We studied the effects of adenosine and adenosine derivatives on adenylate cyclase activity in cultured endothelial cells from bovine pulmonary artery. Basal and stimulated enzyme activities were measured in membrane preparations using [alpha-32P]ATP as the substrate and chromatographic isolation of formed [32P]cAMP. Basal cyclase activity was 11 +/- 1 (mean +/- SEM) pmol/mg protein/min. Forskolin, 5'-guanylylimidodiphosphate (Gpp(NH)p) and (-)isoproterenol stimulated adenylate cyclase in a concentration-dependent manner, producing maximal stimulations of three, seven and four times the basal activity respectively. In the presence of adenosine deaminase, cyclohexyladenosine, an A1 agonist, had no effect on basal and forskolin- or Gpp(NH)p-stimulated activities, whereas 5'-(N-ethyl)-carboxamidoadenosine (NECA), an A2 agonist, had a small stimulatory effect (52% increase over basal). In the presence of IBMX, adenosine and two P-site agonists, 2',5'-dideoxyadenosine (DDA) and 2'-deoxyadenosine-3'-monophosphate (2'-deoxy-3'-AMP), inhibited forskolin (30 microM)-stimulated adenylate cyclase activity with an order of potency of 2'-deoxy-3'-AMP greater than DDA greater than adenosine. DDA and 2'-deoxy-3'-AMP were also able to inhibit cyclase activity stimulated by Gpp(NH)p (10(-5)M) or isoproterenol (10(-6)M) with the same order of potency. Only 2'-deoxy-3'-AMP inhibited the stimulated adenylate cyclase activity by more than 50% (IC50 = 19-32 microM). These findings indicate that (1) long-term cultured endothelial cells from bovine pulmonary artery express A2 and beta-adrenergic receptors which stimulate adenylate cyclase activity through Gs transducer proteins, and (2) the natural compound and P-site agonist, 2'-deoxy-3'-AMP, is a potent inhibitor, and possibly a natural regulator, of adenylate cyclase activity in this tissue.