Modulating effect of adenosine deaminase on function of adenosine A₁ receptors¹

Introduction

Adenosine is a ubiquitous physiological regulator and neuromodulator capable of ultiple physiological actions in various systems^[1]. Adenosine receptors are members of the G-protein-coupled receptor superfamily, and comprise A₁, A_2a, A_2b, and A₃ adenosine receptors, identified by convergent data from molecular, biochemical, and pharmacological studies^[2]. A₁R is widely expressed in the brain, adipose tissue,the testis, and the spinal cord^[3]. Via A₁R, adenosine reduces heart rate^[4], glomerular filtration rate, and renin release in the kidney^[5], induces bronchoconstriction^[6,7] and inhibits lipolysis. A₁R can be coupled to different pertussis toxin-sensitive G proteins^[8–10], which mediate the inhibition of adenylyl cyclase^{[11 ]} and regulate Ca²⁺ and K⁺ channels and inositol phosphate metabolism^[12]. A₁R present two different affinities for agonists, which have classically been attributed to a different coupling to heterotrimeric G proteins^[13], coupled receptor-G protein complexes display high affinity for A₁R agonists, whereas uncoupled receptors display low affinity^[13,14].

Adenosine deaminase (ADA, E.C.3.5.4.4) is an enzyme which catalyzes the hydrolytic deamination of adenosine to inosine. ADA is located both in the cytosol and on the cell membrane. Recent evidence suggest that ecto-ADA had extra-enzymatic and co-stimulatory functional roles. ADA modulates ligand binding and signaling through A₁R on DDT₁MF-2 cells, a smooth muscle cell line^[15]. ADA seems to be necessary for the high affinity binding of agonists to A₁R^[16,17]. In the present study, the effect of ADA on the ligand-mediated regulation of A₁R in HEK293 cells stably expressing human A₁R has been studied.

Materials and methods

Materials High glucose Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum, G418, benzylpenicillin and streptomycin were obtained from Gibco. Restricted enzyme (HindIII, EcoRI, XhoI), T4 DNA ligase, reverse transcriptase enzyme and buffer were purchased from Promega (USA). Pyrobest DNA polymerase and PCR buffer were obtained from TaKaRa (Dalian, China). N⁶-cyclopentyladenosine (CPA), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and 2-Chloro-N⁶-cyclopentyladenosine (CCPA) were obtained from RBI (Natick, MA, USA). Adenosine deaminase was obtained from Roche Biochemicals (Mannheim, Germany). Fluo-3/AM, p-(dipropylsulfamoyl)benzoic acid, pluronic F-127, HEPES, Triton X-100, bovine serum albumin, leupeptin, pepstatin A, aprotinin and PMSF were purchased from Sigma (USA). [³H]DPCPX was purchased from Amersham (USA). Plasmids pcDNA3, pcDNA3.1(+), and Lipofectamine²⁰⁰⁰ were purchased from Invitrogen (USA). Human embryo brain tissue was donated by Huashan Hospital (Shanghai, China). PCR primers were synthesized by Shenyou (Shanghai, China).

Cloning of the human A₁R cDNA Human brain total RNA was obtained from human embryo brain tissue. The total RNA was reverse-transcribed using oligo-dT18 as a primer. With the cDNA, PCR was carried out using the primers A₁R-1SE and A₁R-1AS (Table 1). Gel-purified PCR product was treated with EcoRI/XhoI and subcloned into the EcoRI/XhoI site of the mammalian expression vector pcDNA3. With the subcloned A₁R-pcDNA3 plasmid as template, PCR was carried out using the primers A₁R-2SE and A₁R-2AS (Table 2), which resulted in full length codon sequence cDNA. Gel-purified PCR product was treated with HindIII/EcoRI and cloned into the HindIII/EcoRI site of the mammalian expression vector pcDNA3.1(+).

Production of HEK293 cell lines stably expressing human A₁R HEK293 Cells were transfected with the A₁R-pcDNA3.1(+) expression vector, using the Lipofectamine2000 reagent. Cells were treated with selection medium containing G418 (1 g/L) for 3 weeks to select stably transfected cells displaying neomycin resistance. Between 2 and 3 weeks in the selection process, resistant cells began to appear. They were sought out by serial dilutions and allowed to grow from single cells. Receptor expression of single cell-derived colonies was tested by radioligand binding assay.

Cell culture Human embryonic kidney (HEK293) cells were cultured in DMEM containing streptomycin (100 mg/L), benzylpenicillin (1000 kU/L) and fetal bovine serum (10%). Cells were incubated at 37 °C in 5% CO₂. Stably transfected HEK293 cell lines were cultured in DMEM high glucose medium containing streptomycin (100 mg/L), benzylpenicillin (1000 kU/L), G-418 (200 mg/L), and fetal bovine serum (10%). Cells were incubated at 37 °C in 5% CO₂. For passaging, the cells were detached from the cell culture flask by washing with phosphate-buffered saline (PBS) and brief incubation with trypsin (0.5 g/L)/EDTA (0.2 g/L). The cells were passaged every 3 d.

Membrane preparation The A₁R cells (HEK293 cells stably expressing human A₁R) were lifted from Petri dishes with a cell scraper. Harvested cells were washed twice with ice-cold PBS and centrifuged at 420×g for 5 min at 4 °C. The cell pellet was resuspended with hyponic buffer (Tris-HCl 5 mmol/L, EDTA 2 mmol/L, pH 7.4, leupeptin 1 mg/ L, pepstatin A 1 mg/L, aprotinin 1 mg/L, PMSF 1 mmol/ L) and sonicated (18 s) three times on ice. The homogenate was centrifuged at 960×g for 10 min at 4 °C. The precipitated nucleic fraction was discarded and the supernatant was centrifuged at 40 000×g for 30 min at 4 °C. The pellet was washed with 50 mmol/L Tris-HCl buffer (pH 7.4) and centrifuged again under the same conditions. Finally, the pellet was resuspended in the same buffer, and protein concentration was determined by using the BCA Kit (Pierce) as described previously^[18].

[³H]DPCPX binding assays in intact cells and membranes Binding assays in intact cells were performed in a reaction tube at a density of 2.0×10⁵ cells per tube, using [³H]DPCPX as a radioligand. Cells were resuspended in 50 mmol/L Tris-HCl (pH 7.4) and treated with ADA 65 nmol/L at 4 °C for 30 min^[17]. After this treatment, cells were incubated for 1 h at 37 °C with [³H]DPCPX 0.5 nmol/L for competition assays. Different concentrations of the A₁R agonist CCPA or CPA were used in the competition curves.

Membranes (30–50 µg proteins) from A₁R cells were resuspended in Tris-Cl 50 mmol/L (pH 7.4) and treated with ADA 65 nmol/L at 4 °C for 30 min. Saturation assays were performed at different concentrations of [³H]DPCPX (0.05–10 nmol/L) using unlabeled DPCPX (20 µmol/L) to obtain nonspecific binding. Competition curves were carried out by using [³H]DPCPX 0.5 nmol/L and different concentrations of A₁R agonists CCPA or CPA.

After incubation for 1 h, the binding assays were stopped by rapid filtration through Whatman GF/B filters, and the filters were immediately washed three times with ice-cold buffer. Filters were then transferred to Eppendoff tubes, and scintillation liquid was added to measure the radioactivity.

Intracellular Ca²⁺ measurements Ca²⁺ fluorescence measurements were performed using a NOVOstar plate reader with a pipettor system (BMG labtechnologies, Offenburg, Germany). A₁ cells were harvested with 0.05% trypsin/0.02% EDTA and rinsed with high glucose DMEM containing 10% fetal bovine serum, streptomycin 100 mg/L, and benzylpenicillin 1000 kU/L. Pelleted cells were resuspended in fresh medium and kept under 5% CO₂ at 37 °C for 1 h and vortexed every 15 min. After two washes with Krebs-HEPES buffer, cells were loaded with Fluo-3/AM 5 µmol/L for 30 min containing 1% pluronic F-127 and p-(dipropyl-sulfamoyl)benzoic acid 2.5 mmol/L. Then cells were rinsed 3 times with Krebs-HEPES buffer containing 0.5% bovine serum albumin, the diluted, and evenly plated into 96-well plates at a density of 1×10⁴ cells/well. Microplates were kept at 37 °C for 15 min^[19,20]. Buffer alone or different concentrations of CPA were then injected sequentially into separate wells, and fluorescence intensity was measured at 520 nm for 50 s at 0.2 s intervals. The excitation wavelength was 485 nm.

[Ca²⁺]_i was calculated as follows: [Ca²⁺]_i=K_d (F-F_min)/(F_max-F). F_max refers to fluorescence intensity measured after permeabilization of the cells with 1% Triton X-100. Ten mmol/L EDTA was added to chelate Ca²⁺ and minimum fluorescence intensity was obtained (F_min). A K_d value of 324 nmol/L was used for Fluo-3.

Data analysis Experiments were performed in triplicate. All data were expressed as mean±SD and data were analyzed with the GraphPad Prism 4.0 program (GraphPad Software, San Diego, CA, USA). Student’s t-test was used for statistical analysis.

Table 1 Human A₁ receptor PCR primers.
Full table

Table 2 Representative competition inhibition experiments of CCPA and CPA versus the A₁R antagonist [³H]DPCPX in intact cells and membrane preparations from A₁R cells. R_H is expressed as a mean. K_H, and K_L/IC₅₀ are expressed as a mean with 95% confidence intervals.
Full table

Results

Cloning of the human adenosine A₁R cDNA To obtain the cDNA with the full length codon sequence of human A₁R, a reverse transcription reaction was carried out using the total RNA extracted from human embryo brain tissue. Using cDNA with full length codon sequence of A₁R, a PCR product (1332 bp) was obtained using primers described previously. The PCR product subcloned into a pcDNA3 plasmid and cDNA (981 bp) containing the full A₁R codon sequence was obtained using this subcloned vector as a template. The cDNA was then cloned into the mammalian expression vector pcDNA3.1. Sequence analysis demonstrated that the sequence of the constructed A₁R-pcDNA3.1 expression vector was identical to that of human A₁R cDNA in the gene bank.

Selection of cell clones Colonies of the selected stable integrants were initially analyzed for human A₁R expression by using a receptor binding assay using [³H]DPCPX. From the transfected HEK293 cell clones, one cell line was obtained with specific [³H]DPCPX binding.

Saturation experiments with the adenosine A₁ receptor antagonist [³H]DPCPX As Figure 1 shown, no significant difference was found between the A₁R cells in the absence and presence of ADA with respect to the B_max and K_D values for the A₁R binding sites labeled with [³H]DPCPX. The B_max values for the A₁R cells were 1.819±0.215 and 1.992±0.130 nmol/g of protein (n=3) in the absence and presence of ADA, respectively. The K_D values for the A₁R cells were 1.6±0.2 and 1.3±0.2 nmol/L (n=3) in the absence and presence of ADA, respectively. The nonspecific binding was <5% of the total binding.

Competition experiments of A₁R agonist CCPA or CPA versus the A₁R antagonist [³H]DPCPX Competition experiments with CCPA versus the A₁R antagonist [³H]DPCPX in intact A₁R cells or membrane preparations from A₁R cells incubated previously with ADA showed a significantly better fit with the 2 binding sites model than that with the 1 binding site model (F test, P<0.05). Similar K_H and K_L values were obtained in intact cells or membrane preparations from A₁R cells incubated previously with ADA, and the proportions of the A₁R in the high affinity state (R_H values) were 18%±3% and 20%±5%, respectively (Figure 2 and Table 2). In intact A₁R cells or membrane preparations from A₁R cells in the absence of ADA, a significant better fit for one binding site (R_H =0) was obtained, with the IC₅₀ values very similar to the K_L value obtained in intact cells or membrane preparations previously treated with ADA. Similar results were obtained from competition experiments with CPA versus the adenosine A₁R antagonist [³H]DPCPX in intact A₁R cells or membrane preparations from A₁ cells in the absence of ADA (Figure 3, Table 2).

Effects of A₁R agonist CPA on intracellular calcium level in the presence or absence of ADA An increase in intracellular Ca²⁺ appears to be a universal second messenger signal for a majority of recombinant GPCRs^[20]. A₁R agonists evoked a concentration-dependent and reproducible Ca²⁺ signal at A₁R^[22]. To assess the effect of the presence of ADA on A₁R signal transduction, we performed an intracellular calcium assay. A significant difference was found between the A₁R cells in the absence and presence of ADA with respect to the EC₅₀ values of A₁R agonist CPA. A₁R agonist CPA induced an intracellular [Ca²⁺] increase. The EC₅₀ values for the A₁R cells in the absence and presence of ADA were 94 (38–229) and 10 (5–29) nmol/L, respectively. CPA appears more potent at the A₁ cells in the presence of ADA compared with the A₁R cells in the absence of ADA (Figure 4).

Figure 1 Saturation curve of [³H]DPCPX binding to adenosine A₁R in plasma membrane isolated from A₁R cells untreated (A, B) and treated (C, D) with ADA. Binding assays were performed as described in Methods, using A₁R antagonist [³H]DPCPX as radioligand in a concentration range from 0.05 to 10 nmol/L. A and C: Saturation curve of [³H]DPCPX binding to adenosine A₁ receptors. B and D: Scatchard plot of these data and analysis gave the K_D and B_max values indicated in Results. Data are mean±SD and were obtained from three independent experiments performed in triplicate.

Figure 2 Representative competitive inhibition curve of A₁R agonist CCPA versus the A₁R antagonist [³H]DPCPX in intact cells (A) and membrane preparations (B) from A₁R cells untreated and treated with ADA. Competition assays were done at 0.5 nmol/L [³H]DPCPX and increasing concentrations of CCPA. IC₅₀ values are means (95% confidence intervals) obtained from three independent experiments performed in triplicate.

Figure 3 Representative competitive inhibition curve of A₁R agonist CPA versus the A₁R antagonist [³H]DPCPX in intact cells (A) and membrane preparations (B) from A₁R cells untreated and treated with ADA. Competition assays were done at 0.5 nmol/L [³H]DPCPX and increasing concentrations of CPA. IC₅₀ values are means (95% confidence intervals) obtained from three independent experiments performed in triplicate.

Figure 4 Concentration-response curves for the effect of A₁R agonist CPA on intracellular [Ca²⁺] in A₁R cells untreated and treated with ADA. Mean±SD. n=3. EC₅₀ values is mean (95% confidence intervals) obtained from three independent experiments performed in triplicate.

Discussion

Recently, several evidences demonstrated that cell-surface ADA interacted with A₁ receptors in brain cortex and DDT₁MF-2 cells and that the enzyme was able to modulate ligand binding and signaling through A₁R^[16,21–23]. By immunoprecipitation and affinity chromatography, it was found that ADA and A₁R interacted in pig brain cortical membranes. By means of this interaction ADA led to the appearance of the high-affinity site of the receptor. Thus, it seems that ADA is necessary for coupling A₁R to heterotrimeric G proteins^[16]. In Chinese hamster ovary (CHO) cells, stably transfected with the human adenosine A₁R, it was found that in the presence of ADA, the [³H]DPCPX/cyclohexyladenoine competition curve could be analysed by a two-site model with 93% of the sites having high affinity and the remainder having low affinity. In the absence of ADA, the [³H]DPCPX/cyclohexyladenosine competition curve was well described by a two-site model. Under these conditions, 70% of the binding of [³H]DPCPX was associated with a high-affinity site with the remaining sites having low affinity. Thus there is a clear difference in the potency of cyclohexyladenosine at the high-affinity state^[24]. In the present study, human adenosine A₁R were cloned and stably expressed in the HEK293 cells and the effects of ecto-ADA on adenosine A₁R were studied. By using competitive inhibition assay of adenosine A₁R agonists CCPA or CPA against [³H]DPCPX, we found that A₁R displayed two different affinities for agonists in the presence of ADA. However, A₁R only displayed a low affinity for agonists in the absence of ADA, suggesting that most of the binding of [³H]DPCPX was associated with the low-affinity site under these conditions and that the interaction of ADA with A₁ adenosine receptors led to the appearance of the high-affinity site of the receptor.

To further demonstrate possible interaction between A₁R and ADA, intracellular calcium assay was carried out in the present studies. The results demonstrated that the EC₅₀ value of CPA for inducing increase of intracellular calcium level was lower in the presence of ADA than that in the absence of ADA, suggesting that the presence of ADA may promote signaling through A₁ receptors in the stably transfected HEK293 cells. Our results further support the notion that ADA may promote the signaling through A₁ receptors.

Footnote

Project supported by the National Natural Science Foundation of China (No 30128004) and Shanghai Metropolitan Fund for Research and Development (No 04DZ14005).

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