COMMUNICATION
Peptide Mimics as Substrates for the Intestinal Peptide
Transporter*
Catherine S.
Temple
,
Andrew K.
Stewart
§¶,
David
Meredith
,
Norma A.
Lister
,
Keith M.
Morgan**,
Ian D.
Collier**,
Richard D.
Vaughan-Jones§,
C. A. R.
Boyd

,
Patrick D.
Bailey**, and
J. Ramsey
Bronk
From the
Department of Human Anatomy, University of
Oxford, South Parks Road, Oxford OX1 3QX, § University
Laboratory of Physiology, Parks Road, Oxford OX1 3PT,
Department
of Biology, University of York, P.O. Box 373, York YO1 5YW, and
** Department of Chemistry, Heriot-Watt University, Edinburgh EH14 4AS,
United Kingdom
 |
ABSTRACT |
4-Aminophenylacetic acid (4-APAA), a peptide
mimic lacking a peptide bond, has been shown to interact with a
proton-coupled oligopeptide transporter using a number of different
experimental approaches. In addition to inhibiting transport of labeled
peptides, these studies show that 4-APAA is itself translocated.
4-APAA transport across the rat intact intestine was stimulated 18-fold
by luminal acidification (to pH 6.8) as determined by high performance
liquid chromatography (HPLC); in enterocytes isolated from mouse small
intestine the intracellular pH was reduced on application of 4-APAA, as
shown fluorimetrically with the pH indicator carboxy-SNARF; 4-APAA
trans-stimulated radiolabeled peptide transport in
brush-border membrane vesicles isolated from rat renal cortex; and in
Xenopus oocytes expressing PepT1, 4-APAA produced
trans-stimulation of radiolabeled peptide efflux, and as
determined by HPLC, was a substrate for translocation by this transporter.
These results with 4-APAA show for the first time that the presence of
a peptide bond is not a requirement for rapid translocation through the
proton-linked oligopeptide transporter (PepT1). Further investigation
will be needed to determine the minimal structural requirements for a
molecule to be a substrate for this transporter.
 |
INTRODUCTION |
The rapid uptake of intact small peptides across the brush-border
membrane of the small intestinal epithelium is the major route for
absorption of dietary protein
-amino nitrogen (1). Hitherto, it has
been thought that a number of chemical features, for example free amino
and carboxyl termini, are essential in contributing to substrate
interaction with, and translocation through, the intestinal peptide
transporter. These features include the presence of a peptide bond
within the substrate molecules. Indeed a major review (1) states that
"it is the presence of peptide bonds which make di- and
tripeptide acceptable to the peptide transport systems." Although
previous work (e.g. Ref. 2) has shown that molecules lacking
this feature can inhibit transport of peptides (presumably by substrate
binding), we describe here for the first time rapid transport of a
small totally non-peptidic substrate through the intestinal peptide
transporter. The substrate, 4-aminophenylacetic acid
(4-APAA),1 was selected on
the basis of its chemical structure, it being a potential mimic of a
dipeptide (D-Phe-L-Ala) (Fig.
1) which previously we have shown to be
an excellent substrate for epithelial peptide transport (3, 4).

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Fig. 1.
Chemical structure of a dipeptide (where
R1 and R2 are amino
acid side chains) (A) and 4-aminophenylacetic acid (4-APAA) (B). 4-APAA was designed to mimic the spatial
configuration of a dipeptide. It has the terminal amino and carboxyl
groups in the transconfiguration and is planar, but it does not have a
peptide bond.
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EXPERIMENTAL PROCEDURES |
Rat renal brush-border membrane vesicles were prepared as
described previously (5), and initial rates of labeled peptide transport (influx, efflux) were determined by rapid filtration (4, 6).
Rat intestinal loops in vitro and vascularly perfused small
intestine in situ were used to measure transepithelial
fluxes in the intact small intestine as described previously (3, 7). Luminal pH was changed using a previously published protocol (8). Isolated murine enterocytes were prepared by enzymatic digestion using
haluronidase, and intracellular pH was determined fluorimetrically using carboxy-SNARF (9). Isolated cells were exposed to 20 mM HEPES-buffered Krebs solution containing either the
dipeptide (Phe-Ala) or 4-APAA. The intracellular ratiometric signal
(540 nm excitation, 590 nm/640 nm emission) was calibrated after the experiments by superfusing solutions of 140 mM KCl
containing 10 µM nigericin and buffered to pH 5.5, 7.5, and 9.5 with PIPES, HEPES, and CAPS, respectively. Xenopus
oocyte expression of PepT1 cRNA was as described (10, 11) with
subsequent HPLC detection (3) of transported substrate and correction
for transport in water-injected controls. For efflux assays 4.6 nl (110 fmol) of radiolabeled dipeptide (D-Phe-L-Gln,
0.1 µCi/µl) was injected into each oocyte. The assay was started
after the oocytes were washed, 15 min after injection.
Inhibition constants (Ki) were determined as
described previously (6). Briefly radiolabeled dipeptide influx was corrected for non-mediated transport by subtracting the transport of
radiolabel seen in the presence of saturating unlabeled peptide (10 mM). The ENZFITTER program was then used to calculate the Ki for mediated transport only.
 |
RESULTS |
As part of a screening assay for potential substrates for
epithelial peptide transport we used a preparation from rat renal cortex of apical brush-border membrane vesicles (5). Fig.
2 shows, using this assay, that 4-APAA is
capable both of cis-inhibition and
trans-stimulation of labeled
D-Phe-L-Glu influx and efflux, respectively.
Because in the efflux assay there is no proton electrochemical gradient
(the membrane being voltage-clamped using valinomycin and high
K+ in both intra- and extravesicular compartments and there
being no pH gradient across the membrane) the ability of 4-APAA to
trans-stimulate efflux of peptide must mean that it is
translocated as a substrate (12). Based on this initial observation,
three types of experiments were performed which functionally measure
transport of this molecule in relation to intestinal absorption.

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Fig. 2.
A, cis inhibition of mediated
D-Phe-L-Glu (0.34 µM) influx,
under initial rate, into rat renal cortex brush-border membrane vesicles by 4-APAA. 4-APAA inhibited peptide entry in a
concentration-dependent manner with a Ki
of 6.5 mM. B, trans-stimulation of
labeled D-Phe-L-Glu (0.34 µM)
efflux from rat renal cortex brush-border membrane vesicles in the
presence and absence of external substrates. The concentrations of
external D-Phe-L-Glu (0.48 mM) and
of 4-APAA (13 mM) were twice the estimated
Ki for cis-inhibition of influx. Efflux
was measured (with external and internal pH 5.5 and membrane potential
clamped to zero) over 4 s (i.e. under initial rate
conditions). Data points are shown for control (no external substrate)
and in the presence of peptide or 4-APAA. Note that peptide efflux in
both cases is significantly greater than control (p < 0.02, paired Student's t-test).
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Using isolated rat small intestine in vitro, 4-APAA
was shown using HPLC detection to be transported rapidly across the
intact epithelium at a rate which was very substantially increased by external acidification (pH of lumen decreased from 7.4 to 6.8) characteristic of proton-coupled peptide transport (Table
I). Moreover associated with this
stimulation of transepithelial transport there was "active
accumulation" of 4-APAA within the epithelium to a concentration 3.7 times greater than that in the lumen (Table I). In the intact small
intestine of the rat vascularly perfused in situ there was
also rapid transepithelial transport (at a rate faster than that seen
for the peptide D-Phe-L-Gln); in this
preparation transport of 4-APAA was inhibited significantly by
concomitant addition of 10 mM
D-Phe-L-Gln. Conversely, transport of
D-Phe-L-Gln was inhibited significantly by the
addition of 10 mM 4-APAA (Table I).
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Table I
Transport of peptides and 4-APAA across intact small intestinal
epithelium (rat)
Small intestinal loops were used in vitro to measure the
initial rate of transport from lumen to serosa of peptide or 4-APAA (both at 1 mM luminal concentration). Transport was studied
at pH 7.4 or 6.8 in the lumen as previously described. Note the
stimulation of 4-APAA transport following acidification and the
associated increased accumulation of 4-APAA within the tissue (to a
final concentration 3.7 times greater than that present in the lumen).
Intestinal absorption (nanomoles/min/g dry wt) of peptide
(D-Phe-L-Gln) and 4-APAA was measured by the
vascularly perfused rat small intestine (7). The luminal pH was 6.8, and substrates were perfused through the lumen (single pass) at a
concentration of 1 mM. For inhibition studies, a steady
state rate of transport was established, at 20 min the indicated
inhibitor (10 mM) was added to the lumen and the new
steady-state rate of transport was determined. Transport of substrates
was determined by HPLC analysis of the portal venous effluent.
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When isolated intestinal enterocytes were superfused with a solution
containing Phe-Ala the peptide caused intracellular acidification (as
expected for a substrate for a proton-coupled transporter (10)) (Fig.
3); applied separately, the isostere
4-APAA (2 mM) had the same effect. However in the presence
of a maximal concentration of peptide (20 mM), 4-APAA was
unable to produce further intracellular acidification; similarly in the
presence of a high concentration of 4-APAA (20 mM), the
initial rate and extent of intracellular acidification in response to
the peptide was markedly reduced. These findings strongly suggest that
the peptide and 4-APAA share the same route for influx and that this
pathway is proton-coupled. (Note that at high concentrations 4-APAA
acted as a weak acid (13) producing transient intracellular
acidification and alkalinization following, respectively, its addition
to and removal from the superfusate.)

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Fig. 3.
Effect of dipeptide and 4-APAA separately and
in combination on intracellular pH measured in mouse isolated
enterocytes. Intracellular pH was measured using carboxy-SNARF. In
the presence of supramaximal concentration (20 mM) of
dipeptide no further acidification was induced by the addition of (2 mM) 4-APAA, although on its own 4-APAA caused (reversible)
acidification. At higher concentrations (20 mM) 4-APAA
acted as a weak acid producing transient intracellular acidification
and alkalinization following, respectively, its addition to and removal
from the superfusate. In the presence of 20 mM 4-APAA,
dipeptide failed to elicit additional acidification.
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Fig. 4 directly confirms that 4-APAA is a
substrate for the intestinal peptide transporter PepT1 when this
protein is expressed in Xenopus oocytes. This figure shows
that 4-APAA (measured by HPLC) is transported into PepT1-expressing
oocytes, that 4-APAA inhibits peptide
(D-Phe-L-Gln) uptake into PepT1-expressing
oocytes, and that radiolabeled peptide
(D-Phe-L-Gln) efflux from PepT1-expressing (but
not from water-injected control) oocytes is trans-stimulated by 4-APAA as well as by peptide.

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Fig. 4.
Transport of peptide and 4-APAA into
PepT1-expressing Xenopus oocytes. A, the time
course of 4-APAA (2 mM) influx at pH0 = 6.0 as
determined by HPLC of individual oocytes. Data points are mean ± S.E. (n = 5). Transport into control (water-injected) oocytes has been subtracted. B, 4-APAA inhibition of
mediated D-Phe-L-Gln influx into
PepT1-expressing oocytes. Transport measurements were determined under
initial rate conditions (1 h) at pH0 = 6.0. The
Ki for 4-APAA is approximately 10 mM.
C, trans-stimulation of labeled
D-Phe-L-Gln efflux from Xenopus
oocytes expressing PepT1 in the presence and absence of external
substrate (D-Phe-L-Gln or 4-APAA). The external
pH was 6.0. Data points are mean ± S.E. (n = 5).
The data are significantly different from control *, p < 0.002 for the peptide and for the 4-APAA in PepT1-injected (cross-hatched bars) but not in water-injected (open
bars) controls. Injected oocytes were studied 3 days after cRNA
injection. The concentrations of peptide (10 mM) and 4-APAA
(20 mM) were chosen to be saturating (peptide) and
approximately twice the Ki (4-APAA) for
cis-inhibition of influx (cf. panel B).
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 |
DISCUSSION |
The results from these transport assays demonstrate unambiguously
that the presence of a peptide bond is not necessary for the rapid
translocation of a substrate through the intestinal peptide transporter
(PepT1). Our study leaves open the question as to the minimum
requirements for a molecule to be a substrate. In the case of 4-APAA
the fact that this molecule has both amino- and carboxyl-terminals
separated by 5 carbon atoms as well as being planar (through its
possession of an aromatic ring) may be pertinent. Nevertheless the
findings reported here offer scope for chemical design of drugs which,
despite lacking a peptide bond, will be absorbed rapidly from the small
intestine via this transporter.
 |
ACKNOWLEDGEMENT |
We are grateful to M. A. Hediger for the
PepT1 clone.
 |
FOOTNOTES |
*
This work was supported by the Wellcome Trust.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Medical Research Council training scholar.

To whom correspondence should be addressed. Tel.:
44-1865-272154; Fax: 44-1865-272420; E-mail:
richard.boyd{at}anat.ox.ac.uk.
1
The abbreviations used are: 4-APAA,
4-aminophenylacetic acid; HPLC, high performance liquid chromatography;
PIPES, 1,4-piperazinediethanesulfonic acid; CAPS,
3-(cyclohexylamino)propanesulfonic acid.
 |
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