Viral Kansara

Activity of a sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and mechanism of ascorbate uptake

The objective of this research was to functionally characterize sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and to study the effect of substituted benzene derivatives on the intracellular accumulation of ascorbic acid (AA). Mechanism of AA uptake and transport was delineated. Uptake of [(14)C]ascorbic acid ([(14)C]AA) was studied in the absence and presence of excess unlabelled AA, anion transporter inhibitors, and a series of mono- and di-substituted benzenes. Transepithelial transport of [(14)C]AA across polarized cell membrane has been studied for the first time. Role of cellular protein kinase-mediated pathways on the regulation of AA uptake has been investigated. The cellular localizations of SVCTs were observed using confocal microscopy. Uptake of AA was found to be saturable with a K(m) of 83.2muM and V(max) of 94.2pmol/min/mg protein for SVCT1. The process was pH, sodium, temperature, and energy-dependent. It was under the regulation of cellular protein kinase C (PKC) and Ca(2+)/CaM mediated pathways. [(14)C]AA uptake was significantly inhibited in the presence of excess unlabelled AA and a series of electron-withdrawing group, i.e., halogen- and nitro-substituted benzene derivatives. AA appears to translocate across polarized cell membrane from apical to basal side (A-B) as well as basal to apical side (B-A) at a similar permeability. It appears that SVCT1 was mainly expressed on the apical side and SVCT2 may be located on both apical and basal sides. In conclusion, SVCT has been functionally characterized in MDCK-MDR1 cells. The interference of a series of electrophile-substituted benzenes on the AA uptake process may be explained by their structural similarity. SVCT may be targeted to facilitate the delivery of drugs with low bioavailability by conjugating with AA and its structural analogs. MDCK-MDR1 cell line may be utilized as an in vitro model to study the permeability of AA conjugated prodrugs.

Folic acid transport via high affinity carrier-mediated system in human retinoblastoma cells.

The primary objective of this study was to investigate the expression of a specialized carrier-mediated system for folic acid and to delineate its uptake mechanism and intracellular trafficking in a human derived retinoblastoma cell line (Y-79). Uptake of [3H]Folic acid was determined at various concentrations, pH, temperatures, in the absence of sodium and chloride ions and in the presence of structural analogs, methyltetrahydro folate (MTF) and methotrexate (MTX), vitamins, membrane transport and metabolic inhibitors to delineate the mechanism of uptake. Kinetics of uptake was studied in the presence of various intracellular regulatory pathways; protein kinases A and C (PKA and PKC), protein tyrosine kinase (PTK) and calcium-calmodulin modulators. Reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm the molecular identity of folate carrier systems. The uptake was found to be linear up to 30min. The rate of uptake followed saturation kinetics with apparent Km of 8.29+/-0.74nM, 17.03+/-1.98nM and 563.23+/-115.2nM and Vmax of 393.47+/-9.33, 757.58+/-26.21 and 653.17+/-31.7fmol/(minmg) protein for folic acid, MTF and MTX, respectively. The process was chloride, temperature and energy dependent but sodium and pH independent; inhibited by the structural analogs MTF and MTX but not by structurally unrelated vitamins. Membrane transport inhibitors did not affect the uptake of [3H]Folic acid, however endocytic inhibitor, colchicine, significantly inhibited the [3H]Folic acid uptake indicating the involvement of receptor mediated endocytosis process. PKC, PTK and Ca2+/calmodulin pathways appeared to play important roles in the regulation of folic acid uptake. Molecular evidence of the presence of folate receptor (FR) precursor was identified by RT-PCR analysis. This research work demonstrated, for the first time, the functional and molecular existence of a specialized high affinity carrier-mediated system for folic acid uptake, in human retinoblastoma cells.

Evaluation of an Ex Vivo Model Implication for Carrier-Mediated Retinal Drug Delivery

Purpose: The purpose of this study was to evaluate the implication of an ex vivo model for carrier-mediated retinal drug delivery using an Ussing chamber system. Methods: Dutch Belted Pigmented rabbits weighing 2–2.5 kg were used in these studies. Excised posterior segment tissues (RPE-choroid-sclera and sclera), mounted on the Ussing chamber, were used as an ex vivo model. Transport studies were carried out across sclera and RPE-choroid-sclera (RCS) tissue preparations in the sclera to retina (S → R) and retina to sclera (R → S) directions for 3 hr at 37°C. The model was validated by permeability studies with paracellular and transcellular markers ([3H]mannitol and [14C]diazepam, respectively), tissue viability studies (bioelectrical and biochemical assays), and tissue histology and electron microscopy studies. Functional presence of a carrier-mediated transport system for folic acid (folate receptor alpha) was investigated on the basolateral side of the rabbit retina. Results: Results from bioelectrical, biochemical, and histological evaluation of tissue provide evidence that the RCS tissue preparation remains viable during the period of transport study. Permeability values of [3H]mannitol across sclera were 4.18 ± 1.09 × 10− 5 cm/s (R → S) and 4.11 ± 1.09 × 10− 5 cm/s (S → R) and across RCS were 1.77 ± 0.31 × 10− 5 cm/s (S → R) and 1.60 ± 0.19 × 10− 5 cm/s (R → S). Permeability values of [14C]diazepam across sclera were 2.37 ± 0.38 × 10− 5 cm/s (R → S) and 2.70 ± 0.70 × 10− 5 cm/s (S → R) and across RCS were 3.12 ± 0.12 × 10− 5 cm/s (R → S) and 2.77 ± 0.25 × 10− 5cm/s (S → R). The rate of [3H]folic acid transport across RCS was found to be significantly higher in the S →R direction (16.34 ± 0.94 fmoles min−1 cm−2) as compared with R → S direction (9.38 ± 1.44 fmoles min−1 cm−2) and nearly 10-fold higher across sclera as compared with RCS in both directions. Transport of [3H]folic acid was found to be pH and temperature dependent and was inhibited by 44.5%, 35.1%, and 50.3% in the presence of unlabeled folic acid, 5-methyltetrahydrofolate (MTF), and Methotrexate (MTX). Conclusions: The RCS tissue preparation mounted on the Ussing chamber system, an ex vivo model, can be a useful tool for identification and characterization of carrier-mediated systems present on RPE (a major barrier for retinal drug delivery) and to study carrier-mediated retinal drug delivery via prodrug derivatization.