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Vitamin B1 (thiamine) uptake by human retinal pigment epithelial (ARPE-19) cells: mechanism and regulation. J Physiol 2007 Jul 01;582(Pt 1):73-85 PMID: 17463047 PMCID: PMC2075275

Abstract

Retinal abnormality and visual disturbances occur in thiamine-responsive megaloblastic anaemia (TRMA), an autosomal recessive disorder caused by mutations in the human thiamine transporter-1 (hTHTR-1). Human retinal pigment epithelial cells play a pivotal role in supplying thiamine to the highly metabolically active retina but nothing is known about the mechanism, regulation or biological processes involved in thiamine transport in these cells. To address these issues, we used human-derived retinal pigment epithelial ARPE-19 cells to characterize the thiamine uptake process. Thiamine uptake is energy- and temperature-dependent, pH-sensitive, Na+-independent, saturable at both the nanomolar (apparent Km, 30 +/- 5 nM) and the micromolar (apparent Km, 1.72 +/- 0.3 microM) concentration ranges, specific for thiamine and sensitive to sulfhydryl group inhibition. The diuretic amiloride caused a concentration-dependent inhibition in thiamine uptake, whereas the anti-trypanosomal drug, melarsoprol, failed to affect the uptake process. Both hTHTR-1 and hTHTR-2 are expressed in ARPE-19 cells as well as in native human retinal tissue with expression of the former being significantly higher than that of the latter. Uptake of thiamine was adaptively regulated by extracellular substrate level via transcriptionally mediated mechanisms that involve both hTHTR-1 and hTHTR-2; it was also regulated by an intracellular Ca2+-calmodulin-mediated pathway. Confocal imaging of living ARPE-19 cells expressing TRMA-associated hTHTR-1 mutants (D93H, S143F and G172D) showed various expression phenotypes. These results demonstrate for the first time the existence of a specialized and regulated uptake process for thiamine in a cellular model of human retinal pigment epithelia that involves hTHTR-1 and hTHTR-2. Further, clinically relevant mutations in hTHTR-1 lead to impaired cell surface expression or function of the transporter in retinal epithelial ARPE-19 cells.

Author List

Subramanian VS, Mohammed ZM, Molina A, Marchant JS, Vaziri ND, Said HM

Author

Jonathan S. Marchant PhD Chair, Professor in the Cell Biology, Neurobiology and Anatomy department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

4-Chloromercuribenzenesulfonate
Amiloride
Anemia, Megaloblastic
Biological Transport, Active
Calcium
Calmodulin
Cell Line
Dose-Response Relationship, Drug
Epithelial Cells
Humans
Hydrogen-Ion Concentration
Membrane Transport Proteins
Mutation
Pigment Epithelium of Eye
Protein Transport
RNA, Messenger
Signal Transduction
Sodium
Sulfhydryl Reagents
Temperature
Thiamine
Time Factors
Transcription, Genetic
Transfection



View this publication's entry at the Pubmed website PMID: 17463047
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