Abstract
Purpose: :
The objective of this research was to investigate the role of human sodium dependant multivitamin transporter as a specialized carrier–mediated system for biotin and delineate uptake mechanism and intracellular trafficking of biotin in the human derived retinoblastoma cell line (Y–79).
Methods: :
Human derived retinoblastoma cell line, Y–79, was used for uptake studies. Uptake of [3H]Biotin was determined at various concentrations, pH, temperatures, in the absence of sodium and in the presence of other vitamins and metabolic inhibitors to delineate the mechanism of uptake. Uptake was determined in the presence of various intracellular regulatory pathways (protein kinase A & C, protein tyrosine kinase and calcium–calmodulin) modulators. Reverse transcription polymerase chain reaction (RT–PCR) was performed to confirm the molecular identity of human sodium dependent multivitamin transporter (hSMVT).
Results: :
Uptake of [3H]Biotin in Y–79 cells were found to be saturable at micromolar concentration range, with apparent Km of 8.53 µM and Vmax of 14.12 pmol/min/mg protein, but linear at nanomolar concentration range. Uptake was sodium, pH, temperature and energy dependent but chloride independent; inhibited by the structural analogue desthiobiotin, pantothenic acid and lipoic acid at milimolar concentrations and not at nanomolar concentrations. Uptake of [3H]riboflavin was trans–stimulated by the intracellular biotin. Ca2+/calmodulin pathways appeared to play important roles in the regulation of riboflavin uptake in Y–79 cells via significant reduction in Vmax (66%) and Km (28%) of the uptake process. A human sodium dependant multivitamin transporter, hSMVT, was identified by RT–PCR in Y–79.
Conclusions: :
These studies demonstrated for the first time the existence of a human sodium dependant multivitamin transporter (hSMVT), a specialized carrier–mediated system for biotin uptake, in human derived retinoblastoma cells which can be utilized for transporter targeted retinal drug delivery.
Keywords: retina • retinoblastoma • retinal culture