The present study further examined the functional presence and the signal transduction mechanism(s) for adenosine A(2A) and A(2B) receptors through nitric oxide (NO) and the guanosine 3', 5'-cyclic monophosphate (cGMP) pathway in cultured porcine coronary artery endothelial cells (PCAEC). The application of adenosine receptor agonists, NECA, CGS-21680 and CAD between 10(-7) and 10(-4) M, enhanced the production of NO (measured as nitrite) in a dose-dependent manner. On the basis of EC(50) values, these agonists showed the following order of potency: NECA>CGS-21680>CAD. This order appears to be of the A(2) adenosine receptor subtype. Similarly, the same concentrations of adenosine agonists evoked the production of cGMP in a dose-dependent manner, exhibiting a rank order that is similar to that of NO production. NO synthase inhibitor, N-nitro-L-arginine methylester (L-NAME, 10(-5) M), inhibited the production of NO and cGMP, which was reversed by L-arginine (10(-4) M). Selective A(2A) adenosine receptor antagonists, ZM-241385 and SCH-58261, at 10(-7) M, significantly inhibited the effects of CGS-21680, but only partly inhibited the effect of NECA on NO and cGMP production. Along with the earlier molecular evidence from this laboratory [Am. J. Physiol. 279 (2000) H650], the present data further support the presence of both A(2A) and A(2B) receptors in PCAEC. These results further support that coronary endothelial cells express functional A(2A) and A(2B) adenosine receptors, leading to GMP production through the NO-synthase-linked mechanism. This is the first direct evidence where an A(2B) adenosine receptor has been linked to NO production in cultured endothelial cells and could play a role in coronary artery physiology and pathophysiology.