Modulating effect of adenosine deaminase on function of adenosine A1 receptors
Abstract
Aim: To study the modulating effect of adenosine deaminase (ADA) on yhe
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 KD value and Bmax 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
KD value and Bmax 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 KH value of 0.74 (0.11–4.8) nmol/L (intact cells) or 1.8 (0.25–10) nmol/L
(membrane) and a KL value of 0.94 (0.62–1.41) μmol/L (intact cells) or 0.77 (0.29–
0.99) μmol/L (membrane). The KL value is not significantly different from the IC50
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 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.
Conclusion: 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.
Keywords:
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 KD value and Bmax 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
KD value and Bmax 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 KH value of 0.74 (0.11–4.8) nmol/L (intact cells) or 1.8 (0.25–10) nmol/L
(membrane) and a KL value of 0.94 (0.62–1.41) μmol/L (intact cells) or 0.77 (0.29–
0.99) μmol/L (membrane). The KL value is not significantly different from the IC50
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 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.
Conclusion: 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.