Involvement of P2 purinoceptors in the regulation ofdna synthesis in the neural retina of chick embryo

Abstract

The activation of P2 purinoceptors induces Ca2 mobilization in the earlyembryonic chick neural retina. This purinergic Ca2 response declines parallel with thedecrease in mitotic activity during retinal development. To investigate the role of P2purinoceptors in the regulation of retinal cell proliferation, we studied the effects of the P2purinoceptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2fn2fn2triphosphate ; [Ca2]i, intracellular Ca2 concentration ;DMSO, dimethyl sulfoxide ; E, embryonic day ; EC50, concentration for half maximalexcitation ; F340, fluorescence excited at 340 nm ; F380, fluorescenceexcited at 380 nm ; F340/F380, the ratio of F340/F380 ;FCS, fetal calf serum ; fura-2 AM, fura-2 acetoxymethyl ester ; IC50, concentrationfor half maximal inhibition ; PPADS, pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid ;UTP, uridine 5-triphosphate.,4-disulphonic acid (PPADS), and of the agonist ATP on DNA synthesis in retinal organcultures from embryonic day 3 (E3) chick. Suramin inhibited [3H]-thymidineincorporation in a dose-dependent manner (IC50 : ∼70 μM). PPADS alsoreduced [3H]-thymidine incorporation with maximum inhibition of 46% at 100 μM. Exogenous ATP enhanced [3H]-thymidine incorporation in adose-dependent manner to maximally 200% of control (EC50 : ∼70 μM). Indissociated retinal cultures from E7 chick, both antagonists showed similar inhibitory effects on [3H]-thymidine incorporation without affecting cell viability. In line with theseobservations, the presence of extracellular ATP was demonstrated both in vitro and in vivo. In the medium of E3 retinal organ cultures, the concentration of ATP increased25-fold within 1 h of incubation and this concentration was kept for at least 24 h. In the chickamniotic fluid, the ATP concentration was nearly 3 μM at E3 and declined to 0.15 μM at E7. The results indicate that P2 purinoceptors activated by autocrine or paracrinerelease of ATP are involved in the regulation of DNA synthesis in the neural retina at earlyembryonic stages.