Some Operational Characteristics of Glycine Release in Rat Retina: The Role of Reverse Mode Operation of Glycine Transporter Type-1 (GlyT-1) in Ischemic Conditions

摘要

Rat posterior eyecups containing the retina were prepared, loaded with H-3glycine and superfused in order to determine its release originated from glycinergic amacrine cells and/or glial cells. Deprivation of oxygen and glucose from the Krebs-bicarbonate buffer used for superfusion evoked a marked increase of H-3glycine release, an effect that was found to be external Ca2+-independent. Whereas oxygen and glucose deprivation increased H-3glycine release, its uptake was reduced suggesting that energy deficiency shifts glycine transporter type-1 operation from normal to reverse mode. The increased release of H-3glycine evoked by oxygen and glucose deprivation was suspended by addition of the non-competitive glycine transporter type-1 inhibitor NFPS and the competitive inhibitor ACPPB further suggesting the involvement of this transporter in the mediation of H-3glycine release. Oxygen and glucose deprivation also evoked H-3glutamate release from rat retina and the concomitantly occurring release of the NMDA receptor agonist glutamate and the coagonist glycine makes NMDA receptor pathological overstimulation possible in hypoxic conditions. H-3Glutamate release was suspended by addition of the excitatory amino acid transporter inhibitor TBOA. Sarcosine, a substrate inhibitor of glycine transporter type-1, also increased H-3glycine release probably by heteroexchange shifting transporter operation into reverse mode. This effect of sarcosine was also external Ca2+-independent and could be suspended by NFPS. Energy deficiency in retina induced by ouabain, an inhibitor of the Na+-K+-dependent ATPase, and by rotenone, a mitochondrial complex I inhibitor added with the glycolytic inhibitor 2-deoxy-d-glucose, led to increase of retinal H-3glycine efflux. These effects of ouabain and rotenone/2-deoxy-d-glucose could also be blocked by NFPS pointed to the preferential reverse mode operation of glycine transporter type-1 as a consequence of impaired cellular energy homeostasis. Immunohistochemical studies revealed that glycine transporter type-1, of which reverse mode operation assures H-3glycine release, is expressed in amacrine cells in the inner nuclear and plexiform layers of the retina and also in Muller macroglia cells. We conclude that disruption of the balanced normal/reverse mode operation of glycine transporter type-1 is likely a significant factor contributing to neurotoxic processes of the retina. The possibility to inhibit glycine transporter type-1 mediated glycine efflux by drugs more potently than glycine uptake might offer some therapeutic potential for the treatment of various neurodegenerative disorders of the retina.