Abstract
Purpose: :
Glutamate-induced elevation in intracellular Ca2+ levels has been implicated in the loss of retinal ganglion cells (RGCs) in glaucoma. It is suggested that RGCs respond to excitotoxic injury by activating an extracellular proteolytic cascade involving the components of the plasmin-plasminogen system and culminating in the generation of plasmin. Plasmin, a potent protease cleaves the components of the extracellular matrix (ECM) like laminin resulting in detachment-induced apoptosis of RGCs. The key components of the extracellular proteolytic cascade in the retina are not known. Annexin A2 is a member of a family of Ca2+ dependent phospholipid binding proteins and its affinity to the plasma membrane is enhanced in response to elevated levels of intracellular Ca2+. On the cell surface annexin A2 serves as a proteolytic center by recruiting tissue plasminogen activator (tPA) and plasminogen and increasing the catalytic efficiency of plasmin generation. In this study we used the glutamate-induced Ca2+ influx model to study the cell surface translocation of annexin A2 and its contribution to extracellular proteolytic events in RGC-5 cells.Methods &
Results: :
Immunohistochemistry and immunocytochemistry were used to demonstrate the expression of annexin A2 in the ganglion cell layer of rat retina, primary and transformed RGCs. Glutamate-induced elevation in Ca2+ levels was demonstrated by ratiometric Ca2+ imaging and time lapse confocal microscopy. Western immunoblot and time lapse confocal microscopy were used to show that glutamate mobilizes endogenous and GFP-fused annexin A2 to the extracellular surface by elevating intracellular levels of Ca2+ in a process mediated by the NMDA receptor. By a fluorogenic in vitro plasmin generation assay, we demonstrated that annexin A2 forms a catalytically active plasmin generating complex on glutamate treatment and this activity is inhibited both by an antibody and a competitive peptide directed against the N-terminus of annexin A2. Furthermore site directed mutagenesis studies at the N-terminus of annexin A2 have shown that phosphorylation of Y23 at the N-terminus of annexin A2 by Src kinase is critical and mutation of Y23 to Y23F inhibits the translocation process.
Conclusions: :
These results suggest that glutamate-induced elevation of intracellular Ca2+ results in the translocation of an extracellular proteolytic receptor, annexin A2 leading to degradation of key components of the ECM.
Keywords: ganglion cells • calcium • proteolysis