title: Glutamate acts at NMDA receptors on fresh bovine and on cultured human retinal pigment epithelial cells to trigger release of ATP.
abstract: The photoreceptors lie between the inner retina and the retinal pigment epithelium (RPE). The release of glutamate by the phototoreceptors can signal changes in light levels to inner retinal neurons, but the role of glutamate in communicating with the RPE is unknown. Since RPE cells are known to release ATP, we asked whether glutamate could trigger ATP release from RPE cells and whether this altered cell signalling. Stimulation of the apical face of fresh bovine RPE eyecups with 100 mum NMDA increased ATP levels more than threefold, indicating that both receptors for NMDA and release of ATP occurred across the apical membrane of fresh RPE cells. NMDA increased ATP levels bathing cultured human ARPE-19 cells more than twofold, with NMDA receptor inhibitors MK-801 and d-AP5 preventing this release. Blocking the glycine site of the NMDA receptor with 5,7-dichlorokynurenic acid prevented ATP release from ARPE-19 cells. Release was also blocked by channel blocker NPPB and Ca(2+) chelator BAPTA, but not by cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide or vesicular release inhibitor brefeldin A. Glutamate produced a dose-dependent release of ATP from ARPE-19 cells that was substantially inhibited by MK-801. NMDA triggered a rise in cell Ca(2+) that was blocked by MK-801, by the ATPase apyrase, by the P2Y(1) receptor antagonist MRS2179 and by depletion of intracellular Ca(2+) stores with thapsigargin. These results suggest that glutamate stimulates NMDA receptors on the apical membrane of RPE cells to release ATP. This secondary release can amplify the glutaminergic signal by increasing Ca(2+) inside RPE cells, and might activate Ca(2+)-dependent conductances. The interplay between glutaminergic and purinergic systems may thus be important for light-dependent interactions between photoreceptors and the RPE.
authors.affiliation: Department of Physiology, University of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.
authors.forename: David
authors.initials: D
authors.lastname: Reigada
authors.forename: Wennan
authors.initials: W
authors.lastname: Lu
authors.forename: Claire H
authors.initials: CH
authors.lastname: Mitchell
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000032:analysis / Q000187:drug effects* / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology*; D006801:Humans; D016202:N-Methylaspartate / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000187:drug effects* / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000032:analysis / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014795:Visual Pathways / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; C485668:GRIN1 protein, human; D009419:Nerve Tissue Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate; D000255:Adenosine Triphosphate; D002118:Calcium