Adenine Nucleotides Control Proliferation In Vivo of Rat Retinal Progenitors by P2Y1 Receptor

Abstract

Previous studies demonstrated that exogenous ATP is able to regulate proliferation of retinal progenitor cells (RPCs) in vitro possibly via P2Y₁ 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 P2Y₁ 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 P2Y₁ receptors, reduced cell proliferation by approximately 20 % in P2 rats. Moreover, treatment with MRS 2179 caused an increase in p57^KIP2 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 P2Y₁ receptors regulating transition from G1 to S phase of the cell cycle.

Keywords: Development; P2Y1 receptor; Proliferation; Purines; Retina.