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γ‐aminobutyric acid transporter (BGT‐1) expressed in human astrocytoma U373 MG cells: Pharmacological and molecular characterization and phorbol ester‐induced inhibition
Author(s) -
RuizTachiquín M.E.,
SánchezLemus E.,
SoriaJasso L.E.,
AriasMontaño J.A.,
Ortega A.
Publication year - 2002
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.10258
Subject(s) - phorbol ester , phorbol , astrocytoma , transporter , chemistry , pharmacology , biochemistry , microbiology and biotechnology , cancer research , biology , glioma , gene , signal transduction , protein kinase c
The properties of a transport system specific for γ‐aminobutyric acid (GABA) expressed in human U373 MG astrocytoma cells were examined. The uptake of [ 3 H]GABA was dependent on both extracellular Na + and Cl – ions and was inhibited by (±)‐nipecotic acid, guvacine, and β‐alanine, with a pharmacological profile corresponding to that reported for the human homologue of the GABA/betaine transporter (BGT‐1). Accordingly, [ 3 H]GABA uptake was also inhibited by betaine, and reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis of total RNA from U373 MG cells with specific BGT‐1 primers resulted in the amplification of a 440 bp fragment that was further characterized by restriction analysis and sequencing. In addition, Western blot analysis with anti‐BGT‐1 antiserum revealed the presence of a characteristic 60 kDa band. The primary structure of the human BGT‐1 protein predicts two putative phosphorylation sites for the Ca 2+ /diacylglicerol‐dependent protein kinase (PKC), and treatment of U373 MG cells with the PKC activator phorbol 12‐myristate‐13‐acetate (TPA) led to a concentration‐ and time‐dependent decrease in [ 3 H]GABA uptake. The maximal effect was detected at 2 hr of incubation, to disappear after 4 hr. TPA‐induced reduction in [ 3 H]GABA uptake was reversed by preincubation with staurosporine. Taken together, these results indicate that U373 MG cells express a GABA transporter of the BGT‐1 subtype whose function is regulated by phosphorylation events through PKC. © 2002 Wiley‐Liss, Inc.