Adenosine A2A and A2B receptors in cultured human and porcine coronary artery endothelial cells

We investigated the role of the cAMP link to the signal transduction mechanism coupled with adenosine A(2A) and A(2B) receptors in cultured human coronary artery endothelial cells (HCAEC) and porcine coronary artery endothelial cells (PCAEC). 2-[4-[2-{2-[(4-aminophenyl)methylcarbonylamino] ethylaminocarbonyl}ethyl]phenyl]ethylamino-5'-ethylcarboxamidoadenosine (I-125-PAPA-APEC) (PAPA-APEC) was used to demonstrate the specific binding in PCAEC membranes. The specific binding was saturable and reversible with a maximal number of binding sites (B-max)of 240 fmol/mg protein, and scatchard analysis revealed a single class of binding site with an equilibrium dissociation constant (K-d) of 1.17 +/- 0.035 nM. In competition experiments, adenosine receptor agonists showed the following order of potency (based on IC50): 5'-(N-ethylcarboxamido)adenosine (NECA) greater than or equal to CGS-21680. 2-chloroadenosine. This order appears to be consistent with the A(2) adenosine receptor classification. We also studied the effects of adenosine agonists on the accumulation of cAMP as an indirect approach to show the presence of functional A2 receptors. Similarly, the same adenosine agonists (10(-7)-10(-4) M) elicited the production of cAMP in intact endothelial cells in a dose-dependent manner, exhibiting consistently with the A(2) adenosine receptor classification. A selective A2A adenosine receptor antagonist (ZM-241385, 10(-8) M) significantly inhibited the effect of CGS-21680 on cAMP but only partly inhibited the effect of NECA, suggesting the presence of both A(2A) and A(2B) receptors. Western blot analysis further showed the immunoreactivity of A(2A) and A(2B) receptor at 45 and 36 kDa, respectively, in both HCAEC and PCAEC. Direct evidence for the presence of A(2A) and A(2B) receptors in cultured HCAEC and PCAEC by reverse transcription-polymerase chain reaction (RT-PCR), revealed expected PCR product sizes (205 and 173 bp) for A(2A) and A(2B) receptors in HCAEC and PCAEC, respectively. The data show that adenylate cyclase-coupled adenosine A(2A) and A(2B) receptors are present in coronary endothelial cells.