Gene ablation for PEPT1 in mice abolishes the effects of dipeptides on small intestinal fluid absorption, short-circuit current, and intracellular pH.
PEPT1 function in mouse intestine has not been assessed by means of electrophysiology and methods to assess its role in intracellular pH and fluid homeostasis. Therefore, the effects of the dipeptide glycilsarcosin (Gly-Sar) on jejunal fluid absorption and villous enterocyte intracellular pH (pH(i)) in vivo, as well as on enterocyte[(14)C]Gly-Sar uptake, short-circuit current (I(sc)) response, and enterocyte pH(i) in vitro were determined in wild-type and PEPT1-deficient mice and in mice lacking PEPT1. Immunohistochemistry for PEPT1 failed to detect any protein in enterocyte apical membranes in Slc15a1(-/-) animals. Saturable Gly-Sar uptake in Slc15a1(-/-) everted sac preparations was no longer detectable. Similarly, Gly-Sar-induced jejunal I(sc) response in vitro was abolished. The dipeptide-induced increase in fluid absorption in vivo was also abolished in animals lacking PEPT1. Since PEPT1 acts as an acid loader in enterocytes, enterocyte pH(i) was measured in vivo by two-photon microscopy in SNARF-4-loaded villous enterocytes of exteriorized jejuni in anesthetized mice, as well as in BCECF-loaded enterocytes of microdissected jejunal villi. Gly-Sar-induced pH(i) decrease was no longer observed in the absence of PEPT1. A reversal of the proton gradient across the luminal membrane did not significantly diminish Gly-Sar-induced I(sc) response, whereas a depolarization of the apical membrane potential by high K(+) or via Na(+)-K(+)-ATPase inhibition strongly diminished Gly-Sar-induced I(sc) responses. This study demonstrates for the first time that proton-coupled electrogenic dipeptide uptake in the native small intestine, mediated by PEPT1, relies on the negative apical membrane potential as the major driving force and contributes significantly to intestinal fluid transport.
Chen
Mingmin
M
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany.
Singh
Anurag
A
Xiao
Fang
F
Dringenberg
Ulrike
U
Wang
Jian
J
Engelhardt
Regina
R
Yeruva
Sunil
S
Rubio-Aliaga
Isabel
I
Nässl
Anna-Maria
AM
Kottra
Gabor
G
Daniel
Hannelore
H
Seidler
Ursula
U
D000818:Animals; D001692:Biological Transport; D001826:Body Fluids / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism* / Q000494:pharmacology; D020895:Enterocytes / Q000187:drug effects / Q000378:metabolism*; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007408:Intestinal Absorption / Q000187:drug effects; D007583:Jejunum / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D042282:Microdissection; D036641:Microscopy, Fluorescence, Multiphoton; D000074011:Peptide Transporter 1; D011188:Potassium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D027981:Symporters / Q000172:deficiency* / Q000235:genetics
D004151:Dipeptides; D000074011:Peptide Transporter 1; C489941:Slc15a1 protein, mouse; D027981:Symporters; C004194:glycylsarcosine; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium