Diabetes incidence showed ascending developments lately indicating urgent dependence on new therapeutic real estate agents. MIN6 cells, A3AR, through Ca2+ mediated signaling pathways probably, potentiated glucose-induced insulin secretion. reported that its agonist at high concentrations (100-300 M) boost GSIS from isolated mice islets (6). Based on which signaling pathway triggered in cells, A3AR excitement might trigger different Flupirtine maleate mobile reactions, including apoptosis, cytoprotective, and anti-inflammatory (14,15). These signaling pathways can include Gi or Gq activation of RhoA element and phospholipase D (16), phosphorylation or dephoshphorylation of extracellular signal-regulated kinases 1/2 (6 actually,17) and proteins kinase B (Akt) (15). Knowledge of intracellular mediators of A3AR is vital for detailing of its results in MIN6 cells biology. To the very best of our understanding, primary signaling mediators (cAMP and Ca2+) of the receptor in PBCs never Flupirtine maleate have been determined, however. Analysis of the mediators may provide fresh insight about control of GSIS by this receptor. Thus, in this scholarly study, we targeted to evaluate primary mediators of A3AR signaling in MIN6 cells. Materials AND METHODS Chemicals Ro-20-1724 (a phosphodiesterase inhibitor, Cat: 557502) and MRS 21680 (an A3AR antagonist, Cat: M228) were Flupirtine maleate purchased from Sigma-Aldrich-Merck company (Germany). 1-[2-Chloro-6-[[(3-iodophenyl)methyl]amino]- 9H-purin-9- yl] -1-deoxy-N-methyl- -D- ribo-furanuronamide (Cl-IBMECA, an A3AR agonist, Cat: 1104) was purchased from Tocris Bioscience company (UK). Fura 2/AM (CAS 108964-32-5) was provided from Santa Cruz (USA). Forskolin (Cat: ALX-350-001) and probenecid (Cat: ALX-430-113) were from Enzo Life Sciences. Cell culture supplies like the Dulbecco’s modification of Eagle’s medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin were from Gibco Life Sciences (USA). Cyclic AMP ELISA Kit (Cat: 581001) from Cayman chemical was provided (Michigan, JAG2 USA). Mouse insulin ELISA kit (Cat: 10-1247-01) was purchased from Mercodia (Sweden). Cell culture MIN6 insulinoma cell line was purchased from Iranian Biological Resource Center (IBRC) (Cell No: IBRC “type”:”entrez-nucleotide”,”attrs”:”text”:”C10524″,”term_id”:”1535595″C10524) and maintained in DMEM high glucose (25 mM), 15% FBS, 70 M mercaptoethanol and 1% penicillin/streptomycin under 5% CO2 and humidified atmosphere. MIN6 cells were passaged 10 times and then all assessments were performed. Gene expression assay of A1AR and A3AR Cells (5 105) were lysed to extract total RNA using RNX plus kit (Sinacolon, I.R. Iran). After cDNA synthesize, A1AR and A3AR amplicons were amplified using the following specific primers, forward 5-GGTACAAGACAGTGGTGACTCAG-3 and reverse 5-AGGTTGTTCCAGCCAAACAT-3 for A1AR, forward 5-CCTGTGTGCTGCTGATCTTC-3 and reverse 5-TGAGTGGTAACCGTTCTATATCTGA-3 for A3AR, and forward 5- GTCGGTGTGAACGGATTTG-3 and reverse 5- AGGTCAATGAAGGGGTCGT-3 for GAPDH (as an internal control). The heating protocol was included in holding (10 min at 95 C), cycling (95 C for 15 sec, 60 C for 1 min, repeated for 45 cycle), and melting curve stages (95 C for 15 sec, 60 C for 1 min, and 95 C for 15 sec). Applied Biosystems instrument (ABI 7500 Real-Time PCR System, Foster City, USA) was used for extension of desired amplicons. The results were calculated as relative gene expression level, (2-CT), using the following equations. CT (A1 receptor) = (CT (A1) – CT (GAPDH)) (1) CT (A3 receptor) = (CT (A3) – CT (GAPDH)) (2) CT (For A1 receptor) = CT (A1 receptor) – CT (A1 receptor) = 0 (as control) (3) CT (For A3 receptor) = CT (A3 receptor) – CT (A1 receptor) (4) Cyclic AMP assay Cyclic AMP was assayed according to the previously published protocol (18). 3 105 Cells/well was seeded in 6 well dish, overnight. After that, cells had been pretreated having a selective inhibitor of cAMP-specific phosphodiesterase, Ro-20-1724 (100 M) for 15 min. After that,.