Báo cáo khoa học: Transport of the phosphonodipeptide alafosfalin by the H+/peptide cotransporters PEPT1 and PEPT2 in intestinal and renal epithelial cells doc

Transport of the phosphonodipeptide alafosfalin by the H
+
/peptide
cotransporters PEPT1 and PEPT2 in intestinal and renal epithelial cells
Jana Neumann
1
, Mandy Bruch
1
, Sabine Gebauer
2
and Matthias Brandsch
1
1
Membrane Transport Group, Biozentrum and
2
Institute of Biochemistry, Department of Biochemistry/Biotechnology,
Martin-Luther-University Halle-Wittenberg, Halle, Germany
The interaction of the antibacterial phosphonodipeptide
alafosfalin with mammalian H
+
/peptide cotransporters was
studied in Caco-2 cells, expressing the low-affinity intestinal
type peptide transporter 1 (PEPT1), and SKPT cells,
expressing the high-affinity renal type peptide transporter 2
(PEPT2). Alafosfalin strongly inhibited the uptake of
[
14
C]glycylsarcosine with K
i
values of 0.19 ± 0.01 m
M

and
0.07 ± 0.01 m
M
for PEPT1 and PEPT2, respectively. Sat-
uration kinetic studies revealed that in both cell types ala-
fosfalin affected only the affinity constant (K
t
) but not the
maximal velocity (V
max
) of glycylsarcosine (Gly-Sar) uptake.
The inhibition constants and the competitive nature of
inhibition were confirmed in Dixon-type experiments. Caco-
2 cellsand SKPT cells were also cultured on permeable filters:
apical uptake and transepithelial apical to basolateral flux of
[
14
C]Gly-Sar across Caco-2 cell monolayers were reduced by
alafosfalin (3 m
M
) by 73%. In SKPT cells, uptake of
[
14
C]Gly-Sar but not flux was inhibited by 61%.We found no
evidence for an inhibition of the basolateral to apical uptake
or flux of [
14
C]Gly-Sar by alafosfalin. Alafosfalin (3 m
M
)did

not affect the apical to basolateral [
14
C]mannitol flux.
Determined in an Ussing-type experiment with Caco-2 cells
cultured in Snapwells
TM
, alafosfalin increased the short-
circuit current through Caco-2 cell monolayers. We conclude
that alafosfalin interacts with both H
+
/peptide symporters
and that alafosfalin is actively transported across the intes-
tinal epithelium in a H
+
-symport, explaining its oral avail-
ability. The results also demonstrate that dipeptides where
the C-terminal carboxyl group is substituted by a phosphonic
function represent high-affinity substrates for mammalian
H
+
/peptide cotransporters.
Keywords: alafosfalin; alaphosphin; Caco-2 cells; SKPT
cells; Ussing technique.
Alafosfalin (alaphosphin,
L
-alanyl-
L
-1-aminoethylphos-
phonic acid) is an antibacterial dipeptide analogue where
the carboxyl group at the C-terminal alanine is replaced

with a phosphonic [P(O)(OH)
2
] function. The compound
was one of the most promising aminophosphonic acids
obtained in an extensive study synthesizing more than
300 di- to penta-peptide alanine mimetics with varying
stereometry and different substituents for the C-terminal
carboxyl function [1]. It displays good oral availability,
substantial antibacterial activity mainly against Gram-
negative bacteria and synergism with b-lactam antibiotics
[1–5]. In clinical studies alafosfalin was tested for the
treatment of gastrointestinal [3] and urinary tract infections
[2,4]. Studies demonstrated the competitive effect of food
on its enzymatic breakdown in the intestinal lumen [6]. In
a recent publication Kafarski & Lejczak [7] reviewed the
potential medical importance of aminophosphonic acids
and conclude that, due to their negligible mammalian
toxicity and the fact that they very efficiently mimic
aminocarboxylic acids making them extremely important
antimetabolites, aminophosphonic acids play an important
role in drug development. Very recently Tsopelas and
coworkers assessed [
99m
Tc]alafosfalin as an infection ima-
ging agent and concluded that its distribution characteristics
are advantageous in imaging abdominal and soft tissue
infections [8]. Moreover, alafosfalin has been used as a
selective agent in bacterial growth media for isolation of
strong pathogens like Salmonella [9,10].
Several authors have studied bacterial uptake and

intracellular metabolism of phosphonopeptides. They have
demonstrated the uptake of alafosfalin into bacteria by
peptide permeases located in the cytoplasmic membrane
[1,11–14]. However, there are no detailed studies regarding
the specific uptake mechanism at mammalian cells for
phosphonodipeptides such as alafosfalin.
We hypothesized that these compounds, even though
they do not possess a carboxyl group at the C-terminus, are
substrates for the mammalian peptide transporters PEPT1
and PEPT2. These carriers are not only responsible for the
uptake of nutritional di- and tripeptides across the intestinal
and renal epithelium and in other cell types [15–18], they
also accept several pharmacologically relevant peptidomi-
metics as substrates such as many b-lactam antibiotics,
enzyme inhibitors and d-aminolevulinic acid [17–23]. In the
present study we used the intestinal cell line Caco-2 which
exclusively expresses PEPT1 [22] and the renal SKPT
cell line which expresses PEPT2 but not PEPT1 [22,24,25]
to investigate interaction and transport of alafosfalin with
mammalian peptide transporters.
Correspondence to M. Brandsch, Biozentrum, Martin-Luther-
University Halle-Wittenberg, Membrane Transport Group,
Weinbergweg 22, D-06120 Halle, Germany.
Fax: + 49 345 552 7258, Tel.: + 49 345 552 1630,
E-mail:
Abbreviations: Gly-Sar, glycylsarcosine; PEPT1, intestinal type
peptide transporter 1; PEPT2, renal type peptide transporter 2.
(Received 22 January 2004, revised 18 March 2004,
accepted 24 March 2004)
Eur. J. Biochem. 271, 2012–2017 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04114.x

Materials and methods
Materials
The human colon carcinoma cell line Caco-2 was obtained
from the German Collection of Microorganisms and Cell
Cultures (Braunschweig, Germany). The renal cell line
SKPT-0193 Cl.2, established from isolated cells of rat
proximal tubules, was provided by U. Hopfer (Case
Western Reserve University, Cleveland, OH, USA)
[24,25]. Cell culture media and most supplements were
purchased from Life Technologies, Inc. (Karlsruhe, Ger-
many). Fetal bovine serum was obtained from Biochrom
(Berlin, Germany). Insulin, dexamethasone and glycylsar-
cosine (Gly-Sar) were from Sigma Chemie (Deisenhofen,
Germany).
D
-[1-
14
C]Mannitol (specific radioactivity,
56 mCiÆmmol
)1
) and [glycine-1-
14
C]glycylsarcosine
([
14
C]Gly-Sar; specific radioactivity, 53 mCiÆmmol
)1
)were
supplied from Amersham Biosciences UK, Ltd (Little
Chalfont, UK). Bovine apo-transferrin was purchased from

ICN Biomedicals (Eschwege, Germany).
L
-Alanyl-
L
-
1-aminoethylphosphonic acid (alafosfalin) was from Fluka
(Buchs, Switzerland).
Cell culture
Cells were cultured in 75 cm
2
culture flasks as described
previously [20,24,26–28]. The intestinal cell line Caco-2 was
cultured in minimum essential medium supplemented with
nonessential amino acid solution (1%, v/v), fetal bovine
serum (10%, v/v) and gentamicin (45 lgÆmL
)1
). For uptake
studies, cells were subcultured in 35 mm disposable Petri
dishes (BD Biosciences, Heidelberg, Germany). With a
starting cell density of 83 000 cellsÆcm
)2
, cultures reached
confluence the next day. Uptake experiments were carried
out seven days after seeding. For flux studies, cells were
cultured on permeable polycarbonate TranswellÒ cell
culture inserts (24 mm diameter, 3 lmporesize;Corning
Costar Bodenheim GmbH, Germany) with a starting cell
density of 44 000 cellsÆcm
)2
for 21 days [20].

Culture medium for SKPT cells was Dulbecco’s modified
Eagle’s medium/F12 nutrient mixture (1 : 1, v/v) supple-
mented with fetal bovine serum (10%, w/v), gentamicin
(45 lgÆmL
)1
), epidermal growth factor (10 ngÆmL
)1
), insu-
lin (4 lgÆmL
)1
), dexamethasone (5 lgÆmL
)1
) and apotrans-
ferrin (5 lgÆmL
)1
). Cells were subcultured in Petri dishes
with a starting cell density of 83 000 cellsÆcm
)2
or in
TranswellÒ chambers with a seeding density of 133 000
cellsÆcm
)2
. Uptake and flux studies in SKPT cells were
performed four days after seeding.
Transport studies
Uptake of [
14
C]Gly-Sar was determined at room tempera-
ture [22,24,26,27]. The uptake buffer was 25 m
M

2-(N-morpholino)ethanesulfonic acid/tris(hydroxymethyl)
aminomethane (Mes/Tris, pH 6.0) with 140 m
M
NaCl,
5.4 m
M
KCl, 1.8 m
M
CaCl
2
,0.8m
M
MgSO
4
and 5 m
M
glucose. Uptake experiments were initiated by removing the
culture medium from the dishes, washing the cell mono-
layers twice with 1 mL buffer and adding 1 mL of uptake
buffer containing [
14
C]Gly-Sar and unlabeled compounds.
After incubation for the desired time (typically 10 min)
buffer was removed and the monolayers were quickly
washed four times with ice-cold uptake buffer, dissolved
and prepared for liquid scintillation spectrometry.
Transepithelial flux of [
14
C]mannitol and [
14

C]Gly-Sar
across cell monolayers cultured on permeable filters was
measured at 37 °C at pH 6.0 at the apical (1.5 mL) and 7.5
at the basolateral side (2.6 mL) [20]. After washing the
inserts with buffer, the TranswellÒ chambers were preincu-
bated for 10 min and the transepithelial electrical resistance
was measured. Flux experiments were started by adding
incubation buffer containing radiolabeled compounds
and/or alafosfalin to the donor side (apical or basolateral
compartment). At time intervals of 10, 30, 60 and 120 min,
samples were taken from the receiver compartment and
replaced with fresh buffer. After 2 h, the filters were quickly
washed twice with ice-cold uptake buffer and cut out of the
plastic inserts. Uptake and transepithelial flux of [
14
C]mann-
itol and [
14
C]Gly-Sar were quantified by liquid scintillation
counting. Protein was measured according to the procedure
of Bradford.
Short-circuit current measurement
Caco-2 cell monolayers cultured for 14 days in Snapwells
TM
(1.13 cm
2
,3lm pore size, starting cell seeding density
200 000 cells per well; Corning Costar GmbH) were moun-
ted in Ussing chambers (K. Mussler, Aachen, Germany),
kept at 37 °C and supplied with 5% CO

2
/95% O
2
(v/v).
Experiments were started by adding 5 mL incubation
buffer, pH 6.0, to the mucosal side and 5 mL buffer,
pH 7.5, to the serosal side. An automated voltage/current
clamp apparatus with Ag/AgCl electrodes was used for
continuous monitoring of tissue potential difference, current
and tissue resistance. Chambers were short-circuited 15 min
after mounting the cells. After a further 5–6 min, alafosfalin
was added by replacing 1 mL buffer at the mucosal side by
1mLofa35m
M
solution of alafosfalin (pH 6.0) to obtain
a final alafosfalin concentration of 7 m
M
. Currents with a
resolution of 1 lAÆcm
)2
were recorded every 6 s for a time
period of 5 min.
Data analysis
Each experimental point represents the mean ± SE of three
to six measurements. Inhibition constants (K
i
)werecalcu-
lated from IC
50
values as described previously [24,26].

Flux data were calculated after correction for the amount
removed by linear regression of appearance rates in the
receiver well vs. time [20]. Statistical analysis was performed
by the two-tailed nonparametric U-test. A P <0.05was
considered significant.
Results and Discussion
Affinity of alafosfalin for PEPT1 and PEPT2
Caco-2 and SKPT cell cultures are well established systems
for intestinal or renal peptide transport studies. It has been
unequivocally shown in several investigations, by functional
studies, RT-PCR, Northern blot analyses and using
antibodies, that Caco-2 cells express the low-affinity, high-
Ó FEBS 2004 Alafosfalin transport by H
+
/peptide transporters (Eur. J. Biochem. 271) 2013
capacity (ÔintestinalÕ) type peptide transport system PEPT1
whereas SKPT cells express the high-affinity, low-capacity
(ÔrenalÕ) type system PEPT2 but not PEPT1 [22,24,25]. In
the present investigation, we first determined the inhibition
of [
14
C]Gly-Sar uptake by alafosfalin. Gly-Sar is used as the
reference substrate for peptide transport studies because of
its relatively high enzymatic stability [20,24–27]. Alafosfalin
strongly inhibited [
14
C]Gly-Sar (10 l
M
) uptake in both cell
lines. From the inhibition curves shown in Fig. 1, IC

50
values were calculated, i.e. the alafosfalin concentration
necessary to inhibit the carrier-mediated [
14
C]Gly-Sar
uptake by 50%. From the resulting IC
50
values the apparent
K
i
values of alafosfalin were calculated using K
t
values of
Gly-Sar transport of 580 l
M
for PEPT1 at Caco-2 cells and
71 l
M
for PEPT2 at SKPT cells as described previously
[24,26] (see below). Alafosfalin showed a remarkably high
affinity to both carriers: K
i
values of 194 ± 13 l
M
for
PEPT1 and 74 ± 10 l
M
for PEPT2 were obtained. For
comparison, the inhibition constants (K
i

) of Ala-Ala are
80 ± 10 l
M
for PEPT1 and 6.3 ± 0.3 l
M
for PEPT2 and
those of Ala-Lys are 210 ± 20 l
M
for PEPT1 and
11.7 ± 0.1 l
M
for PEPT2, respectively [24,27,28].
Next, uptake of [
14
C]Gly-Sar (10 l
M
)wasmeasuredfor
10 min at pH 6.0 at two different Gly-Sar concentrations
(50 and 500 l
M
for Caco-2 cells, 20 and 50 l
M
for SKPT
cells) in the presence of increasing amounts of alafosfalin.
The linear, nonmediated transport components, i.e. simple
diffusion plus tracer binding, of 19.2% and 5.1% were
determined with an excess amount of unlabeled Gly-Sar
(31.6 m
M
Gly-Sar for Caco-2 cells and 20 m

M
Gly-Sar for
SKPT cells) and subtracted from total uptake rates. The
results are presented as Dixon plots (Fig. 1 insets). They
reveal linearity at both Gly-Sar concentrations (r
2
values
> 0.99) with lines intersecting above the abscissa in the
forth quadrant as expected for a competitive inhibitor. For
alafosfalin, K
i
values of 140 l
M
for PEPT1 and 73 l
M
for
PEPT2 were calculated from the point of intersection. In a
third series of experiments, the effects of alafosfalin on the
kinetic parameters, K
t
and V
max
, of Gly-Sar uptake were
determined in both cell lines. The uptake of [
14
C]Gly-Sar
(10 l
M
) was measured at increasing concentrations of
unlabeled Gly-Sar in the absence and presence of alafosfalin

in the uptake medium at a fixed concentration of 260 l
M
(Caco-2) or 80 l
M
(SKPT). The Eadie–Hofstee plots
(uptake rate vs. uptake rate/substrate concentration, data
not shown) gave two straight lines for each cell type
intersecting near the ordinate. The maximal velocities of
Gly-Sar uptake (V
max
) in both cell lines were not affected by
alafosfalin. In contrast, the apparent affinity constants of
Gly-Sar uptake (K
t
) were strongly affected by alafosfalin; in
the presence of alafosfalin the K
t
value of Gly-Sar uptake
in Caco-2 cells was 1160 l
M
compared to 580 l
M
in the
absence of the inhibiting phosphonodipeptide. The same
alafosfalin effect was observed in SKPT cells where the V
max
values were 2.2 ± 0.1 nmolÆ(10 min
)1
)Æ(mg protein)
)1

in
the absence and 2.0 ± 0.2 nmolÆ(10 min
)1
)Æ(mg protein)
)1
in the presence of alafosfalin, but the K
t
value of Gly-Sar
uptake was doubled at the alafosfalin concentration close to
its K
i
value from (K
t
¼)71l
M
to 138 l
M
. Both types of
experiments show that the inhibition of Gly-Sar uptake by
alafosfalin is of the competitive type.
The disappearance of alafosfalin from the luminal
compartment during these experiments was determined by
HPLC after incubating the cells for 10, 30, 60 and 120 min
with alafosfalin at a concentration of 1 m
M
. The recovery
rates were 93 ± 0.7% (Caco-2) and 96 ± 0.3% (SKPT)
after 10 min and 81 ± 3% (Caco-2) and 90 ± 0.8%
(SKPT) after 2 h, respectively.
Effect of alafosfalin on transepithelial [

14
C]Gly-Sar flux
PEPT1 and PEPT2 are expressed in the apical membranes
of intestinal (PEPT1) and renal (PEPT1 or PEPT2)
epithelial cells. Absorption of intact di- and tri-peptides
and related mimetics at the intestinal epithelium or their
reabsorption at the renal epithelium requires both their
uptake from the luminal compartment into the cells and
their transport (efflux) across the basolateral membranes to
Fig. 1. Inhibition of [
14
C]Gly-Sar uptake by alafosfalin in Caco-2 cells
(A) and SKPT cells (B). Uptake of 10 l
M
[
14
C]Gly-Sar was measured
at pH 6.0 for 10 min in confluent cell monolayers in the presence of
increasing concentrations of unlabeled alafosfalin (Caco-2: 0–10 m
M
,
SKPT: 0–3.16 m
M
). Uptake of [
14
C]Gly-Sarmeasuredintheabsence
of the inhibitor [Caco-2: 215.3 ± 18.2 pmolÆ(10 min)
)1
Æ(mg pro-
tein)

)1
, SKPT: 241.7 ± 32.1 pmolÆ(10 min)
)1
Æ(mg protein)
)1
]was
designated as 100% (n ¼ 4). Insets: Uptake rate of [
14
C]Gly-Sar
(10 l
M
, 10 min, pH 6.0) was measured at two different concentrations
of unlabeled Gly-Sar: 50 l
M
(d)and500l
M
(s)(Caco-2)and20l
M
(d)and50l
M
(s) (SKPT), respectively, in the presence of increasing
concentrations of alafosfalin (Dixon-plots).
2014 J. Neumann et al.(Eur. J. Biochem. 271) Ó FEBS 2004
the blood side. Basolateral peptide transport systems have
been described both at the intestine and in the kidney [25,
29–31]. Therefore, alafosfalin should not only inhibit the
uptake but also the transepithelial net flux of PEPT1 and
PEPT2 substrates. For such flux studies, Caco-2 and SKPT
cells were cultured on permeable TranswellÒ filters for
21 days (Caco-2) and four days (SKPT), respectively.

After these times, the transepithelial electrical resistances
reached maximal values of 803 ± 29 WÆcm
2
(Caco-2) and
452 ± 13 WÆcm
2
(SKPT). To rule out that alafosfalin has
any negative effect on the integrity of the monolayers, the
transepithelial [
14
C]mannitol flux (10 l
M
) was measured for
up to 2 h in the absence (control) and presence of 3 m
M
alafosfalin. At Caco-2 cells, the apical to basolateral
[
14
C]mannitol flux was 0.07–0.08 ± 0.005%Æcm
)2
Æh
)1
with-
outandwith3m
M
alafosfalin in the uptake medium. At
SKPT cells mannitol flux values of 0.28 ± 0.01%Æcm
)2
Æh
)1

in the absence and 0.27 ± 0.01%Æcm
)2
Æh
)1
in the presence
of 3 m
M
alafosfalin were obtained by linear regression of
flux data. Also, the mean [
14
C]mannitol uptake of
0.05 ± 0.004%Æcm
)2
Æ(2h)
)1
into both cell types was not
affected.
In contrast, alafosfalin (3 m
M
) strongly reduced the
transepithelial flux of [
14
C]Gly-Sar (10 l
M
) across Caco-2
cell monolayers from 165.8 ± 5.5 pmolÆcm
)2
Æh
)1
(¼ 1.11 ± 0.04%Æcm

)2
Æh
)1
) by 73% to 45.1 ± 0.7 pmolÆ
cm
)2
Æh
)1
(Table 1). [
14
C]Gly-Sar uptake into the cells was
also reduced by 73% (Table 1). In SKPT cells flux rates
were 45.1 ± 2.1 pmolÆcm
)2
Æh
)1
(¼ 0.3 ± 0.01%Æcm
)2
Æh
)1
)
in the absence and 45.7 ± 3.2 pmolÆcm
)2
Æh
)1
in the pres-
ence of 3 m
M
alafosfalin. Hence, in SKPT cells we did not
find an inhibitory effect of alafosfalin on the [

14
C]Gly-Sar
flux. The [
14
C]Gly-Sar uptake into the cells, however, was
strongly reduced by alafosfalin by 61% (Table 1).
We also studied basolateral uptake and transepithelial
basolateral to apical flux of Gly-Sar (10 l
M
). In Caco-2 cells,
[
14
C]Gly-Sar flux values were 22.9 ± 0.2 pmolÆcm
)2
Æh
)1
(¼ 0.09 ± 0.001%Æcm
)2
Æh
)1
) without and 19.3 ± 0.3 pmolÆ
cm
)2
Æh
)1
with 3 m
M
alafosfalin (Table 1). In SKPT cells,
the basolateral to apical [
14

C]Gly-Sar flux rates were
35.2 ± 2.5 pmolÆcm
)2
Æh
)1
(¼ 0.14 ± 0.009%Æcm
)2
Æh
)1
)
without and 37.1 ± 2.7 pmolÆcm
)2
Æh
)1
with 3 m
M
alafos-
falin, respectively. Moreover, uptake rates into the cells
were inhibited only insignificantly (Table 1). The results
show that in both cell types basolateral uptake and basolat-
eral to apical flux rates of Gly-Sar were very much lower
compared to the apical to basolateral transport and that the
basolateral [
14
C]Gly-Sar uptake in Caco-2 and SKPT cells
could not be inhibited by an excess amount of alafosfalin.
Hence, we found no evidence for the interaction of alafosfalin
with the putative basolateral peptide transporters.
Transport of alafosfalin – short-circuit current
measurements

Inhibition of PEPT1- and PEPT2-mediated Gly-Sar uptake
and flux by alafosfalin is important new information on
structural requirements of H
+
/peptide symporters. It shows
that phosphonodipeptides interact with mammalian PEPT1
and PEPT2 with high affinity. It does not necessarily mean
that the phosphonodipeptide is actually transported into
the cells. Interaction with PEPT1 and PEPT2, the fact that
bacterial permeases take up alafosfalin [11–14] and the
known oral availability of alafosfalin, can only be regarded
Table 1. Effect of alafosfalin on the transepithelial flux and the intracellular accumulation of [
14
C]Gly-Sar at Caco-2 and SKPT cell monolayers.
Transepithelial flux and uptake of 10 l
M
[
14
C]Gly-Sar from apical to basolateral (J
a-b
) and from basolateral to apical (J
b-a
) side, respectively, was
measured in the absence (control) and presence of 3 m
M
alafosfalin (n ¼ 4–6).
Caco-2 SKPT
Flux
pmolÆcm
)2

Æh
)1
%
Uptake
pmolÆcm
)2
Æ(2h)
)1
%
Flux
pmolÆcm
)2
Æh
)1
%
Uptake
pmolÆcm
)2
Æ(2h)
)1
%
J
a-b
Control 165.8 ± 5.5 100 135.6 ± 2.7 100 45.1 ± 2.1 100 9.6 ± 1.3 100
Alafosfalin 45.1 ± 0.7 27 36.1 ± 1.0 27 45.7 ± 3.2 101 3.7 ± 0.4 39
J
b-a
Control 22.9 ± 0.2 14 7.4 ± 0.2 5.4 35.2 ± 2.5 78 2.8 ± 0.1 29
Alafosfalin 19.3 ± 0.3 12 6.2 ± 0.3 4.6 37.1 ± 2.7 82 2.6 ± 0.1 27
Fig. 2. Stimulatory effect of alafosfalin (7 m

M
) on the short-circuit
current (I
sc
) across Caco-2 cells. Alafosfalin was added 20 min after
mounting the Caco-2 Snapwell
TM
inserts in the Ussing chambers. pH
values were 6.0 at the apical (mucosal) side and 7.5 at the basolateral
(serosal) side. Inset: Total increase of short-circuit current by alafos-
falin (7 m
M
, n ¼ 3), Gly-Sar (10 m
M
, n ¼ 6) and phenylalanine
(10 m
M
, n ¼ 3).
Ó FEBS 2004 Alafosfalin transport by H
+
/peptide transporters (Eur. J. Biochem. 271) 2015
as circumstantial evidence for actual transport. Therefore,
we exploited the electrogenic nature of H
+
/peptide sym-
porters in an Ussing-type of experiment to obtain more
direct evidence. Figure 2 shows the inwardly directed short-
circuit current (I
sc
) across Caco-2 cell monolayers cultured

in SnapwellsÒ and mounted in Ussing chambers. Current
increased rapidly after addition of alafosfalin (7 m
M
)tothe
apical (luminal) compartment by 4.3 ± 0.3 lAÆcm
)2
whereas transepithelial resistance of 175 ± 2 WÆcm
2
remained unchanged. As expected, Gly-Sar but not phenyl-
alanine (both 10 m
M
) also induced an increase in current
(DI
sc
¼ 3.4 ± 0.2 lAÆcm
)2
, Fig. 2 inset). Thwaites and
coworkers reported that net Gly-Sar transport (20 m
M
)
was associated with an increase of the inward short-circuit
current DI
sc
of 6 lAÆcm
)2
[29].
We conclude that the antibacterial phosphonodipeptide
alafosfalin interacts with both mammalian H
+
/peptide

symporters with high affinity. PEPT1 and PEPT2 do not
seem to differentiate very much between a dipeptide and its
derivative where the C-terminal carboxyl group is substi-
tuted by a phosphonic function. Moreover, alafosfalin is
transported electrogenically across Caco-2 cell monolayers,
most likely in a H
+
-symport. This would explain the high
oral availability of small antibacterial peptides of this type.
Phosphonodipeptides are interesting compounds for studies
on structure-affinity relationships of substrates for PEPT1
and PEPT2.
Acknowledgements
This study was supported by Land Sachsen-Anhalt grant 3505 A/
0403 L and by the Federal Ministry of Education and Research grant
# BMBF 0312750 A. We thank Ingelore Hamann for excellent
technical assistance and Prof. Martin Luckner (BioService Halle
GmbH) for his support. This work will form part of the doctoral
thesis of J. N.
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