INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

ADENOSINE IS A LOCAL REGULATOR of many physiological functions; it acts via adenosine receptors (ARs) coupled to G-proteins located in sarcolemma of cells (22). Several AR subtypes have been described on the basis of pharmacological criteria and molecular cloning techniques. On the basis of pharmacological criteria, adenosine has been shown to interact with four receptor subtypes, namely A₁, A_2A, A_2B, and A₃ (7, 19, 21). All the four ARs have been cloned from several animal species, including dog, rat, mouse, and man (19, 22). The A₁ subtype is associated with inhibition of adenylate cyclase activity, whereas the A₂ (A_2A and A_2B) subtype is associated with stimulation of adenylate cyclase activity. ARs are members of the large family of G-protein-coupled receptors whose activation on the cell surface can influence signal transduction through cAMP, calcium, and/or cGMP. For instance, both A_2A and A_2B receptors regulate cAMP production in human umbilical vein and aortic endothelial cells (9, 13). Furthermore, A_2B receptor mRNA has been detected in human aortic endothelial cell (13). The availability of selective ligands has permitted the pharmacological characterization of the A₁ and A_2A receptors. However, the lack of a selective ligand for the A_2B AR has impeded the study of its characterization. The adenosine A_2A receptors are important in the regulation of a variety of cardiovascular functions, including vascular conductance, blood platelet aggregation, and vascular relaxation, among others (20). It has been postulated that endothelium may have a role in the vascular action of adenosine (1). CGS-21680 has been shown to be more than 170-fold selective for adenosine A_2A receptor subtype compared with A_2B (14).

Adenosine and especially A₂-AR agonists appear to act on both endothelium and vascular smooth muscle to cause vasodilation (1). However, despite much investigation, the subtype of AR (presumably A_2A and A_2B) that mediates vasodilation remains unclear. Unlike other ARs, no potent and selective agonist for A_2B receptors has been found, whereas 5'-(N-ethylcarboxamido)adenosine (NECA) remains the most potent A_2B agonist so far (8). [³H]NECA has been used as a radioligand, but its use was associated with multiple artifacts in terms of binding to A_2A, A_2B, A₁, and A₃ ARs (6). The availability of the selective adenosine A_2A receptor radioligand 2-[4-[2-{2-[(4-aminophenyl)methylcarbonylamino]ethylaminocarbonyl}ethyl]phenyl]ethylamino-5'-ethylcarboxamidoadenosine (PAPA-APEC) opens new means to investigate the endothelial AR. Recent development of an anti-peptide antibody to the canine A_2A AR demonstrated the usefulness of such a reagent for studying the tissue distribution and molecular weight differences of A₂ ARs by immunoblotting and immunoprecipitation (16). The present study was undertaken to examine the presence of adenosine A_2A and A_2B receptors through molecular means and the effectiveness of adenosine agonists to directly enhance cAMP production in cultured porcine coronary artery endothelial cells (PCAEC).

	MATERIAL AND METHODS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

Animals. Porcine hearts were obtained from a local slaughterhouse within 10 min of death and transported to the laboratory in oxygenated ice-cold Krebs buffer solution (95% O₂, 5% CO₂) with the following composition (in mM): 1.2 MgSO₄, 118 NaCl, 1.2 KH₂PO₄, 25 NaHCO₃, 2.5 CaCl₂, and 11 glucose (pH 7.4) at 4°C. Branches of left anterior descending and epicardial coronary arteries were dissected and cleaned of fat and connective tissue.

Endothelial cell isolation. Endothelial cells were isolated from porcine coronary artery by collagenase/dispase digestion (18) with minor modifications. The sedimented cells were resuspended in a 75-cm² cell culture flask containing 10 ml M199 medium supplemented with 10% fetal bovine serum (FBS) and 2% antibiotics and incubated at 37°C in 5% CO₂ atmosphere for 11/2 h. Cultured human coronary artery endothelial cells (HCAEC, CC-2585) were purchased from Clonetics (BioWhittaker, Walkersville, MD). Microvascular endothelial cell growth medium (EGM-MV) purchased from Clonetics consisted of 500 ml of endothelial cell basal medium, 10 ng/ml human recombinant epidermal growth factor, 1.0 mg/ml hydrocortisone, 50 mg/ml gentamicin, 50 µg/ml amphotercin-B, 3 mg/ml bovine brain extract, and 25 ml FBS. HCAEC in 5 ml EGM-MV were seeded in a 75-cm² flask (7,000 cells/cm²) and incubated at 37°C in 5% CO₂ atmosphere for 24 h. The incubation medium was replaced with fresh culture medium and thereafter was changed every 2-3 days. Identification of endothelial cells was performed by the factor VIII antigen and the fluorescent probe Dil-acetylated low-density lipoprotein (Dil-Ac-LDL; Bio-medical Technologies, Cambridge, MA), which is taken up preferentially into endothelial cells, and by examination of cellular morphology with phase contrast microscopy (4). Dil-Ac-LDL at a concentration of 10 µg/ml in the normal medium was incubated with the cells for 4 h at 37°C. Cells were washed with fresh medium to eliminate extracellular Dil-Ac-LDL and visualized by means of a fluorescent-phase contrast microscope. Both primary and subcultured cells had typical endothelial cell morphology with strict contact inhibition on phase microscopy. In randomly selected cultures, more than 98% of the cells were found to fluorescence after incubation with Dil-Ac-LDL. The cells between third and fourth passages were used for all experiments. Before experiments, each well was washed three times with 2 ml of buffer A (50 mM Tris · HCl, pH 7.4; 1 mM MgCl₂).

Radioligand binding experiments. [¹²⁵I]PAPA-APEC binding assays were conducted on membranes prepared from PCAEC membrane cells as described by Barrington et. al. (3) with minor modifications. PCAEC membranes were prepared by rinsing the adherent confluent cells twice with buffer B (5 mM Tris · HCl, pH 7.5; 1 mM MgCl₂, 250 mM sucrose, 0.2 U/ml adenosine deaminase). The cells were scraped with a rubber policeman in buffer B containing a protease inhibitor cocktail (10 mM benzamidine HCl, 50 µg/ml phenylmethylsulphonyl fluoride, 5 µg/ml soybean trypsin inhibitor) and sonicated. The homogenate was centrifuged at 105,000 g for 1 h. The pellet was washed twice with buffer C (50 mM HEPES, pH 7.5; 10 mM MgCl₂, 0.2 U/ml adenosine deaminase containing the above-described protease inhibitor cocktail) and finally resuspended in the same washing buffer. The membranes were stored at 70°C until used. Protein concentration was estimated by the Bio-Rad method (5) using gamma globulin as a standard. For binding studies, membranes (50- to 75-µg protein, pretreated with 0.2 U/ml adenosine deaminase) were incubated for 2 h with a gentle shaking at 37°C with [¹²⁵I]PAPA-APEC (100,000 cpm/tube, specific activity 2,200 Ci/mmol) and 50 nM N⁶-cyclopentyladenosine (CPA; to prevent binding to A₁ receptor) in a total volume of 250 µl. The ligand concentration for the saturation experiment varied from 0.01 to 7.5 nM. The nonspecific binding was defined by the addition of 10⁴ M unlabeled CGS-21680. Reaction was terminated by rapid filtration through Whatman GF/B glass fiber filters (soaked in 0.3% polyethyleneimine for at least 1 h) using the Brandel cell harvester followed by three washes with 4 ml ice-cold incubation buffer. The radioactivity on the filters was determined by liquid scintillation counting for 5 min at an efficiency of ~65%. All assays were performed in triplicate. In competition experiments, various competitors [CGS-21680, NECA, 2-chloroadenosine (CAD), CPA, R-phenylisopropyladenosine (R-PIA)] were added at increasing concentrations from 10¹⁰ to 10⁴ M while maintaining the fixed concentration (~1.2 nM) of [¹²⁵I]PAPA-APEC in the same assay conditions as described above. 3-[(3-Cholamidopropl)dimethylammonio]-1-propanesulfonate (CHAPS, 0.01%) was added to all the samples to reduce nonspecific binding. Following incubation period, 4 ml of ice-cold washing buffer C without adenosine deaminase (containing 0.05% CHAPS) was added to each tube to terminate the reaction. The mixture was rapidly filtered through GF/B filters (soaked in 0.3% polyethyleneimine for at least 1 h) using the Brandel cell harvester and washed three times with 4 ml ice-cold washing buffer. Filters were counted for radioactivity in the Packard gamma counter.

Measurements of cAMP. cAMP assay was conducted using intact PCAEC as described by Harper and Brooker (12) with minor modification. Cells grown to confluence in 35-mm 6-well plate culture dishes were washed twice with 2 ml of warm (37°C) Krebs buffer solution as described above. After a 30-min equilibration period inside the incubator, the incubation buffer was replaced with fresh warm (37°C) Krebs buffer solution containing 0.2 U/ml adenosine deaminase to remove endogenous adenosine, indomethacin (10⁵ M) to inhibit cyclooxygenase activity, and IBMX (10⁴ M) to inhibit phosphodiesterases for 20 min at 37°C in 5% CO₂ before the addition of the test drugs. Some experiments were performed in the absence of IBMX. In experiments where adenosine was added as an agonist, no adenosine deaminase was added. Drugs were added to the cells with increasing concentrations of CGS-21680, NECA, or CAD for 20 min at 37°C. In addition, the effect of the agonists (CGS-21680, NECA) on cAMP production was repeated after preincubating the cells for 20 min with a selective A_2A AR antagonist, ZM-241385, at 10⁸ M. The incubation was continued for another 15 min at 37°C in a final volume of 2 ml. The reaction was terminated by aspirating supernatant. The cells were immediately extracted by addition of 0.5 ml of cold 6% trichloroacetic acid (TCA) then scraped with a rubber policeman, and the suspension was collected in an Eppendorf tube. Cells remaining on the plates were recovered by addition of a second volume of 0.5 ml 6% TCA and added to the first extract. Following sonication for 5 s, the cell lysate was centrifuged for 10 min at 2,000 g. For each sample, TCA was extracted from supernatant with 4 volumes of water-saturated ether (anesthetic grade), and the aqueous phase was lyophilized. The cAMP was reconstituted in 0.5 ml sodium acetate buffer (0.05 M, pH 6.2). The intracellular cAMP content was then determined by radioimmunoassay following its acetylation with a cAMP kit in accordance with the instructions of the manufacturer (Biomedical Technologies, Stoughton, MA). The pellet in each Eppendorf tube was resuspended with 100 µl of 0.01 N NaOH, and cell protein concentration was determined as described earlier (5). The content of cAMP was expressed as pmol/mg protein.

Total RNA preparation and reverse transcription polymerase chain reaction. Monolayer cells were rinsed with cold PBS. Total RNA was prepared from a homogeneous population of HCAEC and PCAEC by RNA zol B in accordance with the manufacturer's instruction (Biotecx, Houston, TX). RNA was treated with DNase 1 (GIBCO) to prevent genomic DNA contamination. Reverse transcription-polymerase chain reaction (RT-PCR) was conducted essentially as described by Grillo and Margolis (11) using the Taq PCR core kit (Qiagen). Control reaction was performed simultaneously without RT enzyme. One microgram of total RNA from each sample was subjected to RT-PCR using specific primers for human A_2A and A_2B receptor. PCR primers were synthesized based on the sequence from the AR cDNA. A_2A receptor primer 1 (sense) was 5'-AATCTTCCATTCAGCAGCCCC-3', and primer 2 (antisense) was 5'-GCAAGATCATTCCGCAGCCAC-3'. A_2B receptor primer 1 (sense) was 5'-CAAGTCACTGGCTATGATTGT-3', and primer 2 (antisense) was 5'-ATAGACAATGGGATTGACAAC-3'. Amplification was accomplished using 30 cycles with denaturing temperature at 96°C for 1 min, annealing at 60°C for 1 min, and elongation at 72°C for 1 min. The PCR product (20 µl) was directly analyzed on a 1.5% (wt/vol) ethidium bromide-stained agarose gel. Bands corresponding to the PCR product were then excised from the gel, and the DNA was extracted using -agarase (GIBCO) and ethanol precipitation.

PCR products analysis. Purified PCR fragments were directly sequenced using the PRISM Ready Reaction Dyedeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The sequencing reactions were performed with the A_2A- and A_2B-specific primers used in the RT-PCR reactions. The sequencing reaction products were analyzed on an automated DNA sequencer (model 373A Stretch, Applied Biosystems). Assignment of the PCR products to the respective ARs was conducted using the Basic Local Alignment Search Tool (Blast) search program (NCBI).

Generation of antipeptide antibodies. Antibodies for A_2B were generated by one of the authors (Feoktistov). For the antigenic peptide, the 24-mer peptide KDSATNNCTEPWDGTTNESCCLVK from the presumed second extracellular loop of human A_2B receptor was used. The peptide was synthesized by Chiron Mimitopes Peptide Systems (San Diego, CA) and was conjugated via glutaraldehyde with keyhole limpet hemocyanin and bovine serum albumin. Keyhole limpet hemocyanin-coupled peptide was emulsified with 1:1 Freund's complete adjuvant. This emulsion (100 µg conjugated peptide in 200 µl) was injected into the breast muscle of three 20- to 25-wk-old egg-laying hens. Booster injections of 100 µg keyhole limpet hemocyanin-coupled peptide emulsified in incomplete Freund's adjuvant were administered 2 weeks later. Production of specific anti-peptide antibodies occurred as early as 4-5 wk after the primary immunization, as documented by dot blot assay with bovine serum albumin-coupled peptide. Partially purified chicken IgG was precipitated from chicken yolk by using polyethylene glycol 8,000 within a range of concentrations from 3.5% to 13%. Final purification of antibodies was achieved by affinity chromatography, using bovine serum albumin-conjugated peptide coupled to Sepharose 4B. Affinity media was prepared by using 32.5 mg of bovine serum albumin-conjugated peptide (BSA-to-peptide ratio 2:1, wt/wt) per 1 g of dry CNBr-Sepharose 4B, in accordance with manufacturer's instructions (Pharmacia Biotech, Piscataway, NJ). For affinity purification chicken IgG was collected from 12 eggs, diluted in 100 ml of 10 mM Tris · HCl, pH 7.5, and passed through a 4- to 5-ml column at a speed of 0.1 ml/min at 40°C. The column was then washed with the same buffer and with 10 mM Tris · HCl, pH 7.5, and 0.5 M NaCl until the absorbance at 280 nm of the flowthrough fractions reached a minimum. Antibodies were eluted with 0.2 M glycine, pH 2.2. Fractions containing protein, as determined by monitoring absorbance at 280 nm, were immediately neutralized by the addition of 1 M Tris base. After the protein concentration of pooled fractions was measured, the antibody was aliquoted for storage at 70°C.

Western blot analysis. Western blot analysis was performed in cultured HCAEC and PCAEC membrane fraction using A_2A and A_2B receptor-specific antibodies as described by Marala and Mustafa (16) with minor modifications. The endothelial cell membranes (50 µg) were subjected to 10% SDS-PAGE under either reducing (in the presence of 1,3-mercaptoethanol) or nonreducing conditions (in the absence of 1,3-mercaptoethanol), and the samples were boiled for 3 min, followed by electro-blotting the proteins to an Immobilon membrane (Amicon). For A_2A, the free protein binding sites were blocked by incubating the membrane with PBS (50 mM phosphate buffer, pH 7.5, 150 mM NaCl) containing 5% skimmed milk (PBS-milk) at 40°C overnight. The membranes were then incubated for 2 h at room temperature with PBS-milk containing A_2A receptor antibody (1:1,000 final dilution for receptor antibody) with gentle mixing. The membranes were washed four times for 10 min each time with PBS containing 0.05% Tween 20. Bovine striated membranes (50 mg protein) were used as positive control in all Western blot experiments. The membranes for A_2A receptor were incubated with ¹²⁵I-labeled goat anti-rabbit IgG (500,000 cpm/ml) for 2 h at room temperature with gentle mixing. The membranes were washed two times for 10 min each time with PBS containing 0.05% Tween 20, air dried, and autoradiographed. For A_2B nonspecific protein, binding sites on the membrane were blocked by incubating for 2 h at room temperature or overnight at 40°C in 5% (wt/vol) skimmed milk powder, 0.2% (vol/vol) Tween-20, 100 mM Tris · HCl, pH 7.5, and 0.9% (wt/vol) NaCl. Affinity purified chicken A_2B-specific antibody was incubated with the membrane for 1 h at room temperature at a final concentration of 0.5-1 µg/ml in a fresh blocking solution. The blot was then washed five times with 0.2% (vol/vol) Tween-20, 100 mM Tris · HCl, pH 7.5, 0.9% (wt/vol) NaCl (10 min/wash) before incubation for 1 h with horseradish peroxidase-conjugated rabbit anti-chicken IgG (Sigma) in the blocking solution. The membrane was washed again as described above, and the bands were visualized with an enhanced chemiluminescence method.

Data analysis. The experiments were done in triplicate and repeated two to three times under identical conditions. Data were analyzed using the new version of BIOSOFT KELL, version 5 (KINETIC, EBDA, LIGAND, and LOWRY). The equilibrium dissociation constant (K_d) and the maximal number of binding sites (B_max) of [¹²⁵I]PAPA-APEC were determined from saturation binding isotherm with a nonlinear curve fitting program. Results are expressed as means ± SE. Statistical analysis of the results were carried out by means of analysis of variance followed by t-tests modified for multiple comparisons and were considered significant at the P < 0.05 level.

Chemicals and drugs. IBMX was purchased from Sigma (St. Louis, MO). NECA, CGS-21680, CAD, CPA, and R-PIA were purchased from Research Biochemicals International (Wayland, MA). All other chemicals were of the highest grade quality and purchased from Sigma. All drugs were dissolved in distilled water to make stock solutions with the exceptions of NECA, which was prepared in 50% ethanol at 10² M concentration, and ZM-241385, which was prepared in 20% dimethyl sulfoxide and stored frozen. All stocks were further freshly diluted by 1,000 times or more in distilled water or buffer. The vehicles alone had no effect at levels equivalent to the highest concentrations of agonist and antagonist used.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

Radioligand binding. Binding studies using the relatively specific A_2A receptor ligand [¹²⁵I]PAPA-APEC were performed on PCAEC membranes as described by Barrington et al. (3) in the presence of 50 nM CPA to eliminate any nonspecific binding of [¹²⁵I]PAPA-APEC to the A₁ receptors. Because adenosine was expected to be present in the cell membrane preparation, pretreatment with adenosine deaminase was performed. Specific binding of [¹²⁵I]PAPA-APEC was saturable and dependent upon protein concentration, with a B_max of 240 fmol/mg protein (Fig. 1). Saturation analysis of the binding data revealed a single class of binding sites with a K_d of 1.17 ± 0.035 nM (Fig. 1, inset). Data analysis using a new version of BIOSOFT KELL, version 5 (KINETIC, EBDA, LIGAND, and LOWRY), indicated that a one-component model described the data significantly better than a two-component model (P < 0.05). The linearity (r² = 0.97) of the saturation isotherm/scatchard analysis indicated that the range of A_2A receptor-selective radioligand concentrations used labels only the A_2A receptor as reported by others (3). Competition studies (n = 4-6) of representative AR agonists were evaluated. [¹²⁵I]PAPA-APEC bound to PCAEC membranes could be displaced in a concentration-dependent fashion (10⁷-10⁴ M) by CGS-21680, NECA, and CAD (Fig. 2). The agonists showed the following order of potency: NECA  CGS-21680 > CAD. On the contrary, R-PIA and CPA at concentrations from 10⁷ to 10⁴ M had little or no effect on the displacement binding of [¹²⁵I]PAPA-APEC (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Representative saturation isotherm and scatchard analysis (inset) of 125I-labeled 2-[4-[2-{2-[(4-aminophenyl)methylcarbonylamino] ethylaminocarboxyl}ethyl]phenyl]ethylamino-5'-ethylcarboxamidoadenosine (PAPA-APEC; 0.1 to 7.5 nM) binding to cultured porcine coronary artery endothelial cell (PCAEC) membranes in the presence of 105 M unlabeled PAPA-APEC. The result is typical of four separate experiments and yielded an equilibrium dissociation constant of 1.17 ± 0.0352 nM and a maximal number of binding sites of 240 fmol/mg of protein. B/F, bound/free.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Competitive binding curves for unlabeled adenosine agonists in cultured PCAEC membranes (n = 4). A concentration of 1.2 nM [125I]PAPA-APEC was used in this competition experiment, and the specific binding represented ~75% of the total binding. NECA, 5-(N-ethylcarboxamido)adenosine; CGS, CGS-21680; CAD, 2-chloroadenosine; M, molar.

Accumulation of cAMP. The adenosine agonists (CGS-21680, NECA, CAD) enhanced cAMP production in a dose-dependent manner, and NECA was a more potent agonist than CGS-21680 (Fig. 3A). On the contrary, R-PIA and CPA at concentrations from 10⁷ to 10⁴ M produced little or no effect on the cAMP levels (data not shown). The basal levels of cAMP in PCAEC was 4.96 ± 0.300 pmol/mg of protein (n = 8). Addition of 10⁴ M CGS-21680 increased cAMP formation by threefold (n = 8). The maximal stimulation of cAMP accumulation was about threefold of control at a concentration of 10⁴ M of NECA. Similarly, isoproterenol, used as a positive control, produced a threefold increase in cAMP accumulation in PCAEC at a concentration of 10⁶ M (data not shown). As shown in Fig. 3B, coincubation of cells with 10⁸ M of the A_2A-specific AR antagonist ZM-241385 significantly inhibited cAMP accumulation by CGS-21680 but partly inhibited the effect of NECA. Moreover, NECA at the highest concentration of 10⁴ M increased cAMP production much more than CGS-21680 (P < 0.05) at the corresponding concentration (Fig. 3B). Figure 3C shows the inhibitory effect of increasing concentrations of ZM-241385 on cAMP accumulation as a percentage of the response to 100 µM NECA. The antagonistic effect of ZM-241385 at 10⁸ M, the highest dose that produced maximal blockade of A_2A AR, reduced 100-µM NECA-induced cAMP accumulation by less than 100%, thus supporting the presence of A_2B. These data together suggest that changes in endothelial cAMP levels were mediated through the A_2A and A_2B AR-mediated mechanism.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   A: effect of adenosine agonists on cAMP accumulation in intact cultured PCAEC (n = 4). B: effect of adenosine agonists and the effect of adenosine receptor (AR) antagonist (ZM-241385) on cAMP accumulation in intact cultured PCAEC (n = 4). C: inhibitory effect of increasing concentrations of ZM-241385 on cAMP production as a percentage of the response to 100 µM NECA. Values are means ± SE of triplicate determination. * P < 0.05 and ** P < 0.01, significantly different from zero (basal), respectively.

RT-PCR analysis. Figure 4 shows product analysis of RT-PCR for A_2A and A_2B receptor mRNA both in PCAEC and HCAEC. Predominant amplification products of each A_2A or A_2B primer of the predicted size of 205 and 173 bp, respectively, were evident in the gel both in HCAEC and PCAEC. However, when the PCR procedure was carried out in the absence of RT, neither 205-bp nor 173-bp bands were detected, and there were no other recognizable bands (data not shown). This indicated that the bands originated from mRNA, not from genomic DNA. The arrows on ethidium bromide-stained agarose gel indicate expected PCR product sizes (205 and 173 bp for A_2A and A_2B-receptors, respectively). Sequence analysis of the PCR products using the BLAST search engine demonstrated 99% identities with the human adenosine A_2A (NM-000675) and A_2B (NM-000676) receptor mRNA, respectively.

View larger version (103K): [in this window] [in a new window]   Fig. 4.   Detection of A2A and A2B receptor mRNA in human coronary artery endothelial cells (HCAEC) and PCAEC as assessed by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The arrows indicate expected PCR product sizes (205 and 173 bp for A2A and A2B receptor, respectively). Lanes 1 and 3 are for PCAEC, whereas lanes 2 and 4 are for HCAEC.

Western blot analysis. Western blot analysis of HCAEC and PCAEC membranes was performed for the A_2A and A_2B receptors. Western blot analysis showed a 45-kDa immunogenic band that represents the A_2A receptors in HCAEC and PCAEC (Fig. 5A). There was no difference in the migration pattern of the immunogenic bands when the samples were subjected to SDS-PAGE under reducing or nonreducing conditions. In addition, using anti-A_2B receptor antibody, HCAEC and PCAEC showed one major immunogenic band migrating as 36 ± 1 kDa size (Fig. 5B). Bovine brain striatal membranes were used as a positive control because this tissue has been shown, by our earlier studies as well as by others (2, 15), to contain a high density of ARs.

View larger version (52K): [in this window] [in a new window]   Fig. 5.   Western blot analysis showing the immunoreactivity of A2A and A2B receptor at 45- and 36-kDa mass, respectively, in HCAEC (A) and PCAEC (B). Bovine striated membranes (50 µg protein) were used as positive control (lane 1).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

The primary purpose of this study was to identify the A₂ AR subtype(s) present in cultured HCAEC and PCAEC and to analyze its coupling to cAMP production. We used pharmacological, biochemical, and radioligand binding techniques to study AR subtypes. [¹²⁵I]PAPA-APEC was used to demonstrate the specific binding in PCAEC membranes. Specific binding was saturable, reversible, and dependent upon protein concentration with a B_max of 240 fmol/mg protein. Scatchard analysis of the binding data revealed a single class of binding site with a K_d of 1.17 ± 0.035 nM, and in competition experiments rank order of AR agonists (NECA  CGS-21680 > CAD) supported the existence of A_2B subtype. This order is consistent with the A₂ AR classification. The present results of binding characteristics are in agreement with those observed in bovine striated membranes using [¹²⁵I]PAPA-APEC (2, 3). The little apparent discrepancy between the K_d (1.5 vs. 1.17 nM) for binding in bovine striated membranes and that of PCAEC membranes can be attributed to tissue and species differences, and this is consistent with the differences seen by other investigators (23). We were unable to see the effects of adenosine A₁ receptor agonists (R-PIA, CPA) excluding the presence of A₁ receptors in endothelial cells.

We studied the effects of A₂ AR agonists (CGS-21680, NECA, CAD) on the accumulation of cAMP to determine coupling of receptors to this second messenger. These adenosine agonists stimulated the accumulation of cAMP in the PCAEC in a concentration-dependent fashion as observed in binding study, indicating the coupling of this signal to A₂ AR subtypes as reported by others (10, 12). Moreover, adenosine A_2A-selective antagonist (ZM-241385, 10⁸ M) significantly inhibited the stimulatory effect of CGS-21680 and partly inhibited the effect of NECA, indicating that it is a receptor-mediated effect and supporting the presence of both A_2A and A_2B. It has been shown that CGS-21680 exhibits negligible stimulation of cAMP, whereas NECA shows a strong stimulation of cAMP accumulation, a response attributed to the low-affinity subtype A_2B (15, 16). The high- and low-affinity adenosine A₂ receptors (A_2A and A_2B, respectively) have been shown to be coupled positively to adenylate cyclase via stimulatory guanine nucleotide binding proteins in a variety of systems. Both receptors are associated with an elevation of intracellular cAMP concentrations, but the A_2B subtype is characterized by its low affinity for adenosine analogs in rat striatal membrane (6). Similarly, cAMP formation as a result of adenosine agonists is not inhibited in the presence of 10 nM of the adenosine A₁ receptor-selective antagonist 1,3-dipropyl-8-cyclopentyl-xanthine (DPCPX), further suggesting that there are no adenosine A₁ receptors in PCAEC (data not shown).

Further evidence for the presence of A_2A and A_2B receptors in HCAEC and PCAEC was provided by Western blot analysis with antibody lane 2. With the use of this antibody, Western blot analysis showed a 46-kDa immunogenic band, which represents the A_2A receptor, and 36-kDa, which represents A_2B, as shown by other investigators (16, 20) and by photoaffinity labeling studies (2, 3). Recent studies using Western blotting of membranes from human and porcine cardiac tissues (atria and ventricle) indicated the presence of 45-kDa receptor for A_2A (16). In the past, indirect methods such as activation of adenylate cyclase demonstrated in the present study or organ bath studies were used to identify A_2A receptors in vascular tissues. The development of the antibody against the A_2A and A_2B receptors provides us with an additional tool to study the receptors virtually in any tissue.

Direct evidence for the presence of A_2A and A_2B receptors transcript using RT-PCR showed the presence of bands of the predicted size of 205 and 173 bp for A_2A and A_2B receptors, respectively, in HCAEC and PCAEC. Further analysis and sequencing of the PCR fragments confirm the presence of both A_2A and A_2B receptor mRNA. It is now well established that adenosine A_2A and A_2B receptors are present in HCAEC and PCAEC.

In conclusion, the results presented suggest the presence of both A_2A and A_2B subtypes of AR in cultures of HCAEC and PCAEC. It was previously shown that the coronary endothelial ARs of A₂ subtype are linked to the production of nitric oxide (1). It will be interesting to investigate the subtype of ARs (A_2A and A_2B) that results in the production of nitric oxide in porcine coronary endothelium. This study further provides evidence that adenosine agonists enhance cAMP production in endothelial cells through A_2A and A_2B AR-mediated mechanism. Adenosine appears to act on both endothelium and vascular smooth muscle to cause vasodilation. The endothelial AR has been shown to have great physiological importance. For instance, it has been shown that adenosine stimulates endothelial growth and migration, effects that can be inhibited by adenylyl cyclase inhibition with P-site agonists (17). We believe that this study improves our understanding of the regulation and physiological role of ARs.