Shed human enterocytes as a tool for the study of expression and function of intestinal drug-metabolizing enzymes and transporters.
OBJECTIVE
Intestinal metabolism and transport are now recognized as protective barriers against orally ingested xenobiotics, including drugs. However, in vitro studies of the expression and function of intestinal proteins are hampered by the limited availability of human intestinal tissues. Because enterocytes are constantly shed in large numbers into the gut lumen, this study investigated whether these cells could be collected with a multilumen perfusion catheter and whether they are functionally active.


METHODS
In healthy volunteers, a 20-cm isolated jejunal segment was generated with the perfusion catheter by inflating 2 balloons with air. Shed cells were characterized by fluorescence-activated cell sorting analysis for leukocyte-specific CD45 and enterocyte-specific villin, as well as for apoptosis. Homogenates of the cells were used for reverse transcriptase polymerase chain reaction and Western blotting. Cytochrome P450 enzyme activity was determined with the calcium channel blocker verapamil as a substrate.


RESULTS
On average, 4.83 mg protein and 56.23 million cells were collected from a 20-cm segment during 2 hours. A total of 84.2% of the cells were positive for enterocyte-specific villin, and only 1.6% of the collected cells were positive for CD45. The majority of cells (65.3%) were not in early or late apoptosis or necrosis. In all volunteers, drug-metabolizing enzymes (such as members of the cytochrome P450 family) could be detected as both messenger ribonucleic acid and proteins. Consistent with expression data, formation of verapamil metabolites catalyzed by CYP3A4 and CYP2C was shown.


CONCLUSIONS
The majority of shed human enterocytes collected with a multilumen perfusion catheter were still functionally active and not apoptotic. Harvesting of spontaneously shed enterocytes provides a new tool for studies on expression and function of intestinal proteins.
Glaeser
Hartmut
H
Dr Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany.
Drescher
Siegfried
S
van der Kuip
Heiko
H
Behrens
Christoph
C
Geick
Anke
A
Burk
Oliver
O
Dent
John
J
Somogyi
Andrew
A
Von Richter
Oliver
O
Griese
Ernst-Ulrich
EU
Eichelbaum
Michel
M
Fromm
Martin F
MF
D000328:Adult; D002121:Calcium Channel Blockers / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D020895:Enterocytes / Q000378:metabolism / Q000502:physiology*; D005260:Female; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005838:Genotype; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007583:Jejunum / Q000201:enzymology / Q000502:physiology*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011110:Polymorphism, Genetic; D014700:Verapamil / Q000378:metabolism
D002121:Calcium Channel Blockers; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; C090519:PTPRCAP protein, human; D010750:Phosphoproteins; C026615:villin; D003577:Cytochrome P-450 Enzyme System; D014700:Verapamil; D016650:Fluorescein-5-isothiocyanate