phorbol-12-13-didecanoate and Retinoblastoma

Termination of synaptic glutamate transmission depends on rapid removal of glutamate by neuronal and glial high-affinity transporters. Molecular biological and pharmacological studies have demonstrated that at least five subtypes of Na+-dependent mammalian glutamate transporters exist. Our study demonstrates that Y-79 human retinoblastoma cells express a single Na+-dependent glutamate uptake system with a Km of 1.7 +/- 0.42 microM that is inhibited by dihydrokainate and DL-threo-beta-hydroxyaspartate (IC50 = 0.29 +/- 0.17 microM and 2.0 +/- 0.43 microM, respectively). The protein kinase C activator phorbol 12-myristate 13-acetate caused a concentration-dependent inhibition of glutamate uptake (IC50 = 0.56 +/- 0.05 nM), but did not affect Na+-dependent glycine uptake significantly. This inhibition of glutamate uptake resulted from a fivefold decrease in the transporter's affinity for glutamate, without significantly altering the Vmax. 4Alpha-phorbol 12,13-didecanoate, a phorbol ester that does not activate protein kinase C, did not alter glutamate uptake significantly. The phorbol 12-myristate 13-acetate-induced inhibition of glutamate uptake was reversed by preincubation with staurosporine. The biophysical and pharmacological profile of the human glutamate transporter expressed by the Y-79 cell line indicates that it belongs to the dihydrokainate-sensitive EAAT2/GLT-1 subtype. This conclusion was confirmed by western blot analysis. Protein kinase C modulation of glutamate transporter activity may represent a mechanism to modulate extracellular glutamate and shape postsynaptic responses.

Topics: Amino Acid Transport System X-AG; Anti-Bacterial Agents; Aspartic Acid; ATP-Binding Cassette Transporters; Biological Transport; Carcinogens; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glutamates; Glutamic Acid; Humans; Kainic Acid; Kinetics; Phorbol Esters; Protein Kinase C; Retinoblastoma; Staurosporine; Tumor Cells, Cultured