PURINERGIC AXIS IN CARDIAC BLOOD-VESSELS - AGONIST-MEDIATED RELEASE OF ATP FROM CARDIAC ENDOTHELIAL-CELLS

Purified endothelial cells isolated from guinea pig hearts by enzymatic perfusion were grown in monolayer culture and used to test the ability of a variety of vasoactive agents to stimulate ATP release from these cells. Stimulation of endothelial cells with the peptide agonist bradykinin (1 nmol/L), acetylcholine (1 mu mol/L), serotonin (1 mu mol/L), or adenosine 5'-diphosphate (10 mu mol/L) resulted in the rapid appearance of ATP in the incubation medium determined with the firefly luciferase assay for ATP. Addition of antagonists for muscarinic (atropine, 0.1 mu mol/L) and purinergic (suramin, 100 mu mol/L; reactive blue-2, 100 mu mol/L) receptors suggested that ATP release from these cells was receptor-mediated. Bradykinin-induced release of ATP was rapid (peak <30 seconds at 3 nmol/L bradykinin), dose-dependent (EC(50), 0.18 nmol/L), and diminished with repeated administration of agonist. Desensitization to bradykinin also affected the ability of acetylcholine to induce release and was reversible when cells were returned to growth conditions for short periods. Measurement of released adenyl purines as their fluorescent N-6-ethenopurine derivatives by high-performance liquid chromatography revealed the origin of the purine released to be ATP and confirmed its rapid dephosphorylation. Addition of the purine nucleotide analogues 2-methylthio-ATP (2-methyl-S-ATP), ADP, and py-methylene ATP to endothelial cell cultures resulted in a dose-dependent increase in the appearance of ATP measured in the medium bathing the cells at 30 seconds, suggesting the presence of ATP-induced ATP release. The rank-order of potency for ATP-induced ATP release (2-methyl-S-ATP>ADP>>beta gamma-methylene ATP approximate to(alpha beta-methylene ATP) and its inhibition by suramin and reactive blue-2 suggest a role for the P(2)y receptor in mediating the ATP response. Incubation of cells with [H-3]adenine to label the cellular ATP pool available for release confirmed that addition of ADP stimulated [H-3]ATP release from the cells into the medium in a manner blocked by addition of suramin. Our data are discussed in the light of evidence for a purinergic axis in cardiac blood vessels. (Circ Res. 1994;74:401-407.)