Dipeptide-induced Cl
secretion in proximal tubule cells
Wenwu
Jin and
Ulrich
Hopfer
Department of Physiology and Biophysics, School of Medicine, Case
Western Reserve University, Cleveland, Ohio 44106
 |
ABSTRACT |
During a survey of dipeptides that might be transported by the
renal PEPT2 transporter in proximal tubule cells, we discovered that
acidic dipeptides could stimulate transient secretory anion current and
conductance increases in intact cell monolayers. The stimulatory effect
of acidic dipeptides was observed in several proximal tubule cell lines
that have been recently developed by immortalization of early proximal
tubule primary cultures from the Wistar-Kyoto and spontaneously
hypertensive rat strains and humans, suggesting that this phenomenon is
a characteristic of proximal tubule cells. The electrical current
induced in intact monolayers by Ala-Asp, a representative of these
acidic dipeptides, must represent
Cl
secretion rather than
Na+ or
H+ absorption, because
1) it was
Na+ independent,
2) it showed a pH dependence
different from that of the PEPT2 cotransporter, and
3) it correlated with an
Ala-Asp-induced increase in
Cl
conductance of the
apical membrane in basolaterally amphotericin B-permeabilized
monolayers. The secretory current could be inhibited by stilbene
disulfonates, but not diphenylamine-2-carboxylates, suggesting a
non-cystic fibrosis transmembrane conductance regulator type of
Cl
conductance. The effect
of Ala-Asp was dose dependent, with an apparent 50% effective
concentration of ~1 mM. Ala-Asp also produced intracellular
acidification, suggesting that acidic dipeptides are also substrates
for an H+-peptide cotransporter.
alanine-aspartate; PEPT2 cotransporter; regulated chloride
conductance; salt absorption
 |
INTRODUCTION |
THE KIDNEY PLAYS an essential role in the turnover of
circulating small proteins and peptides. These small proteins and
peptides, present in glomerular filtrate, are a heterogeneous group of
molecules of varied molecular structure, physicochemical properties,
and biological functions. It is now accepted that oligopeptides are hydrolyzed in the proximal lumen to a mixture of amino acids and di-
and tripeptides. The products of the hydrolysis are subsequently absorbed via amino acid as well as peptide transport systems. The
absorptive cells of the renal proximal tubule possess active transport
mechanisms for small peptides (reviewed in Ref. 18). The hydrolysis and
absorption constitute a mechanism of renal conservation of amino acid
nitrogen that might otherwise be lost in the urine. Peptide transport
is unique among the transport systems of renal epithelial cells, in
that it is energized by an electrochemical
H+ gradient (18) rather than the
Na+ gradient that powers
absorption of most nutrients, including amino acids and glucose (5, 9).
Several epithelial cell lines from the proximal tubule of spontaneously
hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats (28) and
humans (20) have been established recently that closely resemble
cells in primary culture and form confluent monolayers on porous
support. The existence of the kidney-specific high-affinity H+-peptide cotransporter (PEPT2)
has been recently reported in one of these cell lines (SKPT0193 Cl.2)
(7). Northern blot analysis has subsequently revealed that the SKPT
cells contain mRNA transcripts that are hybridizable to the PEPT2 cDNA
probe (11). The physiological and pharmacological significance of such
peptide transporter systems has been emphasized (18). However, the
functional aspects of these small peptides have not been fully
characterized. During a survey of dipeptides that might be transported
by the renal PEPT2 transporter in these cell lines, we discovered that
acidic dipeptides could stimulate a secretory anion current or its
equivalent. The present studies characterize the peptide-activated
current in proximal tubule cells.
 |
MATERIALS AND METHODS |
Materials.
Dulbecco's modified Eagle's medium-nutrient mixture F-12 (DMEM-F-12),
human keratinocyte serum-free nutrient growth medium (K-SFM)
supplemented with human epidermal growth factor (5 µg/l) and bovine
pituitary extract (50 mg/l), Hanks' balanced salt solution, trypsin-EDTA, and fetal bovine serum (FBS) were purchased from GIBCO
BRL (Grand Island, NY); dipeptides, amphotericin B,
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS),
nigericin, and bovine serum albumin (BSA) from Sigma Chemical (St.
Louis, MO); 4,4'-dinitro-2,2'-stilbenedisulfonic acid
(DNDS) from Pfaltz and Bauer; and
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) from Molecular Probes (Eugene, OR).
Dichlorodiphenylamine-2-carboxylate (DCDPC) was a gift from Dr. H. J. Lang (Hoechst, Frankfurt, Germany).
Cell culture.
Several proximal tubule cell lines have been recently developed by
immortalization of early proximal tubule primary cultures from the WKY
and SHR rat strains and humans (20, 28). The cell lines were selected
for their ability to form confluent, electrically resistive monolayers,
which represents a major epithelial characteristic. Two rat cell lines
and six human cell lines were tested in this study, including SKPT0193
Cl.2 cells (SHR kidney proximal tubule, isolated January 1993, clone
2), WKPT1292 Cl.8 cells (WKY rat kidney proximal tubule, isolated
December 1992, clone 8), and several HPCT cells (human proximal
convoluted tubule).
All cells were propagated in a humidified atmosphere of 95% air-5%
CO2 at 37°C. Every other day,
the rat cells were fed renal tubule epithelial (RTE) culture medium
[composed of 1:1 DMEM-F-12, with 15 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), 1.2 mg/ml NaHCO3, 5 µg/ml transferrin, 10 ng/ml epidermal growth factor, 4 µg/ml
dexamethasone] supplemented with 5% heat-inactivated FBS. Cells
were grown on collagen-coated 30-mm Millicell-CM filters (Millipore,
Bedford, MA) and passaged by trypsin-EDTA treatment (0.05% trypsin, 1 mM EDTA tetrasodium in Hanks' balanced salt solution without
Ca2+ or
Mg2+) after reaching confluence.
Cell monolayers for measuring the electrical properties were grown on
collagen-coated 12-mm Millicell-CM filters. Electrophysiological
studies were performed after cells became confluent. The passage
numbers were between 30 and 100. The human cell lines were maintained
under the same conditions, except they were fed K-SFM in the absence of
FBS.
Monolayer electrophysiology.
Electrophysiological studies were conducted as previously described
(15). Briefly, transepithelial electrophysiological measurements were
performed in a modified Ussing-type chamber constructed to accept
filters with an outer diameter of 1.2 cm (Analytical
Bioinstrumentation, Cleveland, OH). The chamber was equipped with a
conventional four-electrode system for measuring short-circuit current
(Isc),
transepithelial potential, and conductance. These parameters were
measured with a voltage-clamp module (model 558-C-5, Dept. of
Bioengineering, University of Iowa, Iowa City, IA) that corrects for
fluid resistance and were continuously recorded on a strip chart
recorder and, with the aid of an analog-to-digital converter, also on a
microcomputer. Monolayer conductance was continuously monitored by
application of small (1- to 2-mV) bipolar voltage pulses.
Filters were mounted in the Ussing chamber when cell monolayers became
confluent. The luminal and basal compartments were continuously
perfused with an
-free Ringer solution composed of, unless otherwise indicated, 140 mM NaCl, 4.7 mM
KCl, 2 mM CaCl2, 1.5 mM
MgCl2, 10 mM HEPES, 25 mM
D-glucose, and 0.1% (wt/vol)
BSA. The pH was titrated to 7.4 with tris(hydroxymethyl)aminomethane. Measurements were carried out at 37°C. Peptides were added acutely to the luminal compartment.
Permeabilization of the plasma membrane with amphotericin B.
To specifically evaluate the ion conductance of the apical membrane,
the amphotericin B technique was used (17). Amphotericin B forms pores
in the plasma membrane that are permeable to monovalent inorganic ions
(10). Therefore, when epithelia are perfused on the basolateral side
with 10 µM amphotericin B, the apical membrane becomes rate limiting
for overall transepithelial electrical currents. Amphotericin B was
added in dimethyl sulfoxide, so that the final concentration of
dimethyl sulfoxide was 0.1%. This concentration by itself had no
effects on electrophysiological parameters. To establish a
basolateral-to-apical gradient for
Cl
, the apical compartment
was perfused with a low-Cl
Ringer solution in which NaCl and KCl had been replaced by Na-gluconate and K-gluconate, respectively. To reverse the
Cl
gradient,
low-Cl
Ringer solution was
used in the basolateral compartment. Because the basolateral membrane
was permeable to small monovalent ions and the transepithelial voltage
was clamped at 0 mV, the Cl
gradient across the apical membrane was the sole driving force for
Cl
flux. Measurements were
carried out at 37°C. Dipeptides were added acutely to the luminal
compartment after the permeabilization had stabilized, as judged by
constant Isc and
conductance.
Intracellular pH measurements by BCECF imaging.
SKPT cells were grown to confluence on an Ethicon collagen-coated piece
of Anocell filter that attached over a hole in the center of a plastic
coverslip. Cells were loaded with BCECF by incubation in normal RTE
medium with 10 µM BCECF-AM at room temperature for ~25 min. After
the incubation, cells were placed on ice and washed twice with normal
RTE medium immediately before use.
Cells were placed in a thermostated modified Ussing-type chamber that
was designed for separate apical and basolateral perfusion. Cells were
constantly perfused with the same modified Ringer buffers used in
electrophysiological measurements. The chamber was placed on a
microscope stage, and cells were illuminated through epifluorescence optics alternating the light between 495 and 450 nm. The fluorescence light between 500 and 560 nm was imaged and captured by an intensified charge-coupled device camera. The video images were digitized and
analyzed with Image-1/FL software (Universal Imaging, Media, PA) and
saved on a hard disk as a function of time. Images at the two
wavelengths were each acquired for 0.5 s, and then the ratio image
(495/450) was calculated with acquisition of ratio images every 5 s.
Experiments were carried out at 37°C.
Intracellular pH was calibrated in situ by using the method described
by James-Kracke (13). Briefly, at the end of an experiment, 10 µM
nigericin was added, and then the experimental medium was changed to a
high-K+ salt solution
(K+ replacing
Na+ in experimental buffer) that
was first at pH 5.5 and then at pH 8.5. These two pH extremes gave the
minimum (Rmin) and the maximum
(Rmax) 495/450 ratio,
respectively, thus allowing intracellular pH
(pHi) to be estimated as follows
where
pKa (= 6.98) is
the negative logarithm of the association constant for BCECF (13).
Data analysis.
Values are original recordings expressed as means ± SE;
n is the number of
electrophysiological experiments or cells in intracellular pH
measurements.
 |
RESULTS |
Anion secretion induced by acidic dipeptides.
When intact monolayers of the renal proximal tubule cell line SKPT0193
Cl.2 were perfused with
-free Ringer solution, as illustrated in Fig.
1, Ala-Asp caused an increase in
Isc that
corresponds to anion secretion or cation absorption. The increase was
transient, reaching a maximal value within seconds and returning more
slowly to the baseline level. Ala-Asp-induced anion secretion (or its
equivalent) was dose dependent. Figure 2
shows the dose-response curve, using the peak difference in Isc. The maximal
Isc and 50%
effective concentration were ~11 µA/cm2 and 1.2 mM, respectively.
Similar to Ala-Asp, other acidic dipeptides, such as Ser-Asp and
Gly-Glu, were able to induce similar current increases (Table
1). On the other hand, neutral dipeptides,
such as Ala-Ala and Gly-Sar, showed no stimulatory effects.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 1.
Ala-Asp-induced anion secretion (or its equivalent) in proximal tubule
cells. Short-circuit current
(Isc) was
measured in an intact monolayer of SKPT0193 Cl.2 cells. Cells were
perfused with -free Ringer
solution on apical and basolateral sides. Apical perfusion was stopped
before addition of dipeptide, while basolateral perfusion continued.
Ala-Asp was acutely added to luminal compartment of Ussing chamber to
give a final concentration of 10 mM.
|
|

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 2.
Dose-response curve for Ala-Asp-induced anion secretion.
Isc was measured
in intact monolayers as described in Fig. 1. Ala-Asp was acutely added
to luminal side. Dipeptide-induced change in Isc was measured
as peak difference after addition of Ala-Asp. [Ala-Asp],
Ala-Asp concentration; n = 3.
|
|
The ability of acidic dipeptides to stimulate a secretory current was
also observed in other proximal tubule cell lines. All the cell lines
tested, e.g., the WKPT1292 Cl.8 cell line (derived from WKY rats) and
several HPCT cell lines (human proximal convoluted tubule, isolated May
1994 and June 1995, respectively), showed typical Ala-Asp responses
similar to that in the SKPT0193 Cl.2 cell line (Table
2), suggesting that this phenomenon may be
a characteristic of proximal tubule cells.
To determine the nature of the current induced by acidic dipeptides,
several approaches were taken. In particular, we considered 1) peptide hydrolysis and subsequent
Na+-amino acid cotransport,
2) direct
H+-peptide cotransport, and
3) an indirect, receptor-mediated
change in ion conductance of the apical plasma membrane.
Na+-amino
acid cotransport did not play a role in dipeptide-induced current.
To test the possibility that dipeptides were actually hydrolyzed at the
apical membrane by peptidases and that the hydrolyzation products were
subsequently absorbed through
Na+-amino acid cotransport, two
approaches were taken. First, the effects of amino acids on the
electrical properties of the monolayers were examined. As listed in
Table 1, alanine and aspartate did not induce significant
Isc under the
same experimental conditions, indicating that either
Na+-amino acid cotransporters were
absent in these cell lines or the current through
Na+-amino acid cotransporters was
too small to be detected. Second, Na+ dependence of
dipeptide-induced current was tested.
Na+ was removed from the apical
perfusate. This treatment removed the possible involvement of
Na+-dependent amino acid transport
or apical Na+ channels. As shown
in Fig. 3, the peptide-induced current was totally independent of apical Na+,
indicating that Na+ was not the
charge carrier and Na+-dependent
transport systems were not involved.

View larger version (8K):
[in this window]
[in a new window]
|
Fig. 3.
Ala-Asp-induced
Isc was
independent of extracellular Na+
concentration.
Isc induced by
apical 10 mM Ala-Asp was measured in intact monolayers as in Fig. 1.
Na-Ringer, usual Na+ concentration
on apical and basolateral sides; Na-free, perfusion with
Na+-free Ringer solution on both
sides, where Na+ was substituted
by
N-methyl-D-glucamine;
n = 4.
|
|
Dipeptide-induced
H+ flux and
intracellular pH changes.
The kidney-specific H+-peptide
cotransporter has been identified recently in the SKPT0193 Cl.2 cell
line (7). To sort out the possibility that dipeptide-induced current
might be due to direct H+ influx
through this H+-peptide
cotransporter, a few approaches were taken. We first measured the
effect of extracellular pH on dipeptide-induced
Isc. As shown in
Fig. 4, extracellular pH only slightly
affected the dipeptide-induced electrical current.
Isc was ~40%
higher at pH 6.0 than at pH 7.4 (11.6 ± 2.2 and 8.2 ± 1.0 µA/cm2, respectively). This pH
dependence is very different from that reported for the PEPT2
transporter, which has a 5- to 10-fold stimulation over the same pH
range (7). Therefore, the underlying transport resulting in the
electrical current is likely to be different from that of the PEPT2
transporter.

View larger version (7K):
[in this window]
[in a new window]
|
Fig. 4.
Ala-Asp-induced
Isc was slightly
enhanced by extracellular acidification.
Isc induced by
apical 10 mM Ala-Asp was measured in intact monolayers as in Fig. 1,
except monolayers were perfused with normal Ringer solution (pH 7.4, n = 5) or with acidified Ringer
solution (pH 6.0, n = 6).
|
|
To directly assess H+ flux, we
then measured the peptide-induced pH changes and converted them to
H+ flux. As shown in Fig.
5, apical Ala-Asp could induce a sustained decrease in intracellular pH: from pH 7.20 to 7.09 in the presence of
normal apical Na+ concentration
(Fig. 5A) and to pH 6.5 in the
absence of apical Na+ (Fig.
5B). The intracellular pH recovered
after the washout of Ala-Asp from the apical side, provided
Na+ was present (cf. Fig. 5,
A and
B). The higher steady-state pH in
the presence of apical Na+ and the
Na+ dependence of the recovery are
consistent with the known role of apical
Na+/H+
exchange for eliminating cellular acid equivalents. The
Na+ independence of the
peptide-induced H+ influx suggests
the presence of H+-peptide
cotransport and is consistent with the finding of the PEPT2
cotransporter in these cells (7).

View larger version (19K):
[in this window]
[in a new window]

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5.
Ala-Asp-induced intracellular acidification. Intracellular pH
(pHi) was measured with
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein fluorescence ratio imaging. Bars, length of apical perfusion with 5 mM
Ala-Asp. A: monolayer on Anocell
filter perfused on both sides with Ringer solution with normal
Na+ concentration (140 mM);
n = 15. B: monolayer on Anocell filter perfused with Na+-free Ringer
solution, where Na+ was replaced
by
N-methyl-D-glucamine;
n = 19.
|
|
Interestingly, there is a clear difference in the time course of the
peptide-induced pH changes and the electrical flux (cf. Figs. 1 and
5A), suggesting that the underlying
transport processes are different. The decrease in cellular pH reached
steady state 3-5 min after the start of apical perfusion with
Ala-Asp and was then sustained, implying sustained
H+ influx. In contrast, the
electrical flux peaks within a few seconds and then declines with a
half time of ~5 min. It is likely that the pH changes largely
reflect H+-dipeptide cotransport,
inasmuch as the magnitude of the peptide-induced H+ flux is similar to the
dipeptide flux measured isotopically and reported earlier (7; see
DISCUSSION).
Dipeptide-induced current is mediated by
Cl
.
Ruling out the involvement of apical
Na+-amino acid cotransport and
H+-peptide cotransport in
dipeptide-induced anion secretion, the hypothesis was that an ion
conductance in the apical plasma membrane was activated by those
dipeptides. To specifically study the electrical properties of the
apical plasma membrane, cells in the monolayer were permeabilized on
the basolateral side with amphotericin B, which forms pores for small,
monovalent ions. With basolateral permeabilization, the ion flux across
the apical membranes can be specifically studied by controlling the ion
composition of the perfusates on both sides. Amphotericin B
permeabilization did not significantly affect the basal
Isc and monolayer
conductance, because the tight junction in parallel with the apical
plasma membrane usually determines the total conductance. In
permeabilized cells, acidic dipeptides caused transient changes in
conductance that fully explain the
Isc observed in
intact cells (Fig. 6). The corresponding
stimulatory effect on
Isc depended on
the direction of a Cl
gradient across the apical membrane, i.e., the driving force (Fig.
7), suggesting that
Cl
was the main carrier of
the charge flux across the apical membrane.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Ala-Asp-induced anion secretion in basolaterally permeabilized cells.
Intact monolayers were mounted and perfused as in Fig. 1, except
low-Cl Ringer solution was
used as apical perfusate to establish a basolateral-to-apical Cl gradient. Basolateral
plasma membranes were then permeabilized to monovalent ions with 10 µM amphotericin B in 0.1% DMSO. Ala-Asp (final concentration 5 mM)
was added acutely to luminal compartment of Ussing chamber.
Gm, monolayer
conductance.
|
|

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 7.
Ala-Asp-induced
Isc was dependent
on Cl gradient. Cell
monolayers were perfused as in Fig. 6, except for orientation of
Cl gradients, as indicated
below. Basolateral plasma membranes were permeabilized with 10 µM
amphotericin B in 0.1% DMSO. Ala-Asp (final concentration 5 mM) was
added acutely to luminal compartment of Ussing chamber. bas > ap,
Basolateral-to-apical Cl
gradient across apical membranes
(low-Cl Ringer solution on
apical side); ap > bas, apical-to-basolateral Cl gradient across apical
membranes (low-Cl Ringer
solution on basal side); bas = ap, no
Cl gradients (normal
Cl concentration on both
sides).
|
|
To further characterize the nature of the ion conductance activated by
these dipeptides, the relationship between current and voltage
(I-V curve) was measured for the
dipeptide-stimulated ion conductance. Figure
8 shows
I-V relationships for permeabilized monolayers with and without Ala-Asp stimulation. Ion selectivity was
derived from the reversal potential data interpreted on the basis of
the Henderson diffusion
equation.1 As
shown in Table 3, there was a nonselective
ion conductance under the basal condition that most likely represents
tight junctional permeability; in contrast, Ala-Asp activated an ion
conductance that was about four times more anion than
cation selective.

View larger version (16K):
[in this window]
[in a new window]

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 8.
Current-voltage (I-V) relationship
for Ala-Asp-induced ion conductance. Intact monolayers were perfused
with a basolateral-to-apical Cl gradient as in Fig. 6.
Basolateral plasma membranes were then permeabilized to monovalent ions
with 10 µM amphotericin B in 0.1% DMSO. Ala-Asp (final concentration
5 mM) was added acutely to luminal compartment of Ussing chamber.
A:
I-V relationship at 3 time points,
i.e., before Ala-Asp addition ( ), at peak
Isc change after
Ala-Asp addition ( ), and after washout of Ala-Asp ( ).
B: curve representing Ala-Asp-induced
current: Isc = Isc( ) Isc( ).
|
|
To substantiate the finding of dipeptide-induced apical
Cl
conductance, some
general inhibitors for Cl
channels were tested. Ala-Asp was added to the apical side of the
Ussing chamber before and after apical perfusion with stilbene disulfonates and diphenylamine-carboxylate derivatives (DIDS, DNDS, and
DCDPC, respectively). Because DIDS covalently reacts with amino groups,
a Ringer solution buffered by
/CO2 instead of HEPES was used in the experiments with this inhibitor (23 mM
NaHCO3 bubbled with 5%
CO2 at 37°C). As shown in Fig.
9, dipeptide-induced
Isc could be
inhibited by 1 mM apical DIDS but was relatively insensitive to 2.5 mM
DNDS and 100 µM DCDPC. Furthermore, the inhibition by DIDS was
irreversible; i.e., washout of apical DIDS could not restore an
Ala-Asp-induced
Isc, indicating a
covalent modification of apical
Cl
channels by DIDS.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 9.
Ala-Asp-induced
Isc could be
inhibited by Cl channel
inhibitors. Isc
induced by apical 5 mM Ala-Asp was measured in intact monolayers as in
Fig. 1. Ala-Asp was added acutely to apical side in absence (control)
or presence of apical 1 mM DIDS, 2.5 mM
4,4'-dinitro-2,2'-stilbenedisulfonic acid (DNDS), or 100 µM dichlorodiphenylamine-2-carboxylate (DCDPC).
|
|
Dipeptide-induced activation of apical
Cl
conductance is receptor mediated.
The activation of apical Cl
conductance by dipeptides suggests that regulatory events occurred.
However, it is possible that the acidic dipeptides were transported
into the cells by the H+-peptide
cotransporter and that the activation of
Cl
conductance was
initiated inside the cells. To evaluate this possibility, the acidic
dipeptide-induced Cl
conductance was measured in the presence of neutral dipeptides, which
could be transported through
H+-peptide cotransport and thus
acted as competitive substrates with acidic dipeptides. As shown in
Fig. 10, the stimulatory effect of
submaximal 1 mM Ala-Asp was not changed by the presence of supramaximal
15 mM Gly-Sar, a neutral dipeptide, suggesting that the activation of
Cl
conductance was
initiated extracellularly.

View larger version (8K):
[in this window]
[in a new window]
|
Fig. 10.
Ala-Asp-induced
Isc was not
affected by Gly-Sar. Cell monolayers were perfused with a
basolateral-to-apical Cl
gradient as in Fig. 6. Basolateral plasma membranes were permeabilized to monovalent ions with 10 µM amphotericin B in 0.1% DMSO, 1 mM Ala-Asp was added acutely to luminal compartment of Ussing chamber in
absence (control) or presence (+Gly-Sar) of 15 mM Gly-Sar, and peak
differences in
Isc induced by
Ala-Asp were measured; n = 3.
|
|
The exact mechanisms by which dipeptides activate apical
Cl
conductance are not
known, and specific receptors for dipeptides have not been reported.
However, these acidic dipeptides may structurally resemble certain
bioactive ligands and, thereby, may be able to bind to certain known,
if not novel, receptors. The time courses of transient responses of
Isc and monolayer
conductance also suggested that this regulation may occur through a
receptor. Several efforts were attempted to characterize such
receptors. A few agonists were tested for their abilities to stimulate
apical Cl
conductance
(Table 1). Endorphin, which is an endogenous bioactive peptide and
contains acidic Gly-Glu as the last two COOH-terminal amino acids,
failed to stimulate similar
Isc and
conductance changes. Aspartate and glutamate also showed no stimulatory
effects.
N-methyl-D-aspartic acid (NMDA), on the other hand, showed a stronger stimulatory effect on
Isc and
conductance with similar characteristics, i.e., large transient
increases in Isc
and conductance. ATP is another potent agonist to activate a transient
Cl
conductance in the
apical plasma membrane in these cells (12). ATP stimulation is
apparently through the activation of purinergic receptors in the apical
membrane, and pertussis toxin-sensitive G proteins are involved. It is
not clear whether acidic dipeptides, NMDA, and ATP activate the same
Cl
conductances. However,
after NMDA or ATP stimulation, Ala-Asp failed to further increase
Isc or
conductance (Fig. 11), suggesting that
common components were shared by all three agonists, most likely the
apical Cl
conductance.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 11.
Ala-Asp failed to increase
Isc and monolayer
conductance in
N-methyl-D-aspartate
(NMDA)-prestimulated cells. Intact cell monolayers were perfused with
-free Ringer solution on apical
and basolateral sides. NMDA and Ala-Asp were added acutely to luminal
compartment of Ussing chamber (arrows). Final concentration for NMDA
and Ala-Asp was 5 mM.
|
|
 |
DISCUSSION |
The present study showed that small acidic peptides could induce an
apical Cl
conductance in
proximal tubule cells. The study also provided evidence that acidic
dipeptides are substrates for
H+-peptide cotransporter(s) that
are present in the proximal tubule cells.
The subject of renal handling of oligopeptides is undergoing extensive
study. It has been established that
H+-peptide cotransport systems
exist in the kidney and are of physiological importance. The peptide
transport systems are independent of
Na+ and are electrogenic, and
concentrative peptide uptake is driven by a transmembrane
electrochemical H+ gradient (18).
The kidney cell line SKPT0193 Cl.2 constitutively expresses the
kidney-specific high-affinity
H+-peptide cotransporter (7). The
dipeptide uptake for Gly-Sar was found to be mediated by the PEPT2
system with a half-maximal concentration of 67 µM and a maximal
transport velocity of 0.12 nmol · min
1 · mg
protein
1. Consistent with
this observation, we found that Gly-Sar and Ala-Asp could induce
Na+-independent intracellular
acidification with comparable rates: the rate of pH change
(
pH/
t) was ~0.03/min and
0.04/min for Gly-Sar and Ala-Asp, respectively. However, the
H+ influx with Ala-Asp does not
account for the
Isc observed in electrophysiological studies. Conversion of the dipeptide-induced rates
of pH change indicates negligible electrical currents of maximally 0.8 µA/cm2, which is <10% of the
actually observed peak electrical currents and actually within the
noise level of typical monolayer electrophysiological recordings. The
initial dipeptide-induced pH changes are ~0.04-0.1
pH/min
(Fig. 5). These values convert to
H+ flux
current2
of ~0.8 µA/ cm2. A similar
conclusion is reached by converting the maximal rate of uptake of
Gly-Sar given above, which with conservative estimates yields maximally
an Isc of 1 µA/cm2 (28). Other differences
in the characteristics of dipeptide-induced electrical flux and pH
changes would also support two separate processes that are going on
simultaneously, such as differences in the time courses and pH
dependence.
Apart from being absorbed and digested in the proximal tubule cells,
small peptides may also act as bioactive ligands that could bind to
specific membrane receptors with certain physiological or pathological
consequences. However, the functional characterization of these small
peptides in proximal tubules has been rare. Our data showed that acidic
dipeptides were able to induce a transient Cl
secretion in
short-circuited proximal tubule cells, suggesting that certain small
peptides might play a role in the regulation of proximal tubular
handling of Cl
and, as a
result, possibly Na+ and water as
well. It is worthwhile to emphasize that the stimulatory effects of
acidic dipeptides are different from those of the anionic amino acids.
It has been reported that the flux of certain excitatory amino acids in
brain tissues is associated with the activation of a ligand-gated
Cl
conductance (26). It
appears that the dipeptide-induced
Cl
conductance is different
from that activated by glutamate or aspartate, because these amino
acids by themselves did not have any observable stimulatory effect
under our experimental conditions. On the basis of the
Na+ independence of the
dipeptide-induced current, we can also exclude major contributions from
electrogenic anionic amino acid transporters that are driven by an
Na+ gradient (16).
Although ~50% of the filtered
Cl
is reabsorbed in the
proximal tubule, the cellular mechanism of
Cl
transport is poorly
understood (5). An inside-outside electrochemical gradient for
Cl
exists due to several
Cl
accumulation mechanisms,
including Cl
/formate
exchange in the apical membrane (1, 2, 4) and Cl
/
exchange in the basolateral membrane (22). Given this electrochemical
Cl
gradient across the
plasma membrane, opening of apical
Cl
channels results in
Cl
secretion.
Apical Cl
conductance is
generally not present in proximal tubule cells in the basal state.
However, several reports demonstrate Cl
conductance in renal
cortical brush-border membranes, with contributions of adenosine
3',5'-cyclic monophosphate/protein kinase A-dependent and
-independent transporters (19, 25). Patch-clamp studies have suggested
that renal proximal convoluted tubule cells possess an apical
Cl
channel that could be
activated by parathyroid hormone (23). However, this conclusion is not
definite, because the cells lacked fully differentiated transport
polarity. Furthermore, the existence of an adenosine
3',5'-cyclic monophosphate-activated and
5-nitro-2-(3-phenylpropylamino)benzoate-sensitive Cl
channel has been
recently reported for the apical membrane of microperfused proximal
convoluted tubules (27). Molecular biology has made some progress in
cloning a so-called ClC family of voltage-gated Cl
channels, of which
several members have been found in kidney (14, 24). However, the exact
location and physiological relevance of these
Cl
channels have yet to be
established in proximal tubule cells.
Our studies relied on newly established proximal tubule cell lines that
closely resembled cells in primary culture and were able to form
confluent monolayers on porous support. These cell lines showed
retention of typical differentiated proximal tubular phenotypes (28).
The high electrical resistance of monolayers formed by the cell lines
allows the use of conventional electrophysiology to quantify ion
channel activity and their location on apical or basolateral plasma
membrane. Consistent with earlier observations, the present study
demonstrates a regulated apical
Cl
conductance in proximal
tubule cells, with negligible anion-selective conductance in the basal
state.
Stilbene disulfonates appear to inhibit most known epithelial
Cl
channels, with the
exception of cystic fibrosis transmembrane conductance regulator (CFTR)
(3, 8). CFTR can be inhibited by DCDPC. The specificity of different
blockers may vary significantly in different tissues. Our data showed
that acidic dipeptide-induced Cl
secretion was sensitive
to DIDS, but not to DCDPC, suggesting a non-CFTR type of
Cl
channel. Further
characterization is required to determine the nature of this
Cl
channel.
The physiological roles for these
Cl
secretory effects of
dipeptides in proximal tubules are not known. The opening of apical Cl
channels in vivo is
unlikely to result in net NaCl secretion but would be expected to
decrease NaHCO3 absorption on the
basis of the following sequence of events:
1) depolarization of membrane potential due to apical Cl
efflux, 2) decreased
NaHCO3 efflux from the basolateral
membrane because of a lower membrane potential, and
3) decreased apical Na+/H+
exchange because of a higher steady-state cellular pH as a result of
decreased
efflux.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Heart, Lung, and Blood
Institute Grant HL-41618.
 |
FOOTNOTES |
1
The Henderson diffusion equation (21) is as
follows: 
=
RT/F[(Ub
Vb)
(Ua
Va)]/[(Ub + Vb)
(Ua + Va)]ln[(Ub + Vb)/(Ua + Va)], where U =
Ccation (total monovalent
cation concentrations), V =
Canion (total monovalent
inorganic anion concentrations), and superscripts a and b refer to
apical and basolateral sides, respectively. Other ions (divalent
cations, gluconate) are assumed to be impermeant. In the experiment
shown in Fig. 8, Ua = Ub = 144.7 mM,
Va = 7 mM, and
Vb = 151.7 mM.
2
The expected area-normalized electrical current
(Isc in
µA/cm2) through
H+-peptide cotransporter is
identical to the H+ influx that
can be estimated from the pH change (
pH/min) as follows:
Isc =
pH/
t × F ×
× vol/area, where
F is Faraday's constant,
is
buffer capacity, and vol/area gives the height of the cells. The buffer
capacity was estimated from pH changes after perfusion of weak acids or
bases (6) as 5 mM/pH unit at ~pH 7.1 (D. E. Orosz, unpublished
observations). A rate of pH change of 0.1/min converts to 0.8 µA/cm2, using 10 µm as
estimated average cell height on the basis of electron micrographs
(28).
Address for reprint requests: U. Hopfer, Dept. of Physiology and
Biophysics, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4970.
Received 27 January 1997; accepted in final form 8 July 1997.
 |
REFERENCES |
1.
Alpern, R. J.
Apical membrane chloride/base exchange in the rat proximal convoluted tubule.
J. Clin. Invest.
79:
1026-1030,
1987[Medline].
2.
Alpern, R. J.
Cell mechanisms of proximal tubule acidification.
Physiol. Rev.
70:
79-114,
1990[Free Full Text].
3.
Anderson, M. P.,
D. N. Sheppard,
H. A. Berger,
and
M. J. Welsh.
Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L1-L14,
1992[Abstract/Free Full Text].
4.
Baum, M.
Effect of luminal chloride on cell pH in rabbit proximal tubule.
Am. J. Physiol.
254 (Renal Fluid Electrolyte Physiol. 23):
F677-F683,
1988[Abstract/Free Full Text].
5.
Berry, C. A.,
and
F. C. Rector, Jr.
Renal transport of glucose, amino acids, sodium, chloride, and water.
In: The Kidney (4th ed.), edited by B. M. Brenner,
and F. C. Rector, Jr.. Philadelphia, PA: Saunders, 1991, chapt. 7, p. 245-282.
6.
Boyarsky, G.,
M. B. Ganz,
R. B. Sterzel,
and
W. F. Boron.
pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of
.
Am. J. Physiol.
255 (Cell Physiol. 24):
C844-C856,
1988[Abstract/Free Full Text].
7.
Brandsch, M.,
C. Brandsch,
P. D. Prasad,
V. Ganapathy,
U. Hopfer,
and
F. H. Leibach.
Identification of a renal cell line that constitutively expresses the kidney-specific high affinity H+/peptide cotransporter.
FASEB J.
9:
1489-1495,
1995[Abstract/Free Full Text].
8.
Cabantchik, Z. I.,
and
R. Greger.
Chemical probes for anion transporters of mammalian cell membranes.
Am. J. Physiol.
262 (Cell Physiol. 31):
C803-C827,
1992[Abstract/Free Full Text].
9.
Christensen, H. N.
Role of amino acid transport and countertransport in nutrition and metabolism.
Physiol. Rev.
70:
43-77,
1990[Free Full Text].
10.
De Kruijff, B.,
and
R. A. Demel.
Polyene antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. 3. Molecular structure of the polyene antibiotic-cholesterol complexes.
Biochim. Biophys. Acta
339:
57-70,
1974[Medline].
11.
Ganapathy, M. E.,
M. Brandsch,
P. D. Prasad,
V. Ganapathy,
and
F. H. Leibach.
Differential recognition of
-lactam antibiotics by intestinal and renal peptide transporters, PEPT 1 and PEPT 2.
J. Biol. Chem.
270:
25672-25677,
1995[Abstract/Free Full Text].
12.
Hopfer, U.,
and
W. Jin.
Physiological role for P-glycoprotein and purinergically stimulated Cl conductance in proximal tubule cells (Abstract).
FASEB J.
11:
A300,
1997.
13.
James-Kracke, M. R.
Quick and accurate method to convert BCECF fluorescence to pHi: calibration in three different types of cell preparations.
J. Cell. Physiol.
151:
596-603,
1992[Medline].
14.
Jentsch, T. J.,
W. Gunther,
M. Pusch,
and
B. Schwappach.
Properties of voltage-gated chloride channels of the ClC gene family.
J. Physiol. (Lond.)
482:
19S-25S,
1995[Medline].
15.
Jin, W.,
and
U. Hopfer.
Purinergic-mediated inhibition of Na+-K+-ATPase in proximal tubule cells: elevated cytosolic Ca2+ is not required.
Am. J. Physiol.
272 (Cell Physiol. 41):
C1169-C1177,
1997[Abstract/Free Full Text].
16.
Kanai, Y.,
S. Nussberger,
M. F. Romero,
W. F. Boron,
S. C. Hebert,
and
M. A. Hediger.
Electrogenic properties of the epithelial and neuronal high affinity glutamate transporter.
J. Biol. Chem.
270:
16561-16568,
1995[Abstract/Free Full Text].
17.
Kirk, K. L.,
and
D. C. Dawson.
Basolateral potassium channel in turtle colon: evidence for single-file ion flow.
J. Gen. Physiol.
82:
297-329,
1983[Abstract].
18.
Leibach, F. H.,
and
V. Ganapathy.
Peptide transporters in the intestine and the kidney.
Annu. Rev. Nutr.
16:
99-119,
1996[Medline].
19.
Lipkowitz, M. S.,
and
R. G. Abramson.
Modulation of the ionic permeability of renal cortical brush-border membranes by cAMP.
Am. J. Physiol.
257 (Renal Fluid Electrolyte Physiol. 26):
F769-F776,
1989[Abstract/Free Full Text].
20.
Orosz, D. E.,
M. B. Finesilver,
W. Jin,
P. G. Woost,
P. S. Frisa,
M. I. Resnick,
J. W. Jabobberger,
J. G. Douglas,
and
U. Hopfer.
Immortalization and characterization of immortalized early proximal tubule cells derived from human kidneys (Abstract).
J. Am. Soc. Nephrol.
6:
774,
1995.
21.
Schultz, S. G.
Basic Principles of Membrane Transport. Oxford, UK: Cambridge University Press, 1980.
22.
Seki, G.,
S. Taniguchi,
S. Uwatoko,
K. Suzuki,
and
K. Kurokawa.
Effect of parathyroid hormone on acid/base transport in rabbit renal proximal tubule S3 segment.
Pflügers Arch.
423:
7-13,
1993[Medline].
23.
Suzuki, M.,
T. Morita,
K. Hanaoka,
Y. Kawaguchi,
and
O. Sakai.
A Cl
channel activated by parathyroid hormone in rabbit renal proximal tubule cells.
J. Clin. Invest.
88:
735-742,
1991[Medline].
24.
Takeuchi, Y.,
S. Uchida,
F. Marumo,
and
S. Sasaki.
Cloning, tissue distribution, and intrarenal localization of ClC chloride channels in human kidney.
Kidney Int.
48:
1497-1503,
1995[Medline].
25.
Vayro, S.,
and
N. L. Simmons.
An effect of Ca2+ on the intrinsic Cl
conductance of rat kidney cortex brush border membrane vesicles.
J. Membr. Biol.
150:
163-173,
1996[Medline].
26.
Wadiche, J. I.,
S. G. Amara,
and
M. P. Kavanaugh.
Ion fluxes associated with excitatory amino acid transport.
Neuron
15:
721-728,
1995[Medline].
27.
Wang, T.,
A. S. Segal,
G. Giebisch,
and
P. S. Aronson.
Stimulation of chloride transport by cAMP in rat proximal tubules.
Am. J. Physiol.
268 (Renal Fluid Electrolyte Physiol. 37):
F204-F210,
1995[Abstract/Free Full Text].
28.
Woost, P. G.,
D. E. Orosz,
W. Jin,
P. S. Frisa,
J. W. Jacobberger,
J. G. Douglas,
and
U. Hopfer.
Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive (SHR) and normotensive (WKY) rats.
Kidney Int.
50:
125-134,
1996[Medline].
AJP Cell Physiol 273(5):C1623-C1631
0363-6143/97 $5.00
Copyright © 1997 the American Physiological Society