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1 Department of Pharmacology, School of Medicine and 2 Department of Biology, East Carolina University, Greenville, North Carolina 27858; and 3 Department of Cardiology, Vanderbilt University Medical Center, Nashville, Tennessee 37232
We investigated the role of the cAMP link to the signal transduction
mechanism coupled with adenosine A2A and A2B
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
(125I-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 (Bmax) of 240 fmol/mg protein, and scatchard
analysis revealed a single class of binding site with an equilibrium
dissociation constant (Kd) 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) 
CGS-21680 > 2-chloroadenosine. This order appears to be
consistent with the A2 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
7-104 M) elicited the production of cAMP
in intact endothelial cells in a dose-dependent manner, exhibiting
consistently with the A2 adenosine receptor classification.
A selective A2A adenosine receptor antagonist
(ZM-241385, 108 M) significantly inhibited the effect of
CGS-21680 on cAMP but only partly inhibited the effect of NECA,
suggesting the presence of both A2A and A2B
receptors. Western blot analysis further showed the immunoreactivity of
A2A and A2B receptor at 45 and 36 kDa, respectively, in both HCAEC and PCAEC. Direct evidence for
the presence of A2A and A2B receptors in
cultured HCAEC and PCAEC by reverse transcription-polymerase chain
reaction (RT-PCR), revealed expected PCR product sizes (205 and 173 bp)
for A2A and A2B receptors in
HCAEC and PCAEC, respectively. The data show that adenylate cyclase-coupled adenosine A2A and A2B receptors
are present in coronary endothelial cells.
vascular endothelium; cyclic nucleotides; coronary circulation
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