Mrs1220

hA₁ and hA₃ AR functional experiments were carried out in CHO-A₁ and CHO-A₃#18 cell line, respectively. The day before the assay, the cells were seeded on the 96-well culture plate (Falcon 353072). The cells were washed with wash buffer (Nutrient Mixture F12 Ham’s (Sigma N6658) for hA₁ AR; Dulbecco’s modified eagle’s medium nutrient mixture F-12 ham (Sigma D8062) for hA₃ AR; 25 mM Hepes; pH = 7.4). Wash buffer was replaced by incubation buffer (Mixture F12 Ham’s (Sigma N6658) for hA₁ AR; Dulbecco’s modified eagle’s medium nutrient mixture F-12 ham (Sigma D8062) for hA₃ AR; 25 mM Hepes, 20 µM Rolipram (Sigma R6520); pH = 7.4). Test compounds and XAC (Sigma X103) or MRS1220 (Sigma M228) as reference compound for hA₁ and hA₃ AR, respectively, were added and incubated at 37 °C for 15 min. Afterward, 0.1 µM of 5′-(N-Ethilcarboxamido) adenosine (NECA) (Sigma E2387) was added and incubated at 37 °C for 10 min. FSK (Sigma F3917) was added and incubated at 37 °C for 5 min. After incubation, the amount of cAMP was determined using cAMP Biotrak Enzyme immunoassay (EIA) System Kit (GE Healthcare RPN225).

For in vitro studies AOPCP (50 μM; α,β-Methyleneadenosine 5′-diphosphate) was used as a CD73 inhibitor (M8386; Sigma, Saint Louis, MO) and MRS1220 (#1217; 10 μM) as a selective antagonist of A₃AR [44 (link)]. Vincristine (Vc; 100 nM; #1257) was used as a drug substrate of the MRP1 transporter and LY294002 (LY 50 nM; #1130) as a PI3K inhibitor. All products were purchased from Tocris Biosciences, Minneapolis, MN. PD98059 (PD 50 μM; #167869, Calbiochem) was used as a MAPK inhibitor. For in vivo studies we used MRS1220 (0.15 mg/kg/72 hrs) and Vc (0.3 mg/kg/72 hrs) in NOD/SCID-IL2Rg^null mouse.