Cellular resistance to Evans blue toxicity involves an up-regulation of a phosphate transporter implicated in vesicular glutamate storage.
It has recently been suggested that the brain-specific Na+-dependent phosphate inorganic co-transporter (BNPI) is able to support glutamate transport and storage in synaptic vesicles. A procedure for measuring the vesicular pool of glutamate is described and was used to select cell lines according to their glutamate storage capacity. Two cell lines were selected: C6BU-1, with a large intracellular glutamate storage capacity, and NG108-15, devoid of it. Their contents in BNPI mRNA were compared by RT-PCR. We found that both cell lines had BNPI, but in addition C6BU-1 alone expresses the other isoform, DNPI. We also carried out a clonal selection of NG108-15 cells in the presence of the dye Evans blue, a competitive inhibitor of vesicular glutamate transport, very toxic for cells in culture. It was assumed that only those that sequester and eliminate the drug by overexpressing a vesicular glutamate transporter would survive. We found that the NG108-15 clones resistant to Evans blue had an increased storage capacity for glutamate. These cells also up-regulated the BNPI isoform of the phosphate transporter as shown by RT-PCR and northern blot.
Israël
M
M
Laboratoire de Neurobiologie Cellulaire et Moléculaire, CNRS, Gif-sur-Yvette, France.
Tomasi
M
M
Bostel
S
S
Meunier
F M
FM
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002531:Cerebellum / Q000166:cytology; D004396:Coloring Agents / Q000633:toxicity; D004351:Drug Resistance / Q000502:physiology; D005070:Evans Blue / Q000633:toxicity*; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D020033:Protein Isoforms; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D013574:Synaptosomes / Q000378:metabolism
D002352:Carrier Proteins; D004396:Coloring Agents; D020033:Protein Isoforms; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D018698:Glutamic Acid; D005070:Evans Blue