γ-aminobutyric acid transporter (BGT-1) expressed in human astrocytoma U373 MG cells: Pharmacological and molecular characterization and phorbol ester-induced inhibition

• Published in: Journal of neuroscience research 2002-07, Vol.69 (1), p.125-132
• Main Authors: Ruiz-Tachiquín, M.-E., Sánchez-Lemus, E., Soria-Jasso, L.-E., Arias-Montaño, J.-A., Ortega, A.
• Format: Article
• Language: English
• Subjects: astrocytoma; Astrocytoma - genetics; Astrocytoma - metabolism; Base Sequence; Betaine - metabolism; Carcinogens - pharmacology; Carrier Proteins - antagonists & inhibitors; Carrier Proteins - biosynthesis; Carrier Proteins - genetics; Dose-Response Relationship, Drug; GABA; GABA transporters; GABA uptake; gamma-Aminobutyric Acid - metabolism; gamma-Aminobutyric Acid - pharmacology; glia; Humans; Molecular Sequence Data; Phorbol Esters - pharmacology; PKC; Protein Kinase C - metabolism; Sequence Homology, Nucleic Acid; Tumor Cells, Cultured; U373 MG cells
• ISSN: 0360-4012; 1097-4547
• DOI: 10.1002/jnr.10258

The properties of a transport system specific for γ‐aminobutyric acid (GABA) expressed in human U373 MG astrocytoma cells were examined. The uptake of [3H]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, [3H]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 Ca2+/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 [3H]GABA uptake. The maximal effect was detected at 2 hr of incubation, to disappear after 4 hr. TPA‐induced reduction in [3H]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.