA 3 -R cDNA revealed a full-lengthtranscript complete with initiation and termination codons, aswell as 5 ¢  and 3 ¢  untranslated regions. ClustalW alignment of theeA 3 -R cDNA indicated that this receptor cDNA had a high degreeof sequence similarity with that of other mammalian A 3  receptortranscripts. The position of the start codon was conserved in thealignment of the equine, human, ovine, and canine A 3 -R cDNA,respectively. However, while the termination signal was con-served for the equine, human, and ovine, the canine terminationsignal was located 15 base pairs upstream. Furthermore, BLASThomology screening of the deduced amino acid sequence of theeA 3 -R with that of other mammalian A 3 -R proteins revealed asimilar degree of homology among species (Fig. 1). ClustalW formatted alignments Sheep.A3.Prot Human.A3.Prot K9.A3.Prot Equine A3  10 20 30 40 50 60 70  M P V N  S  T A  V  S  W T S  V T Y I T V E I L I G L C A I V G N V L V I W V V K L N P S L Q T T T F Y F I V S L A L A D I A V G V L V M P L A IM P  N  N  S  T A L S  L  A  N  V T Y I T  M  E I  F  I G L C A I V G N V L V I  C  V V K L N P S L Q T T T F Y F I V S L A L A D I A V G V L V M P L A IM  A  V N  G  T A L  L L  A  N  V T Y I T V E I L I G L C A I V G N V L V I W V V K L N P S L Q T T T F Y F I V S L A L A D I A V G V L V M P L A IM P V N  G  T A L S  W  A  T A  T Y I T V E I L I G L C A I V G N V L V I W V V K L N P S L Q T T T F Y F I  I  S L A L A D I A V G V L V M P L A I M P V N T A L S A . V T Y I T V E I L I G L C A I V G N V L V I W V V K L N P S L Q T T T F Y F I V S L A L A D I A V G V L V M P L A I Sheep.A3.Prot Human.A3.Prot K9.A3.Prot Equine A3  80 90 100 110 120 130 140  V I S L G  V  T I H F Y S C L F M T C L  M  L I F T H A S I M S L  - -  L A I A V D R Y L R V K L T V R Y  R  R V T T Q R R I W L A L G L C W L V SV  V  S L G I T I H F Y S C L F M T C L  L  L I F T H A S I M S L  - -  L A I A V D R Y L R V K L T V R Y  K  R V T T  H  R R I W L A L G L C W L V SV I S L G I T I  Q  F Y  N  C L F M T C L  L  L I F T H A S I M S L  - -  L A I A V D R Y L R V K L T V R Y  R  R V T T Q R R I W L A L G L C W L V SV I S L  S  I T I H F Y S C L  L  M T C L  M  L  V  F T H A S I M S L  S F  L A I A V D R Y L R V K L T  I  R Y  K  R V T T Q R R I W L A L G L C W L V S V I S L G I T I H F Y S C L F M T C L . L I F T H A S I M S L L A I A V D R Y L R V K L T V R Y . R V T T Q R R I W L A L G L C W L V S Sheep.A3.Prot Human.A3.Prot K9.A3.Prot Equine A3  150 160 170 180 190 200 210  F L V G L T P M F G W N M K L  S  S  A D E N - L  T F L  P  C  R  F  R  S V M R M D Y M V Y F S F F  L  W I  L V  P L V V M C A I Y  F  D I F Y  I  I R N  R  LF L V G L T P M F G W N M K L T S  E Y H R N V  T F L  S  C Q F  V  S V M R M D Y M V Y F S F  L  T W I  F  I P L V V M C A I Y L D I F Y  I  I R N K LF L V G L T P M F G W N M K L T S  E H Q R N V  T F L  S  C Q F  S  S V M R M D Y M V Y F S F F T W I  L  I P L V V M C A I Y L D I F Y  V  I R N K LF L V G L T P M F G W N  K  K L T S  G S K N - - D S  L  A  C Q F  R  S V  V S  M D Y M V Y F S F F T W I  F  I P L  A  V M  S  A I Y L D I F  F V  I R N K L F L V G L T P M F G W N M K L T S . . T F L . C Q F S V M R M D Y M V Y F S F F T W I I P L V V M C A I Y L D I F Y . I R N K L Sheep.A3.Prot Human.A3.Prot K9.A3.Prot Equine A3  220 230 240 250 260 270 280  S Q  S F  S  G  S  R  E T G A F Y G R E F K T A K S L  L  L V L F L F A L  C  W L P L S I I N C I  L  Y F  D  G  Q  V P Q  T  V L Y L G I L L S H A N S M M NS  L  N  L  S  N  S K E T G A F Y G R E F K T A K S L F L V L F L F A L S W L P L S I I N C I  I  Y F  N  G E V P Q  L  V L Y  M  G I L L S H A N S M M N N  Q N  F  S  S  S K E T G A F Y G R E F K T A K S L F L V L F L F A  F  S W L P L S I I N C I  T  Y F  H  G E V P Q  I I  L Y L G I L L S H A N S M M NS Q N  S  S  G  S K E T G A F Y G R E F K T A K S L F L V L F L F A L S W L P L S I I N C I  T  Y F  N  G E V P Q  T  V L Y L G I L L S H A N S M M N S Q N S . S K E T G A F Y G R E F K T A K S L F L V L F L F A L S W L P L S I I N C I Y F . G E V P Q V L Y L G I L L S H A N S M M N Sheep.A3.Prot Human.A3.Prot K9.A3.Prot Equine A3  290 300 310 320 330 340 350  P I V Y A Y K I K K F K E T Y L L I L K  A  C  V M  C  Q P  S  K  S  M  D  P  S  T  E  Q T S E P I V Y A Y K I K K F K E T Y L L I L K  A  C  V V  C  H P  S D S  L  D  T  S  I  E  K N S E P I V Y A Y K I K K F K E T Y L L I  F  K  T Y M I  C  Q S  S D S  L  D  S  S  T  EP I V Y A  C  K I K K F K E T Y L L I L K  T  C  M I  C  H R  S D  C F G L K H R V E N RP I V Y A Y K I K K F K E T Y L L I L K . C . . C . S D S . D S E Fig. 1.  ClustalW protein alignments of the equine A 3  receptor protein with that of other species.258  C. I. Brandon  et al.   2006 The Authors. Journal compilation    2006 Blackwell Publishing Ltd  Evaluation of eA 3  receptor expression The eA 3 -R/HEK clones were screened for membrane expressionutilizing radioligand binding assays to detect displaceablebinding of 0.25 n M  [ 125 I]AB-MECA in the presence of 10  l M IB-MECA. Ten individual plaque-isolated clones were screenedand compared with nontransfected HEK cells, which served asnegative controls. From these 10 clones, four were identified thatexpressed the eA 3 -R as indicated by the specific binding of [ 125 I]AB-MECA (clones eA 3  2.0 B, eA 3  2.0 C, eA 3  1.0 D, eA 3  1.0F; Fig. 2a). Receptor density was calculated and indicatedexpression levels of 14.28 (eA 3  2.0 B), 3.84 (eA 3  2.0 C), 8.92(eA 3  2.0 D), and 3.95 (eA 3  2.0 F) fmol/mg protein bound(Fig. 2b). Adenylate cyclase assays To determine that the heterologously expressed equine A 3 recep-tor functionally coupled to the intracellular G-protein complex,adenylate cyclase assays were performed. Initially, the fourselected clones identified from the binding assays were screenedfor adenylate cyclase activity using the A 3  selective agonist IB-MECA. Results from these experiments indicated that three of thefour clones functionally coupled to adenylate cyclase as indicatedby inhibition of [ 3 H]cAMP accumulation. IB-MECA EC 50  valuesfor clones A 3  2.0 B, A 3  2.0 C, and A 3  1.0 D, were 0.10 n M (0.004–0.26), 2.92 n M  (1.79–4.76), and 1.54 n M  (1.08–2.21),respectively (data not shown). As clone A 3  2.0 B was shown tohave the highest affinity for IB-MECA in these cyclase assays, itwas utilized in an additional adenylate cyclase experiment usingthe selective A 3  antagonist MRS1220 (1  l M ) to document A 3 receptor mediation of IB-MECA-induced inhibition of adenylatecyclase. Addition of MRS1220 resulted in a rightward shift of theIB-MECA concentration–response curve (Fig. 3). Equilibrium competition binding On the basis of the data from binding and adenylate cyclaseassays, one clone (eA 3  2.0 B) was chosen to determine the rankorder of agonist and antagonist potencies in equilibriumcompetition binding assays. In these experiments, three adeno-sine agonists were tested (IB-MECA, NECA, and CGS21680), aswell as three antagonists (MRS1220, ZM241386, and 8-para-sulfophenyltheophylline) to determine their respective rankorders of potency. Nonlinear regression analysis of these datarevealed that the three agonists fit a one-site competition model Fig. 2.  (a) Radioligand binding assay using eA 3 -R/HEK cell mem-branes to determine receptor expression. Membrane fractions wasincubated with 0.25 n M  [ 125 I]AB-MECA in the presence and absenceof 10  l M  IB-MECA to define specific binding. The binding reaction wasincubated for 45 min at room temperature. Membrane fractions werethen harvested via rapid filtration in ice cold Tris and radioactivitydetermined. (b). Replot of the histogram in 2A indicating specificmembrane receptor densities; data are expressed in fmol/mg proteinbound. Fig. 3.  Equine A 3  receptor adenylate cyclase assays in the presence andabsence of 1  l M  MRS1220. Cells were plated in 6-well microtiter, grownto 75–80% confluence and preloaded with 1.2  l Ci/mL [ 3 H]Adenine.Cells were then treated with the respective concentrations of IB-MECAwith or without 1  l M  MRS1220, subsequently lysed, and [ 3 H]cAMPassayed. Table inset depicts EC 50  values and 95% confidence intervals(n M ). Data were analyzed using nonlinear regression using GraphPadPrism software. Equine adenosine A 3  receptor   259   2006 The Authors. Journal compilation    2006 Blackwell Publishing Ltd
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