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

Abstract

When retinal cell cultures were mechanically scratched, cell growth over the empty area was observed. Only dividing and migrating, 2 M6-positive glial cells were detected. Incubation of cultures with apyrase (APY), suramin, or Reactive Blue 2 (RB-2), but not MRS 2179, significantly attenuated the growth of glial cells, suggesting that nucleotide receptors other than P2Y 1 are involved in the growth of glial cells. UTPγS but not ADPβS antagonized apyrase-induced growth inhibition in scratched cultures, suggesting the participation of UTP-sensitive receptors. No decrease in proliferating cell nuclear antigen (PCNA + ) cells was observed at the border of the scratch in apyrase-treated cultures, suggesting that glial proliferation was not affected. In apyrase-treated cultures, glial cytoplasm protrusions were smaller and unstable. Actin filaments were less organized and alfa-tubulinlabeled microtubules were mainly parallel to scratch. In contrast to control cultures, very few vinculin-labeled adhesion sites could be noticed in these cultures. Increased Akt and ERK phosphorylation was observed in UTP-treated cultures, effect that was inhibited by SRC inhibitor 1 and PI3K blocker LY294002. These inhibitors and the FAK inhibitor PF573228 also decreased glial growth over the scratch, suggesting participation of SRC, PI3K, and FAK in UTP-induced growth of glial cells in scratched cultures. RB-2 decreased dissociated glial cell attachment to fibronectin-coated dishes and migration through transwell membranes, suggesting that nucleotides regulated adhesion and migration of glial cells. In conclusion, mechanical scratch of retinal cell cultures induces growth of glial cells over the empty area through a mechanism that is dependent on activation of UTP-sensitive receptors, SRC, PI3K, and FAK.

The Healthy and Diseased Retina Seen through Neuron–Glia Interactions

“…ATP may contribute to the formation of glial scars by regulating both the proliferation and migration of Müller cells [300]. Accordingly, the activation of UTP-sensitive P2Y2/4 receptors induces the growth of glial cells through a mechanism involving the PI3K, SRC, and FAK signaling pathways in mechanically scratched retinal cultures [301]. When purified retinal glial cultures are used, both cell adhesion and migration are decreased by P2 receptor antagonists [301].…”

The Healthy and Diseased Retina Seen through Neuron-Glia Interactions

“…ATP may contribute to the formation of glial scars by regulating both proliferation and migration of Müller cells [303]. Accordingly, activation of UTP-sensitive P2Y2/4 receptors induces the growth of glial cells through a mechanism involving PI3K, SRC and FAK signaling pathways in mechanically scratched retinal cultures [304] . When purified retina glial cultures are used, both cell adhesion and migration are decreased by P2 receptor antagonists [304].…”

The Healthy and Diseased Retina Seen through Neuron-Glia Interactions

“…ATP may contribute to the formation of glial scars by regulating both proliferation and migration of Müller cells [304]. Accordingly, activation of UTP-sensitive P2Y2/4 receptors induces the growth of glial cells through a mechanism involving PI3K, SRC and FAK signaling pathways in mechanically scratched retinal cultures [305] . When purified retina glial cultures are used, both cell adhesion and migration are decreased by P2 receptor antagonists [305].…”