Gamma-aminobutyric acid transporter (BGT-1) expressed in human astrocytoma U373 MG cells: pharmacological and molecular characterization and phorbol ester-induced inhibition.
The properties of a transport system specific for gamma-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 beta-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.
Ruiz-Tachiquín
M-E
ME
Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México D.F., México.
Sánchez-Lemus
E
E
Soria-Jasso
L-E
LE
Arias-Montaño
J-A
JA
Ortega
A
A
D001254:Astrocytoma / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001622:Betaine / Q000378:metabolism*; D002273:Carcinogens / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D050485:GABA Plasma Membrane Transport Proteins; D006801:Humans; D008969:Molecular Sequence Data; D010703:Phorbol Esters / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured; D005680:gamma-Aminobutyric Acid / Q000378:metabolism / Q000494:pharmacology
D002273:Carcinogens; D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D010703:Phorbol Esters; C072207:betaine plasma membrane transport proteins; D001622:Betaine; D005680:gamma-Aminobutyric Acid; D011493:Protein Kinase C