Dipeptide-induced Clminus 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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 HCO<SUP>−</SUP><SUB>3</SUB>-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
pH<SUB>i</SUB> = p<IT>K</IT><SUB>a</SUB> − log (R<SUB>max</SUB> − R)/(R − R<SUB>min</SUB>)
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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Anion secretion induced by acidic dipeptides. When intact monolayers of the renal proximal tubule cell line SKPT0193 Cl.2 were perfused with HCO<SUP>−</SUP><SUB>3</SUB>-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 HCO<SUP>−</SUP><SUB>3</SUB>-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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Isc and conductance increase induced by acidic dipeptides

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Rat strain and species independence of Ala-Asp-induced Cl- secretion in proximal tubule cell lines

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 (bullet ), at peak Isc change after Ala-Asp addition (black-square), and after washout of Ala-Asp (black-triangle). B: curve representing Ala-Asp-induced current: Delta Isc = Isc(black-square) - Isc(bullet ).

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Ion selectivity for Ala-Asp-induced ion conductance

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 HCO<SUP>−</SUP><SUB>3</SUB>/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 HCO<SUP>−</SUP><SUB>3</SUB>-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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (Delta pH/Delta 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 Delta 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-/HCO<SUP>−</SUP><SUB>3</SUB> 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 HCO<SUP>−</SUP><SUB>3</SUB> 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: Delta psi  = -RT/F[(Ub - Vb- (Ua - Va)]/[(Ub + Vb- (Ua + Va)]ln[(Ub + Vb)/(Ua + Va)], where U = Sigma Ccation (total monovalent cation concentrations), V = Sigma 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 (Delta pH/min) as follows: Isc = Delta pH/Delta t × F × beta  × vol/area, where F is Faraday's constant, beta  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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
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 HCO<SUP>−</SUP><SUB>3</SUB>. 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 beta -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