scholarly journals Development of a Rat Sandwich-Cultured Hepatocytes Model Expressing Functional Human Organic Anion Transporting Polypeptide (OATP) 1B3: A Potential Screening Tool for Liver-Targeting Compounds

2021 ◽  
Vol 24 ◽  
pp. 475-483
Author(s):  
Taleah Farasyn ◽  
Chao Xu ◽  
Wei Yue
Keyword(s):  
Biliary Excretion ◽  
In Vitro Model ◽  
Organic Anion ◽  
Rat Hepatocytes ◽  
Drug Uptake ◽  
Liver Targeting ◽  
Organic Anion Transporting Polypeptide ◽  
Vitro Model ◽  
In Cells

Purpose: Organic anion transporting polypeptide (OATP) 1B3 transports many clinically important drugs, including statins, from blood into the liver. It exclusively expresses in human liver under normal physiological conditions. There is no rodent ortholog of human OATP1B3. Tissue targeting of therapeutic molecules mediated by transporters, including liver-targeting via liver-specific OATPs, is an emerging area in drug development. Sandwich-cultured primary hepatocytes (SCH) are a well characterized in vitro model for assessment of hepatic drug uptake and biliary excretion. The current study was designed to develop a novel rat SCH model expressing human OATP1B3 to study the hepatic disposition of OATP1B3 substrates. Methods: Primary rat hepatocytes transduced with adenoviral vectors expressing FLAG-tagged OATP1B3 (Ad-OATP1B3), a control vector Ad-LacZ, or that were non-transduced were cultured in a sandwich configuration. FLAG immunoblot and immunofluorescence-staining determined expression and localization of OATP1B3. Uptake of [3H]-cholecystokinin octapeptide (CCK-8), a specific OATP1B3 substrate, was determined. Taurocholate (TC) is a substrate routinely used in SCH to assess biliary excretion via bile canaliculi (BC) and is also a substrate of OATP1B3. [3H]-TC accumulation in cells+BC, cells, biliary excretion index (BEI) and in vitro Clbiliary were determined using B-CLEAR® technology. Results: OATP1B3 protein was extensively expressed and primarily localized on the plasma membrane in day 4 Ad-OATP1B3-transduced rat SCH. [3H]-CCK-8 accumulation in cells+BC was significantly greater (~5-13 folds, p<0.001) in day 4 SCH with vs. without Ad-OATP1B3-transduction. Expressing OATP1B3 in rat SCH significantly increased [3H]-TC accumulation in cells+BC and cells, without affecting BEI and in vitro Clbiliary. Conclusions: Rat SCH expressing human OATP1B3-is a novel in vitro model allowing simultaneous assessment of hepatic uptake, hepatocellular accumulation and biliary excretion process of a human OATP1B3 substrate. This model could be a potential tool for screening for liver-targeting compounds mediated by OATP1B3.

Biliary excretion of fluorescent cholephiles in hepatocyte couplets: An in vitro model for hepatobiliary and hepatotoxicity studies

1990 ◽  
Vol 4 (4-5) ◽  
pp. 452-457 ◽  
Author(s):  
J.H. Fentem ◽  
B. Foster ◽  
C.O. Mills ◽  
R. Coleman ◽  
J.K. Chipman
Keyword(s):  
Biliary Excretion ◽  
In Vitro Model ◽  
Vitro Model

Increased susceptibility of fat-laden Zucker-rat hepatocytes to bile acid-induced oncotic necrosis: An in vitro model of steatocholestasis

2005 ◽  
Vol 145 (5) ◽  
pp. 247-262 ◽  
Author(s):  
Gregory E. Kobak ◽  
Rolf Dahl ◽  
Michael W. Devereaux ◽  
Eric Gumpricht ◽  
Maret Traber ◽  
...  
Keyword(s):  
Bile Acid ◽  
In Vitro Model ◽  
Rat Hepatocytes ◽  
Zucker Rat ◽  
Vitro Model ◽  
Increased Susceptibility

An In Vivo/In Vitro Study of Allyl Alcohol Toxicity Using Enzyme Inhibitors

1993 ◽  
Vol 21 (1) ◽  
pp. 38-42
Author(s):  
Romana Pulci ◽  
Donatella Moneta ◽  
Philippe Dostert ◽  
Marco Brughera ◽  
Giovanna Scampini ◽  
...  
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Allyl Alcohol ◽  
In Vitro Model ◽  
Enzyme Inhibitors ◽  
In Vitro Study ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Vitro Model

The aim of this study was to verify an in vitro model of hepatotoxicity, designed to assess the production of reactive species from biologically-inert chemicals through their metabolic transformation. One example is allyl alcohol, which produces acrolein through the action of the enzyme alcohol dehydrogenase. Acrolein is a highly hepatotoxic aldehyde which is detoxified to acrylic acid by aldehyde dehydrogenase (ALDH). A deficiency of this enzyme, common in some Asian populations, can give rise to pathological conditions of hepatotoxicity. Isolated rat hepatocytes were incubated with allyl alcohol with and without cyanamide, a known inhibitor of ALDH. The toxicity of allyl alcohol, assessed on the basis of release of glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) into the culture medium, was dramatically increased by the addition of cyanamide. In vivo, the same treatment scheme was used in rats treated with allyl alcohol with or without cyanamide pretreatment. It was also demonstrated that allyl alcohol toxicity is dramatically enhanced by the addition of an aldehyde dehydrogenase inhibitor, as shown by plasma levels of hepatic enzymes (GOT, GPT and LDH) and by histological findings. We believe that this in vitro model, involving the use of enzyme inhibitors, could be useful for verification of the hypothesis that hepatotoxins, such as acrolein, are produced from some pharmaceutical and other chemical compounds.

Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis

2014 ◽  
Vol 89 (6) ◽  
pp. 979-990 ◽  
Author(s):  
Gisel S. Miszczuk ◽  
Ismael R. Barosso ◽  
Andrés E. Zucchetti ◽  
Andrea C. Boaglio ◽  
José M. Pellegrino ◽  
...  
Keyword(s):  
In Vitro Model ◽  
Rat Hepatocytes ◽  
Cultured Rat Hepatocytes ◽  
Vitro Model

Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration

1999 ◽  
Vol 277 (1) ◽  
pp. G12-G21 ◽  
Author(s):  
Xingrong Liu ◽  
Edward L. LeCluyse ◽  
Kenneth R. Brouwer ◽  
Liang-Sheng L. Gan ◽  
John J. Lemasters ◽  
...  
Keyword(s):  
Biliary Excretion ◽  
Organic Anion ◽  
Rat Hepatocytes ◽  
Immunoblot Analysis ◽  
Isolated Hepatocytes ◽  
Cultured Hepatocytes ◽  
Bile Canaliculi ◽  
Standard Buffer ◽  
Sandwich Configuration

The objective of the present investigation was to examine the functional reestablishment of polarity in freshly isolated hepatocytes cultured between 2 layers of gelled collagen (sandwich configuration). Immunoblot analysis demonstrated that the canalicular multispecific organic anion transport protein (multidrug resistance-associated protein, Mrp2) was partially maintained in day 5 hepatocytes cultured in a sandwich configuration. Fluorescein-labeled taurocholate and carboxydichlorofluorescein were excreted into and concentrated in the bile canalicular lumen of day 5sandwich-cultured hepatocytes, resulting in formation of fluorescent networks in standard buffer (intact bile canaliculi). Confocal microscopy studies demonstrated that 1) carboxydichlorofluorescein that had concentrated in the canalicular lumen was released into the incubation buffer in the presence of Ca2+-free buffer (disrupted bile canaliculi), and 2) rhodamine-dextran, an extracellular space marker, was only able to diffuse into the canalicular lumen in the presence of Ca2+-free buffer. The cumulative uptake of [3H]taurocholate in day 5 sandwich-cultured hepatocytes was significantly higher in standard buffer compared with Ca2+-free buffer, due to accumulation of taurocholate in canalicular spaces. When [3H]taurocholate was preloaded in the day 5sandwich-cultured hepatocytes, taurocholate efflux was greater in Ca2+-free compared with standard buffer. The biliary excretion index of taurocholate, equivalent to the percentage of retained taurocholate in the canalicular networks, increased from ∼8% at day 0 to ∼60% at day 5 in sandwich-cultured hepatocytes. In summary, hepatocytes cultured in a collagen-sandwich configuration for up to 5 days establish intact canalicular networks, maintain Mrp2, reestablish polarized excretion of organic anions and bile acids, and represent a useful in vitro model system to investigate the hepatobiliary disposition of substrates.

scholarly journals Na+-independent phosphate transport in Caco2BBE cells

2014 ◽  
Vol 307 (12) ◽  
pp. C1113-C1122 ◽  
Author(s):  
Eduardo Candeal ◽  
Yupanqui A. Caldas ◽  
Natalia Guillén ◽  
Moshe Levi ◽  
Víctor Sorribas
Keyword(s):  
In Vitro Model ◽  
Actinomycin D ◽  
Phosphate Transport ◽  
Alkaline Ph ◽  
Anion Exchangers ◽  
Independent Components ◽  
Vitro Model ◽  
Inhibition Pattern ◽  
In Cells

Pi transport in epithelia has both Na+-dependent and Na+-independent components, but so far only Na+-dependent transporters have been characterized in detail and molecularly identified. Consequently, in the present study, we initiated the characterization and analysis of intestinal Na+-independent Pi transport using an in vitro model, Caco2BBE cells. Only Na+-independent Pi uptake was observed in these cells, and Pi uptake was dramatically increased when cells were incubated in high-Pi DMEM (4 mM) from 1 day to several days. No response to low-Pi medium was observed. The increased Pi transport was mainly caused by Vmax changes, and it was prevented by actinomycin D and cycloheximide. Pi transport in cells grown in 1 mM Pi (basal DMEM) decreased at pH > 7.5, and it was inhibited with proton ionophores. Pi transport in cells incubated with 4 mM Pi increased with alkaline pH, suggesting a preference for divalent phosphate. Pi uptake in cells in 1 mM Pi was completely inhibited only by Pi and partially inhibited by phosphonoformate, oxalate, DIDS, SITS, SO42−, HCO3−, and arsenate. This inhibition pattern suggests that more than one Pi transporter is active in cells maintained with 1 mM Pi. Phosphate transport from cells maintained at 4 mM Pi was only partially inhibited by phosphonoformate, oxalate, and arsenate. Attempts to identify the responsible transporters showed that multifunctional anion exchangers of the Slc26 family as well as members of Slc17, Slc20, and Slc37 and the Pi exporter xenotropic and polytropic retrovirus receptor 1 are not involved.

An in vitro model for investigation of vitamin A effects on wound healing

2021 ◽  
pp. 1-6
Author(s):  
Hoda Keshmiri Neghab ◽  
Mohammad Hasan Soheilifar ◽  
Gholamreza Esmaeeli Djavid
Keyword(s):  
Wound Healing ◽  
Endothelial Cell ◽  
Vitamin A ◽  
In Vitro Model ◽  
Cellular Proliferation ◽  
Endothelial Cell Migration ◽  
Normal Fibroblast ◽  
Anti Inflammatory ◽  
Vitro Model

Abstract. Wound healing consists of a series of highly orderly overlapping processes characterized by hemostasis, inflammation, proliferation, and remodeling. Prolongation or interruption in each phase can lead to delayed wound healing or a non-healing chronic wound. Vitamin A is a crucial nutrient that is most beneficial for the health of the skin. The present study was undertaken to determine the effect of vitamin A on regeneration, angiogenesis, and inflammation characteristics in an in vitro model system during wound healing. For this purpose, mouse skin normal fibroblast (L929), human umbilical vein endothelial cell (HUVEC), and monocyte/macrophage-like cell line (RAW 264.7) were considered to evaluate proliferation, angiogenesis, and anti-inflammatory responses, respectively. Vitamin A (0.1–5 μM) increased cellular proliferation of L929 and HUVEC (p < 0.05). Similarly, it stimulated angiogenesis by promoting endothelial cell migration up to approximately 4 fold and interestingly tube formation up to 8.5 fold (p < 0.01). Furthermore, vitamin A treatment was shown to decrease the level of nitric oxide production in a dose-dependent effect (p < 0.05), exhibiting the anti-inflammatory property of vitamin A in accelerating wound healing. These results may reveal the therapeutic potential of vitamin A in diabetic wound healing by stimulating regeneration, angiogenesis, and anti-inflammation responses.

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