Modulation of human glutamate transporter activity by phorbol ester.

Abstract

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.

• Authors: R Ganel, C E Crosson
• Journal: Journal of neurochemistry (J Neurochem) Vol. 70 Issue 3 Pg. 993-1000 (Mar 1998) ISSN: 0022-3042 [Print] England
• PMID: 9489718 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• ATP-Binding Cassette Transporters
• Amino Acid Transport System X-AG
• Anti-Bacterial Agents
• Carcinogens
• Enzyme Inhibitors
• Glutamates
• Phorbol Esters
• 3-hydroxyaspartic acid
• phorbol-12,13-didecanoate
• Aspartic Acid
• Glutamic Acid
• 2-methylglutamic acid
• dihydrokainic acid
• Protein Kinase C
• Staurosporine
• Kainic Acid

Topics

• ATP-Binding Cassette Transporters (metabolism)
• Amino Acid Transport System X-AG
• Anti-Bacterial Agents (pharmacology)
• Aspartic Acid (analogs & derivatives, pharmacology)
• Biological Transport (drug effects)
• Carcinogens (pharmacology)
• Dose-Response Relationship, Drug
• Enzyme Inhibitors (pharmacology)
• Glutamates (pharmacology)
• Glutamic Acid (pharmacokinetics)
• Humans
• Kainic Acid (analogs & derivatives, pharmacology)
• Kinetics
• Phorbol Esters (pharmacology)
• Protein Kinase C (metabolism)
• Retinoblastoma
• Staurosporine (pharmacology)
• Tumor Cells, Cultured (drug effects, enzymology)