Cytoskeletal scaffolds regulate riboflavin endocytosis and recycling in placental trophoblasts.

Microfilaments and microtubules (MT) play a vital role in cellular endocytic processes. The present study evaluates the role of these cytoskeletal elements in the apical internalization and postendocytic fate of riboflavin (RF) in placental trophoblasts (BeWo cells). Biochemical modification of the actin and microtubule network by (1) okadaic acid (OA), which disrupts MT-based vesicular trafficking; (2) cytochalasin D and latrunculin B, which promote actin depolymerization; and (3) 2,3-butanedione monoxime (BDM), which inhibits myosin-actin interaction, was confirmed by immunofluorescence microscopy using actin- and tubulin-specific antibodies. Furthermore, involvement of the molecular motors dynein and kinesin was assessed in the presence of (1) sodium orthovanadate, which inhibits dynein-ATPase activity and (2) adenosine 5'-(beta,gamma-imido)triphosphate tetralithium salt hydrate, a non-hydrolyzable ATP analog, which results in defective kinesin-driven processes. RF internalization consequent to cytoskeletal alterations was compared with that of a clathrin-dependent endocytic marker ([125I]-transferrin [TF]), a caveolae-mediated endocytic substrate ([3H]-folic acid [FA]), and a fluid-phase endocytic marker ([125I]-horse radish peroxidase [HRP]). Apical recycling and bidirectional transport of RF and TF was measured following cytoskeletal alterations. Results indicate that uptake of RF, TF, FA and HRP are markedly reduced (approximately 30-65%) in the presence OA and BDM, suggesting differential sensitivities to modification of kinesin-driven microtubules. However, actin depolymerization negatively affected HRP endocytosis alone, while RF, FA and TF internalization remained unchanged. Disturbances in protein phosphorylation cascades also influenced apical recycling while net ligand transport across monolayers remained unaffected. In conclusion, apical RF trafficking in placental cells is tightly regulated by microtubules and supported by accessory actin involvement.

Extracellular glucose concentration alters functional activity of the intestinal oligopeptide transporter (PepT-1) in Caco-2 cells.

The objective of this study was to determine the effect of different cell culture media glucose concentrations on the functional activity of PepT-1 in Caco-2 cells. Uptake kinetics of Gly-Sar into Caco-2 cells that were maintained in iso-osmotic media containing 25 or 5.5 mM glucose were determined in the presence and absence of amino acid-selective chemical modifiers and dithiothreitol. Inhibition of Gly-Sar uptake into Caco-2 cells was measured in the presence of dipeptides and xenobiotics exhibiting various binding affinities for the PepT-1. The effect of extracellular glucose on PepT-1 gene expression was assessed using comparative RT-PCR. Long-term exposure of Caco-2 cells to 25 mM glucose reduced maximum transport capacity for Gly-Sar uptake without altering PepT-1 gene expression. In contrast, binding affinity of Gly-Sar and other dipeptides or xenobiotics was not significantly changed. Chemical modification of Lys and Tyr residues decreased V(max), while Cys modification increased the maximum transport capacity of the carrier. Preincubation of Caco-2 cells with dithiothreitol restored PepT-1 activity in cells maintained at 25 mM glucose. In conclusion, cell culture media containing 25 mM glucose decreases maximum transport capacity of PepT-1 in Caco-2 cells without affecting substrate recognition, at least in part, mediated via an oxidative pathway.

High glucose concentration in isotonic media alters caco-2 cell permeability.

Caco-2 cell permeability was evaluated in isotonic media containing high (25 mM) or physiological (5.5 mM) glucose concentrations. Transepithelial electrical resistance (TEER) and membrane fluidity were measured to assess glucose-induced alterations in physical barrier properties. In parallel, distribution of the actin filament (F-actin) and zonula occludens-1 (ZO-1) proteins was assessed by confocal microscopy. Transepithelial fluxes of mannitol, hydrocortisone, digoxin, and glycyl sarcosine (Gly-Sar) that permeate the intestinal mucosa by various pathways were measured to quantify the effect of glucose-induced changes on Caco-2 cell permeability. High glucose decreased maximum TEER of cell monolayers by 47%, whereas membrane fluidity at the hydrophobic core and lipid/polar head interphase was significantly increased. F-actin distribution in high glucose cells appeared more diffuse while ZO-1 was unchanged. Mannitol and hydrocortisone fluxes across Caco-2 cells cultured in high glucose increased by 65% and 24%, respectively. In addition, high glucose decreased the maximum transport capacity (Vmax) of PepT-1. P-glycoprotein activity, however, was unchanged. In conclusion, high extracellular glucose concentration in isotonic media significantly alters physical barrier properties of Caco-2 cell monolayers, which predominantly affects transepithelial transport of solutes permeating the cell barrier by paracellular and transcellular passive diffusion and facilitated transport mediated by the proton-dependent oligopeptide transporter (PepT-1).

cAMP-Coupled riboflavin trafficking in placental trophoblasts: a dynamic and ordered process.

Riboflavin (RF, vitamin B(2)), an essential micronutrient central to cellular metabolism through formation of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors, is internalized, at least in part, via a proposed receptor-mediated endocytic (RME) process. The purpose of this study was to delineate the cellular RF distribution using human placental trophoblasts and evaluate the regulatory role of cAMP in this process. Subcellular fractionation and three-dimensional confocal microscopy analyses were carried out to define the RF accumulation profile. Biochemical assays evaluating the cAMP dependence of this pathway were also performed. This study records an intracellular RF distribution pattern that shows dynamic accumulation of the ligand predominantly in the endosomal and lysosomal compartments and to a lesser extent in the Golgi and mitochondria. In contrast, transferrin (TF) colocalizes rapidly within endosomes with minimal accumulation in the other organelles. The temporal and spatial distribution of RF and TF colocalized with unique markers of the endocytic machinery provides added morphological evidence in support of the RME process with ultimate translocation to the mitochondrial domain. Colocalized staining with the Golgi also suggests a possible recycling or exocytic mechanism for this ligand. Furthermore, this study demonstrates cAMP regulation of the putative ligand-bound RF receptor and its association into endocytic vesicles. Delineating the dynamics of the process governing cellular RF homeostasis presents an untapped resource that can be further exploited in improving our current understanding of nutritional biology and fetal growth and development, and perhaps in targeting the endogenous system for developing novel therapeutic approaches.

Recognition, co-internalization, and recycling of an avian riboflavin carrier protein in human placental trophoblasts.

Absorption of riboflavin (RF) across membrane barriers is essential to cellular oxidation reduction processes. Riboflavin carrier protein (RCP), a 37-kDa secretory protein, is proposed to play an important role in RF absorption, although information on the mammalian ortholog remains unclear. This study alludes to the existence of a mammalian RF carrier protein and further characterizes its carrier role and fate using avian RCP in human placental trophoblast (BeWo), another mammalian cell line, monkey kidney (COS-1), and the avian control, chicken hepatic (LMH/2A) cells. The presence of RCP and its involvement in RF internalization was analyzed by immunofluorescence and immunobinding assays using chicken RCP (cRCP) antibodies. In the presence of anti-cRCP, cellular RF uptake is significantly decreased (5% of control) in BeWo cells. Kinetic analyses of intracellular accumulation of (125)I-cRCP revealed a J(max) and K(m) of 28.56 +/- 2.70 pmol/mg protein/min and 142.43 +/- 82.16 nM, respectively, in BeWo cells and 75.14 +/- 7.6 pmol/mg protein/min and 104.37 +/- 23.96 nM in the species-specific control, LMH/2A cells. Subcellular fractionation studies revealed colocalization of both radiolabeled RF and cRCP within endosomal and lysosomal fractions, further elucidating RCP's role in trafficking RF through the cell. Following intracellular release of RF from the carrier complex, the protein is either subject to lysosomal breakdown or is conserved via recycling mechanisms for continued RF sequestration and uptake. In summary, mammalian placental trophoblasts exhibit specific carrier protein dependence that sequesters and essentially mediates RF internalization via the proposed receptor-mediated endocytic pathway.

In vitro and pharmacophore-based discovery of novel hPEPT1 inhibitors.

PURPOSE: The human proton-coupled small peptide carrier (hPEPT1) is a low-affinity, high-capacity transporter with broad substrate specificity. We have taken an iterative in vitro and in silico approach to the discovery of molecules with hPEPT1 affinity. METHODS: A pharmacophore-based approach was taken to identifying hPEPT1 inhibitors. The well-characterized and relatively high affinity ligands Gly-Sar, bestatin, and enalapril were used to generate a common features (HIPHOP) pharmacophore. This consisted of two hydrophobic features, a hydrogen bond donor, acceptor, and a negative ionizable feature. RESULTS: The pharmacophore was used to search the Comprehensive Medicinal Chemistry (CMC) database of more than 8000 drug-like molecules and retrieved 145 virtual hits mapping to the pharmacophore features. The highest scoring compounds within this set were selected and tested in a stably transfected CHO-hPepT1 cell model. The antidiabetic repaglinide and HMG CoA reductase inhibitor fluvastatin were found to inhibit hPEPT1 with sub-millimolar potency (IC(50) 178 +/- 1.0 and 337 +/- 4 microM, respectively). The pharmacophore was also able to identify known hPEPT1 substrates and inhibitors in further database mining of more than 500 commonly prescribed drugs. CONCLUSIONS: This study demonstrates the potential of combining computational and in vitro approaches to determine the affinity of compounds for hPEPT1 and, in turn, provides insights into key molecular interactions with this transporter.

Functional characterization of the peptide transporter PEPT2 in primary cultures of human upper airway epithelium.

This study characterizes the expression and function of the peptide transporter hPepT2 (SLC15A2) in differentiated primary cultures of human upper airway lung epithelia obtained from six human donors. Genotype analysis of a SNP in exon 15 of hPepT2 genotypes in six donors revealed an expected distribution of the two main variants present at similar frequency (two AA homozygotes, two BB homozygotes, and two AB heterozygotes). Real-time PCR analysis of the hPepT2 mRNA message revealed no significant differences among genotypes. hPEPT2 was expressed on the apical membrane in all donor specimens, demonstrated by cell surface biotinylation and Western analysis (104 kD). We then compared transepithelial transport of the prototypical substrate (3)H-glycylsarcosine in all donor cultures in the absence and presence of known inhibitors of hPEPT2 to ascertain the phenotype of functionally expressed hPepT2 in the upper airway epithelium. An array of inhibitors included dipeptides, beta-lactam antibiotics, bestatin, and ACE inhibitors. hPEPT2 exhibited saturable Michaelis-Menten-type kinetic parameters for GlySar, corroborating previously reported values for K(T) and J(max). Donor-to-donor variation of transport for different substrates did not correlate with hPepT2 haplotypes in this sample cohort. These findings demonstrate functional hPEPT2 transporter expression in primary cultures of human lung epithelial cells. hPEPT2-mediated transport could serve as a strategy for noninvasive systemic delivery of peptides and peptidomimetics drugs.