Mechanisms by which extracellular ATP and UTP stimulate the release of prostacyclin from bovine pulmonary artery endothelial cells

Abstract

Extracellular ATP and UTP caused increases in the concentration of cytoplasmic free calcium ([Ca 2+] i) and the intracellular level of inositol 1,4,5-trisphosphate (IP 3), a second messenger for calcium mobilization, prior to the release of prostacyclin (PGI 2) from cultured bovine pulmonary artery endothelial (BPAE) cells. The agonist specificity and dose-dependence were similar for nucleotide-mediated increases in IP 3 levels, [Ca 2+] i and PGI 2 release. An increase in [Ca 2+]; and PGI 2 release was observed after addition of ionomycin, a calcium ionophore, to BPAE cells incubated in a calcium-free medium. The addition of ATP to the ionomycin-treated cells caused no further increase in [Ca 2+] i or PGI 2 release. The inability of ATP to cause an increase in [Ca 2+] i or PGI 2 release in ionomycin-treated cells was apparently due to the ionomycin-dependent depletion of intracellular calcium stores since the subsequent addition of extracellular calcium caused a significant increase in both [Ca 2+] i and PGI 2 release. Introduction of BAPTA, a calcium buffer, into BPAE cells inhibited ATP-mediated increases in [Ca 2+] i and PGI 2 release, further evidence that PGI 2 release is dependent upon an increase in [Ca 2+] i. The increase in [Ca 2+] i elicited by ATP apparently caused the activation of a calmodulin-dependent phospholipase A 2 since trifluoperazine, an inhibitor of calmodulin, and quinacrine, an inhibitor of phospholipase A 2, prevented the stimulation of PGI 2 release by ATP. Furthermore, ATP caused the specific hydrolysis of [ 14C]arachidonyl-labeled phosphatidylcholine and the generation of free arachidonic acid, the rate-limiting substrate for PGI 2 synthesis, prior to the release of PGI 2 from BPAE cells. These findings suggest that the increase in PGI 2 release elicited by ATP and UTP is at least partially dependent upon a phospholipase C-mediated increase in [Ca 2+] i and the subsequent activation of a phosphatidylcholine-specific phospholipase A 2. ATP analogs modified in the adenine base or phosphate moiety caused PGI 2 release with a rank order of agonist potency of adenosine 5′- O-(2-thiodiphosphate) (ADPβS) > 2-methylthioATP (2-MeSATP) > ATP, whereas α, β methyleneATP and β, γ methyleneATP had no effect on PGI 2 release.