Modulating effect of adenosine deaminase on function of adenosine A1 receptors.
OBJECTIVE
To study the modulating effect of adenosine deaminase (ADA) on the adenosine A1 receptor (A1R) in HEK293 cells stably expressing the human A1R.


METHODS
cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned A1R cDNA was sequenced and stably expressed in HEK293 cells. The modulating effect of adenosine deaminase on A1R was studied by using [3H]DPCPX binding assay and an intracellular calcium assay.


RESULTS
HEK293 cells stably expressing human A1R were obtained. Saturation studies showed that the 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 respectively, 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]DPCPX were unaffected by ecto-ADA. In the case of [3H]DPCPX competition curves obtained from intact cells or membranes, A1R agonist 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 (membrane) and a K(L) value of 0.94 (0.62+/-1.41) micromol/L (intact cells) or 0.77 (0.29+/-0.99) micromol/L (membrane). The K(L) value is not significantly different from the IC50 value of 0.84(0.57+/-1.23) micromol/L (intact cells) or 0.84 (0.63+/-1.12) micromol/L (membrane) obtained in the absence of ecto-ADA. 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 EC50 value of CPA were 10 (5+/-29) nmol/L and 94 (38+/-229) nmol/L in the presence or absence of ecto-ADA, respectively.


CONCLUSIONS
A1R stably expressed in the HEK293 cells display a low affinity for agonists in the absence of ADA and high and low affinities for agonists in the presence of ADA. The presence of ADA may promote the signaling through the adenosine A1 receptor in HEK293 cells.
Sun
Wan-Chun
WC
Department of Pharmacology, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Cao
Yan
Y
Jin
Lei
L
Wang
Li-Zhen
LZ
Meng
Fan
F
Zhu
Xing-Zu
XZ
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D058907:Adenosine A1 Receptor Agonists; D058916:Adenosine A1 Receptor Antagonists; D000243:Adenosine Deaminase / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004622:Embryo, Mammalian; D006801:Humans; D007668:Kidney / Q000166:cytology* / Q000378:metabolism; D011869:Radioligand Assay; D043682:Receptor, Adenosine A1 / Q000378:metabolism*; D015398:Signal Transduction; D014970:Xanthines / Q000494:pharmacology
D058907:Adenosine A1 Receptor Agonists; D058916:Adenosine A1 Receptor Antagonists; D043682:Receptor, Adenosine A1; D014970:Xanthines; C058394:2-chloro-N(6)cyclopentyladenosine; C048599:N(6)-cyclopentyladenosine; C051360:1,3-dipropyl-8-cyclopentylxanthine; D000243:Adenosine Deaminase; D000241:Adenosine; D002118:Calcium