Characterization of the dopamine transporter gene expression and binding sites in cultured human amniotic epithelial cells.
In this study we sought to investigate whether the dopamine transporter, DAT, and its binding sites are expressed in the human amniotic epithelial cells (HAEC) using reverse transcription-polymerase chain reaction (RT-PCR) and radioligand binding studies, respectively. The RT-PCR findings showed that HAEC expressed DAT mRNA with 100% homology to the human brain DAT. Saturation binding studies using [3H]mazindol showed a high affinity DAT binding site with K(D) and B(max) values of 12.32+/-1.67 nM and 82.7+/-9.74 fmol/mg protein, respectively. Competition experiments showed that selective DAT blockers are potent displacers of [3H]mazindol binding. The rank order of potency of the competing drugs is consistent with the pharmacology of the DAT. The present results provide compelling evidence that HAEC natively express the DAT mRNA and binding sites. More importantly, these results may suggest that HAEC is an appropriate human cell model for studying dopamine release and uptake processes and potential ligands at these sites.
Elwan
Mohamed A
MA
Department of Inherited Metabolic Diseases, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan. melwan@emory.edu
Ishii
Takashi
T
Sakuragawa
Norio
N
D000650:Amnion / Q000166:cytology / Q000378:metabolism*; D001665:Binding Sites; D002478:Cells, Cultured; D050483:Dopamine Plasma Membrane Transport Proteins; D004847:Epithelial Cells / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger / Q000378:metabolism; D011869:Radioligand Assay; D020133:Reverse Transcriptase Polymerase Chain Reaction
D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C493214:SLC6A3 protein, human