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Aim:To study the modulating effect of adenosine deaminase(ADA)on yheadenosine A_1 receptor(A_1R)in HEK293 cells stably expressing the human A_1R.Methods:cDNA was amplified by RT-PCR using total RNA from human embryobrain tissue as the template.The PCR products were subcloned into the plasmidpcDNA3 and cloned into the plasmid pcDNA3.1.The cloned A_1R cDNA wassequenced and stably expressed in HEK293 cells.The modulating effect of ad-enosine deaminase on A_1R was studied by using[~3H]DPCPX binding assay andan intracellular calcium assay.Results:HEK293 cells stably expressing humanA_1R were obtained.Saturation studies showed that the K_D value and B_(max)valueof[~3H]DPCPX were 1.6±0.2 nmol/L and 1.819±0.215 nmol/g of protein respectively,in the absence of ecto-ADA respectively,and 1.3±0.2 nmol/L and 1.992±0.130nmol/g of protein in the presence of ecto-ADA respectively,suggesting that theK_D value and B_(max)value of[~3H]DPCPX were unaffected by ecto-ADA.In the caseof[~3H]DPCPX competition curves obtained from intact cells or membranes,A_1Ragonist CCPA/[~3H]DPCPX competition curve could be fitted well to a one-sitemodel in the absence of ecto-ADA and a two-site model in the presence of ecto-ADA with a K_H value of 0.74(0.11-4.8)nmol/L(intact cells)or 1.8(0.25-10)nmol/L(membrane)and a K_L value of 0.94(0.62-1.41)μmol/L(intact cells)or 0.77(0.29-0.99)μmol/L(membrane).The K_L value is not significantly different from the IC_(50)value of 0.84(0.57-1.23)μmol/L(intact cells)or 0.84(0.63-1.12)μmol/L(membrane)obtained in the absence of ecto-ADA.Similar results were obtained from theCPA/[~3H]DPCPX competition curve in the absence or presence of ecto-ADA onintact cells or membranes.Intracellular calcium assay demonstrated that the EC_(50)value of CPA were 10(5-29)nmol/L and 94(38-229)nmol/L in the presence orabsence of ecto-ADA,respectively.Conclusion:A_1R stably expressed in theHEK293 cells display a low affinity for agonists in the absence of ADA and highand low affinities for agonists in the presence of ADA.The presence of ADA maypromote the signaling through the adenosine A_1 receptor in HEK293 cells.
Aim: To study the modulating effect of adenosine deaminase (ADA) on yheadenosine A_1 receptor (A_1R) in HEK293 cells stably expressing the human A_1R.Methods: cDNA was amplified by RT-PCR using total RNA from human embryo tissue as the template. PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3. The cloned A_1R cDNA was sequenced and stably expressed in HEK293 cells. The modulating effect of ad-enosine deaminase on A_1R was studied by using [~ 3H] DPCPX binding assay andan Results: HEK293 cells stably expressing humanA_1R were obtained. Saturation studies showed that K_D value and B_ (max) value of [~ 3H] DPCPX were 1.6 ± 0.2 nmol / L and 1.819 ± 0.215 nmol / g of protein respectively, in the absence of ecto-ADA and 1.3 ± 0.2 nmol / L and 1.992 ± 0.130 nmol / g of protein in the presence of ecto-ADA respectively, suggesting that the K_D value and B_ (max) value of [~ 3H] were unaffected by ecto-ADA. the caseof [~ 3H] DPCPX competit ion curves obtained from intact cells or membranes, A_1Ragonist CCPA / [~ 3H] DPCPX competition curve could be fitted well to a one-site model in the absence of ecto-ADA and a two-site model in the presence of ecto-ADA with a K_H value of 0.74 (0.11-4.8) nmol / L (intact cells) or 1.8 (0.25-10) nmol / L of membrane and a K_L value of 0.94 (0.62-1.41) μmol / L of intact cells or 0.77 0.29-0.99) μmol / L (membrane). The K_L value is not significantly different from the IC 50 value of 0.84 (0.57-1.23) μmol / L (intact cells) or 0.84 (0.63-1.12) μmol / L Similar results were obtained from the CPA / [~ 3H] DPCPX competition curve in the absence or presence of ecto-ADA on intact cells or membranes. Intracellular calcium assay demonstrated that the EC_ (50) value of CPA were 10 (5-29) nmol / L and 94 (38-229) nmol / L in the presence orabsence of ecto-ADA, respectively.Conclusion: A_1R stably expressed in theHEK293 cells display a low affinity for agonists in the absence of ADA and highand low affiniti es foragonists in the presence of ADA. The presence of ADA maypromote the signaling through the adenosine A_1 receptor in HEK293 cells.