ATP‐induced proliferation of developing retinal cells: regulation by factors released from postmitotic cells in culture

Abstract

ATP is an important mitogen in the developing retina and its proliferative response decreases as chick retinal cells differentiate in culture. Both non-stimulated or ATP-induced proliferative response was abolished if cycling cells were cocultured with cells from older embryos or cultured with conditioned medium (CM) from postmitotic cells. The effect of CM was dose-dependent and reversible, as removal of CM from the cultures restored both basal and ATP-induced incorporation of [3H]-thymidine. The effect of CM was also dependent on the developmental stage of the retina used to prepare the medium. As tissues from older embryos were used, inhibition of the basal and ATP-induced proliferative response of the cells increased. Similar inhibition of ATP-induced increase in [3H]-thymidine incorporation was observed using CM from purified glial cultures. Neither ARL 67156, an ecto-ATPase inhibitor, prevented nor TGF-beta1 and TGF-beta2 mimicked the inhibitory effect of conditioned medium. Incubation of cells with CM or ATP for 24 h completely abolished the formation of [3H]-phosphoinositides induced by ATP. These effects were blocked by the P2 receptor antagonist PPADS and were not observed with dialysed CM, suggesting that agonist-dependent desensitization of P2 receptors occurred in cultures incubated with CM. However, removal of small molecules such as nucleotides by dialysis did not affect the decline in the proliferative activity induced by CM, suggesting that desensitization is not responsible for the conditioned medium-dependent cell cycle arrest of early developing retinal cells in culture. These results suggest that factors released from postmitotic cells induce the arrest of retinal cells in the mitotic state, a phenomenon that is concomitant with agonist-dependent P2 receptor desensitization.

Involvement of nucleotides in glial growth following scratch injury in avian retinal cell monolayer cultures

“…Although MRS 2179 did not affect the growth of glial cells over the scratched area in cultures at the stages between E8C7 and E8C10, this compound blocked, in a dose-dependent manner, the increase in the incorporation of [ 3 H]-thymidine induced by 500 μM ADP in retinal cultures at E7C2 (Online Resource 2). Moreover, no effect of 50 μM ADPβS on the incorporation of [ 3 H]-thymidine in retinal cultures at E8C8 was observed, corroborating previous evidences showing that activation of P2Y 1 receptors by ATP induces the proliferation of glial progenitors in early developing, but not in more differentiated retinal cultures [20,21].…”

“…Previously, Sanches et al [20] demonstrated that ATP through the activation of P2Y 1 receptors induces the proliferation of retinal progenitors in culture, an effect that could be observed until the fourth day after the onset of retinal cell cultures [21]. However, besides their effect on the proliferation of glial progenitors, several experimental data suggest that nucleotides are involved in the growth of glial cells in the reactive gliosis that occurs when adult nervous tissue is injured [33,34].…”

“…Although affecting different cell populations, both ADP and UTP induce cell proliferation in the early developing retina [13,20,21,23,24]. UTP also stimulates the proliferation of astrocytes from the cerebral cortex [35].…”

“…As opposed to early developing neuronal progenitors that are sensitive to UTP, no proliferation of late developing glial progenitors is observed by activation of UTP-sensitive nucleotide receptors in monolayer cultures or cultured explants of chick embryo retinas [20,24]. The proliferative response of glial cells in culture to ATP decreases, as the cultured cells differentiate, and no effect of this nucleotide is observed 4 days after the onset of cultures obtained from retinas of 7-day-old chick embryos or 1-day-old mice [21,23].…”

“…During development, evidence for the presence of P2Y 1 receptors in glial progenitors was obtained in chick embryo retinal cultures and early postnatal rodent retina [20][21][22][23]. Activation of these receptors by ATP or ADP induces the proliferation of glial progenitors by a mechanism involving PKC, MAPK, and PI3K/AKT pathways [23][24][25][26].…”