Purinergic-induced signaling in C11-MDCK cells inhibits the secretory Na-K-Cl cotransporter

Tatyana A. Brindikova,3 Nathalie Bourcier,2 Brian Torres,4 Dimitri Pchejetski,2,3 Michel Gekle,5 Georgy V. Maximov,3 Valérie Montminy,1 Paul A. Insel,4 Sergei N. Orlov,2,3,* and Paul Isenring1,*

1Nephrology Group, Research Center L'Hôtel-Dieu de Québec, Department of Medicine, Faculty of Medicine, Laval University, Québec G1R 2J6; 2Research Centre l'Hôtel-Dieu de Montréal, Centre Hospitalier de l'Université de Montréal, Montréal, Quebec, Canada H2W 1T8; 3Faculty of Biology, MV Lomonosov Moscow State University, Moscow, Russia; 4Department of Pharmacology, University of California, San Diego, La Jolla, California 92093; and 5Department of Physiology, University of Wurzburg, D97070 Wurzburg, Germany

Submitted 25 August 2002 ; accepted in final form 4 August 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purinergic inhibition of Na-K-Cl cotransport has been noted in various renal epithelial cells derived from the collecting tubule, including Madin-Darby canine kidney (MDCK) cells. In recent studies, we have observed purinergic inhibition of Na-K-Cl cotransport in C11-MDCK subclones ({alpha}-intercalated-like cells). Interestingly, Na-K-Cl cotransport activity was also detected in C7-MDCK subclones (principal-like cells) but was not affected by ATP. In this investigation, we have transfected the human Na-K-Cl cotransporter (huNKCC1) in both C11 and C7 cells to determine whether these differences in NKCC regulation by ATP were due to cell-specific purinoceptor signaling pathways or to cell-specific isoforms/splice variants of the transporter. In both cell lines, we found that endogenous as well as huNKCC1-derived cotransport activity was restricted to the basolateral side. In addition, we were able to show that extracellular application of 100 µM ATP or 100 µM UTP abolished NKCC activity in both mock- and huNKCC1-transfected C11 cells but not in mock- and huNKCC1-transfected C7 cells; in C11 cells, intriguingly, this inhibition was not affected by inhibitors of RNA and protein synthesis and occurred even though expression levels of UTP-sensitive P2Y2-, P2Y4-, and P2Y6-purinoceptors were not different from those observed in C7 cells. These results suggest that C11 cells express an undetermined type of UTP-sensitive P2-purinoceptors or a unique P2Y-purinoceptor-triggered signaling cascade that leads to inhibition of NKCC1.

P2-purinoceptors; Na-K-Cl cotransporter; C7- and C11-Madin-Darby canine kidney cells


EPITHELIAL CELLS along the collecting duct regulate free water reabsorption and acid secretion through various hormones and neurotransmitters, including atrial natriuretic peptide, bradykinin, catecholamines, miner-alocorticoids, prostanoids, and vasopressin (5). Under certain circumstances, P2-purinoceptor agonists (ATP, UTP, ADP, and UDP) can also regulate ion transport in these cells by activating various signaling cascades (1, 7).

Two types of P2-purinoceptors have been described to date: P2X-purinoceptors that correspond to ligand-gated cation channels and P2Y-purinoceptors that are coupled to heterotrimeric G proteins (8, 40). Both the P2X- and P2Y-purinoceptors are known to occur in several isoforms, most of which exhibit wide tissue distribution. These proteins are also diffusely distributed along various nephron segments (2, 32, 35, 37, 38, 41, 42).

In peripheral blood, ectonucleotidases maintain circulating levels of ATP <10 nM (21). At this concentration, renal P2-purinoceptors on serosal membranes cannot be activated; the ID50(ATP) for these proteins is generally >1 µM (8). However, nucleosides can act in paracrine and autocrine ways, reaching high concentrations in the peritubular space after sympathetic stimulation or exposure to various osmotic, mechanical, and ischemic stresses (3, 21, 24, 26, 29, 36).

The transport systems that are influenced by nucleosides are largely unknown. Recently, we have observed that ATP and UTP led to inhibition of Na-K-Cl cotransport in Madin-Darby canine kidney (MDCK) cells, as well as in collecting duct cells derived from rat and rabbit (9, 10). It is noteworthy that in MDCK cells, the Na-K-Cl cotransporter (NKCC) was not influenced by changes in intracellular cAMP and cGMP or by modulators of transepithelial ion fluxes, e.g., angiotensin II, bradykinin, dopamine, methacholine, and vasopressin (10, 27).

Two populations of cells have been recently isolated from commercially available stocks of MDCK cells: the C7- and C11-MDCK cells (11). The C7 subtypes resemble principal cells; they have high transepithelial electrical resistance (Rte), are peanut lectin negative, maintain intracellular pH (pHi) at 7.39, and have a large K conductance. The C11 subtypes, on the other hand, resemble intercalated cells; they have low Rte, are peanut lectin positive, maintain pHi at 7.16, and have large Cl and H conductances (11).

We have recently demonstrated that both C7- and C11-MDCK cells possess ATP-triggered signaling pathways as well as a bumetanide-sensitive NKCC, presumably the ubiquitously distributed NKCC1, which in epithelial cells is localized basolaterally (16, 34). However, we observed that the inhibitory effect of ATP on the canine NKCC (caNKCC) occurred in C11 cells only (27). These results suggest among various possibilities that C7 cells 1) have a different NKCC (e.g., a NKCC1 splice-variant, the kidney-specific NKCC2 or a novel isoform), 2) express a posttranslationally modified NKCC1, or 3) utilize different P2-sensitive signaling pathways (27).

In the present study, we have measured and localized ATP-sensitive NKCC activity in wild-type (wt) and in human NKCC (huNKCC1)-transfected C7 and C11 cells. We were able to show that NKCC activity is expressed basolaterally in all cell lines and that the effect of ATP on heterologous and endogenous Na-K-Cl cotransport was similar. These results suggest that C7 and C11 cells express the same NKCC isoform but that they have different P2-activated transduction mechanisms.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. C7-MDCK, C11-MDCK, and wt MDCK cells (also called MDCK-D1) were obtained from commercially available stocks. For genomic and RNA isolation, they were maintained in culture as previously described (14). For functional studies, they were maintained in DMEM supplemented with 2.5 g/l sodium bicarbonate, 2 g/l HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (DMEMPLUS) and were plated onto 24-well plates or 1-cm2 permeable inserts (Corning Brand transwell plate inserts, Fisher Scientific, Montreal, QC, Canada) at a density of 1.25 x 103 cell/cm2.

Transfection and selection of cell lines. Subconfluent C7 and C11 cells were transfected with 40 µg of cDNAs (pJB20M or huNKCC1 cloned in pJB20M; see Refs. 15-17) using the Effectene transfection reagent (Qiagen, Mississauga, ON, Canada). They were then selected for 12 days by adding 0.6 mg/ml G418 to the DMEMPLUS. After this period, individual colonies were randomly chosen (1 from pJB20M-transfected C7 cells, 6 from huNKCC1/pJB20M-transfected C7 cells, 1 from pJB20M-transfected C11 cells, and 5 from huNKCC1/pJB20M-transfected C11 cells) and passaged onto 24-well plates for amplification in G418-supplemented DMEMPLUS medium. huNKCC1-transfected cells with the highest cotransport activity were used for the other studies (Table 1).


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Table 1. Na-K-Cl cotransport in control, mock-transfected, and huNKCC1-transfected C7 and C11 cells

 

General conditions for flux measurements. Cells grown on impermeable plastic supports (24-well plates) were used initially to allow measurements of NKCC activity in an extended number of samples. Cell monolayers grown on permeable transwell inserts were used afterwards to determine whether NKCC activity in MDCK cells was localized on the apical vs. the basolateral membrane. Unless mentioned otherwise, all experiments were done at 37°C and all solutions were at pH 7.4.

Flux studies in 24-well plates. Confluent cells were washed twice in 150 mM NaCl, 1 mM MgCl2/CaCl2, and 10 mM HEPES-Tris (medium A) and were incubated for 30 min in 140 mM NaCl, 5 mM KCl, 1 mM MgCl2/CaCl2, 5 mM D-glucose, and 20 mM HEPES-Tris (medium B). They were then reincubated for 15 min in medium B supplemented with 0.5-1 µCi/ml 86RbCl and 100 µM ouabain ± 10 µM bumetanide; in previous studies, we have shown that 86Rb influx is linear during this time interval (10). Influxes were terminated by four washes in an ice-cold solution containing 100 mM MgCl2 and 10 mM HEPES-Tris buffer (medium C). Cells were subsequently incubated in 1% SDS, and radioactivity of the lysed material was counted with a liquid scintillation analyzer. Influxes were calculated as V = A/am, where A is the total count in the sample (counts per minute), a is 86Rb-specific radioactivity in the flux medium (counts per minute per nanomole), and m is the protein content (in milligrams).

Rb fluxes across basal and apical membranes. Monolayers were grown on transwell inserts until Rte values, determined with an EVOM device (World Precision Instruments, Sarasota, FL), became stable (~400 and 3,000 {Omega}·cm2 for C11 and C7 monolayers, respectively). The monolayers were then preincubated for 30 min in DMEM containing 2% calf serum, 110 mM NaCl, 5.3 mM KCl, 0.8 mM MgSO4, 1.8 mM CaCl2, 0.9 mM NaH2PO4, and 30 mM NaHCO3 (medium E); during this time interval, both the apical and basolateral surfaces were exposed to the medium. In some experiments, ATP (100 µM), UTP (100 µM), BAPTA-AM (20 µM), actinomycin (1 µg/ml), or cycloheximide (1 µg/ml) were added to medium E; to determine the time course of the ATP effect on NKCC activity, preincubation in medium E was 40 min (instead of 30) and ATP was added during the last 0, 2.5, 5, 10, 20, 30, or 40 min.

After these various maneuvers, cells were transferred to 12-well plates and bathed in another medium E containing ~0.5 µCi/ml 86Rb, ±100 µM ouabain, ±10 µM bumetanide, ±100 µM ATP; here, ATP was added on both surfaces of the monolayers, whereas 86Rb, ouabain, and bumetanide were applied to either the apical or the basolateral surface. In prior studies, we observed that 86Rb flux in C7 monolayers (from both the serosal and mucosal media) is linear during the first 15 min (4). Accordingly, incubation in the flux medium was 10 min or less. For most studies, it was precisely 10 min, whereas for the kinetic studies presented in Fig. 2, it was 2 min, so measurements would also reflect the immediate effect of ATP on NKCC activity. All influxes were terminated by transferring plates to an ice-cold water bath and washing cells four times in ice-cold medium C. Solubilization was carried out in 1% SDS, and radioactivity was counted as above except that ion fluxes were normalized per monolayer area.



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Fig. 2. Kinetics of Na-K-Cl cotransport inhibition by ATP in C7 and C11 monolayers. Cells preincubated for 40 min in medium E with 20 µM BAPTA-AM ({bullet}, curve 2) or without ({circ}, curve 1) were reincubated 0 to 40 min in medium E supplemented with 100 µM ATP. They were then incubated for 2 min in medium E containing 1 µCi/ml 86RbCl + 100 µM ouabain ± 10 µM bumetanide. ATP and BAPTA-AM were added to the serosal and mucosal sides, whereas 86Rb, ouabain, and bumetanide were added to the serosal solution only. Results are expressed as means ± SE of quadruplicates.

 

Short current measurements. Transwell inserts containing monolayers of C7 cells were mounted between halves of an Ussing chamber (Warner Instrument, Hamden, CT). Fluid in each half was connected via KCl-agar bridges to voltage and current electrodes and clamped at 0 mV using an EC-825 epithelial voltage-clamp amplifier (Warner Instruments). Basolateral and apical solutions containing 2% calf serum and HEPES-NaHCO3-buffered DMEM (medium D) were circulated by airlifting with 5% CO2. Total charge movement was calculated as , where t = time after addition of epinephrine and Isc is short-circuit current (see Ref. 4 for more details).

RNA and protein synthesis. Amounts of newly synthesized RNA and proteins were determined in both wt C7 and C11 cells following previously described procedures (28). Briefly, RNA synthesis was determined from cellular incorporation of [3H]uridine and protein synthesis from cellular incorporation of [3H]leucine. Experiments were conducted in the presence or absence of actinomycin, which inhibits RNA synthesis, or cycloheximide, which inhibits protein synthesis.

PCR studies. These experiments were also performed as previously described (14). Briefly, PCR products were generated by using P2Y-purinoceptor-specific primers (shown in Table 2) at 20 µM and various DNA templates derived from wt C7, C11, or MDCK-D1 cells. These templates were as follows: 0.5 µg of cDNA synthesized from 2 mg of random-primed total RNA (C7 and C11 cells) and 100 ng of genomic DNA (MDCK-D1 cells). The PCR reactions were conducted as described in the legend of Fig. 5.


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Table 2. Primers used for PCR amplification

 


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Fig. 5. PCR products with P2Y-purinoceptors-specific primers in C7-MDCK, C11-MDCK, and MDCK-D1 cells. Lanes 1 and 2, RT-PCR using total RNA from C7 cells and C11 cells, respectively. Lane 3, PCR using genomic DNA from MDCK-D1 cells. In A, B and C, intra-exonic primers specific to P2Y1-, P2Y2-, and P2Y11-purinoceptors were used (expected sizes were 422 bp, 539 bp, and 234 bp, respectively). In D, intraexonic primers were specific to GADPH (expected size was 233 bp). The final PCR buffer composition was 50 mM KCl, 10 mM Tris·HCl, pH 8.3, 2.5 mM MgCl2, 0.2 mM dNTPs, and 2.5 U Amplitaq Gold polymerase. Samples (25 µl) were treated as follows: 10-min activation at 95°C, 35 thermocycles (1 min at 95°C, 1 min at 58°C, and 1 min at 72°C), and 10-min final extension at 72°C. PCR products were subjected to ethidium bromide-stained 1.8% agarose gel electrophoresis.

 

Chemicals and reagents. DMEM, calf serum, and other reagents for cell culture were purchased from GIBCO Laboratories (Burlington, ON, Canada). ATP, epinephrine, UTP, adenosine, BAPTA-AM, ouabain, bumetanide, and trypsin were from Sigma-RBI (St. Louis, MO). [3H]uridine and [3H]leucine were from Amersham (Oakville, ON, Canada). 86RbCl was from DuPont (Boston, MA) and Isotope (St. Petersburg, Russia). Salts and buffers were from Sigma and Anachemia Science (Montreal, QC, Canada), and molecular biology reagents or kits were from Invitrogen (Burlington, ON, Canada) and Applied Biosystems.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Selection of huNKCC1-transfected cell lines (plastic supports). As mentioned in METHODS, seven transfected C7 cell lines (six with huNKCC1 and one with the vector alone) and six transfected C11 cell lines (five with huNKCC1 and one with the vector alone) were randomly chosen and amplified over several passages; these cell lines are called C7-NKCC1, C7-mock, C11-NKCC1, and C11-mock, respectively. Nontransfected C7 and C11 cell lines (called C7-wt and C11-wt) were also amplified in parallel to serve as controls. After a few weeks in culture, cell lines were characterized by measuring bumetanide-sensitive fluxes (in the presence of ouabain).

As shown in Table 1, the transfection procedure itself does not alter the functional properties of cell lines in regard to ion-mediated fluxes. For example, the bumetanide-sensitive component of 86Rb influx is higher (and of similar magnitude) in both C7-wt and C7-mock cells compared with C11-wt or C11-mock cells. Similarly, the ouabain- and bumetanide-resistant component of 86Rb influx is similar between C7-mock and C7-NKCC1 (36 ± 6 vs. 38 ± 5 nmol·mg protein-1·15 min-1) or between C11-mock and C11-NKCC1 (19 ± 3 vs. 23 ± 4).

Regarding NKCC activity in C7- and C11-transfected cells (see Table 1), bumetanide-sensitive influxes in five out of six C7-NKCC1 cell lines and in four out of five C11-NKCC1 cell lines were found to be significantly higher than those of the nontransfected or mock-transfected counterparts. On average, the increases in cotransporter-specific fluxes are 1.5-fold for C7-NKCC1 cells and 1.8-fold for C11-NKCC1 cells. Based on these measurements, clones D and A were used for subsequent investigations.

Characterization of Na-K-Cl cotransport in wt C7 and C11 cells grown as monolayers. In these studies, ouabain is shown to decrease the rate of 86Rb influx only when added in the serosal medium (Table 3). Because the Na-K ATPase pump in all epithelial cells studied so far is restricted to the basolateral side, ouabain was used in our studies to confirm the polarity of the monolayer. With the addition of bumetanide, 86Rb influx decreases further at the basolateral side (40% in C7 cells and 80% in C11 cells) but is still unaffected at the apical side.


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Table 3. Rates of 86Rb influx across basolateral and apical membranes of C7 and C11 monolayers

 

Bumetanide and other high-ceiling diuretics are able to inhibit Na-independent K-Cl cotransport. However, K-Cl cotransport is known to be more sensitive to furosemide than to bumetanide (20). In addition, we have recently shown that 500 µM furosemide in the presence of 10 µM bumetanide and 100 µM ouabain has no effect on the rate of 86Rb influx in MDCK cells (10), as well as in C7 and C11 cells (data not reported). For the current investigations, hence, K-Cl cotransport activity does not contribute to basolateral K influx by MDCK cells.

It is noteworthy that when grown on permeable inserts, C11 cells exhibit higher rates of Na-K-Cl cotransport than C7 cells (Table 3), whereas when grown on impermeable support, both cell lines exhibit comparable rates (Table 1). These results suggest that polarization of epithelial cells occurring on porous supports leads to change in surface expression and/or functional activity of NKCC. Polarization has been shown to affect other transport systems as well (4, 23, 25, 31).

Characterization of Na-K-Cl cotransport in huNKCC1-transfected C7 and C11 cells grown as monolayers. Table 4 shows that transfection of C7 and C11 cells with huNKCC1 leads to an elevation of the bumetanide-sensitive 86Rb activity (3- and 2-fold increase, respectively) on the basolateral side. On the apical side, however, no significant changes are observed compared with mock-transfected cells.


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Table 4. Contransporter-specific 86Rb influx rates in mock- and huNKCC1-transfected C7 and C11 cells

 

In several secretory epithelia, including monolayers of MDCK (6) and C7 cells (4), bumetanide applied basolaterally inhibits solute secretion triggered by epinephrine and other activators of cAMP signaling. In the present study, we compared the kinetics of epinephrine-induced changes in Isc as a means of confirming further the polarized expression of transfected huNKCC1. Here, C11 monolayers were not investigated because their Rte was not sufficiently high to yield detectable epinephrine-induced Isc (4).

Figure 1 shows that the maximal value of transepithelial current (Isc-max) across C7 monolayers was not increased by transfection with huNKCC1 (compare Fig. 1B with Fig. 1, A and C). On the other hand, a two- to threefold rise in total charge movement (QT) was observed (compare Fig. 1B with Fig. 1, A and D). Basolateral application of bumetanide diminishes QT by 20-30% (Fig. 1, D and E, open bars) and abolishes the increase in QT observed after transfection of C7 cells with huNKCC1 (Fig. 1, D and E, filled bars). As anticipated, the addition of bumetanide on the apical side does not affect epinephrine-induced changes in Isc (data not shown).



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Fig. 1. Changes in short-circuit current (Isc) after basolateral application of epinephrine. A: mock-transfected C7 cells. B: huNKCC1-transfected C7 cells. C: maximum increase in short-circuit current transepithelial current (Isc-max) for both C7-mock and C7-NKCC1 cells. D and E: total transepithelial charge movement (QT) for mock- and huNKCC1-transfected C7 cells. Isc-max and QT were calculated as illustrated in A and as described previously (4). Bumetanide was added to the serosal solution at a concentration of 10 µM for 2 min before epinephrine was added. Results are expressed as means ± SE from 5 experiments.

 

Kinetics of the ATP effect on Na-K-Cl cotransport activity in monolayers of wt C7 and C11 cells. The kinetics of this effect was determined after 0- to 40-min preincubations in medium E + ATP ± BAPTA-AM. As seen in Fig. 2 (curve 1), a 2-min preincubation of C7 monolayers with ATP lead to an approximately twofold activation in NKCC activity, whereas 10-min to 40-min preincubation do not yield appreciable changes relative to baseline. In C11 cells, a transient activation is also observed after 2 min; compared with baseline, however, a decrease in 86Rb influx starts to manifest after 10 min and is maximal after 30 min. Pretreatment of cells with the intracellular Ca chelator BAPTA-AM prevents the transient ATP-induced activation in both C7 and C11 cells but not the delayed inhibition in C11 cells (Fig. 2, curve 2). For the studies presented below, accordingly, ATP was generally incubated 30 min with the monolayers before the 10-min fluxes.

Effect of ATP on Na-K-Cl cotransport activity in mock- and huNKCC1-transfected cells grown as monolayers. As seen in Fig. 3B, a 30-min preincubation with ATP leads to a sharp inhibition of NKCC activity in C11-mock cells (Fig. 3B), similar to what was observed in C11-wt cells (Fig. 2). In these experiments, additionally, we can see that ATP completely abolishes the increase in cotransport activity that occurs in C11 cells transfected with huNKCC1. For C7 cells, on the other hand, neither the endogenous Na-K-Cl cotransport activity nor the huNKCC1-mediated increase in cotransport activity is affected by ATP (Fig. 3A).



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Fig. 3. Effect of ATP on Na-K-Cl cotransport in monolayers of MDCK cells. A: mock-transfected and huNKCC1-transfected C7 cells. B: mock-transfected and huNKCC1-transfected C11 cells. Na-K-Cl cotransport activity was measured as rates of ouabain-resistant, bumetanide-sensitive 86Rb influxes across basolateral membranes. Except in control experiments, ATP was added to both serosal and mucosal solutions 30 min before the flux measurements. 86Rb (0.5 µCi/ml) and ouabain (100 µM) ± bumetanide (10 µM) were added to the serosal solution only. The bumetanide-sensitive component of 86Rb influxes in mock-transfected cells not exposed to ATP (43.3 ± 2.9 and 6.2 ± 0.5 nmol·cm-2·10 min-1 in C11 and C7 monolayers, respectively) was assigned a 100% value. Results are expressed as means ± SE of triplicates from 3 to 4 experiments.

 

Effect of UTP and adenosine on Na-K-Cl cotransport in huNKCC1-transfected C11 cells grown as monolayers. Extracellular ATP has been reported to interact with ionotropic P2X- and P2Y-purinoceptors (1), UTP with different types of P2Y-purinoceptors (8, 39), and adenosine, which is derived from ATP catabolism, with P1-purinoceptors. Hence, various nucleotides can be used to determine types or classes or purinoceptors express in a given cell type. As illustrated in Fig. 4, activity of huNKCC1-transfected C11 cells is completely blocked by UTP but unaffected by adenosine. These results suggest that inhibition of NKCC in C11 cells is mediated by P2Y-purinoceptors.



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Fig. 4. Effect of purinoceptor agonists on Na-K-Cl cotransport in monolayers of huNKCC1-transfected C11 cells. Na-K-Cl cotransport activity was measured as rates of ouabain-resistant, bumetanide-sensitive 86Rb influxes across basolateral membranes. ATP, UTP, and adenosine (100 µM) were added to both serosal and mucosal solutions 30 min before the flux measurements. 86Rb (0.5 µCi/ml) and ouabain (100 µM) ± bumetanide (10 µM) were added to the serosal solution only. Results are presented as means ± SE of quadruplicates from 3 experiments.

 

P2Y-expression in C7 and C11 cells. The inhibitory effect of ATP on NKCC activity could result from an interaction between nucleosides and P2Y-purinoceptor subtypes that are present in C11 cells but not in C7 cells. To verify this hypothesis, we conducted PCR experiments to identify which subtypes are expressed by each cell line. Not surprisingly, we were able to amplify several P2Y-purinoceptors, from the genomic DNA of C7 or C11 cells, e.g., P2Y1, P2Y2, and P2Y11 (Fig. 5; see also Refs. 41, 42) as well as P2Y4, P2Y6, P2Y12, and P2Y13 (results not shown). On the other hand, we were not able to detect important differences in the subset of P2Y-purinoceptors expressed by C7 or C11 cells. In addition, expression of UTP-sensitive P2Y-purinoceptors was weak or even absent in both cell lines (results only shown for P2Y2).

Effect of inhibitors of macromolecular synthesis on Na-K-Cl cotransport in wt C11 cells grown as monolayers. The delayed inactivation of NKCC by ATP in C11 cells (see Fig. 2) suggests (among various possibilities) that P2-purinoceptor-mediated pathways induce de novo synthesis of various proteins that can lead to inhibition of NKCC. Table 5 shows that actinomycin and cycloheximide decrease RNA and protein synthesis by ~30- and 4-fold, respectively, but that neither compound abolishes the ATP-induced inhibition of Na-K-Cl cotransport activity.


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Table 5. Effect of actinomycin D and cycloheximide on RNA/protein synthesis and Na-K-Cl cotransport activity in monolayers of C11 cells

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In recent studies, we have demonstrated that extracellular ATP is able to inhibit NKCC in renal epithelial cells derived from canine, rabbit, and rat collecting ducts (10, 27). We have also demonstrated that inhibition of NKCC by ATP occurs in C11- but not in C7-MDCK cells (27). In this work, we have examined the effect of ATP on mock-transfected MDCK cells as well as on huNKCC1-transfected MDCK cells to determine whether the C11 subclone, which resembles intercalated cells, expresses a unique P2-purinoceptor-sensitive NKCC or a unique P2-purinoceptor signaling pathway.

Several lines of evidence suggest that the NKCC expressed in C7 and C11 cells corresponds to the ubiquitous secretory isoform (22) and not to the absorptive NKCC2 (18, 19, 30) or to another NKCC isoform. 1) For both mock-transfected C7 and C11 subclones, bumetanide-sensitive cotransport is localized on the basolateral side (Table 3); in addition, epinephrine-induced change in Isc (Fig. 1) is sensitive to basolateral (but not to apical) application of bumetanide. 2) Other groups have previously identified NKCC1 basolaterally in the collecting duct (12, 13), a nephron segment from which MDCK cells are presumably derived (33). 3) HuNKCC1 transfected in C7- or C11-MDCK cells is also delivered basolaterally (Table 4). 4) In each cell line, the exogenous NKCC behaves like the endogenous transporter in response to purinergic activation; that is, huNKCC1 is inhibited by ATP in C11 cells but not in C7 cells (Fig. 3).

The results presented in this study suggest that NKCC1 is the isoform present in both C7 and C11 cells, but they do not allow ruling out the possibility that each cell type processes the same transporter variably, leading to NKCC1s with cell-specific posttranslational features. Differences in processing could indeed account for the variant behavior of NKCC1 in MDCK subclones. However, because anti-huNKCC1 and/or caNKCC antibodies are not available, and because cell-specific differences in processing could actually have no functional impact, it is important to examine the P2-purinoceptor-triggered pathway upstream of NKCC directly.

Results in this study also do not allow ruling out the possibility that differences in expression levels of endogenous caNKCC and/or exogenous huNKCC1 account for cell line-specific behaviors of the cotransporters. In monolayers of C7 cells, for example, NKCC-mediated 86Rb fluxes are generally lower than in those of C11 cells, whereas the huNKCC1-dependent increase in 86Rb fluxes is more important in the C11 monolayers (see Tables 3 and 4). Given these functional differences, quantitative measurements of huNKCC1 expression at the protein and/or RNA level would have been a useful adjunct. As mentioned above, however, NKCC1-specific antibodies are not available; in addition, it was also not possible to measure expression of huNKCC1 by RT-PCR due to technical difficulties.

Through this investigation, we have found that the effect of ATP on NKCC1 in C11 cells can be reproduced by UTP (Fig. 4). Because all P2X-purinoceptors except P2X7, which is restricted to macrophages, are insensitive to UTP and because several P2Y-purinoceptors (P2Y2, P2Y4, and P2Y6) are equally sensitive to UTP and ATP (8, 21, 39), the ATP-triggered NKCC inhibition in C11 cells could have been caused by activation of P2Y-receptors. As shown in Fig. 5, however, our studies showed that both C7 and C11 cells synthesize the same subsets of P2Y-purinoceptors. Hence, it appears that C11 cells express undetermined UTP-sensitive P2-purinoceptor subtypes or cell-specific postreceptor events that account for the ATP-induced effect.

In recent studies, we have begun to assess the role of postreceptor events, those referred to above, after identifying signaling intermediates in MDCK cells that affect NKCC activity or are affected by ATP. For example, we have shown that protein kinase C leads to inhibition of NKCC in wt C7 and C11 cells and that concentration of intracellular Ca, production of cAMP, and activation of enzymes such as phospholipase C, p42/p44 mitogen-activated protein kinase (MAPK), and JNK1 MAPK are increased by ATP; interestingly, activation of these MAPK occurs in C11 but not C7 cells (9, 27). However, we have found that suppression of protein kinase C with various agents or of p42/p44 MAPK with PD98059 does not preclude or affect purinergic inhibition of NKCC (9, 10). Hence, other signaling intermediates account for the ATP effect reported here, as well as in previous studies.

In this work, we have explored other postreceptor events that could potentially underlie the ATP effect by determining the role intracellular of Ca and that of various de novo transcribed or translated gene products. The latter events were especially important to investigate given the delayed kinetics of NKCC inactivation observed in C11 cells. Once more, however, these additional investigations were inconclusive. Indeed, we were able to show that the ATP effect on NKCC activity is BAPTA-AM insensitive (Fig. 2) and that it is not prevented by pretreating cells with inhibitors of RNA or of protein synthesis (Table 5).

During the course of the kinetics studies shown in Fig. 2, we observed that a brief exposure of MDCK cells to ATP has a very different effect compared with that of a longer exposure; that is, NKCC is transiently activated in both C7 and C11 cells. Quite interestingly, we were also able to show that under such circumstances, this activation is BAPTA-AM sensitive (Fig. 2). These results are consistent with stimulation of NKCC1 by Ca-raising stimuli as observed in various cell types (34) and suggest that ATP can influence NKCC activity through different pathways. On the other hand, they do not account for the delayed effect of ATP on NKCC.

Independently of P2Y-signaling pathways, postreceptor events could also involve changes in intracellular Cl or cell volume, both of which can affect the normal operation of NKCC (15, 16). For example, ATP could modulate Cl fluxes by altering the activity of other transport systems such as ion channels. On the basis of available data, however, this possibility appears unlikely. For example, we have recently shown that purinergic inhibition of NKCC in MDCK cells is not affected by changes in cell volume or by 30-min preincubations in Cl-free medium (9).

The exact origin of MDCK cells is unknown. Interestingly, C7 subclones behave like principal cells and C11 subclones like {alpha}-intercalated cells (11), suggesting that MDCKs originate from the collecting tubule. Interestingly, NKCC may play an important role in this specific nephron segment by maintaining the volume or the ionic content of individual cells and by promoting NH4 transport (Isenring et al., unpublished data; see also Ref. 13). During osmotic or ischemic stresses, for example, a regulatory decrease in the activity of this transporter (through ATP release) could prevent deleterious increases in cell volume or proton influx.

In conclusion, we have shown that ATP regulates NKCC differentially in C7 and C11 subclones because of cell-specific P2Y-purinoceptors or cell-specific postreceptor events and not because of differences in the NKCC protein per se. Further studies are required to determine the molecular mechanisms of P2Y-induced inhibition of NKCC1 in C11 cells and the implication of this effect in normal renal physiology, as well as in the pathophysiology of various diseases.


    DISCLOSURES
 
This study was supported by The Kidney Foundation of Canada, the Russian Foundation for Fundamental Sciences, and the Canadian Institute of Health and Research.


    ACKNOWLEDGMENTS
 
We acknowledge the precious advice of Dr. N. O. Dulin and D.-F. Guo, as well as the editorial assistance of Ovid M. Da Silva.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. Isenring, L'Hôtel-Dieu de Québec Research Center, 10 rue McMahon (Rm. 3852), QC, Canada G1R 2J6 (E-mail: paul.isenring{at}crhdq.ulaval.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* S. N. Orlov and P. Isenring contributed equally to this work. Back


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