Abstract
Purpose: :
In glaucoma, the elevation of intraocular pressure (IOP) places a mechanical strain on cells of the posterior eye. While damage to and death of retinal ganglion cells (RGCs) are responsible for the loss of vision, the signaling cascades that link increased mechanical strain to RGC damage are not well understood. Previous work demonstrates that stimulation of the P2X7 receptor for ATP on RGCs can raise calcium and injure the neurons, and that elevated IOP can lead to an increase in extracellular ATP levels. This study explores the mechanically sensitive release of ATP from retinal cells and probes the identity of cell type and pathway responsible for this release.
Methods: :
Retinal cells were obtained from juvenile Long Evans rats following approved protocols. ATP release was examined from both a mixed population of dissociated retinal cells and from isolated immunopanned RGCs. Cells were swollen by adding 30 % water to the solution and ATP levels were monitored on-line using the luciferin-luciferase assay. To apply stretch, cells were grown on a silicone substrate in a specially designed chamber and pressure was elevated on one side to provide mild deformation of the membrane and the attached cells. A sample of extracellular solution was obtained 2 min later and ATP content assayed.
Results: :
Swelling mixed retinal cells led to a 12-fold increase in extracellular ATP that was inhibited by pannexin blockers carbenoxolone (10 µm; 43%) and probenicid (1 mM; 64%). Swelling induced a similar ATP release from isolated retinal ganglion cells that was also blocked by carbenoxelone and probenicid, suggesting ganglion cells themselves were responsible for at least some of the ATP release through pannexin channels. In both cases release was detected by the initial measurement within 30 sec. To examine effects of cell strain without swelling, cells attached to a silicone substrate were stretched by application of 20 mm Hg. Both mixed retinal cells and isolated ganglion cells released ATP upon stretch. Carbenoxolone blocked 62% of the release from mixed retinal cells and 91% from isolated ganglion cells.
Conclusions: :
Mechanical strain induces a release of ATP from retinal ganglion cells. The conduit for this ATP release is likely pannexin channels. While it remains to be determined whether this response to stretch contributes to the pathology seen in glaucoma, this study has identified one of the first responses of ganglion cells to mechanical stress.
Keywords: ganglion cells • intraocular pressure • adenosine