Effects of adenosine and analogs on adenylate cyclase activity in cultured bovine aortic endothelial cells.

We studied the effects of adenosine and analogs on adenylate cyclase (AC) activity in membranes from long-term cultured bovine aortic endothelial cells, using [alpha-32]ATP as substrate and chromatographic separation of [32P]cAMP. Compared to our previous findings in cultured bovine pulmonary arterial endothelial cells (Legrand et al., Biochem Pharmacol 38: 423-430, 1989), the present results were qualitatively and quantitatively comparable between the two cell types. In aortic cells, AC activity was stimulated in a concentration-dependent manner by isoproterenol, forskolin and 5'-guanylylimidodiphosphate (Gpp(NH)p), by 2.6-, 5.2- and 4.8-fold respectively. The A2 adenosine agonist 5'-(N-ethyl)-carboxamidoadenosine induced a smaller (60%) increase of AC activity. Adenosine (10(-3) M) partially inhibited (30%) the Gpp(NH)p-stimulated AC activity. Similarly, adenosine partially reversed, but 2',5'-dideoxyadenosine (DDA) totally blocked (IC50: 540 microM), the forskolin-induced stimulation of AC activity. DDA and 2'-deoxyadenosine-3'-monophosphate (2'-deoxy-3'-AMP) also inhibited the isoproterenol-induced stimulation of AC activity (IC50: 350 and 23 microM respectively). Adenosine-induced inhibition of stimulated AC activity does not appear to involve adenosine A1 receptors since the specific A1 agonist cyclohexyladenosine did not reverse forskolin stimulation of AC activity. Instead, it suggests a direct action of adenosine on the catalytic subunit of the adenylate cyclase (P site). We conclude that membranes from long-term cultured bovine aortic endothelial cells, express beta-adrenergic and adenosine A2 receptors coupled to adenylate cyclase activation. The two P site agonists, DDA and 2'-deoxy-3'-AMP, and, with a weaker effect, adenosine itself, inhibited the activated cyclase at the P site. The natural nucleotide 2'-deoxy-3'-AMP was a strong inhibitor in aortic cell types (as in pulmonary arterial endothelial cells) and may possibly act as a modulator of adenylate cyclase in these cells.