scholarly journals Cholesterol stimulates the cellular uptake of L-carnitine by the carnitine/organic cation transporter novel 2 (OCTN2)

2020 ◽  
pp. jbc.RA120.015175
Author(s):  
Lu Zhang ◽  
Ting Gui ◽  
Lara Console ◽  
Mariafrancesca Scalise ◽  
Cesare Indiveri ◽  
...  
Keyword(s):  
Cellular Uptake ◽  
Organic Cation ◽  
Transmembrane Protein ◽  
Organic Cation Transporter ◽  
Cholesterol Content ◽  
Dependent Manner ◽  
Intact Cells ◽  
Lipid Species ◽  
Carnitine Uptake ◽  
In Cells

The carnitine/organic cation transporter novel 2 (OCTN2) is responsible for the cellular uptake of carnitine in most tissues. Being a transmembrane protein OCTN2 must interact with the surrounding lipid microenvironment to function. Among the main lipid species that constitutes eukaryotic cells, cholesterol level is highly dynamic under a number of physio-pathological conditions. This work describes how plasma membrane cholesterol modulates OCTN2 transport of L-carnitine in human embryonic kidney 293 cells overexpressing OCTN2 (OCTN2-HEK293) and in proteoliposomes harboring human OCTN2. We manipulated the cholesterol content of intact cells, assessed by thin layer chromatography, through short exposures to empty and/or cholesterol-saturated methyl-β-cyclodextrin (mβcd), whereas free cholesterol was used to enrich reconstituted proteoliposomes. We measured OCTN2 transport using [3H]L-carnitine, and expression levels and localization by surface biotinylation and western blotting. A 20-minute preincubation with mβcd reduced the cellular cholesterol content and inhibited L-carnitine influx by 50% in comparison to controls. Analogously, the insertion of cholesterol in OCTN2-proteoliposomes stimulated L-carnitine uptake in a dose-dependent manner. Carnitine uptake in cells incubated with empty mβcd and cholesterol-saturated mβcd to preserve cholesterol content was comparable to controls, suggesting that the mβcd effect on OCTN2 was cholesterol dependent. Cholesterol stimulated L-carnitine influx in cells by markedly increasing the affinity for L-carnitine and in proteoliposomes by significantly enhancing the affinity for Na+ and, in turn, the L-carnitine maximal transport capacity. Because of the antilipogenic and antioxidant features of L-carnitine, the stimulatory effect of cholesterol on L-carnitine uptake might represent a novel protective effect against lipid-induced toxicity and oxidative stress.

scholarly journals Extended Intake of Mulberry Leaf Extract Delayed Metformin Elimination via Inhibiting the Organic Cation Transporter 2

Pharmaceutics ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 49 ◽  
Author(s):  
Hyun Wook Huh ◽  
Young-Guk Na ◽  
Ki-Hyun Bang ◽  
Sung-Jin Kim ◽  
Minki Kim ◽  
...  
Keyword(s):  
Organic Cation ◽  
Organic Cation Transporter ◽  
Dependent Manner ◽  
Pharmacokinetics And Pharmacodynamics ◽  
Concentration Dependent Manner ◽  
Major Health Problem ◽  
Major Health ◽  
Mulberry Leaf ◽  
Organic Cation Transporter 2 ◽  
Hyperglycemic Effect

Diabetes mellitus (DM) has become a major health problem in most countries of the world. DM causes many complications, including hyperglycemia, diabetic ketoacidosis, and death. In Asia, mulberry has been used widely in the treatment of DM. Combination of drugs with herbal medicine may reduce the unwanted side effects caused by drugs. In this study, the influence of extended mulberry leaves extract (MLE) intake on metformin (Met) was evaluated in terms of pharmacokinetics and pharmacodynamics in DM-induced rats. Three week-treatment of MLE alone produced the anti-hyperglycemic effect (around 24%) if compared to the control. Interestingly, Met administration after MLE treatment for 3 weeks enhanced about 49% of the anti-hyperglycemic effect of Met. In addition, the extended intake of MLE potentiated the anti-hyperglycemic effect of Met on various concentrations. This potentiated anti-hyperglycemic effect of Met appears to be due to the pharmacokinetic change of Met. In this study, 3 week-treatment of MLE reduced the elimination of Met in DM-induced rats. In addition, MLE reduced the human organic cation transporter 2 (hOCT2) activity in a concentration-dependent manner. Thus, these findings suggest that MLE lowered the elimination of Met via inhibiting the hOCT2.

scholarly journals Erythrocyte membrane protein band 3: its biosynthesis and incorporation into membranes.

1981 ◽  
Vol 91 (3) ◽  
pp. 637-646 ◽  
Author(s):  
E Sabban ◽  
V Marchesi ◽  
M Adesnik ◽  
D D Sabatini
Keyword(s):  
Plasma Membrane ◽  
Electrophoretic Mobility ◽  
Messenger Rna ◽  
Transmembrane Protein ◽  
Band 3 ◽  
In Vitro Translation ◽  
Intact Cells ◽  
Amino Terminal ◽  
In Cells

Band 3, a transmembrane protein that provides the anion channel of the erythrocyte plasma membrane, crosses the membrane more than once and has a large amino terminal segment exposes on the cytoplasmic side of the membrane. The biosynthesis of band 3 and the process of its incorporation into membranes were studied in vivo in erythroid spleen cells of anemic mice and in vitro in protein synthesizing cell-free systems programmed with polysomes and messenger RNA (mRNA). In intact cells newly synthesized band 3 is rapidly incorporated into intracellular membranes where it is glycosylated and it is subsequently transferred to the plasma membrane where it becomes sensitive to digestion by exogenous chymotrypsin. The appearance of band 3 in the cell surface is not contingent upon its glycosylation because it proceeds efficiently in cells treated with tunicamycin. The site of synthesis of band 3 in bound polysomes was established directly by in vitro translation experiments with purified polysomes or with mRNA extracted from them. The band-3 polypeptide synthesized in an mRNA-dependent system had the same electrophoretic mobility as that synthesized in cells treated with tunicamycin. When microsomal membranes were present during translation, the in vitro synthesized band-3 polypeptide was cotranslationally glycosylated and inserted into the membranes. This was inferred from the facts that when synthesis was carried out in the presence of membranes the product had a lower electrophoretic mobility and showed partial resistance to protease digestion. Our observations indicate that the primary translation product of band-3 mRNA is not proteolytically processed either co- or posttranslationally. It is, therefore, proposed that the incorporation of band 3 into the endoplasmic reticulum (ER) membrane is initiated by a permanent insertion signal. To account for the cytoplasmic exposure of the amino terminus of the polypeptide we suggest that this signal is located within the interior of the polypeptide. a mechanism that explains the final transmembrane disposition of band 3 in the plasma membrane as resulting from the mode of its incorporation into the ER is presented.

Diferric transferrin regulates transferrin receptor 2 protein stability

Blood ◽  
2004 ◽  
Vol 104 (13) ◽  
pp. 4287-4293 ◽  
Author(s):  
Martha B. Johnson ◽  
Caroline A. Enns
Keyword(s):  
Transferrin Receptor ◽  
Iron Homeostasis ◽  
Transmembrane Protein ◽  
Hereditary Hemochromatosis ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Diferric Transferrin ◽  
In Cells ◽  
Transferrin Receptor 2

Abstract Transferrin receptor 2 (TfR2) is a type 2 transmembrane protein expressed in hepatocytes that binds iron-bound transferrin (Tf). Mutations in TfR2 cause one form of hereditary hemochromatosis, a disease in which excessive absorption of dietary iron can lead to liver cirrhosis, diabetes, arthritis, and heart failure. The function of TfR2 in iron homeostasis is unknown. We have studied the regulation of TfR2 in HepG2 cells. Western blot analysis shows that TfR2 increases in a time- and dose-dependent manner after diferric Tf is added to the culture medium. In cells exposed to diferric Tf, the amount of TfR2 returns to control levels within 8 hours after the removal of diferric Tf from the medium. However, TfR2 does not increase when non–Tf-bound iron (FeNTA) or apo Tf is added to the medium. The response to diferric Tf appears to be hepatocyte specific. Real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis shows that TfR2 mRNA levels do not change in cells exposed to diferric Tf. Rather, the increase in TfR2 is attributed to an increase in the half-life of TfR2 protein in cells exposed to diferric Tf. Our results support a role for TfR2 in monitoring iron levels by sensing changes in the concentration of diferric Tf.

scholarly journals Calcium-dependent biosynthesis of platelet-activating factor by submandibular gland cells

1991 ◽  
Vol 276 (1) ◽  
pp. 175-182 ◽  
Author(s):  
T Dohi ◽  
K Morita ◽  
S Kitayama ◽  
A Tsujimoto
Keyword(s):  
Submandibular Gland ◽  
Platelet Activating Factor ◽  
Dependent Manner ◽  
Additional Mechanism ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Calcium Dependent ◽  
Gland Cells ◽  
Stimulation Of ◽  
In Cells

Isolated dog submandibular gland cells synthesize platelet-activating factor (PAF) when stimulated with acetylcholine (ACh). This production of PAF was concentration- and time-dependent, and was inhibited by pretreatment with anticholinergic agents. PAF that had accumulated in cells through prior stimulation with ACh vanished rapidly on addition of atropine. Phenylmethanesulphonyl fluoride produced an accumulation of PAF in non-stimulated cells and greatly potentiated further ACh-induced accumulation. PAF production and [14C] arachidonic acid (AA) liberation induced by ACh were increased by higher concentrations of extracellular Ca2+, and ACh failed to stimulate PAF formation in the absence of Ca2+, although ACh still stimulated the liberation of [14C]AA without Ca2+. Both the Ca2+ ionophore ionomycin in intact cells and Ca2+ (at concentrations greater than or equal to 300 nM) in digitonin-permeabilized cells facilitated PAF formation. 1-O-Alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase activity rapidly increased in cells incubated with ACh or ionomycin. These results suggest, at least, that the stimulation of a remodelling pathway is involved in the increased PAF synthesis induced by ACh. Dithiothreitol-insensitive cholinephosphotransferase activity was also activated by ACh. However, the activation of both enzymes by ACh was transient, in spite of the fact that ACh-stimulated PAF formation was continuous. This may suggest that additional mechanism(s) other than the activation of these enzymes play an important role in controlling PAF synthesis. The present study provides further evidence that the exocrine submandibular gland cells of dogs have the capacity to increase PAF turnover upon stimulation in a Ca(2+)-dependent manner and retain PAF within the cells partly associated with the membrane and partly released into the cytosol.

scholarly journals Functional significance of conserved cysteines in the human organic cation transporter 2

2012 ◽  
Vol 303 (2) ◽  
pp. F313-F320 ◽  
Author(s):  
Ryan M. Pelis ◽  
Yodying Dangprapai ◽  
Yaofeng Cheng ◽  
Xiaohong Zhang ◽  
Jennifer Terpstra ◽  
...  
Keyword(s):  
Organic Cation ◽  
Chinese Hamster Ovary Cells ◽  
Transmembrane Helix ◽  
Chinese Hamster ◽  
Organic Cation Transporter ◽  
Disulfide Bridge ◽  
Cell Surface Expression ◽  
Extracellular Loop ◽  
Intact Cells ◽  
Organic Cation Transporter 2

The significance of conserved cysteines in the human organic cation transporter 2 (hOCT2), namely the six cysteines in the long extracellular loop (loop cysteines) and C474 in transmembrane helix 11, was examined. Uptake of tetraethylammonium (TEA) and 1-methyl-4-phenypyridinium (MPP) into Chinese hamster ovary cells was stimulated >20-fold by hOCT2 expression. Both cell surface expression and transport activity were reduced considerably following mutation of individual loop cysteines (C51, C63, C89, C103, and C143), and the C89 and C103 mutants had reduced Michaelis constants ( Kt) for MPP. The loop cysteines were refractory to interaction with thiol-reactive biotinylation reagents, except after pretreatment of intact cells with dithiothreitol or following cell membrane solubilization. Reduction of disulfide bridge(s) did not affect transport, but labeling the resulting free thiols with maleimide-PEO2-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the Kt value for MPP. In contrast, the Kt value associated with TEA transport was reduced sevenfold in the C474A mutant, and the C474F mutant failed to transport TEA. This study shows that some but not all of the six extracellular loop cysteines exist within disulfide bridge(s). Each loop cysteine is important for plasma membrane targeting, and their mutation can influence substrate binding. The effect of C474 mutation on TEA transport suggests that it contributes to a TEA binding surface. Given that TEA and MPP are competitive inhibitors, the differential effects of C474 modification on TEA and MPP binding suggest that the binding surfaces for each are distinct, but overlapping in area.

scholarly journals Cellular Uptake of Imatinib into Leukemic Cells Is Independent of Human Organic Cation Transporter 1 (OCT1)

2013 ◽  
Vol 20 (4) ◽  
pp. 985-994 ◽  
Author(s):  
Anne T. Nies ◽  
Elke Schaeffeler ◽  
Heiko van der Kuip ◽  
Ingolf Cascorbi ◽  
Oliver Bruhn ◽  
...  
Keyword(s):  
Cellular Uptake ◽  
Organic Cation ◽  
Organic Cation Transporter ◽  
Leukemic Cells ◽  
Organic Cation Transporter 1
Sign in / Sign up

Export Citation Format

Share Document