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Expression and regulation of transmembrane transporters in healthy intestine and gastrointestinal diseases

Hruz, Petr. Expression and regulation of transmembrane transporters in healthy intestine and gastrointestinal diseases. 2006, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_7487

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Abstract

Transmembrane transporters mediate energy dependent or independent translocation of drugs,
potentially toxic compounds, and of various endogenous substrates such as bile acids and
bilirubin across membranes. In this thesis the focus is on two classes of transporters, the ATPbinding
cassette (ABC) transporters, which mediate ATP dependent transport and the solute
carriers (SLC) which use electrochemical gradients for their transport. The transporters are
expressed on membranes of cells of excretory organs (e.g. kidney, liver) and protective barriers
(e.g. intestine, blood brain barrier) and influence therefore the absorption, distribution and
elimination of compounds. They may reduce the intracellular concentration of drugs.
Transport activity of transmembrane transporters in the intestine depends on the expression
level and distribution along the intestine. Transport activity of transporters might be influenced by
other factors such as genetic variations, which may present with a dysfunctional phenotype (e.g.
single nucleotide polymorphisms; SNPs), or certain disease states, which might adaptively
regulate transporter expression on the transcriptional and posttranscriptional level.
The first aim of this thesis was a systematic site-specific analysis of the expression of several
ABC transporters and solute carriers along the intestinal tract. Following that, regulation of
hepatobiliary transporters in the human intestine during obstructive cholestasis was evaluated.
Studies were performed in close collaboration with the Department of Gastroenterology
(University Hospital of Basel).
As described in Chapter 2, transporter mRNA expression was analyzed by real time PCR
(Taqman), a method that was previously developed in our laboratory to quantify the expression
of transporters using standard curves. Protein expression was assessed by
immunohistochemistry, bile acids plasma concentrations were measured by capillary gas
chromatography (U. Beuers, Munich).
The results presented in this thesis include systematic site specific analysis of quantitative
expression of the human multidrug resistance transporters, such MDR1 (ABCB1), breast cancer
resistance transporter (BCRP; ABCG2) and multidrug resistance associated transporters
(MRP1-5; ABCC1-5) as well as the apical sodium dependent bile acid transporter ASBT
(SLC10A2) along the the intestinal tract. These data are shown in Chapter 3. As the mRNA
expression levels of the investigated transporters change along the intestinal tract, these
findings might be of interest to develop target strategies for orally administered drugs.
Also, this systematic site specific analysis of MDR transporters serves as a preparation for a
prospective clinical study in patients with Inflammatory Bowel Disease (IBD), which will
investigate MDR transporter gene expression in intestinal biopsies (intestinal epithelial cells,
intestinal macrophages) in newly diagnosed IBD patients compared to treatment refractory IBD
patients, patients in remission and disease free controls. This study design will help to evaluate,
if MDR transporters vary due to interindividual differences, inflammatory processes and/or
pharmacological treatment and might serve as an explanation for patients with IBD not
responding to drug treatment.
Intestinal macrophages play a central role in the orchestration of innate immune response
reactions in the gut. As anti-inflammatory as well as immunosuppresive drugs such as
glucocorticoids, methotrexate, cyclosporine, 6-mercaptopurine and sulfasalazine, which all of
them are used in the treatment of IBD patients, are substrates of MDR transporters, a method
for isolation of CD14+ peripheral blood cells (monocytes), their ex vivo cultivation and
differentiation into macrophages was established. In Chapter 4, first results demonstrate the
influence of the differentiation process of monocytes into monocyte derived macrophages
(MDM) and the stimulation of MDM with bacterial products (LPS) on MDR transporter
expression. At present, the effect of budesonide, methotrexate, 6-mercaptopurine and
sulfasalazine on the transporter expression in ex vivo cultivated human MDM is evaluated.
Adaptive regulation of hepatobiliary transport systems during obstructive cholestasis with a
disrupted enterohepatic circulation has been demonstrated in the intestine only for rodents
before. The results presented in Chapter 3 showed, that ASBT, which contributes substantially
to the enterohepatic circulation of bile acids by their reabsorption from the intestine, is
adaptivelly regulated in the human duodenum during obstructive cholestasis. Our findings are of
clinical importance as we have shown for the first time that ASBT is expressed in the human
duodenum. These results may indicate species specific differences to rodents, and that changes
in the ASBT gene expression can be measured in the duodenum during obstructive cholestasis.
Adaptive regulation of ASBT in the intestine has clinical implications for the bile acid
homeostasis and also for the lipid metabolism.
BCRP mediates energy dependent efflux of drugs and potentially toxic compounds, and of
various endogenous substrates such as bile acids. Here, expression of human BCRP mRNA
was shown to be highest in the duodenum with a continuos decrease along the intestinal tract
down to the rectum. BCRP mRNA and protein expression in the duodenum was found to be
decreased during obstructive cholestasis when compared to control subjects and BCRP
expression increased after reconstitution of bile flow. In consequence, reduced intestinal BCRP
expression during obstructive cholestasis might influence the accumulation of bile acids, foodderived
carcinogens and the pharmacokinetics of various drugs that are transported by BCRP.
In an isolated project with the background of observed therapy resistance to antibiotics in the
treatment of patients with chronic prostatitis, MDR expression in prostatic tissue in regard to
inflammation was evaluated in 50 patients that underwent transurethral resection of prostate. In
this study group, neither inflammation nor localization of inflammation in prostate tissue (acinar
versus interstitial) influenced MDR transporter expression.
Advisors:Drewe, Jürgen
Committee Members:Huwyler, Jörg and Beglinger, Christoph
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Ehemalige Einheiten Pharmazie > Klinische Pharmazie (Drewe)
UniBasel Contributors:Drewe, Jürgen and Huwyler, Jörg and Beglinger, Christoph
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:7487
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:127
Language:English
Identification Number:
Last Modified:22 Apr 2018 04:30
Deposited On:13 Feb 2009 15:32

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