Proton-Driven Dipeptide Uptake in Primary Cultured
Rabbit Conjunctival Epithelial Cells
Sujit K. Basu,1 Ian S. Haworth,1 Michael B. Bolger,1 and Vincent H. L Lee1'2
characterize proton-driven carrier-mediated dipeptide uptake in primary cultured
conjunctival epithelial cells of the pigmented rabbit using j3-alanyl-L-histidine (L-carnosine) as a
model dipeptide substrate.
PURPOSE. TO
METHODS. Uptake of tritiated L-carnosine was monitored using conjunctival epithelial cells on days
6 through 8 in culture on a filter support. The structural features of dileucine stereoisomers and
cephalexin contributing to interaction with the dipeptide transporter were evaluated by computer
modeling and inhibition of tritiated L-carnosine uptake.
RESULTS. Uptake
of L-carnosine by primary cultured conjunctival epithelial cells in the presence of
an inwardly directed proton gradient showed directional asymmetry (favoring apical uptake by a
factor of five), temperature dependence, and saturability correlated with substrate concentration,
with a Michaelis-Menten constant (KJ of 0.3 ± 0.03 mM and a maximum uptake rate (Y ) of
22.0 ±1.0 picomoles per milligram protein per minute. L-Carnosine uptake was optimal at pH 6.0
and was reduced by 60% and 35%, respectively, by 50 /mMp-trifluoromethoxyphenylhydrazone (a
proton ionophore) and by acid preloading with 50 mM NH4C1. The constituent amino acids did not
inhibit L-carnosine uptake. L-Carnosine uptake was inhibited, however, from 50% to 80% by other
dipeptides and structurally similar drugs such as bestatin, j3-lactam antibiotics, and angiotensinconverting enzyme inhibitors. The LL, LD, or DL forms of the dipeptide Leu-Leu inhibited tritiated
L-carnosine uptake by approximately 60%, 40%, and 70%, respectively. By contrast, the DD form did
not inhibit uptake. Results from computer modeling suggest that an appropriate dipeptide Nterminal to C-terminal distance and a favorable orientation of the side chains may be important for
svibstrate interaction with the conjunctival dipeptide transporter.
CONCLUSIONS. Uptake of the dipeptide L-carnosine in primary cultured pigmented rabbit conjunctival
epithelial cells is probably mediated by a proton-driven dipeptide transporter. This transporter may
be used for optimizing the uptake of structurally similar peptidomimetic drugs. (Invest Ophthalmol
Vis Set. 1998;39:2365-2373)
ipeptide transporters are proton-coupled transport
systems that mediate the absorption of dipeptides and
tripeptides in the intestinal1'2 and renal3'4 epithelial
cells. Since the isoforms PepTl,5'6 PepT2,78 and PHT19 were
cloned, dipeptide transporters have been the focus of intensive
research in drug delivery. This interest is because of their
essential role in the absorption of several important peptidomimetic drugs, including j3-lactam antibiotics, 310 " angiotensin-converting enzyme (ACE) inhibitors,1213 renin inhibitors,14
and the antitumor drug bestatin.1516 Several of these drugs
D
From the 'Departments of Pharmaceutical Sciences and Ophthalmology, Schools of Pharmacy and Medicine, University of Southern
California, Los Angeles.
Supported in part by Grant EY10421 (VHLL) from the National
Institutes of Health, Bethesda, Maryland; the Gavin S. Herbert Professorship (VHLL); the PhRMA Undergraduate Fellowship (VHLL) from
the Pharmaceutical Research and Manufacturers of America Foundation, Inc., Washington, DC; and the USP Fellowship (SKB) from the
United States Pharmacopeia Inc., Rockville, Maryland.
Submitted for publication March 23, 1998; revised June 18, 1998;
accepted July 31, 1998.
Proprietary interest category: N.
Reprint requests: Vincent H. L. Lee, Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California,
1985 Zonal Avenue, Los Angeles, CA 90033Investigative Ophthalmology & Visual Science, November 1998, Vol. 39, No. 12
Copyright © Association for Research in Vision and Ophthalmology
Downloaded From: http://iovs.arvojournals.org/ on 06/17/2017
may also be important in the treatment of such diseases as
ocular bacterial infections17 and glaucoma.18
Work in our laboratory has revealed that the rabbit conjunctiva is permeable to a variety of molecules, including cidofovir,19 j8-adrenergic antagonists,20 arginine vasopressin,21
and fluorescein isothiocyanate-labeled dextrans ranging from
4,400 to 167,000 in molecular weight.22 Moreover, the conjunctiva seems to possess transporter systems for nucleosides,23 arginine derivatives (nitric oxide synthase inhibitors),24,25 and monocarboxylates. In 1995, Sun et al.:
reported that j3-alanyl-L-histidine (L-carnosine), a naturally occurring hydrolysis-resistant dipeptide that has been used to
characterize dipeptide uptake in the small intestine28'29 and in
the kidney,4'29 was transported across the conjunctival tissue
by a carrier-mediated, pH-dependent process. The purpose of
the present study was to demonstrate the functional existence
of a dipeptide transporter and to delineate dipeptide uptake
characteristics in primary cultured rabbit conjunctival epithelial cells. This cell culture model has been shown to exhibit
drug transport characteristics comparable with those in the
isolated tissue, insofar as /3-adrenergic antagonists are concerned.20 Among the uptake characteristics evaluated were
directionality, pH dependence, temperature dependence, concentration dependence, stereoselectivity, and substrate specificity. The experimental results of stereoselective uptake were
2365
2366
Basu et al.
IOVS, November 1998, Vol. 39, No. 12
Guiding Principles in the Care and Use of Animals (DHEW, NIH
80-23) and the tenets of the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research.
A
Primary Culture of Rabbit Conjunctival Epithelial
Cells
COO
1. Structures of dileucine dipeptide (A) and cephalosporins (cephalexin [stmcture 1] and cefadroxil [structure 2])
(B). Torsion angles scanned in conformational analysis are
indicated by arrows.
FIGURE
The protocol developed by Saha et al.30 was used. Briefly, the
conjunctiva was excised and incubated in 0.2% protease XTV
(Sigma) at 37°C for 90 minutes. Epithelial cells were scraped
off, suspended in a minimum essential medium (S-MEM) containing 10% fetal bovine serum and 0.5 mg/ml deoxyribonuclease I (Sigma), and centrifuged at 200g for 10 minutes at room
temperature. The cells were washed twice with S-MEM containing 10% fetal bovine serum, filtered through a 40-jam cell
strainer, pelleted at 200g for 10 minutes, and resuspended in
PC-1 medium supplemented with 2 mM L-glutamine, 100 U/ml
penicillin, 100 jag/ml streptomycin, 50 /xg/ml gentamicin, and
1 ptg/ml fungizone. The isolated cells were plated in wells
coated with a mixture of 15 /xg/cm2 rat tail type I collagen and
5 ixg/cm2 nbronectin (both obtained from Collaborative Biomedical Products, Bedford, MA), at a density of 1.5 X 106
cells/cm2 (day 0) and cultured in a humidified atmosphere of
5% CO2 and 95% air at 37°C. The volumes of apical and
basolateral culture media were 0.5 ml and 1.5 ml, respectively.
Potential difference (PD) and transepithelial resistance (R^
were monitored from day 4 onward, to assess viability and
barrier tightness. The cell layers were used for uptake experiments after reaching peak bioelectric parameters (i?t > 1.0
kCL/cm2; PD > 10 mV) from day 6 through day 8.
Uptake of L-Carnosine by Primary Cultured
Conjunctival Epithelial Cell Layers
Unless otherwise specified, all uptake experiments were performed in a humidified atmosphere of 5% CO2 and 95% air at
501
rationalized using computer modeling of the dipeptide conformers.
40
MATERIALS AND METHODS
Materials
[3H] L-carnosine (5 Ci/mmol) was purchased from Moravek
Biochemicals (Brea, CA). Unlabeled L-carnosine, cephalexin,
bestatin, captopril, and enalapril were purchased from Sigma
(St. Louis, MO). Enalaprilat was a generous gift from Merck
Research Laboratory (Whitehouse Station, NJ). Cyclacillin and
cefadroxil were kindly provided by Takeda Chemical Industries (Osaka, Japan) and Bristol-Myers Squibb Pharmaceutical
Research Institute (Princeton, NJ), respectively. Cell culture
reagents and supplies were obtained from Life Technologies
(Grand Island, NY). PC-1, a low protein defined medium used
for cell growth, was obtained from BioWhittaker (Walkersville,
MD). Tissue culture-treated wells (Transwells; 0.4 jam, 12 mm
outer diameter) were obtained from Costar (Cambridge, MA).
All other chemicals were of the highest purity available commercially.
Animals
Male Dutch-belted pigmented rabbits, weighing 2.5 to 30 kg,
were purchased from Irish Farms (Norco, CA). The investigations using rabbits described in this report conformed to the
Downloaded From: http://iovs.arvojournals.org/ on 06/17/2017
I
30
"55
O
E
3 20
3
re
Q.
10
10
20
30
40
50
60
Time (min)
2. Time course of L-carnosine uptake by primary cultured pigmented rabbit conjunctival epithelial cells (apical pH
6.0; basolateral pH 7.4). Uptake was initiated using 10 jaM
L-carnosine spiked with 20 jaCi/ml [3H]L-carnosine. Each point
represents the mean ± SEM value of six to eight determinations from two cultures. Where no error is shown, the error is
within the symbol.
FIGURE
Dipeptide Uptake in the Conjunctiva
IOVS, November 1998, Vol. 39, No. 12
TABLE 1. Directionality, Temperature Dependence, and
Energy Dependence of the Conjunctival Dipeptide
Transport System
Uptake
Conditions
Apical, 37°C
+ 100 fjM DNP
+ 50 JLLM FCCP
+ 50 mM NH4C1
Apical, 4°C
Basal, 37°C
(pmoles/mg protein/min)*
2.47
1.01
0.90
1.66
0.14
0.45
±
±
±
±
±
±
0.21
0.08
0.07
0.28
0.02
0.04
(100)
(41% ± 3)t
(36% ± 3)t
(67% ± l l )
(6% ± l)f
(18% ± 2)t
* In the presence of an inwardly directed proton gradient (apical
pH 6.0, basolateral pH 7.4). Uptake was initiated using 10 /xM Lcarnosine spiked with 20 jaCi/ml [3H]L-carnosine. Values are mean ±
SEM with percentages in parentheses; n = 6-8 from two cultures.
f Statistical significance determined by Student's f-test by comparing with the apical uptake at 37°C. P < 0.05 was considered statistically significant.
DNP, 2,4-dinitrophenol; FCCP, /Hrifluoromethoxyphenylhydra-
37°C in the presence of an inwardly directed proton gradient
(apical pH 6.0; basolateral pH 7.4) from the apical side. Before
each experiment, the cell layers were washed with bicarbonated Ringer's solution at an osmolality of 300 mOsm/kg. The
buffer contained 116.4 mM NaCl, 5.4 mM KC1, 0.78 mM
NaH2PO4, 25 mM NaHCO3, 1.80 mM CaCl2, 0.81 mM MgSO4,
555 mM glucose, and 15 mM HEPES (7V-[2-hydroxyethyl]piperazine-/V'-[2-ethane sulfonic acid]; pH 7.4 and 8.5), MES
(2-[A^morpholino]ethanesulfonic acid; pH 55-6.5), or citric
acid (pH 4.0-50). Uptake was initiated by spiking the apical or
basolateral fluid with 20 /uCi/ml [3H]L-carnosine and an appropriate amount of unlabeled substrate. After a predetermined
period, uptake was terminated by suctioning off the dosing
solution and immersing the cell layers in ice-cold bicarbonated
Ringer's solution (pH 7.4). The cell layers were then solubilized in 0.5 ml 0.5% Triton X-100 solution. Ten microliters of
the solution was taken for protein assay by the method of
Bradford31 using a protein assay kit (Bio-Rad, Hercules, CA)
with bovine serum albumin as a standard. The remainder of the
sample was mixed with 5 ml scintillation cocktail (EconoSafe;
Research Products International, Mount Prospect, IL) for measurement of radioactivity in a liquid scintillation counter (Beckman, Fullerton, CA).
2367
workstation (Mountain View, CA) by scanning the t/<l) [N(l>
Ca(l)-C(l)-N(2)] and the [(j>(2) C(l)-N(2)-Ca(2)-C(2)] backbone torsion angles (Fig. 1A) from 0° to 360° in 30° increments. For each conformation a minimization of 2000 steps
was performed, with the omega torsion angle [Ca(l)-C(l)N(2)-Cc<2)] fixed at 180° to maintain the planar geometry and
trans configuration of the peptide bond (-HN-CO-) (Fig. 1A).
A distance-dependent dielectric constant (e = AT, where r is
the interatomic distance) was used in all the calculations to
simulate a solvent environment. A cephalexin starting structure was built using the simulation program Hyperchem; (Hypercube, Gainesville, FL), and a preliminary minimization was
performed using the MM+ molecular mechanics force field in
the software.34 The structure was then subjected to conformational analysis under conditions similar to those described for
the dileucine dipeptides. The w(l) and co(3) backbone torsion
angles were scanned from 0° to 360° in 30° increments (Fig.
IB), with the w(2) torsion angle fixed at 180°. Potential energy
was plotted against the $(l)-t/<2) or co(l)-co(3) torsion angle
pair for each of the 12 X 12 = 144 conformations of dileucine
dipeptides or cephalexin, respectively. To determine the potential for the preferred conformers of each dileucine dipeptide tofitinto cephalexin binding sites on the dipeptide transporter, their similarity in shape with the N- or the C-terminal
dipeptide segment of cephalexin was determined using a least
squares rigid body fit algorithm in the software (Quanta).
Statistical Analysis
Drug uptake data are presented as the mean ± SEM. Statistical
significance was determined by Student's /-test for unpaired
sample assuming equal variance; P < 0.05 was considered
significant. Statistical differences among multiple different
groups were determined by one-way analysis of variance, and
group means were contrasted for significant differences using
the Fisher's PLSD post hoc test; P < 0.05 was considered
™
1 ••
Computer Modeling of Dileucine Dipeptides and
Cephalexin
To understand further the molecular basis of conjunctival
dipeptide uptake, we performed a conformational analysis of
the four dileucine dipeptides (LL, LD, DL, and DD) and of
cephalexin. The equivalent study was not performed with
cefadroxil, because it is structurally similar to cephalexin, the
only change being the addition of a />-hydroxyl group on the
aromatic ring (Fig. IB). The starting structures of zwitterionic
dileucine dipeptides were built using a commercial software
program (Quanta, ver. 4.0; Biosym/Molecular Simulation, San
Diego, CA). Conformational analysis of each dileucine dipeptide was performed using the CHARMm (version 30) comprehensive empiric force field32'33 on a Silicon Graphics Indigo2
Downloaded From: http://iovs.arvojournals.org/ on 06/17/2017
4.0
5.0
6.0
Apical solution pH
3. Effect of pH of apical bathing fluid on L-carnosine
uptake by primary cultured pigmented rabbit conjunctival epithelial cells (basolateral pH 7.4). Uptake was initiated using 10
/LtM L-carnosine spiked with 20 ju-Ci/ml [3H]L-carnosine. Each
point represents the mean ± SEM value of six to eight determinations from two cultures. Statistical significance was tested
by one-way analysis of variance. Group means were contrasted
for significant differences using Fisher's PLSD post hoc test.
Statistical significance was set at P < 0.05. * P < 0.05; ** P <
0.01; and not significant (NS), P > 0.05, when compared with
uptake at pH 6.0.
FIGURE
2368
Basu et al.
IOVS, November 1998, Vol. 39, No. 12
significant. Assumption of normal distribution and equal variance was also tested and the significance confirmed by nonparametric statistical analysis (Mann-Whitney rank sum test for
comparing two groups), the Kruskal-Wallis analysis of variance
on Ranks (for comparing many groups), or both, using commercial software (StatView; Abacus Concepts, Berkeley, CA).
Overall
4°C
(Diffusional)
RESULTS
Kinetics of L-Carnosine Uptake
Directionality and Energy Dependence. L-Carnosine
uptake in conjunctival epithelial cells was linear for the first 10
minutes, reaching a plateau at approximately 60 minutes (Fig.
2). Therefore, in all subsequent experiments, 10 minutes was
chosen as the observation period. Apical L-carnosine uptake
was five times higher than basolateral uptake (P < 0.05; Table
1). Moreover, apical uptake at 37°C was 17 times higher than
that at 4°C (P < 0.05) and was inhibited by 60% in the
presence of 100 juM 2,4-dinitrophenol, a metabolic inhibitor.
pH Dependence. Within the pH range of 4.0 to 8.5,
L-carnosine uptake was maximum at pH 6.0 (Fig. 3). Uptake
was reduced by more than 60% by pretreating the cells with a
proton ionophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone, at a final concentration of 50 juM in bicarbonated
Ringer's solution (P < 0.05; Table 1). Moreover, uptake was
inhibited by more than 30% after intracellular acidification by
prepulsing the conjunctival epithelial cells for 30 minutes with
50 mM NH4C1 in Na+- and HCO3~-free bicarbonated Ringer's
solution (P < 0.05; Table 1).
Concentration Dependence. L-carnosine uptake by the
conjunctival epithelial cells was saturable within the concentration range of 0.02 mM to 2 mM (Fig. 4A). Eadie-Hofstee
analysis (Fig. 4B) of the uptake data revealed a MichaelisMenten constant (K ) of 0.3 ± 0.03 mM and a maximum
uptake rate (Vma) of 22.0 ± 1 . 0 picomoles per milligram
protein per minute (r2 = 0.96).
Substrate Selectivity. The substrate selectivity of dipeptide uptake by conjunctival epithelial cells was studied by
measuring the inhibitory effect of unlabeled amino acids and
dipeptides on the uptake of tritiated L-carnosine (Fig. 5). LHistidine and /3-alanine, the constituent amino acids of L-carnosine, at 10 mM, did not affect tritiated L-carnosine uptake
(P > 0.05). By contrast, 10 mM unlabeled L-carnosine decreased [3H]L-carnosine uptake by 80% (P < 0.05; Fig. 5).
Gly-Sar, Gly-Phe, Gly-Val, and Leu-Leu in the L-configviration, all
at 10 mM, also inhibited L-carnosine uptake by at least 60%
(P < 0.05; Fig. 5). The peptidomimetics cyclacillin, an aminopenicillin antibiotic; cephalexin and cefadroxil, aminocephalosporin antibiotics; captopril ([25]-Ar-mercapto-2-methylpropionyl]-L-proline) and enalapril ([S]-l-[iV-(l-[ethoxycarbonyl]-3phenylpropyl)-L-alanyl]-L-proline), ACE inhibitors; and bestatin
([(25,3i?)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine) inhibited L-carnosine uptake by 50% to 80% (P < 0.05; Fig. 5). By
contrast, thyrotropin-releasing hormone (TRH; L-pyroglutamylL-histidyl-L-prolinamide), a tripeptide without a free carboxylic
acid group, did not inhibit the uptake of L-carnosine (P > 0.05;
Fig. 5).
Stereoselectivity. The stereoselectivity of dipeptide uptake in the conjunctival epithelial cells was studied by measuring the inhibitory effect of unlabeled Gly-Phe and Leu-Leu on
the uptake of tritiated L-carnosine (Table 2). As can be seen,
Downloaded From: http://iovs.arvojournals.org/ on 06/17/2017
Carrier-mediated
0.5
A
1.0
1.5
2.0
mM of L-carnosine
25 -r
^.20
I
5
-
B
15
30
45
60
75
V/S (pmoles/mg protein/min/mM)
4. Concentration dependence of L-carnosine uptake
by primary cultured pigmented rabbit conjunctival epithelial
cells. (A) L-Carnosine uptake in the presence of an inwardly
directed proton gradient (apical 6.0; basolateral pH 7.4). Uptake was initiated using a denned concentration of L-carnosine
spiked with 20 jaCi/ml [3H]L-carnosine. Each point represents
the mean ± SEM value of six to eight determinations from two
cultures. Overall uptake (•) was corrected for simple diffusion
determined at 4°C (O) to yield carrier-mediated uptake (A),
which was fitted to Michaelis-Menten kinetics by nonlinear
regression analysis. (B) Eadie-Hofstee plot of carrier-mediated
L-carnosine uptake.
FIGURE
Gly-L-Phe was twice as inhibitory as Gly-D-Phe. For the leucine
dipeptides, Leu-L-Leu (59%) was slightly more inhibitory than
Leu-D-Leu (43%). Surprisingly, i>Leu-Leu was even more inhibitory (72%). By contrast, D-Leu-D-Leu did not inhibit L-carnosine
uptake (P > 0.05).
Rationalization of Stereoselective Dipeptide
Uptake Using Computer Modeling
Li et al.35 have reported a correlation between the dipeptide
NC distance (the distance between the N and C termini) for the
neutral forms of four divaline stereoisomers (LL, LD, DL, and
DD) and their interaction with the dipeptide transporter. To
investigate whether the same association holds true for other
dipeptides, we performed computer modeling studies with
dileucine stereoisomers (LL, LD, DL, and DD). Our results
Dipeptide Uptake in the Conjunctiva
IOVS, November 1998, Vol. 39, No. 12
2369
TRH
Enalaprilat
Enalapril
Captopril
Bestatin
Cephalexin
Cefadroxil
Cyclacillin
L-Leu-L-Leu
Gly-L-Phe
Gly-Sar
Gly-L-Val
Carnosine
L-Histidine
R-Alanine
Control
80
100
120
L-Carnosine Uptake (% Control)
5. Effect of unlabeled amino acids, excess unlabeled L-carnosine, unlabeled dipeptides, and drugs structurally similar to dipeptides and tripeptides at 10 mMfinalconcentration
on [3H] L-carnosine uptake by primary cultured pigmented rabbit conjunctival epithelial cells
(apical pH 6.0; basolateral pH 7.4). Uptake was initiated using 10 fxM L-carnosine spiked with
20 ju,Ci/ml [3H]L-carnosine. Each column represents the mean ± SEM value of six to eight
determinations from two cultures. Statistical significance was determined by Student's t-test;
*P < 0.05 was considered statistically significant. TRH, thyrotropin-releasing hormone; N.S.,
not significant.
FIGURE
suggest that each zwitterionic dileucine dipeptide exists in two
conformer populations, characterized by the i//(l)-<K2) torsion
angle pairs shown in Table 3 and referred to as conformer (I)
and (II). The NC distance of the two conformer populations of
each dileucine dipeptide is also shown in Table 3- Two minimum energy conformers were also obtained for cephalexin
(for w(l), w(2), and w(3) torsion angles, see note to Table 3).
TABLE 2. Stereoselectivity of the Conjunctival
Dipeptide Transport System
Uptake
Substrates*
Control (apical, 37°C)
+ Gly-L-Phe
+ Gly-i>Phe
+ Leu-Leu
+ Leu-D-Leu
+ D-Leu-Leu
+ D-Leu-D-Leu
(pmoles/mg protein/min)f
1.05 ± 0.03
0.18 ± 0.02
0.34 ± 0.08
0.43 ± 0.11
0.60 ± 0.14
0.29 ± 0.03
0.91 ± 0.22
(100)
(17 ± 2)+
(32 ± 8)*
(41 ± 10)*
(57 ± 13)*
(28 ± 3)*
(87 ± 21)NS
* Final substrate concentration 10 mM.
t In the presence of an inwardly directed proton gradient (apical
pH 6.0, basolateral pH 7.4). Uptake was initiated using 10 /xM Lcarnosine spiked with 20 /xCi/ml [3H]L-carnosine. Values are mean ±
SEM, with percentages in parentheses; n = 6-8 from two cultures.
* Represents statistical significance determined by Student's ?-test
by comparing with the apical uptake at 37°C. P < 0.05 was considered
statistically significant.
NS, Statistically not significant.
Downloaded From: http://iovs.arvojournals.org/ on 06/17/2017
The cephalexin conformers are probably similar to the conformation recognized and transported by the dipeptide transporter, because deformation of the rigid cephalexin would be
energetically expensive. Therefore, we computed the similarity in shape of cephalexin N- and C-terminal dipeptide segments with each dileucine conformer. The LL dileucine conformer (I) closely overlays the N-terminal dipeptide fragment
(from the a-amino group to the carbonyl group of the /3-lactam
ring) of cephalexin (Table 3, Fig. 6). Although the DD dileucine
conformer (I) has an NC distance comparable with that of the
LL dileucine conformer (I), it poorly overlays the same region
of cephalexin. Overlay of all other preferred dileucine dipeptide conformers on the N- and the C-terminal fragment of
cephalexin produced much higher root mean square deviations than that of the LL dileucine conformer (I) (Table 3). The
overlay of all the dileucine dipeptide conformer (I) structures
on the N-terminal fragment of cephalexin showed that the best
fit was for the LL conformer and least fit with the DD conformer, with the fit for DL and LD conformers being intermediate (Fig. 6).
DISCUSSION
A major finding in the present study is that a proton-coupled
dipeptide transporter probably exists on the apical side of the
conjunctival epithelial cells rabbit that mediates the transport
of dipeptides and peptidomimetics. Evidence for a predominantly apical location is the fivefold higher uptake of L-carnosine from the apical than the basolateral side of the conjunc-
2370
Basu et al.
IOVS, November 1998, Vol. 39, No. 12
3. Preferred Conformers of Zwitterionic Dileucine Dipeptides and Their Root Mean Square Deviation from
Cephalexin
TABLE
Conformer (n)f
Conformer (I)*
Leucine
Dipeptides
NC
Distance^
• <!)
• CO
Leu-Leu
Leu-r>Leu
D-Leu-Leu
i>Leu-i>Leu
5.98
5.51
5.50
5.83
180
120
240
240
210
150
210
150
RMSD
RMSD
(DS
0011
0.035
0.374
0.340
0.338
0.034
0.376
0.339
0.337
NC
Distance^
• CD
• c»
4.74
5.13
5.12
4.81
300
300
60
60
210
150
210
150
RMSD
RMSD
(D§
0.666
0.663
0.683
0.689
0.666
0.663
0.685
0.691
* Conformation of dileucine having the longer NC distance of the two conformer populations.
t Conformation of dileucine dipeptides having the shorter NC distance.
t The distance in Angstrom (A) between the nitrogen of the a-amino group and the carbon of the carboxyl group of dipeptides.
§ Root mean square deviation in Angstrom (A) from the cephalexin conformer [w (1) = 90°, co (2) = 180°, w (3) = 60°, E = 30.6 kcal/mol].
|| Root mean square deviation in Angstrom (A) from cephalexin conformer [w (1) = 180°, w (2) = 180°, co (3) = 60°, E = 30.9 kcal/mol].
NC, N-terminal to C-terminal; RMSD, root mean square deviation. t//(l) and $(2) values are in degrees.
tival epithelial cells (Table 1). The first hint for the involvement
of a nonpassive component in the uptake of L-carnosine
(2.85 ± 0.19 picomoles per milligram protein per minute) by
primary cultured conjunctival epithelial cells was the 18-fold
difference compared with the uptake of the paracellular transport marker 14C-mannitol (0.16 ± 0.03 picomoles per milligram protein per minute). L-Carnosine uptake was not affected
by its constituent amino acids, L-His and /3-Ala (Fig. 5), consistent with the substrate selectivity of a dipeptide transporter.
Conjunctival dipeptide uptake was energy dependent, indicated by the 94% and 60% inhibition obtained on lowering the
temperature to 4°C and on treating the cells with 100 JUJVI
2,4-dinitrophenol (Table 1). Moreover, L-carnosine uptake was
driven by a proton gradient that was optimal at pH 6.0 (Fig. 3)
and was inhibited by pretreating the conjunctival epithelial
cells with 50 /xM of the protonophore p-trifluoromethoxyphenylhydrazone and by preacidifying them with 50
mM NH4C1 (Table 1). The pH optimum for dipeptide transport
has been reported to be dependent on the substrates studied.
Steel et al.36 reported that, for PepTl expressed in Xenopus
laevis oocytes, maximum uptake for basic, neutral, and acidic
dipeptides occurred at pH 6.0, 55, and 5.0 of the bathing fluid,
respectively. The maximum uptake of L-carnosine, itself a basic
dipeptide, by primary cultured rabbit conjunctival epithelial
cells also occurred at pH 6.0 (Fig. 3).
As shown in Table 2, the conjunctival dipeptide transporter can tolerate one amino acid of a dipeptide in the Dconfiguration. To investigate further the role of chirality of the
constituent amino acids on the ability of a dipeptide substrate
to interact with the transporter, Leu-Leu was chosen because
all four chiral compounds are commercially available. Alteration of the chirality of one amino acid in Leu-Leu did not
abolish its ability to interact with the dipeptide transporter.
However, when both amino acids were in the D-configuration,
the ability of Leu-Leu to interact with the dipeptide carrier
system was abolished (Table 2). These data are consistent with
the stereoselectivity of the dipeptide transporter in the human
intestinal epithelial cell line Caco-2 for the dipeptide ValVal,37'38 the tripeptide Val-Val-Val,39 and the peptidomimetic
cephalexin.10
Detailed structural information about the substrate binding domain of the dipeptide transporter is unlikely to be forthcoming in the near future because of the inherent complexity
Downloaded From: http://iovs.arvojournals.org/ on 06/17/2017
of determining the structure of a transmembrane protein. As an
alternative, we used a computer modeling approach to gain
insight into the stereoselective inhibition of L-carnosine uptake
by dileucine dipeptides and especially the exclusion of the DD
dileucine from interaction with the conjunctival dipeptide
transporter. We found that each of the dileucine dipeptides
and cephalexin have two preferred conformer populations.
Superimposition of the preferred conformers of each dileucine
dipeptide with the N-terminal dipeptide segment of cephalexin
revealed lower root mean square deviations for the LL zwitterionic dileucine conformer (I) but not for the DD conformer (I)
with a comparable NC distance, which suggests that the dipeptide NC distance alone cannot account for the differential
uptake of these molecules. This finding agrees with that of Li et
al.,35 who found that the NC distance alone could not explain
the potential for a tripeptide (Val-Val-Val) to interact with the
intestinal dipeptide transporter. Therefore, it is possible that an
appropriate NC distance and a favorable orientation of the side
chains are needed for the recognition of a substrate by the
dipeptide transporter.
In addition to the Gly- and Leu-containing dipeptides,
peptidomimetics such as bestatin, /3-lactam antibiotics, and
ACE inhibitors also inhibited the uptake of L-carnosine by
primary cultured conjunctival epithelial cells to varying extents
(Fig. 5). The involvement of the dipeptide transporter in the
uptake of bestatin, itself a leucine derivative, by rabbit intestinal brush border membranes has been reported. 1516 Tomita et
al.16 have shown that bestatin inhibits cephradine uptake in a
competitive manner. Inui et al.15 have shown that the uptake
of bestatin itself is inhibited by cephalosporins and dipeptides
but not by amino acids. Of the three ACE inhibitors studied,
enalapril was 1.5 times more inhibitory than captopril (Fig. 5).
This finding is consistent with the observation made by Kitagawa et al.,13 in that ACE inhibitors significantly inhibited the
uptake of cefroxadine uptake by rabbit small intestinal brush
border membrane vesicles in the order of captopril < enalapril. Moreover, Thwaites et al.12 have found that the inhibition
offered by enalapril is approximately 1.5 times more than that
of captopril for [l4C]Gly-Sar uptake into Caco-2 cell monolayers. Furthermore, the active ACE inhibitor enalaprilat failed to
inhibit L-carnosine uptake, whereas the prodrug enalapril offered significant inhibition (Fig. 5). This finding is consistent
with the results of Swaan et al.,40 who have reported that
IOVS, November 1998, Vol. 39, No. 12
Dipeptide Uptake in the Conjunctiva
2371
FIGURE 6. Overlay of conformer (I) of each dilelucine dipeptide (LL in green, DL in red, LD in blue, and DD in yellow} on the
N-terminal dipeptide fragment of cephalexin (white).
enalapril is transported by intestinal dipeptide transporter in a
carrier-mediated manner, whereas enalaprllat is transported by
passive diffusion. The reason for the difference in interaction
between enalapril and enalaprilat with the dipeptide transport
system is thought to be the negative influence of the second
free carboxylic acid group in enalaprilat.40 The inhibitory results of cefadroxil and cyclacillin are particularly interesting,
because they may shed some light on the relative abundance of
the dipeptide transporter isoforms PepTl and PepT2 in the
conjunctiva! epithelial cells. Ganapathy et al. ll have shown
that cyclacillin is approximately 9 times more selective of
PepTl than of PepT2, whereas cefadroxil is approximately 14
times more selective of PepT2 than of PepTl based on its 50%
inhibitory concentration (IC50; substrate concentration required for 50% inhibition of uptake) and K (inhibition constant) values. Similar studies for conjunctival epithelial cells
are under way. Yamashita et al.9 have recently cloned a peptide-histidine transporter PHT1 from brain and retina. L-Carnosine was found, by competitive inhibition study, to interact
Downloaded From: http://iovs.arvojournals.org/ on 06/17/2017
with this transport system. Nevertheless, PHT1 was probably
not involved in the conjunctival epithelial uptake of L-carnosine, because histidine, a substrate for PHT1, did not significantly inhibit L-carnosine uptake in conjunctival epithelial
cells (Fig. 5).
Whether a dipeptide transporter is involved in the transepithelial transport of TRH is controversial. In the present
study, we found no evidence for the interaction of TRH with
the conjunctival dipeptide transport system. Thus, our finding is consistent with that of Gan et al.41 and of Thwaites et
al.,42 who detected predominantly non-carrier-mediated
paracellular transport of TRH across Caco-2 cell monolayers.
By contrast, Walter and Kissel''3 observed saturable carriermediated transport of TRH in addition to passive paracellular transport in a certain clone of Caco-2. Their findings are
in agreement with the observation of Tanaka et al.44 that
TRH was taken up in rat intestinal mucosal cells by a dipeptide transporter.
2372
Basu et al.
In conclusion, a proton-coupled dipeptide transport system probably exists in the conjunctival epithelial cells to mediate the uptake of model dipeptide L-carnosine. Gly-L-Sar, GlyL-Phe, Gly-L-Val, and Leu-L-Leu and peptidomimetics such as
bestatin, /3-lactam antibiotics, and ACE inhibitors that may be
important in ocular therapeutics significantly inhibited conjunctival L-carnosine uptake. The present study sets the stage
for confirmation of the molecular identity of the dipeptide
transporter isoform in the conjunctival epithelial cells using
techniques based on molecular biology.
IOVS, November 1998, Vol. 39, No. 12
17.
18.
1920.
Acknowledgments
The authors thank Wilson Meng for advice and helpful suggestions on
computer modeling and John Xu for technical assistance in uptake
studies.
21.
22.
References
1. Thwaites DT, Brown CDA, Hirst BH, Simmons NL. Transepithelial
glycylsarcosine transport in intestinal Caco-2 cells mediated by
expression of H+-coupled carrier at both apical and basal membranes. / Biol Chem. 1993;268:7640-7642.
2. Saito H, Inui K. Dipeptide transporters in apical and basolateral
membranes of the human intestinal cell line Caco-2. Am]Physiol.
1993;265:G289-G294.
3. Daniel H, Adibi SA. Transport of j3-lactam antibiotics in kidney brush border membrane. Determinants of their affinity for
the oligopeptide/H+ symporter. / Clin Invest. 1993:92:22152223.
4. Ganapathy V, Leibach FH. Peptide transport in rabbit kidney.
Biochim Biophys Acta. 1982;691:362-366.
5- Fei YJ, Kanai Y, Nussberger S, et al. Expression cloning of a
mammalian proton-coupled oligopeptide transporter. Nature.
1994;368:563-566.
6. Liang R, Fei YJ, Prasad PD, et al. Human intestinal H+/peptide
cotransporter. Cloning, functional expression, chromosomal localization. / Biol Chem. 1995:270:6456-6463.
7. Boll M, Herget M, Wagner A, et al. Expression cloning and functional characterization of the kidney cortex high-affinity protoncoupled peptide transporter. Proc Natl Acad Sci USA. 1996;93:
284-289.
8. Liu W, Liang R, Ramamoorthy S, et al. Molecular cloning of PEPT2,
a new member of the H+/peptide cotransporter family, from
human kidney. Biochim Biophys Acta. 1995;1235:46l-466.
9. Yamashita T, Shimada S, Guo W, et al. Cloning and functional
expression of a brain peptide/histidine transporter. / Biol Chem.
1997:272:10205-10211.
10. Dantzig AH, Bergin L. Uptake of the cephalosporin, cephalexin, by
a dipeptide transport carrier in human intestinal cell line, Caco-2.
Biochim Biophys Acta. 1990;1027:211-217.
11. Ganapathy ME, Brandsch M, Prasad PD, Ganapathy V, Leibach FH.
Differential recognition of /3-lactam antibiotics by intestinal and
renal peptide transporters, PEPT1 and PEPT2.JBiol Chem. 1995;
270:25672-25677.
12. Thwaites DT, Cavet M, Hirst BH, Simmons NL. Angiotensin-converting enzyme (ACE) inhibitor transport in human intestinal epithelial (Caco-2) cells. Br J Pharmacol. 1995;114:981-986.
13. Kitagawa S, Takeda J, Kaseda Y, Sato S. Inhibitory effects of
angiotensin-converting enzyme inhibitor on cefroxadine uptake by
rabbit small intestinal brush border membrane vesicles. Biol
PharmBull. 1997;20:449-451.
14. Kramer W, Girbig F, Gutjahr U, et al. Interaction of renin inhibitors
with the intestinal uptake system for oligopeptides and j8-lactam
antibiotics. Biochim Biophys Acta. 1990;1027:25-30.
15. Inui K, Tomita Y, Katsura T, Okano T, Takano M, Hori R. H+coupled active transport of bestatin via the dipeptide transport
system in rabbit intestinal brush-border membranes. / Pharmacol
Exp Ther. 1992;260:482-486.
16. Tomita Y, Katsura T, Okano T, Inui K, Hori R. Transport mechanisms of bestatin in rabbit intestinal brush-border membranes: role
Downloaded From: http://iovs.arvojournals.org/ on 06/17/2017
23.
24.
25.
26.
27.
28.
29-
30.
31.
32.
33.
34.
35.
36.
37.
38.
of H+/dipeptide cotransport system. J Pharmacol Exp Ther. 1990;
252:859-862.
Ooishi M, Miyao M. Antibiotic sensitivity of recent clinical isolates
from patients with ocular infections. Ophthalmologica. 1997;
211(Suppl 1): 15-24.
Abrams KL, Brooks DE, Laratta LJ, Barnhill MA, Frazier D. Angiotensin converting enzyme system in the normal canine eye: pharmacological and physiological aspects./ Ocul Pharmacol. 1991;
7:41-51.
Hosoya K, Lee VHL. Cidofovir transport in the pigmented rabbit
conjunctiva. Curr Eye Res. 1997; 16:693-697.
Saha P, Uchiyama T, Kim KJ, Lee VHL. Permeability characteristics
of primary cultured rabbit conjunctival epithelial cells to low
molecular weight drugs. Curr Eye Res. 1996;15:1170-1174.
Sun L, Basu SK, Kim KJ, Lee VHL. Arginine vasopressin transport
and metabolism in the pigmented rabbit conjunctiva. EurJPharnt
Sci. 1998;6:47-52.
Horibe Y, Hosoya K, Kim KJ, Ogiso T, Lee VHL. Polar solute
transport across the pigmented rabbit conjunctiva: size dependence and the influence of 8-bromo cyclic adenosine monophosphate. Pharm Res. 1997;l4:1246-1251.
Hosoya K, Horibe Y, Kim KJ, Lee VHL. Nucleoside transport
mechanisms in the pigmented rabbit conjunctiva. Invest Ophthalmol Vis Sci. 1998;39:372-377.
Hosoya K, Horibe Y, Kim KJ, Lee VHL. Na+-dependent L-arginine
transport in the pigmented rabbit conjunctiva. Exp Eye Res. 1997;
65:547-553.
Hosoya K, Horibe Y, Kim KJ, Lee VHL. Carrier-mediated transport
of NG-nitro-L-arginine, a nitric oxide synthase inhibitor, in the
pigmented rabbit conjunctiva. / Pharmacol Exp Ther. 1998;285:
223-227.
Horibe Y, Hosoya K, Kim KJ, Lee VHL. Carrier-mediated transport
of monocarboxylate drugs in the pigmented rabbit conjunctiva.
Invest Ophthalmol Vis Sci. 1998;39:372-377.
Sun L, Kim KJ, Lee VHL. Carrier-mediated L-carnosine transport in
the pigmented rabbit conjunctiva [ARVO Abstract]. Invest Ophthalmol Vis Sci. 1995;36(4):S699.
Matthews DM, Addison JM, Burston D. Evidence for active transport of the dipeptide carnosine (/3-alanyl-L-histidine) by hamster
jejunum in vitro. Clin Sci Mol Med. 1974;46:693-705.
Ganapathy V, Leibach FH. Role of pH gradient and membrane
potential in dipeptide transport in intestinal and renal brush-border membrane vesicles from the rabbit. / Biol Chem. 1983;258:
14189-14192.
Saha P, Kim KJ, Lee VHL. A primary culture model of rabbit
conjunctival epithelial cells exhibiting tight barrier properties.
Curr Eye Res. 1996;15:ll63-ll69.
Bradford MM. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem. 1976;72:248-254.
Nilsson L, Clore GM, Gronenborn AM, Brunger AT, Karplus M.
Structure refinement of oligonucleotides by molecular dynamics
with nuclear Overhauser effect interproton distance restraints:
application to 5' d(C-G-T-A-C-G)2. /Mol Biol. 1986; 188:455-475.
Brunger AT, Clore GM, Gronenborn AM, Karplus M. Three-dimensional structure of proteins determined by molecular dynamics
with interproton distance restraints: application to crambin. Proc
Natl Acad Sci USA. 1986;83:3801-3805.
Allinger NL. Conformational analysis: 130. MM2, a hydrocarbon
force field utilizing Vt and V2 torsional terms. J Am Chem Soc.
1977:99:8127-8134.
Li J, Tamura K, Lee C-P, Smith PL, Borchardt RT, Hidalgo IJ.
Structure-affinity relationship for the oligopeptide transporter in
Caco-2 cells. Pharm Res. 1996;13:S24l.
Steel A, Nussberger S, Romero MF, Boron WF, Boyd CAR, Hediger
MA. Stoichiometry and pH dependence of die rabbit proton-dependent oligopeptide transporter VepTl.J Physiol. 1997:498:563-569Hidalgo IJ, Bhatnagar P, Lee C-P, Miller J, Cucullino G, Smith PL.
Structural requirements for interaction with the oligopeptide
transporter in Caco-2 cells. Pharm Res. 1995;12:317-319Tamura K, Bhatnagar PK, Takata JS, Lee C-P, Smith PL, Borchardt
RT. Metabolism, uptake, and transepithelial transport of the diaste-
IOVS, November 1998, Vol. 39, No. 12
reomers of Val-Val in the human intestinal cell line, Caco-2. Pbarm
Res. 1996;13:1213-1218.
39. Tamura K, Lee C-P, Smith PL, Borchardt RT. Metabolism, uptake, and
transepithelial transport of the stereoisomers of Val-Val-Val in the
human intestinal cell line, Caco-2. Pbarm Res. 1996;13:l663-l667.
40. Swaan PW, Stehouwer MC, Tukker JJ. Molecular mechanism for
the relative binding affinity to the intestinal peptide carrier. Comparison of three ACE-inhibitors: enalapril, enalaprilat, and lisinopril. Biochim Biophys Acta. 1995;1236:31-38.
41. Gan L, Niederer T, Eads C, Thakker D. Evidence for predominantly
paracellular transport of thyrotropin-releasing hormone across
Caco-2 cell monolayers. Biochem Biophys Res Comtnun. 1993;
197:771-777.
Downloaded From: http://iovs.arvojournals.org/ on 06/17/2017
Dipeptide Uptake in the Conjunctiva
2373
42. Thwaites DT, Simmons NL, Hirst BH. Thyrotropin-releasing hormone (TRH) uptake in intestinal brush-border membrane vesicles:
comparison with proton-coupled dipeptide and Na+-coupled glucose transport. Pbarm Res. 1993; 10:667- 67343. Walter E, Kissel T. Transepithelial transport and metabolism of
thyrotropin-releasing hormone (TRH) in monolayers of a human
intestinal cell line (Caco-2): evidence for an active transport component? Pharm Res. 1994; 11:1575-1580.
44. Tanaka K, Fujita T, Yamamoto Y, Murakami M, Yamamoto A,
Muranishi S. Enhancement of intestinal transport of thyrotropinreleasing hormone via a carrier-mediated transport system by
chemical modification with lauric acid. Biochim Biophys Acta.
1996;1283:119-126.