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

Purified endothelial cells isolated from guinea pig hearts by enzymati c perfusion were grown in monolayer culture and used to test the abili ty of a variety of vasoactive agents to stimulate ATP release from the se cells. Stimulation of endothelial cells with the peptide agonist br adykinin (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 app earance of ATP in the incubation medium determined with the firefly lu ciferase assay for ATP. Addition of antagonists for muscarinic (atropi ne, 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 cell s 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. Dese nsitization to bradykinin also affected the ability of acetylcholine t o 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-m ethylene 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 releas e. The rank-order of potency for ATP-induced ATP release (2-methyl-S-A TP>ADP>>beta gamma-methylene ATP approximate to(alpha beta-methylene A TP) and its inhibition by suramin and reactive blue-2 suggest a role f or the P(2)y receptor in mediating the ATP response. Incubation of cel ls with [H-3]adenine to label the cellular ATP pool available for rele ase confirmed that addition of ADP stimulated [H-3]ATP release from th e cells into the medium in a manner blocked by addition of suramin. Ou r data are discussed in the light of evidence for a purinergic axis in cardiac blood vessels. (Circ Res. 1994;74:401-407.)