CGS 21680

BRET experiments were conducted as previously described [36 (link)]. 20 µg of the transfected cells (100 µl) were distributed in duplicates into a black 96-well plate with black bottom for fluorescence and a white 96-well plate with white bottom for bioluminescence measurements. Signals were detected by a Mithras LB 940 fluorimeter calculating the integration for bioluminescence using filters for 440–500 nm (485 nm maximum emission of bioluminescence), and for fluorescence with a filter for 510–590 nm (530 nm maximum emission of EYFP). To confirm equal expression of Rluc and increasing expression of EYFP, for each sample bioluminescence and fluorescence was measured before starting the experiment. EYFP fluorescence was defined as the fluorescence of the sample minus the fluorescence of cells expressing only Rluc-tagged receptors. For BRET, 5 µM coelenterazine-H (PJK, Kleinblittersdorf, Germany) was added to the samples and measurements were performed after 1 min (net BRET determination) and after 10 min (Rluc luminescence quantification). Net BRET was defined as the bioluminescence of the sample minus the bioluminescence of cells expressing only Rluc-tagged receptors. BRET signals were determined by calculating the ratio of the light emitted by EYFP over the light emitted by the Rluc A. milliBRET unit (mBU) is the BRET ratio × 1000. Curves were fitted using nonlinear regression. Displacement studies were performed in triplicates at a constant BRET ratio, around the BRET₅₀ of the A_2BAR-Rluc/A_2AAR-YFP pair, and increasing amounts of the A_2BAR. For testing the effects of compounds on heteromerization of A_2AAR and A_2BAR the agonists adenosine (100 µM), NECA (100 µM), CGS-21680 (A_2AR, 10 µM), BAY60-6583 (A_2BR, 10 µM), and the antagonist PSB-603 (A_2BR, 500 nM) were added for 60 min to cells which expressed A_2BAR-Rluc and A_2AAR-YFP, around the BRET₅₀ value. The final DMSO concentration in these experiments was 2.5 % and experiments were performed in triplicates.

Mice were anesthetized by i.p. injection of pentobarbital sodium (50 mg kg^-1) and hearts were excised. Right and left atria were dissected from isolated A_2A-AR transgenic and wild type (WT) mice hearts and mounted in an organ bath. Left atrial preparations were continuously electrically stimulated (field stimulation) with each impulse consisting of 1 Hz, with a voltage of 10–15% above threshold and 5 ms duration. Right atrial preparations were allowed to contract spontaneously. The bathing solution contained (in mM) NaCl 119.8, KCl 5.4, CaCl₂ 1.8, MgCl₂ 1.05, NaH₂PO₄ 0.42, NaHCO₃ 22.6, Na₂EDTA 0.05, ascorbic acid 0.28 and glucose 5.0, continuously gassed with 95% O₂ and 5% CO₂ and maintained at 35°C resulting in a pH of 7.4. Signals detected via an isometric force transducer were amplified and continuously recorded. CGS 21680, ZM 241385or isoproterenol (1 μM each) were cumulatively applied with approximately 20 min for each compound. Contraction experiments were performed after addition of adenosine deaminase (1 μg ml^-1) and DPCPX (1 μM) to avoid interference from endogenous adenosine or A₁ adenosine receptor activation.

In lymphocytes, the potency of the well-known A_2A and A₃ adenosine agonists CGS 21680 and Cl-IB-MECA (0.1 nM–1 μM) was investigated. Cells were seeded in 96-well white half-area microplate (Perkin Elmer, Boston, MA, USA) in a stimulation buffer composed of Hank Balanced Salt Solution, 5 mM HEPES, 0.5 mM Ro 20-1724, 0.1% BSA. Forskolin (1 μM) was used to stimulate adenylyl cyclase activity after the addition of Cl-IB-MECA. cAMP levels were then quantified by using the AlphaScreen cAMP Detection Kit (Perkin Elmer) following the manufacturer’s instructions. At the end of the experiments, plates were read with the Perkin Elmer EnSight Multimode Plate Reader.

(1) SCH 58261 (an A_2AR antagonist) was microinjected into the rVLM of EA-treated DSHRs (n = 9). The vehicle (DMSO) was administered into other sites of the rVLM in random order in five of these nine animals. (2) The rVLM of DSHRs treated with sham-EA (n = 9) was administered with CGS-21680 (an A_2AR agonist). The vehicle (DMSO) was randomly microinjected into the different rVLM sites in five of the nine rats. (3) In separate rats in normotensive groups, SCH 58261 and its vehicle (n = 4) or CGS-21680 and its vehicle (n = 4) were microinjected into different sites of the rVLM in random order.

Adenosine A_2AR agonist, CGS-21680 (0.4 mM) (35 (link)); adenosine A_2AR antagonists, SCH 58261 (1 mM) (36 (link), 37 (link)), and the vehicle for these drugs, dimethylsulfoxide (DMSO). All chemicals were purchased from Sigma Aldrich (St. Louis, MO, United States). Affinities, specificities, and dosages for each drug are documented in the cited references. The solution of each drug or vehicle contained 0.5% pontamine sky blue for histological examination of the injection site after the experiment. Microinjection of the vehicle into the rVLM and the drugs into surrounding regions of the rVLM provided chemical and anatomical controls.

7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261; a selective A_2AR antagonist; Tocris, USA) and 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS21680; a selective A_2AR agonist; Tocris, USA) were dissolved in saline containing 10% dimethylsulfoxide (DMSO) and administered systemically by intraperitoneal (i.p.) injection at the doses of 0.1 and 0.2 mg/kg, respectively (in a volume of 1 mL/kg) immediately after the fear conditioning session, unless otherwise specified. The doses and concentrations used in this study were chosen based on previous studies (see [11 (link), 15 (link), 21 (link)]). For the electrophysiological experiments testing the modification of adenosine A₁ receptor (A₁R) function, we used the closest but stable chemical analog of adenosine, 2-chloroadenosine (CADO, from Sigma-Aldrich, Portugal) in a previously validated concentration range of 0.1–1 μM [22 (link)] as well as the selective A₁R antagonist, 1,3-dipropylcyclopentlxanthine (DPCPX, from Tocris), used in a supra-maximal and selective concentration of 100 nM [23 (link)]. It should be noted that although CADO can activate A₁R and A_2AR, the fact that A_2AR are devoid of effects in the control of basal synaptic transmission [24 (link), 25 (link)], allows using CADO to selectively probe the efficiency of A₁R to control hippocampal synaptic transmission [22 (link), 23 (link)].