Adenosine A(2A) and A(2B) receptors in cultured human and porcine coronaryartery endothelial cells

We investigated the role of the cAMP link to the signal transduction mechan ism coupled with adenosine A(2A) and A(2B) receptors in cultured human coro nary artery endothelial cells (HCAEC) and porcine coronary artery endotheli al cells (PCAEC). 2-[4-[2-{2-[(4-aminophenyl)methylcarbonylamino] ethylamin ocarbonyl}ethyl]phenyl]ethylamino-5'-ethylcarboxamidoadenosine (I-125-PAPA- APEC) (PAPA-APEC) was used to demonstrate the specific binding in PCAEC mem branes. The specific binding was saturable and reversible with a maximal nu mber of binding sites (B-max)of 240 fmol/mg protein, and scatchard analysis revealed a single class of binding site with an equilibrium dissociation c onstant (K-d) of 1.17 +/- 0.035 nM. In competition experiments, adenosine r eceptor 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) adenosin e receptor classification. We also studied the effects of adenosine agonist s 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 rece ptor classification. A selective A2A adenosine receptor antagonist (ZM-2413 85, 10(-8) M) significantly inhibited the effect of CGS-21680 on cAMP but o nly partly inhibited the effect of NECA, suggesting the presence of both A( 2A) and A(2B) receptors. Western blot analysis further showed the immunorea ctivity 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) recep tors 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 pres ent in coronary endothelial cells.