Cellular disposition of arabinogalactan in primary cultured rat hepatocytes

Tanaka, Tetsuro; Fujishima, Yuko; Hamano, Shinya; Kaneo, Yoshiharu

doi:10.1016/j.ejps.2004.04.014

Cited by 18 publications

(9 citation statements)

References 30 publications

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“…The value of K a for pullulan was similar to that for asialofetuin, however, it was smaller than that for arabinogalactan. 7) Following the binding of pullulan at 0°C, EDTA treatment resulted in the loss of 54.4Ϯ2.1% of the total bound molecules, indicating dissociation of pullulan from the cell surface. The requirement of Ca 2ϩ for the binding of asialoglycoprotein to the ASGPR has been established.…”

Section: Discussionmentioning

confidence: 99%

“…4) For arabinogalactan, the nonspecific process was represented by a zero-order kinetic model. 7) We represented the pullulan uptake process in cultured rat liver parenchymal cells as involving two types of pathways, specific and nonspecific (Chart 1).…”

Section: Discussionmentioning

confidence: 99%

“…Values for the uptake parameters, except for k b,app and k int,app , were smaller than those for asialofetuin, which were determined previously. 7) Values of B ss , I ss,app , and k int,ss for pullulan were 12%, 6%, and 9%, respectively, of those for asialofetuin. The value of k b,app for pullulan (1.25Ϯ0.6 min Ϫ1 ) was larger than that for asialofetuin (0.424Ϯ0.119 min…”

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confidence: 99%

“…The value of k int for pullulan was smaller than those found for asialofetuin and arabinogalactan. 7) This number was used as the initial value for subsequent computer calculations, as described by Yamaoka et al 12) The data for both binding and internalization at 37°C were fitted to Eqs. 5 and 6, respectively.…”

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Uptake of Pullulan in Cultured Rat Liver Parenchymal Cells

Tanaka

Hamano

Fujishima

et al. 2005

Biological & Pharmaceutical Bulletin

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Polysaccharides are an attractive drug carrier directed to the liver because they are distributed in the liver after intravenous injection. Pullulan is a polysaccharide consisting of three a-1,4-linked glucose molecules, polymerized by a-1,6-linkages to the terminal glucose, and has been employed as a carrier of human interferon-b and plasmid DNA to the liver. 1,2) We previously investigated the biodisposition of pullulan in rats and demonstrated that the asialoglycoprotein receptor (ASGPR) contributes to hepatic distribution.3) Furthermore, intracellular disposition of polysaccharides has been examined in rat liver parenchymal and nonparenchymal cells and studies have shown that binding and internalization to parenchymal cells was inhibited by asialofetuin, indicating that ASGPR is involved in the intracellular disposition of pullulan. 4)In this study, we examined the uptake of pullulan, including the binding process followed by internalization in cultured rat liver parenchymal cells, and discuss the possibility of pullulan as a passive or active drug carrier via receptor mediated endocytosis (RME). MATERIALS AND METHODSMaterials Pullulan (MW 58200) was supplied by Kuraray (Japan). Arabinogalactan and asialofetuin were purchased from Sigma (St. Louis, MO. U.S.A.). Fluorescein isothiocyanate was obtained from Wako Pure Chemical (Osaka, Japan). All other reagents were the highest grade commercially available.Radio-Labeling of Pullulan The tyramine derivative of pullulan was prepared according to a previous report. 4)Briefly, pullulan was dissolved in 10% acetone in water and cooled in an ice bath. Then, 10% CNBr in acetone was added and the mixture stirred for 2 min. Tetraethylamine in acetone was added to the solution. After 2 min, tyramine in 0.1 M NaHCO 3 containing 0.5 M NaCl was added and the reaction mixture stirred overnight at 4°C. The mixture was dialyzed against water and the macromolecular fraction obtained as a white powder. Animals Wistar male rats (6 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). The rats were maintained on a commercially balanced stock diet (Oriental Yeast Co. Ltd., Tokyo, Japan) with water ad libitum. Before experimentation, each rat was anesthetized with pentobarbital. All aspects of the study were performed according to the Fukuyama University guidelines for animal experiments.Isolation and Culture of Rat Liver Parenchymal Cells Hepatocytes were isolated from male Wistar rats (7 weeks old) by the collagenase perfusion method.6) The hepatocytes were centrifuged at 50ϫg and the resulting pellet washed with Hanks' medium containing 2.4 mM CaCl 2 and 10 mM HEPES (pH 7.4). The cells were fractionated on Percoll density gradients, which resulted in a greater than 98% viability, as verified by a trypan blue exclusion test. The cells were then resuspended in William's Medium E containing dexamethasone (1 µM), insulin (0.1 µM), and 10% FBS. The cells (1ϫ10 6 cells) were plated in a 35-mm collagen type I coated culture dish (Iwaki, Funabashi, Japan) and incubated in hu...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Uptake of Pullulan in Cultured Rat Liver Parenchymal Cells

Tanaka

Hamano

Fujishima

et al. 2005

Biological & Pharmaceutical Bulletin

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“…16 Therefore, a synthetic method capable of stereoselectively forming the a-anomers is needed. Compared to D-glucose or D-fructose, recently L-arabinose was particularly used either as a pharmacophore of new bioactive compounds 17,18 or as a moiety of polymer chain of drug carrier, 19 and resulted in an increase in trans-membrane permeability. In this context, the present paper reports a stereoselective synthetic route of N-(a-L-arabinofuranos-1-yl)-Lamino acids.…”

Section: Introductionmentioning

confidence: 99%