H+-peptide cotransport in the human bile duct epithelium cell line SK-ChA-1

2002 ◽  
Vol 283 (1) ◽  
pp. G222-G229 ◽  
Author(s):  
Ilka Knütter ◽  
Isabel Rubio-Aliaga ◽  
Michael Boll ◽  
Gerd Hause ◽  
Hannelore Daniel ◽  
...  
Keyword(s):  
Bile Duct ◽  
Apical Membrane ◽  
Aminolevulinic Acid ◽  
Intestinal Type ◽  
Peptide Transport ◽  
Peptide Transporter ◽  
Clinical Aspects ◽  
Biliary Duct ◽  
Human Bile ◽  
In Cells

This study describes for the first time the presence of H+-peptide cotransport in cells of the bile duct. Uptake of [glycine-1-14C]glycylsarcosine ([14C]Gly-Sar) in human extrahepatic cholangiocarcinoma SK-ChA-1 cells was stimulated sevenfold by an inwardly directed H+ gradient. Transport was mediated by a low-affinity system with a transport constant ( K t) value of 1.1 mM. Several dipeptides, cefadroxil, and δ-aminolevulinic acid, but not glycine and glutathione, were strong inhibitors of Gly-Sar uptake. SK-ChA-1 cells formed tight, polarized monolayers on permeable membranes. The transepithelial electrical resistance was 856 ± 29 Ω × cm2. The transepithelial flux of [14C]Gly-Sar in apical-to-basolateral direction exceeded the basolateral-to-apical flux 11-fold. Uptake was 20-fold higher from the apical side. RT-PCR analysis using primer pairs specific for the intestinal-type peptide transporter (PEPT1) or kidney-type (PEPT2) revealed that the transport system expressed in SK-ChA-1 and also in cells of the native rabbit bile duct is PEPT1. Immunohistochemistry localized PEPT1 to the apical membrane of cholangiocytes of mouse extrahepatic biliary duct. We conclude that the cells of the mammalian extrahepatic biliary tract epithelium express the intestinal-type H+-peptide cotransporter in their apical membrane. SK-ChA-1 cells represent a convenient model to study the physiological and clinical aspects of peptide transport in cholangiocytes.

Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line

2001 ◽  
Vol 281 (1) ◽  
pp. G191-G199 ◽  
Author(s):  
Carsten Uhd Nielsen ◽  
Jan Amstrup ◽  
Bente Steffansen ◽  
Sven Frokjaer ◽  
Birger Brodin
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Line ◽  
Peptide Transport ◽  
Peptide Transporter ◽  
Intestinal Cell ◽  
Intestinal Cell Line ◽  
Epidermal Growth ◽  
Human Intestinal Cell Line ◽  
In Cells

The human intestinal cell line Caco-2 was used as a model system to study the effects of epidermal growth factor (EGF) on peptide transport. EGF decreased apical-to-basolateral fluxes of [14C]glycylsarcosine ([14C]Gly-Sar) up to 50.2 ± 3.6% ( n = 6) of control values. Kinetic analysis of the fluxes showed that maximal flux ( V max) of transepithelial transport decreased from 3.00 ± 0.17 nmol · cm−2 · min−1 in control cells to 0.50 ± 0.07 nmol · cm−2 · min−1 in cells treated with 5 ng/ml EGF ( n = 6, P < 0.01). The apparent Michaelis-Menten constant ( K m) was 2.71 ± 0.31 mM ( n= 6) in control cells and 1.89 ± 0.28 mM ( n = 6, not significantly different from control) in EGF-treated cells. Similarly, apical uptake of [14C]Gly-Sar decreased in cells treated with EGF, with an ED50 value of 0.36 ± 0.06 ng/ml ( n = 6) EGF and a maximal inhibition of 80 ± 0.02% ( n = 6). V maxdecreased from 2.61 ± 0.4 to 1.06 ± 0.1 nmol · cm−2 · min−1( n = 3, P < 0.05), whereas K m remained constant. Basolateral Gly-Sar uptake showed no changes in V max or K m after EGF treatment ( n = 3). RT-PCR showed a decrease in hPepT1 mRNA (using glucose-6-phosphate dehydrogenase mRNA as control) in cells treated with EGF. Western blotting indicated a decrease in hPepT1 protein in cell lysates. We conclude that EGF treatment decreases Gly-Sar transport in Caco-2 cells by decreasing the number of peptide transporter molecules in the apical membrane.

scholarly journals Transcriptional regulation of theN-acetylglucosaminyltransferase V gene in human bile duct carcinoma cells (HuCC-T1) is mediated by Ets-1.

1999 ◽  
Vol 274 (1) ◽  
pp. 554
Author(s):  
Rujun Kang ◽  
Hiroyuki Saito ◽  
Yoshito Ihara ◽  
Eiji Miyoshi ◽  
Nobuto Koyama ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Bile Duct ◽  
Bile Duct Carcinoma ◽  
Carcinoma Cells ◽  
Duct Carcinoma ◽  
Human Bile ◽  
V Gene

Indicial response functions of growth and remodeling of common bile duct postobstruction

2004 ◽  
Vol 286 (3) ◽  
pp. G420-G427 ◽  
Author(s):  
Quang Dang ◽  
Hans Gregersen ◽  
Birgitte Duch ◽  
Ghassan S. Kassab
Keyword(s):  
Bile Duct ◽  
Common Bile Duct ◽  
Wall Thickness ◽  
Time Course ◽  
Quantitative Description ◽  
Biliary Duct ◽  
Stress And Strain ◽  
Response Functions ◽  
Mechanical Remodeling ◽  
Scheduled Time

Biliary duct obstruction is an important clinical condition that stems from cholelithiasis, the neoplasm in the wall or, most commonly, gallbladder stones. The objective of this study is to understand the structural and mechanical remodeling of the common bile duct (CBD) postobstruction. Porcine CBD was ligated near the duodenum that increased the duct's pressure from 6.4 to 18.3 cmH2O in the first 12 h and to 30.7 cmH2O after 32 days. The remodeling process was studied after 3 h, 12 h, 2 days, 8 days, and 32 days ( n = 5 in each group) after obstruction. One additional animal in each group was sham operated. At each scheduled time, the time course of change of morphometry (diameter, length, wall thickness, etc.) and mechanical properties (stress, strain, etc.) was documented. It was found that the diameter increased by about threefold and the wall thickness of the CBD doubled in the 32-day group compared with the sham group ( P < 0.001). The stress and strain increased initially with increase in pressure but recovered to near the control values by day 32 due to the structural and mechanical adaptations. Hence, the net effect of the structural and mechanical remodeling is to restore the stress and strain to their homeostatic values. Furthermore, the strain recovers more rapidly and more completely than stress. Finally, the remodeling data were expressed mathematically in terms of indicial response functions (IRF), i.e., change of a particular feature of a CBD in response to a unit step change of the pressure. The IRF approach provides a quantitative description of the remodeling process in the CBD.

Regulators of Apoptosis in Cholangiocarcinoma

2005 ◽  
Vol 129 (4) ◽  
pp. 481-486
Author(s):  
Nirag C. Jhala ◽  
Selwyn M. Vickers ◽  
Pedram Argani ◽  
Jay M. McDonald
Keyword(s):  
Bile Duct ◽  
Biliary Tract ◽  
P53 Gene ◽  
Tumor Location ◽  
Biliary Duct ◽  
Early Event ◽  
Tumor Differentiation ◽  
P53 Gene Mutation ◽  
P53 Expression ◽  
Poorly Differentiated

Abstract Context.—Dysregulation of mediators of apoptosis is associated with carcinogenesis. For biliary duct cancers, p53 gene mutation is an important contributor to carcinogenesis. Mutations in the p53 gene affect transcription of the Fas gene, resulting in lack of Fas expression on cell membrane. It has been previously shown that cloned Fas-negative but not Fas-positive human cholangiocarcinoma cells are resistant to anti–Fas-mediated apoptosis and develop tumors in nude mice. In addition, interferon gamma induces Fas expression in Fas-negative cholangiocarcinoma cells and makes them susceptible to apoptosis. Therefore, it becomes important to characterize immunophenotypic expression of p53 and Fas in normal and neoplastic human tissues of the biliary tract to further understand the pathogenesis of the disease. To date, human studies to characterize differences in immunophenotypic expression of the Fas protein between intrahepatic and extrahepatic biliary duct cancers and in their precursor lesions have not been performed. Objective.—To report the immunophenotypic expression of p53 and Fas expression in various stages in the development of bile duct cancers (intrahepatic and extrahepatic tumor location) and their association with tumor differentiation. Design.—Thirty bile duct cancer samples (13 intrahepatic and 17 extrahepatic) from 18 men and 12 women who ranged in age from 44 to 77 years (mean age, 65.6 years) were retrieved from the surgical pathology files. Hematoxylin-eosin–stained slides were evaluated for the type and grade of tumor and dysplastic changes in the biliary tract epithelium. Additional slides were immunohistochemically stained with p53 and anti–Fas mouse monoclonal antibody. The pattern of Fas distribution and percentage of cells positive for p53 and Fas expression were determined. Results.—The percentage of Fas-expressing cells is significantly (P = .01) more frequently noted in extrahepatic tumors compared with intrahepatic tumors. Furthermore, Fas expression decreased from dysplastic epithelium to cholangiocarcinoma (P = .01), and this decreasing trend continued from well to poorly differentiated tumors. Nuclear p53 expression was not identified in normal and dysplastic epithelium but was noted in 30% of carcinomas (P = .02). Conclusion.—Fas expression is an early event in pathogenesis of bile duct cancers. Immunophenotypic expression of Fas is associated with well to moderately differentiated tumors but not with poor tumor differentiation.

Towards a structural understanding of drug and peptide transport within the proton-dependent oligopeptide transporter (POT) family

2011 ◽  
Vol 39 (5) ◽  
pp. 1353-1358 ◽  
Author(s):  
Simon Newstead
Keyword(s):  
High Resolution ◽  
Anticancer Agents ◽  
Targeted Delivery ◽  
Drug Transport ◽  
Structural Information ◽  
Sequence Similarity ◽  
The Body ◽  
Peptide Transport ◽  
Peptide Transporter ◽  
Starting Point

One of the principal aims of modern drug design is the targeted delivery of drugs within the body, such as to the central nervous system, combined with their exclusion from the liver and kidneys, which break down foreign molecules and subsequently eliminate them. Many of the commonly prescribed drugs are transported into cells and across the plasma membrane via endogenous membrane transporters, whose principal roles are the uptake of essential nutrients for metabolism. In many cases, such drug transport is serendipitous as they are simply mistaken as ‘natural’ compounds. Many of these transporters could, however, be targeted more efficiently, improving drug absorption, distribution and retention. The molecular details of these drug–transporter interactions, however, are at best poorly understood, in large part through the absence of any high-resolution structural information. To address this issue, we recently determined the structure of a prokaryotic peptide transporter, PepTSo from Shewanella oneidensis, which shares a high degree of sequence similarity and functional characteristics with the human PepT1 and PepT2 proteins. PepT1 and PepT2 contribute significantly to the oral bioavailability and pharmacokinetic properties of a number of important drug families, including antibiotics, antivirals and anticancer agents. The crystal structure of PepTSo provides the first high-resolution model of a drug importer and provides the starting point for understanding drug and peptide transport within the human body.

Increased biliary secretion of cysteinyl-leukotrienes in human bile duct obstruction

1996 ◽  
Vol 25 (5) ◽  
pp. 725-732 ◽  
Author(s):  
Lothar Richter ◽  
Norbert Hesselbarth ◽  
Klaus Eitner ◽  
Klaus Schubert ◽  
Hans Bosseckert ◽  
...  
Keyword(s):  
Bile Duct ◽  
Biliary Secretion ◽  
Bile Duct Obstruction ◽  
Human Bile ◽  
Cysteinyl Leukotrienes ◽  
Duct Obstruction

scholarly journals Computed Tomography Imaging in Lemmel Syndrome: A Report of Two Cases

2019 ◽  
Vol 9 ◽  
pp. 23
Author(s):  
Giulia Frauenfelder ◽  
Annamaria Maraziti ◽  
Vincenzo Ciccone ◽  
Giuliano Maraziti ◽  
Oliviero Caleo ◽  
...  
Keyword(s):  
Computed Tomography ◽  
Abdominal Pain ◽  
Bile Duct ◽  
Common Bile Duct ◽  
Acute Abdominal Pain ◽  
Imaging Modality ◽  
Biliary Duct ◽  
History Of ◽  
The Common ◽  
Solid Lesions

Lemmel syndrome is a rare and misdiagnosed cause of acute abdominal pain due to a juxtapapillary duodenal diverticulum causing mechanical obstruction of the common bile duct. Frequently, patients suffering from Lemmel syndrome have a history of recurrent access to the emergency room for acute abdominal pain referable to a biliopancreatic obstruction, in the absence of lithiasis nuclei or solid lesions at radiological examinations. Ultrasonography (US) may be helpful in evaluation of upstream dilatation of extra-/intra-hepatic biliary duct, but computed tomography (CT) is the reference imaging modality for the diagnosis of periampullary duodenal diverticula compressing the intrapancreatic portion of the common bile duct. Recognition of this entity is crucial for targeted, timely therapy avoiding mismanagement and therapeutic delay. The aim of this paper is to report CT imaging findings and our experience in two patients affected by Lemmel syndrome.

scholarly journals Glycans in the intestinal peptide transporter PEPT1 contribute to function and protect from proteolysis

2017 ◽  
Vol 312 (6) ◽  
pp. G580-G591 ◽  
Author(s):  
Tamara Stelzl ◽  
Kerstin E. Geillinger-Kästle ◽  
Jürgen Stolz ◽  
Hannelore Daniel
Keyword(s):  
Protein Function ◽  
Proteolytic Cleavage ◽  
Glycosylation Site ◽  
Proteinase K ◽  
Peptide Transport ◽  
Peptide Transporter ◽  
Electrophysiological Recordings ◽  
Transport Velocity ◽  
Flux Measurements ◽  
The Individual

Despite the fact that many membrane proteins carry extracellular glycans, little is known about whether the glycan chains also affect protein function. We recently demonstrated that the proton-coupled oligopeptide transporter 1 (PEPT1) in the intestine is glycosylated at six asparagine residues (N50, N406, N439, N510, N515, and N532). Mutagenesis-induced disruption of the individual N-glycosylation site N50, which is highly conserved among mammals, was detected to significantly enhance the PEPT1-mediated inward transport of peptides. Here, we show that for the murine protein the inhibition of glycosylation at sequon N50 by substituting N50 with glutamine, lysine, or cysteine or by replacing S52 with alanine equally altered PEPT1 transport kinetics in oocytes. Furthermore, we provide evidence that the uptake of [14C]glycyl-sarcosine in immortalized murine small intestinal (MODE-K) or colonic epithelial (PTK-6) cells stably expressing the PEPT1 transporter N50Q is also significantly increased relative to the wild-type protein. By using electrophysiological recordings and tracer flux studies, we further demonstrate that the rise in transport velocity observed for PEPT1 N50Q is bidirectional. In line with these findings, we show that attachment of biotin derivatives, comparable in weight with two to four monosaccharides, to the PEPT1 N50C transporter slows down the transport velocity. In addition, our experiments provide strong evidence that glycosylation of PEPT1 confers resistance against proteolytic cleavage by proteinase K, whereas a remarkable intrinsic stability against trypsin, even in the absence of N-linked glycans, was detected. NEW & NOTEWORTHY This study highlights the role of N50-linked glycans in modulating the bidirectional transport activity of the murine peptide transporter PEPT1. Electrophysiological and tracer flux measurements in Xenopus oocytes have shown that removal of the N50 glycans increases the maximal peptide transport rate in the inward and outward directions. This effect could be largely reversed by replacement of N50 glycans with structurally dissimilar biotin derivatives. In addition, N-glycans were detected to stabilize PEPT1 against proteolytic cleavage.

Agonist-induced polarized trafficking and surface expression of the adenosine 2b receptor in intestinal epithelial cells: role of SNARE proteins

2004 ◽  
Vol 287 (5) ◽  
pp. G1100-G1107 ◽  
Author(s):  
Lixin Wang ◽  
Vasantha Kolachala ◽  
Baljit Walia ◽  
Srividya Balasubramanian ◽  
Randy A. Hall ◽  
...  
Keyword(s):  
Adenosine Receptor ◽  
Intestinal Inflammation ◽  
Apical Membrane ◽  
Snare Proteins ◽  
Cell Fractionation ◽  
Camp Synthesis ◽  
Agonist Stimulation ◽  
A2b Receptor ◽  
In Cells

Adenosine, acting through the A2b receptor, induces vectorial chloride and IL-6 secretion in intestinal epithelia and may play an important role in intestinal inflammation. We have previously shown that apical or basolateral adenosine receptor stimulation results in the recruitment of the A2b receptor to the plasma membrane. In this study, we examined domain specificity of recruitment and the role of soluble N-ethylmaleimide (NEM) attachment receptor (SNARE) proteins in the agonist-mediated recruitment of the A2b receptor to the membrane. The colonic epithelial cell line T84 was used because it only expresses the A2b-subtype adenosine receptor. Cell fractionation, biotinylation, and electron microscopic studies showed that the A2b receptor is intracellular at rest and that apical or basolateral adenosine stimulation resulted in the recruitment of the receptor to the apical membrane. Upon agonist stimulation, the A2b receptor is enriched in the vesicle fraction containing vesicle-associated membrane protein (VAMP)-2. Furthermore, in cells stimulated with apical or basolateral adenosine, we demonstrate a complex consisting of VAMP-2, soluble NEM-sensitive factor attachment protein (SNAP)-23, and A2b receptor that is coimmunoprecipitated in cells stimulated with adenosine within 5 min and is no longer detected within 15 min. Inhibition of trafficking with NEM or nocodazole inhibits cAMP synthesis induced by apical or basolateral adenosine by 98 and 90%, respectively. cAMP synthesis induced by foskolin was not affected, suggesting that generalized signaling is not affected under these conditions. Collectively, our data suggest that 1) the A2b receptor is intracellular at rest; 2) apical or basolateral agonist stimulation induces recruitment of the A2b receptor to the apical membrane; 3) the SNARE proteins, VAMP-2 and SNAP-23, participate in the recruitment of the A2b receptor; and 4) the SNARE-mediated recruitment of the A2b receptor may be required for its signaling.

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