betadex and oxophenylarsine

Ingested proteins are absorbed from the intestinal lumen via the paracellular and/or transcellular pathways, depending on their physicochemical properties. In this study, we investigated the absorption pathway(s) of ovalbumin (OVA), an egg white-allergen, as well as the mechanisms of aspirin-facilitated OVA absorption in rats. In situ intestinal re-circulating perfusion experiments showed that the absorption rate of fluorescein isothiocyanate (FITC)-labeled OVA in the distal intestine was higher than that for a marker of non-specific absorption, FITC-dextran (FD-40), and that colchicine, a general endocytosis inhibitor, suppressed OVA absorption. In the distal intestine, bafiromycin A1 and phenylarsine oxide inhibited the OVA absorption rate, whereas mehyl-β-cyclodextrin exerted no significant effects. Thus, OVA is preferentially absorbed from the distal intestine via the paracellular and receptor- and clathrin-mediated endocytic pathways. Furthermore, aspirin increased OVA absorption in the presence or absence of colchicine, indicating that aspirin facilitated OVA absorption by inducing intestinal barrier disruption and paracellular permeability.

Topics: Allergens; Animals; Arsenicals; Aspirin; beta-Cyclodextrins; Colchicine; Dextrans; Endocytosis; Fluorescein-5-isothiocyanate; Intestinal Absorption; Intestine, Small; Macrolides; Male; Ovalbumin; Rats, Sprague-Dawley; Serum Albumin, Bovine

In this paper, we characterize the uptake mechanism of fluorescein isothiocyanate-labeled human immunoglobulin G (FITC-hIgG) in opossum kidney (OK) epithelial cells, which have been shown to express megalin and cubilin. Confocal immunofluorescence microscopy showed the punctate expression of the neonatal Fc receptor FcRn in the cytoplasm, but not on the cell surface membrane. Temperature- and energy-dependent uptake of FITC-hIgG was observed at pH 7.4 but not at pH 6.0, indicating that the internalization of FITC-hIgG might not be due to FcRn, which has a binding affinity for IgG under acidic conditions. Under physiological pH conditions, human and bovine serum γ-globulin decreased FITC-hIgG uptake in a concentration-dependent manner. In addition, FITC-hIgG uptake was inhibited by various megalin and/or cubilin ligands including albumin, cytochrome c, transferrin and gentamicin. Endosomal acidification inhibitors (bafilomycin A(1) and chloroquine) significantly decreased the uptake of FITC-hIgG. Clathrin-dependent endocytosis inhibitors (phenylarsine oxide and chlorpromazine) decreased FITC-hIgG uptake. Potassium depletion and hypertonicity, conditions known to inhibit clathrin-dependent endocytosis, also decreased FITC-hIgG uptake. In contrast, caveolin-dependent endocytosis inhibitors (nystatin and methyl-β-cyclodextrin) did not decrease, but rather increased the uptake of FITC-hIgG. These observations suggest that the internalization of FITC-hIgG in OK cells might be, at least in part, due to megalin/cubilin-mediated, clathrin-dependent endocytosis.

Topics: Animals; Arsenicals; beta-Cyclodextrins; Cattle; Cell Line; Chlorpromazine; Clathrin; Endocytosis; Epithelial Cells; Fluorescein-5-isothiocyanate; gamma-Globulins; Histocompatibility Antigens Class I; Humans; Hydrogen-Ion Concentration; Immunoglobulin G; Kidney; Ligands; Low Density Lipoprotein Receptor-Related Protein-2; Microscopy, Confocal; Opossums; Receptors, Cell Surface; Receptors, Fc

Methylene blue (MB), a common cell stain, has been shown to inhibit nitric oxide synthase and guanylate cyclase, which has led to the recent use of MB in nitric oxide signaling studies. This study documents the effects of MB on 2-deoxyglucose (2DG) uptake in L929 fibroblast cells where uptake is controlled by a single glucose transporter, GLUT 1. MB significantly activates cytochalasin B-inhibitable glucose transport in a dose dependent fashion within 10 min. A maximal stimulation of up to 800% was achieved by 50 microM MB after a 45-min exposure. The Vmax of transport increased without a change in the Km, which was accomplished without a significant change in the GLUT 1 content. The reduced form of MB, did not stimulate 2DG uptake and potassium ferricyanide, an extracellular redox agent, prevented both the staining and stimulatory effects of MB suggesting MB is reduced at the cell surface before it enters L929 cells. Phenylarsine oxide did not block cell staining as noted in other cells lines, but it did inhibit both basal and MB-stimulated 2DG uptake. Likewise, methyl-beta-cyclodextrin, an agent used to remove membrane cholesterol, blocked both the staining and stimulatory effects of MB. The AMP analog, AICAR, inhibited rather than activated basal 2DG uptake, and it did not alter MB-stimulated uptake suggesting that AMP kinase activation is not critical to the MB effect. Wortmannin, an inhibitor of PI kinase, had no effect on MB-stimulated 2DG uptake. These data provide additional insight into the acute regulation of GLUT 1 transport activity in L929 cells.

Topics: Adenosine Triphosphate; Androstadienes; Animals; Arsenicals; beta-Cyclodextrins; Cytochalasin B; Deoxyglucose; Dose-Response Relationship, Drug; Ferricyanides; Fibroblasts; Glucose Transporter Type 1; Methylene Blue; Mice; Wortmannin

In order to investigate activation and internalization of c-kit we created a functional c-kit-EGFP chimera by inserting EYFP (enhanced yellow fluorescent protein) within the extracellular domain of c-kit immediately downstream of the signal sequence, SS-EYFP-kit. This location was chosen because the C-terminal fusion of EGFP to c-kit unexpectedly caused constitutive activation of the c-kit tyrosine kinase. As analysed in fixed cells and by real time imaging in vivo, SCF induced activation led to internalization of the fusion construct and translocation to punctate structures resembling vesicles. Analysis of the internalization process by time lapse imaging revealed high mobility and discontinuous movement of these vesicles and their predominantly radial tracks. Two subsets of vesicles were observed: Traffic of the majority of vesicles was directed from the periphery to the center of the cell and most likely represents the internalization of activated receptor molecules via the endosomal pathway. However, some vesicular structures were observed to move towards the periphery of the cell and probably contain newly synthesized protein to replace internalized receptor molecules. The calculated velocity of moving vesicles ranged from 0.05 to 0.2 microm per se. Vesicle formation upon SCF induced dimerization of the receptor was strictly dependent on kinase activity of c-kit. Treatment of cells with phenylarsine oxide, an agent blocking receptor internalization, prior to SCF stimulation resulted in abrogation of the translocation of the chimera to vesicles whereas accumulation of vesicles was observed when cells were treated with proteasome inhibitors. Cholesterol depletion of the cell membrane by methyl-beta-cyclodextrin resulted in dose dependent reduction of receptor internalization indicating that c-kit may be present in lipid rafts or that intact lipid rafts are required for efficient internalization of the receptor. Using the induction of vesicular structures as a sign of efficient internalization of the receptor analysis of mutant c-kit constructs deficient either in activation of PI3-Kinase or Src revealed that internalization of c-kit is dependent on recruitment of Src but not PI3-Kinase.

Topics: Animals; Arsenicals; beta-Cyclodextrins; Blotting, Western; Cell Line; Cholesterol; Cloning, Molecular; COS Cells; Cyclodextrins; Cysteine Endopeptidases; Dimerization; Endocytosis; Endosomes; Enzyme Activation; Flow Cytometry; Green Fluorescent Proteins; Luminescent Proteins; Membrane Microdomains; Microscopy, Fluorescence; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Structure, Tertiary; Protein Transport; Proto-Oncogene Proteins c-kit; Recombinant Fusion Proteins; Time Factors; Tyrosine