Previous studies demonstrated that exogenous ATP is able to regulate proliferation of retinal progenitor cells (RPCs) in vitro possibly via P2Y receptor, a G protein-coupled receptor. Here, we evaluated the function of adenine nucleotides in vivo during retinal development of newborn rats. Intravitreal injection of apyrase, an enzyme that hydrolyzes nucleotides, reduced cell proliferation in retinas at postnatal day 2 (P2). This decrease was reversed when retinas were treated together with ATPγ-S or ADPβ-S, two hydrolysis-resistant analogs of ATP and ADP, respectively. During early postnatal days (P0 to P5), an increase in ectonucleotidase (E-NTPDase) activity was observed in the retina, suggesting a decrease in the availability of adenine nucleotides, coinciding with the end of proliferation. Interestingly, intravitreal injection of the E-NTPDase inhibitor ARL67156 increased proliferation by around 60 % at P5 rats. Furthermore, immunolabeling against P2Y receptor was observed overall in retina layers from P2 rats, including proliferating Ki-67-positive cells in the neuroblastic layer (NBL), suggesting that this receptor could be responsible for the action of adenine nucleotides upon proliferation of RPCs. Accordingly, intravitreal injection of MRS2179, a selective antagonist of P2Y receptors, reduced cell proliferation by approximately 20 % in P2 rats. Moreover, treatment with MRS 2179 caused an increase in p57 and cyclin D1 expression, a reduction in cyclin E and Rb phosphorylated expression and in BrdU-positive cell number. These data suggest that the adenine nucleotides modulate the proliferation of rat RPCs via activation of P2Y receptors regulating transition from G1 to S phase of the cell cycle.
Adenine nucleotides through P2Y receptor stimulation are known to control retinal progenitor cell (RPC) proliferation by modulating expression of the p57, a cell cycle regulator. However, the role of Gi protein-coupled P2Y and P2Y receptors also activated by adenine nucleotides in RPC proliferation is still unknown. Gene expression of the purinergic P2Y subtype was detected in rat retina during early postnatal days (P0 to P5), while expression levels of P2Y were low. Immunohistochemistry assays performed with rat retina on P3 revealed P2Y receptor expression in both Ki-67-positive cells in the neuroblastic layer and Ki-67-negative cells in the ganglion cell layer and inner nuclear layer. Nonetheless, P2Y receptor expression could not be detected in any stratum of rat retina. Intravitreal injection of PSB 0739 or clopidogrel, both selective P2Y receptor antagonists, increased by 20 and 15%, respectively, the number of Ki-67-positive cells following 24 h of exposure. Moreover, the P2Y receptor inhibition increased cyclin D1 and decreased p57 expression. However, there were no changes in the S phase of the cell cycle (BrdU-positive cells) or in mitosis (phospho-histone-H3-positive cells). Interestingly, an increase in the number of cyclin D1/TUNEL-positive cells after treatment with PSB 0739 was observed. These data suggest that activation of P2Y receptors is required for the successful exit of RPCs from cell cycle in the postnatal rat retina.
Literature data demonstrate already that the presence of adenine nucleotides in the extracellular environment induces cell death that leads to several retinopathies. As said, the objective is to carry out a systematized review of the last decade, relating purinergic signaling to the outcome of cell death and retinopathies. It is possible to identify different mechanisms that occur through the activation of purinergic receptors. The exacerbated activation of the P2X7 receptor is mainly involved in the apoptotic death pathway, and this response is due to the dysregulation of some components in the intracellular environment, such as the Ca2+ ion, CD40, MiR-187, and influence of mononuclear macrophages. The A2A receptor is involved in increasing levels of cytokines and promoting inflammatory processes. The data presented can be used as a basis to better understand the mechanisms of death in retinopathies, in addition to proposing therapeutic strategies with the potential to be transposed to several other models.
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