Cell experiment:

10×106 MNCs from each group are re-suspended in 2 mL RPMI 1640. Cell suspensions are added with carboxy-fluorescein diacetate, succinimidyl ester (CFSE, final concentration 2.5 μM) and thoroughly mixed. After incubation in the dark for 15 min at 37℃, the staining process is quenched by adding 10 mL ice-cold complete RPMI 1640 (containing 10% FBS) and incubated on ice for 5 min. Then cells are washed twice with RPMI 1640. Cell pellets are re-suspended in complete RPMI 1640 (containing 10% FBS). The stained MNCs (1×106 cells/mL, 1 mL/well) are cultured in triplicates in 24-well culture plates in the dark at 37℃. Each well is supplied with 50 μL of Concanavalin A (ConA, final concentration 5 μg/mL) or 50 μL of P0 peptide (final concentration 10 μg/mL). 72 h later, cells are collected and stained with PE-labeled anti-rat CD4 antibody for 30 min at 4℃. Finally, cells are analyzed with a flow cytometer.

Animal experiment:

Female Lewis rats aged 6-8 weeks (body weight, 140-160 g) are housed under specific pathogen-free conditions in the local animal facility with free access to water and food. Administration of CGS21680 (at a dose of 1 mg/kg in PBS) starts on day 5 p.i. Rats in experimental group are injected with CGS21680 intraperitoneally (i.p.) every two days until the end of the experiments. Rats in control group are given equal volume of PBS in the same way. The doses (1 mg/kg/i.p.) and the treatment regimen (every two days, start on day 5 p.i.) are determined.

• J Ethnopharmacol (2022): 115661. PMID:36002086

CGS 21680 HCl is a selective agonist of A2 adenosine receptor with IC50 value of 22nM [1].

In rat striatal membranes, CGS 21680 potently prevents ligands from binding to A2 adenosine receptors with IC50 value of 22nM. In the binding assay, CGS 21680 shows no effect to other putative neurotransmitter/neuromodulator sites in brain membranes such as adrenergic, dopamine and serotonin. In the rat heart model, CGS 21680 is effective in increasing coronary flow with EC25 value of 2nM [1].

In vivo, the hydrochloride salt of CGS 21680 has potent efficacy to decrease blood pressure in the anesthetized normotensive rat with ED50 value of 9μg/kg. It also causes heart rate increasing up to 15%. Besides that, CGS 21680 HCl significantly lower the blood pressure at dose of 10mg/kg in the spontaneously hypertensive rat. A transient elecation of heart rate is also observed at 30 min in the model [1].

References:
[1] Hutchison A J, Webb R L, Oei H H, et al. CGS 21680C, an A2 selective adenosine receptor agonist with preferential hypotensive activity. Journal of Pharmacology and Experimental Therapeutics, 1989, 251(1): 47-55.