ATP-induced Cell Permeabilization in the Rat Inner Retina

Abstract

Abstract: : Purpose:The P2X₇ receptor belongs to a family of cation channels which are activated by ATP. Different from other P2 receptors, prolonged activation of P2X₇ can result in an additional opening of a membrane pore that is permeable to molecules up to 900 Da and has been shown to induce cell death in the immune system. We have previously reported that P2X₇ receptors are expressed in the rat retina and located in the inner nuclear and ganglion cell layer (GCL). The present study was aimed to investigate if the P2X₇ receptor can act as a mediator of cell permeabilization in the rat retina and to identify the putative cellular target(s) of its effect. Methods:Fluorescent imaging was performed on flat-mounted retinas isolated from Brown Norway rats. As an indicator of cell permeabilization we used the fluorescent dye YO-PRO-1 (MW 375Da). The dye enters into the cells through large pores such like those opened by prolonged or sustained stimulations of the P2X₇ subunit. After entering a cell, YO-PRO-1 binds to DNA, thus providing a stable labeling of the activated cells. Results:Different agonists for P2 receptors (ATP, BzATP, ATPγS, ADP and UTP) were tested for their ability to cause cell permeabilization in the inner rat retina. Among them, only high concentrations of ATP (500µM) and BzATP (100µM) were able to induce accumulation of YO-PRO-1 in cells of the GCL. This effect was blocked by the P2-antagonists suramin and PPADS (100µM). The two set of experiments together supported the hypothesis that cell permeabilization in the retina can be induced by activation of the P2X₇ subunit. In order to identifying the retinal cell types affected by ATP-induced permeabilization, we used ‘in vivo' labeling techniques. Ganglion cells were retrogradely labeled by red fluorescent beads injected in P1-animal superior colliculus or by application of the rhodamine-biotin-conjugated dextran crystals to the nerve stump of dissected eye balls. Microglia was labeled with Cy3-conjugated ILB-4, Müller cells by uptake of the dye red Mitotracker, and other glial cells by calcein-AM. With these tools we were able to identify microglial cells as targets of P2X₇–induced cell permeabilization. Conclusions:Despite the strong expression of P2X₇ subunit by different cell types in the inner rat retina, prolonged stimulation of this subunit elicits permeabilization only in microglial cells. The different activation properties in different retinal cell types suggests that P2X₇ is involved in multiple physiological and pathological states of the mammalian retina.

Keywords: receptors: pharmacology/physiology • neurotransmitters/neurotransmitter systems • microglia