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Published on: February 15, 2017
ATP-induced non-neuronal cell permeabilization in the rat inner retina
Barbara Innocenti1, Sylke Pfeiffer, Eberhart Zrenner
1Laboratory of Cell Physiology and Molecular Biology, Department of Experimental Ophthalmology, University Eye Hospital, D-72076 Tuebingen, Germany. barbara.innocenti@uth.tmc.edu
Abstract:
The P2X7 subtype holds a special position among P2X receptors because of its ability to act both as a classical, ligand-gated ion channel, and as a permeabilization pore that can induce cell death under prolonged activation by ATP. We have shown previously that, in rat retina, P2X7 receptors are located in the inner nuclear layer and ganglion cell layer (GCL). The present study was aimed at finding whether retinal P2X7 receptors can act as a mediator of cell permeabilization and, if so, at identifying the cellular target(s) of this effect. As an indicator of cell permeabilization, we used the fluorescent dye YO-PRO-1 (molecular weight, 375 Da), which enters cells only through large pores like those opened by prolonged or sustained stimulation of P2X(7) receptors and binds to DNA, providing a stable labeling of the activated cells. Different agonists for P2 receptors were tested for their ability to cause cell permeabilization in flat-mounted rat retinas. Among them, only high concentrations of ATP (500 microM) and BzATP (2',3'-O-(4-benzoyl-benzoyl)-ATP triethylammonium) (100 microM) were able to induce accumulation of YO-PRO-1 in the GCL and in the nerve fiber layer, suggesting that different cell types were responding to P2X7 stimulation. This effect was blocked by the P2 antagonists suramin and PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid) and by the P2X7-selective inhibitor Brilliant Blue G. To identify the retinal cell types affected by ATP-induced permeabilization, we used in vivo labeling techniques. Our data clearly reveal that prolonged stimulation of P2X7 receptors elicits permeabilization exclusively in microglial cells but not in neurons of the inner retina.
Insights
The P2X7 receptor in rat retinas can open pores and allow substances like YO-PRO-1 dye to enter cells. This study found that P2X7 receptor activation specifically permeabilizes microglial cells, not retinal neurons.
Area of Science:
- Neuroscience
- Cell Biology
- Ophthalmology
Background:
- P2X7 receptors are unique ion channels involved in cell death.
- Retinal P2X7 receptors are found in the inner nuclear and ganglion cell layers.
- The function of P2X7 receptors in mediating cell permeabilization in the retina is not fully understood.
Purpose of the Study:
- To investigate if retinal P2X7 receptors mediate cell permeabilization.
- To identify the specific retinal cell types targeted by P2X7 receptor-induced permeabilization.
Main Methods:
- Utilized the fluorescent dye YO-PRO-1 to indicate cell permeabilization.
- Administered various P2 receptor agonists to flat-mounted rat retinas.
- Employed P2 receptor antagonists and a P2X7-selective inhibitor to block effects.
- Used in vivo labeling techniques to identify affected cell types.
Main Results:
- High concentrations of ATP and BzATP induced YO-PRO-1 uptake in the ganglion cell layer and nerve fiber layer.
- This P2X7 receptor-mediated permeabilization was blocked by suramin, PPADS, and Brilliant Blue G.
- Prolonged P2X7 receptor stimulation resulted in permeabilization exclusively in microglial cells.
Conclusions:
- Retinal P2X7 receptors can mediate cell permeabilization.
- Microglial cells are the primary cellular targets of P2X7 receptor-induced permeabilization in the rat retina.
- This finding highlights a specific role for P2X7 receptors in retinal microglial function.
