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J. Biol. Chem., Vol. 281, Issue 42, 31317-31325, October 20, 2006

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Transient Activation and Delayed Inhibition of Na⁺,K⁺,Cl^– Cotransport in ATP-treated C11-MDCK Cells Involve Distinct P2Y Receptor Subtypes and Signaling Mechanisms^*

Olga A. Akimova, Alexandra Grygorczyk, Richard A. Bundey, Nathalie Bourcier, Michael Gekle^¶, Paul A. Insel^||, and Sergei N. Orlov¹

From the Centre de Recherche, Centre Hospitalier de l'Université de Montréal-Technopôle Angus, Montreal, Quebec H1W 4A4, Canada, the Departments of Pharmacology and ^||Medicine, University of California, San Diego, La Jolla, California 92093, and the ^¶Department of Physiology, University of Wurzburg, 97070 Wurzburg, Germany

Received for publication, March 6, 2006 , and in revised form, July 10, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In C11-MDCK cells, which resemble intercalated cells from collecting ducts of the canine kidney, P2Y agonists promote transient activation of the Na⁺,K⁺,Cl^– cotransporter (NKCC), followed by its sustained inhibition. We designed this study to identify P2Y receptor subtypes involved in dual regulation of this carrier. Real time polymerase chain reaction analysis demonstrated that C11-MDCK cells express abundant P2Y₁ and P2Y₂ mRNA compared with that of other P2Y receptor subtypes. The rank order of potency of agents (ATP UTP >> 2-(methylthio)-ATP (2MeSATP); adenosine 5'-[-thio]diphosphate (ADPS) inactive) indicated that P2Y₂ rather than P2Y₁ receptors mediate a 3–4-fold activation of NKCC within the first 5–10 min of nucleotide addition. NKCC activation in ATP-treated cells was abolished by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin (CaM) antagonists trifluoroperazine and W-7, and KN-62, an inhibitor of Ca²⁺/CaM-dependent protein kinase II. By contrast with the transient activation, 30-min incubation with nucleotides produced up to 4–5-fold inhibition of NKCC, and this inhibition exhibited a rank order of potency (2MeSATP > ADPS > ATP >> UTP) typical of P2Y₁ receptors. Unlike the early response, delayed inhibition of NKCC occurred in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-loaded cells and was completely abolished by the P2Y₁ antagonists MRS2179 and MRS2500. Transient activation and delayed inhibition of NKCC in C11 cell monolayers were observed after the addition of ATP to mucosal and serosal solutions, respectively. NKCC inhibition triggered by basolateral application of ADPS was abolished by MRS2500. Our results thus show that transient activation and delayed inhibition of NKCC in ATP-treated C11-MDCK cells is mediated by Ca²⁺/CaM-dependent protein kinase II- and Ca²⁺-independent signaling triggered by apical P2Y₂ and basolateral P2Y₁ receptors, respectively.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Na⁺,K⁺,Cl^– cotransporters (NKCC),² which belong to the superfamily of Cl^–-coupled monovalent cation cotransporters, provide electroneutral symport of monovalent ions and are selectively inhibited by bumetanide and other "high ceiling" diuretics. Two isoforms of this carrier have been cloned from vertebrate cDNA libraries. The NKCC1 isoform is expressed in all cell types studied thus far (1), including the basolateral membrane of epithelial cells from Madin-Darby canine kidneys (MDCK) (2); this isoform contributes to cell volume control (3) and adjustment of [Cl^–]_i above values predicted by Nernst equilibrium potential (4, 5) and might affect renal function via Cl^–_i-mediated regulation of vascular smooth muscle cell contraction (6, 7). In contrast to NKCC1, three alternatively spliced NKCC2 isoforms are found exclusively on the apical membrane of renal epithelial cells from the macula densa and thick ascending limb of Henle's loop, playing a key role in salt reabsorption and tubuloglomerular feedback regulation of renal function (8, 9).

Epithelial cells in the collecting ducts adjust salt reabsorption and acid secretion through the actions of various hormones and neurotransmitters, including vasopressin, bradykinin, atrial natriuretic peptide, prostanoids, mineralocorticoids, and catecholamines (10). Under certain circumstances, extracellular nucleotides, such as ATP, UTP, and ADP, also regulate ion transport in the renal epithelium by activating various signaling cascades (11, 12). Two major types of nucleotide receptors have been described: P2X receptors that are ligand-gated cation channels and P2Y receptors that are coupled to heterotrimeric G proteins (13, 14). Several subtypes of these receptors have been detected in renal epithelial cells along the nephron (15–19).

Searching for P2 receptor-sensitive ion transporters in renal epithelial cells, we observed that transient (5–10-min) application of extracellular ATP activates, whereas sustained exposure inhibits, NKCC in MDCK cells and in epithelial cells from the collecting ducts of rats and rabbits (20–22). Two populations of cells have been isolated from commercial stocks of MDCK cells: C7- and C11-MDCK cells (23). C7 cells have high transepithelial electrical resistance (R_te), are peanut lectin-negative, maintain pH_i at 7.39, and have a large K⁺ conductance, thus resembling principal cells from the collecting ducts. C11 cells, on the other hand, resemble intercalated cells; they have low R_te, are peanut lectin-positive, maintain pH_i at 7.16, and have large Cl^– and H⁺ conductances (23). Both C7 and C11 cells exhibit several identical mediators of ATP-induced intracellular signaling, including transient activation of NKCC; however, the inhibitory action of ATP on NKCC has been documented only in C11 cells (22, 24).

We reported that inhibition of NKCC in ATP-treated cells does not affect transmembrane gradient of monovalent cations (20), implying a key role of P2Y-rather than P2X-mediated signaling. In the current study, we sought to identify the P2Y receptor subtypes and downstream intermediates of intracellular signaling involved in the dual regulation of NKCC in C11 cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture—C11-MDCK cells were cultured in Dulbecco's modified Eagle's medium supplemented with 2.5 g/liter sodium bicarbonate, 2 g/liter HEPES, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (Invitrogen). The cells were passaged upon reaching subconfluent density by treatment in Ca²⁺- and Mg²⁺-free Dulbecco's phosphate-buffered saline with 0.1% trypsin and scraped from the flasks with a rubber policeman. Dispersed cells were counted and inoculated at 1.25 x 10³ cell/cm².

Measurement of K⁺ (⁸⁶Rb) Influx—Subconfluent cells growing in 24-well plates were washed twice with 2 ml of phosphate-buffered saline and incubated for 30 min at 37 °C in 1 ml of medium A or B with or without the test compounds. Medium A contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 5 mMD-glucose, and 20 mM HEPES-Tris (pH 7.4). In medium B, NaCl, KCl, and MgCl₂ were replaced by equimolar amounts of sodium gluconate, potassium gluconate, and MgSO₄, respectively. In 30 min, nucleotide and other test compounds were added, and the cells were incubated for up to 40 min. Then the medium was replaced by 0.25 ml of medium A containing 1 µCi/ml ⁸⁶Rb, 50 µM ouabain with or without 10 µM bumetanide. ⁸⁶Rb uptake was terminated by the addition of 2 ml of ice-cold medium C containing 100 mM MgCl₂ and 10 mM HEPES-Tris buffer (pH 7.4). The cells were then placed on ice, washed four times with 2 ml of ice-cold medium C, and lysed with 1 ml of 1% SDS, 4 mM EDTA mixture. The radioactivity of the cell lysate was measured with a liquid scintillation analyzer. ⁸⁶Rb influx was calculated as V = A/amt, where A is the radioactivity in the sample (cpm), a is the specific radioactivity of ⁸⁶Rb (K⁺) (cpm/nmol) in the incubation medium, m is the protein content in the sample (mg), and t is the incubation time (min). As shown previously, the kinetics of ⁸⁶Rb uptake in the presence of ouabain is linear for at least 20 min (21). Unless otherwise indicated, an incubation time of 5 min was used to determine the initial rate of K⁺ influx. NKCC activity was estimated as the rate of ouabain-resistant, bumetanide-sensitive ⁸⁶Rb influx.

To examine the role of basolateral versus apical location of P2Y receptors in NKCC regulation, we employed monolayers of C11 cells seeded on 1-cm² permeable inserts (Corning brand transwell plate inserts; Fisher) as described previously (25). Because of the basolateral location of NKCC in C11 monolayers (22), we added ⁸⁶Rb and bumetanide to the serosal side only.

Intracellular Cl^– Content—Intracellular Cl^– content was measured by ³⁶Cl distribution between C11 cells and extracellular medium under steady-state conditions, as described previously (4), and calculated as V = A/am, where A is the radioactivity of the cell lysate (cpm), m is the protein content (mg), and a is the specific radioactivity of the incubation medium (cpm/nmol).

Extracellular ATP Content—Extracellular ATP content was measured by assaying luciferase-dependent luminescence. For this purpose, 50 µl of incubation medium containing 100 µM ATP and up to 100 µM 6-N,N'-diethyl-,-dibromomethylene-D-adenosine-5-triphosphate (ARL 67156) was mixed with dilution buffer and luciferase in accordance with the ATP bioluminescent assay kit instructions (Sigma), and luminescence was measured with a TD 20/20 luminometer (Turner Designs, Inc., Sunnyvale, CA). In preliminary experiments, we found that the intensity of luminescence was linear up to an ATP concentration of 10 µM and was only slightly affected by ARL 67156 (2,395 ± 31 and 2,168 ± 59 cpm in 10-fold diluted samples containing 100 µM ATP and 100 µM ATP plus 100 µM ARL 67156, respectively).

[Ca²⁺]_i Measurement in Single Cells by Fluorescence Ratio Imaging—The cells grown on coverslips were incubated during 30 min in medium A containing 5 µM fura 2-AM with or without 20 µM BAPTA-AM. Then cells were washed, placed in the bottom of a laminar flow-through chamber mounted on the stage of a Nikon inverted microscope (Eclipse TE300; Nikon, Tokyo, Japan), and perfused with control or Ca²⁺-free medium A. The cells were illuminated at 340 and 380 nm with a 100-watt mercury lamp and interference filters. Images at 510-nm emitted light were acquired via a x40 objective at the rate of 1 ratio image/4 s. For more details, see Refs. 26 and 27.

Total RNA—Total RNA was isolated from cells seeded in 75-cm² flasks by Trizol reagent (Invitrogen). First strand cDNA synthesis was carried out with 1 µg of total RNA and random primers using SuperScript First-Strand Synthesis 11904-018 (Invitrogen) as recommended by the manufacturer.

Real Time PCR—Real time PCR was performed on 2 ng of reverse-transcribed RNA with a QuantiTect SYBR kit (Qiagen, Valencia, CA) in conjunction with forward/reverse primers for canine P2Y receptor subtypes shown in our previous papers (22, 27) and manufactured by Integrated DNA Technologies (Coralville, IA). An Opticon monitor system (MJ Research, Hercules, CA) was used for thermocycling and detection. Primer specificity was confirmed by melting curve analysis, gel electrophoresis of PCR products, and sequencing. cDNA synthesized in the absence of reverse transcriptase was used as a negative control. RNA from dog brain and lymphocytes were used as positive controls.

Western Blot Analysis—Western blot analysis was performed in accordance with a previously described protocol (27) using anti-P2Y₁ and anti-P2Y₂ antibodies from Alomone Laboratories (Jerusalem, Israel). Antibody preabsorption to peptide antigen controls (P2Y₁, RALIYKDLDNSPLRRKS; P2Y₂, KPAYGTTGLPRAKRKSVR) were used to validate antibody specificity.

Statistics—The data were analyzed by Student's t test or the t test for dependent samples, as appropriate. Significance was defined as p < 0.05.

Chemicals—ATP, UTP, 2MeSATP, ADPS, ouabain, bumetanide, indomethacin, ARL 67156, and 2'-deoxy-N⁶-methyladenosine-3',5'-biphosphate (MRS2179) were obtained from Sigma; BAPTA-AM, trifluoroperazine, W-7, KN-62, and KN-92 were from Calbiochem; ⁸⁶RbCl and H³⁶Cl were from DuPont; salts, D-glucose, and buffers were from Sigma and Anachemia (Montreal, Canada). 2'-Iodo-N⁶-methyl-(N)-metanocarba-2'-deoxyadenosine-3',5'-biphosphate (MRS2500) was kindly provided by Dr. K. A. Jacobson (University of North Carolina, Chapel Hill, NC).

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Kinetics of modulation of the rate of 86Rb influx in C11 cells by ATP. Curve 1, medium contains 50 µM ouabain; curve 2, medium contains ouabain + 10 µM bumetanide; curve 3, NKCC (ouabain-resistant, bumetanide-sensitive 86Rb influx-1,2). Cells were preincubated for 30 min in medium A, and ATP was added at a final concentration of 100 µM for the times indicated. This medium was then aspirated, and medium A containing 86Rb plus ouabain with or without bumetanide was added for 5 min. Mean ± S.E. values from experiments performed in triplicate are shown.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effect of Intracellular Cl^–—Table 1 shows that Cl^–_i depletion of C11 cells, produced by substitution of extracellular Cl^– with gluconate, activated NKCC by 8-fold but did not affect bumetanide-resistant ⁸⁶Rb influx. These data are consistent with a negative feedback regulation of NKCC by Cl^–_i as documented in other cell types (1). Cl^–_i depletion completely abolished NKCC activation seen after 5-min treatment of cells with ATP, whereas the inhibitory action of a 30-min exposure to ATP was preserved. On the other hand, the base-line values of NKCC in control conditions (medium A) are 3-fold less than total ouabain-resistant ⁸⁶Rb influx (Fig. 1, Table 1), which complicates precise estimation of down-regulation of the carrier. Considering this, we explored the mechanism of transient activation and delayed inhibition of NKCC by treating cells with ATP and other P2Y agonists for 5 min in Cl^–-rich medium and 30 min in Cl^–-depleted medium, respectively.

Dose-dependent Modulation of NKCC by Nucleotides—Nucleotides activate P2Y₁ receptors cloned from human, bovine, rat, and mouse cDNA libraries with a rank order of potency 2MeSATP ATP ADPS UTP (28, 29). These observations contrast with the rank order of potency UTP ATP > 2MeSATP of cells transfected with P2Y₂ receptors cloned from mammalian cells (28), including MDCK cells (30). We thus compared the ability of such compounds to promote rapid activation and delayed inhibition of NKCC. Analysis of dose-dependent action of nucleotides, especially with long term application, is complicated by their degradation by ectonucleotidases. To overcome this problem, we tested ARL 67156, a compound that inhibits ecto-ATPase activity (31, 32). Fig. 3 illustrates that by 40 min of incubation, ATP concentration in the incubation medium was decreased from 100 to 13 ± 2 µM and that ARL 67156 dose-dependently attenuated ATP degradation.

We did not observe any significant modulation of NKCC in C11 cells treated for 30 min with 100 µM ARL 67156 (data not shown). ARL 67156 did not significantly affect the dose-dependent action of the nonhydrolyzable ADP analog ADPS but sharply potentiated the inhibitory action of three other tested nucleotides on the activity of the carrier (Fig. 4). Thus, for example, half-maximal inhibition of NKCC in the absence and presence of ARL 67156 occurred at ATP concentrations of 30 and 1 µM, respectively. In the presence of ARL 67156, the rank order of potency of NKCC inhibition after 30 min of nucleotide addition was 2MeSATP > ADPS > ATP >> UTP (Fig. 4b).

The presence of 100 µM ARL 67156 did not affect the dose-dependent activation of NKCC caused by 5-min exposure to ATP (data not shown). In contrast to the inhibitory action, the NKCC activation exhibited similar sensitivity to ATP and UTP; 2MeSATP was much less effective, whereas ADPS had no effect (Fig. 5). Viewed collectively, these results suggest that transient activation and delayed inhibition of NKCC in C11 cells are triggered by P2Y₂ and P2Y₁ receptors, respectively.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. P2Y receptor expression in C11 cells. A, real time PCR was performed using P2Y subtype-specific primer pairs in conjunction with SYBR Green dye I. mRNA abundance was normalized to glyceraldehyde-3-phosphate dehydrogenase expression and presented relative to P2Y2 receptors. Mean ± S.E. values from experiments performed in triplicate are shown. B, Western blot using anti-P2Y1 and anti-P2Y2 antibodies (Ab) in the absence and presence of peptide antigens (Ag).

View larger version (14K): [in this window] [in a new window]   FIGURE 3. Effect of ARL 67156 on ATP concentration in the incubation medium. Cells seeded in 24-well plates were incubated in 0.5 µl of medium A containing 100 µM ATP and ARL 67156 at the concentrations indicated. After 40 min, 50-µl aliquots of medium were collected, and ATP concentration was measured as indicated under "Materials and Methods." Mean ± S.E. values from experiments performed in duplicate are shown.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Concentration-dependent effect of 30-min incubation with ATP, ADPS, UTP, and 2MeSATP on NKCC in C11 cells in the absence (a) or presence (b) of 100 µM ARL 67156. Cells were preincubated for 1 h in medium B. Nucleotides and ARL 67156 were added during the last 30 min of incubation. NKCC was measured in medium A as indicated in the legend to Fig. 1. NKCC activity in nucleotide-untreated cells was taken as 100%. Mean values from experiments performed in triplicate are shown.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. Concentration-dependent effect of 5-min incubation with ATP, ADPS, UTP, and 2MeSATP on NKCC in C11 cells. After 30 min of preincubation in medium A, the medium was aspirated, and medium A containing 1 µCi/ml 86Rb and 50 µM ouabain with or without 10 µM bumetanide and nucleotides at the indicated concentrations was added for 5 min. NKCC activity in nucleotide-untreated cells was set at 100%. Mean ± S.E. values from experiments performed in triplicate are shown.

To further verify asymmetrical location of P2Y₂ and P2Y₁ involved in transient activation and delayed inhibition of NKCC, we treated C11 monolayers with ADPS or UTP. Data obtained with nonpolarized cells showed that ADPS, a nonhydrolyzable P2Y₁ agonist, inhibited but did not activate NKCC, whereas UTP was a potent activator but not an inhibitor of NKCC (Figs. 4 and 5). Table 3 shows that activation of NKCC occurred after 5-min addition of UTP but not ADPS to the solution on the mucosal side. As noted above, the selective P2Y₁ antagonist MRS2500 did not affect transient activation of NKCC but completely abolished delayed inhibition of this carrier in ATP-treated nonpolarized C11 cells (Fig. 6). Neither basal nor apical application of MRS2500 abolished activation of NKCC by apical UTP. In contrast, significant inhibition of NKCC occurred in response to basolateral MRS2500 after a 30-min application of ADPS but not UTP to the serosal solution.

View this table: [in this window] [in a new window]   TABLE 3 Effect of ADP S and MRS2500 on NKCC activity in C11 cell monolayers For experiment A, after a 1-h incubation in medium A, 1 µCi/ml 86Rb, 50 µM ouabain with or without 10 µM bumetanide were added to the serosal solution for 5 min, whereas 10 µM ADPS, 10 µM UTP, and 3 µM MRS2500 were added to apical or basolateral surfaces of monolayers. For experimental B, cells were incubated for 1 h in Cl–-depleted medium B and 10 µM ADPS or 10 µM UTP with or without 3 µM MRS2500 µM were added to apical or basolateral surfaces of monolayers during the last 30 min of incubation. The medium was then aspirated, and medium A containing 50 µM ouabain with or without 10 µM bumetanide was added for 5 min to the apical and basolateral surface of the monolayers, respectively. Serosal medium also contained 1 µCi/ml 86Rb. NKCC activity in ADPS- and MRS2500-untreated cells was taken as 100%. Mean ± S.E. values from experiments performed in quadruplicate are shown.

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Effect of MRS2179 and MRS2500 on transient activation (curves 1 and 2) and delayed inhibition (curves 3 and 4) of NKCC after 5 and 30 min of treatment with ATP, respectively. Curves 1 and 2, after 30 min of preincubation in medium A, the medium was aspirated, and medium A containing 1 µCi/ml 86Rb and 50 µM ouabain with or without 10 µM bumetanide, MRS2179, or MRS2500 and 100 µM ATP was added for 5 min. Curves 3 and 4, cells were preincubated for 1 h in medium B with or without MRS2179 or MRS2500, and 200 µM ATP was added during the last 30 min of incubation. Then this medium was aspirated, and medium A containing 1 µCi/ml 86Rb and 50 µM ouabain with or without 10 µM bumetanide was added for 5 min. NKCC activity in control cells was taken as 100%. Mean ± S.E. values from experiments performed in quadruplicate are shown.

View larger version (26K): [in this window] [in a new window]   FIGURE 7. Transient activation (a) and delayed inhibition (b) of NKCC after 5 and 30 min of treatment with ATP applied to apical and basolateral surfaces of monolayers of C11 cells. a, after a 1-h incubation in medium A, 1 µCi/ml 86Rb and 50 µM ouabain with or without 10 µM bumetanide were added to the serosal solution for 5 min, whereas 200 µM ATP was added to apical (ATPap), basolateral (ATPbas), or both (ATPap + basATPap + bas) surfaces of monolayers. b, cells were incubated for 1 h in Cl–-depleted medium B, and 200 µM ATP was added to apical, basolateral, or both surfaces of monolayers during the last 30 min of incubation. The medium was then aspirated, and medium A containing 50 µM ouabain with or without 10 µM bumetanide was added for 5 min to the apical and basolateral surface of the monolayers, respectively. Serosal medium also contained 1 µCi/ml 86Rb. NKCC activity in ATP-untreated cells was taken as 100%. Mean ± S.E. values from experiments performed in quadruplicate are shown.

CAMKII mediates Ca²⁺_i-induced signaling triggered by diverse stimuli (37). We observed that 30-min incubation with KN-62, a cell-permeable inhibitor of CAMKII, completely abolished NKCC activation triggered by 5-min exposure to ATP, whereas its inactive analogue KN-92 decreased the activatory action of ATP by only 20% (Fig. 11). Neither CaM antagonists nor KN-62 affected NKCC inhibition seen after 30-min exposure to ATP (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Consistent with previously reported data (22), we observed that ATP affects NKCC in C11-MDCK cells in a biphasic manner; transient activation seen during the initial 5–10 min of ATP addition was followed by inhibition of the carrier after 30-min incubation of cells with ATP (Fig. 1, Table 1). In the current study, we analyzed the mechanism of this phenomenon and draw three major conclusions (Fig. 12). First, transient activation and delayed inhibition of NKCC in ATP-treated C11 cells is triggered by P2Y₂ and P2Y₁ receptors, respectively. Second, P2Y₁ and P2Y₂ receptors involved in dual regulation of NKCC are located on the basolateral and apical membranes of C11 cells, respectively. Third, transient activation of NKCC is mediated by elevation of [Ca²⁺]_i and CAMKII, whereas delayed inhibition of the carrier is a Ca²⁺_i-independent phenomenon. Below, we discuss evidence supporting these conclusions.

Real time PCR showed that C11 cells express P2Y₁ and P2Y₂ receptor mRNAs but a negligible amount of mRNA for P2Y₁₁, P2Y₁₂, and P2Y₁₄ and no detectable levels of P2Y₄, P2Y₆, and P2Y₁₃ mRNA (Fig. 2). The half-maximal inhibition of NKCC caused by 30-min incubation with 2MeSATP, ADPS, ATP, and UTP in the presence of the ecto-ATPase inhibitor ARL 67156 occurs at concentrations of 0.2, 0.8, 1.0, and >100 µM, respectively (Fig. 4b), results that are consistent with a rank order of potency for activation of cloned P2Y₁ (2MeSATP ATP ADPS UTP) rather than P2Y₂ (UTP ATP > 2MeSATP; ADPS inactive) and/or P2Y₁₁ (ATP > 2MeSATP; UTP inactive) receptors (28, 29, 38, 39). By contrast, ADPS had no effect, and 2MeSATP was much less effective in the transient activation of NKCC (IC₅₀ 100 µM) compared with ATP and UTP, both of which had similar apparent affinities (IC₅₀ 0.3 µM) (Fig. 5), results that are consistent with activation of P2Y₂ receptors. The conclusions regarding the distinct roles of P2Y₁ and P2Y₂ receptors are also supported by data obtained with P2Y₁ antagonists MRS2179 and MRS2500; both compounds abolished inhibition of NKCC caused by 30-min exposure of C11 cells to ATP but did not affect transient activation of the carrier (Fig. 6).

View larger version (9K): [in this window] [in a new window]   FIGURE 8. Representative records of the effect of 100µM ATP on the F340/F380 ratio in control (1 and 2) and BAPTA-loaded (3) C11 cells measured in control (1) or in Ca2+-free medium A (2 and 3). In Ca2+-free medium, CaCl2 was substituted by the addition of 0.1 mM EGTA.

Similar to [Ca²⁺]_i elevation (Fig. 8), transient activation of NKCC by ATP was partially suppressed in the absence of extracellular Ca²⁺ and was completely abolished in cells loaded with the intracellular Ca²⁺ chelator BAPTA (Fig. 9a). Transient activation of NKCC in ATP-treated cells was also inhibited by the CaM antagonists trifluoroperazine and W-7 (Fig. 10) over concentration ranges that block their interactions with CaM-Ca²⁺ complexes in a cell-free system (36) and was completely abolished in the presence of KN-62, an inhibitor of CAMKII (Fig. 11). These results and prominent inositol 1,4,5-trisphosphate release in ATP-treated C11 cells (24) lead us to conclude that transient activation of NKCC by ATP is mediated by a P2Y₂-phospholipase C-inositol 1,4,5-trisphosphate-[Ca²⁺]_i-CaM-CAMKII signaling cascade (Fig. 12).

Using C11 cells, we demonstrated that ATP completely abolished an increment of bumetanide-sensitive ⁸⁶Rb uptake triggered by transfection with human NKCC1 (22). Importantly, analysis of NKCC1 cDNA from different species did not reveal regions that fulfill the criteria for phosphorylation by CAMKII (42, 43), suggesting that CAMKII activates NKCC1 via phosphorylation of an unidentified regulator of the carrier activity. Because Ca²⁺_i depletion sharply attenuated an increment of NKCC activity seen after preincubation of cells in Cl^–-free medium (Fig. 9b), this downstream intermediate of [Ca²⁺]_i-CaM-CAMKII signaling may also be involved in sensing intracellular Cl^– concentration and in negative feedback regulation of the carrier's activity. An alternative hypothesis is that elevation of [Ca²⁺]_i and activation of CAMKII opens Ca²⁺-activated Cl^– channels, which activates NKCC via lowering of [Cl^–]_i. However, this hypothesis is not supported by data showing a lack of effect of 5-min incubation with ATP on Cl^–_i content (Table 2).

View larger version (28K): [in this window] [in a new window]   FIGURE 9. Effect of Ca2+o and intracellular Ca2+ chelator BAPTA on transient activation (a) and delayed inhibition (b) of NKCC after 5 and 30 min of treatment with ATP, respectively. a, cells were incubated for 1 h in medium A, and 20 µM BAPTA-AM was added during the last 30 min of incubation. This medium was aspirated, and control or Ca2+-free medium A containing 1 µCi/ml 86Rb, 50 µM ouabain with or without 10 µM bumetanide with or without 200 µM ATP was added for 5 min. b, cells were incubated for 1 h in control or Ca2+-free Cl–-depleted medium B; 20 µM BAPTA-AM and 200 µM ATP were added during the last 60 and 30 min of incubation, respectively. The medium was then aspirated, and medium A containing 1 µCi/ml 86Rb and 50 µM ouabain with or without 10 µM bumetanide was added for 5 min. In Ca2+-free medium, CaCl2 was substituted by the addition of 0.1 mM EGTA. Mean ± S.E. values from experiments performed in triplicate are shown.

View larger version (20K): [in this window] [in a new window]   FIGURE 10. Effect of CaM antagonists on NKCC activity in control and ATP-treated C11 cells. Cells were incubated for 1 h in medium A, and trifluoroperazine (TFP) and W-7 were added during the last 30 min of incubation. Then the medium was aspirated, and control or Ca2+-free medium A containing 1 µCi/ml 86Rb and 50 µM ouabain with or without 10 µM bumetanide with or without 200 µM ATP was added for 5 min. NKCC activity in the absence of ATP and CaM antagonists was set at 100%. Mean ± S.E. values from experiments performed in quadruplicate are shown.

View larger version (27K): [in this window] [in a new window]   FIGURE 11. Effect of KN-62 and KN-92 on NKCC in control and ATP-treated C11 cells. Cells were preincubated for 30 min in medium A with or without 10 µM KN-62 and KN-92. The same volume of medium A containing 1 µCi/ml 86Rb and 50 µM ouabain with or without 20 µM bumetanide with or without 200 µM ATP was then added for 5 min. NKCC activity in the absence of ATP, KN-62, and KN-92 was taken as 100%. Mean ± S.E. values from experiments performed in quadruplicate are shown.

View larger version (14K): [in this window] [in a new window]   FIGURE 12. Intermediates of intracellular signaling involved in transient activation and delayed inhibition of NKCC in C11 cells. a.m. and b.m., apical and basolateral membrane, respectively; DAG, diacylglycerol; ER, endoplasmic reticulum; Gq/11, heterotrimeric GTP-binding proteins;?, unknown steps; and , activation and inhibition, respectively.

Is there a physiological significance of dual regulation of NKCC by P2Y receptors? Ectonucleotidases maintain a circulating blood level of ATP < 10 nM (48, 49), a concentration at which renal P2Y receptors on serosal membranes are not likely to be activated (13). However, nucleotides can act in both a paracrine and autocrine manner, reaching high concentrations in the peritubular space after sympathetic stimulation or exposure to osmotic, mechanical, and ischemic stresses (50, 51). These stimuli might be sufficient for transient elevation of [ATP]_o (or perhaps [UTP]_o), P2Y₂-mediated activation of NKCC, and modulation of transepithelial ion movement. In contrast, sustained elevation of [ATP]_o, causing P2Y₁-mediated NKCC inhibition, is likely to occur in pathological conditions, resulting in injury-mediated release of intracellular ATP or inhibition of ecto-ATPases. Further studies will be needed to define downstream intermediates of [Ca²⁺]_i-CaM-CAMKII and Ca²⁺-independent signaling involved in P2Y₂-mediated activation and P2Y₁-mediated inhibition of NKCC and to define the physiological and pathophysiological roles of these P2Y receptor-regulated phenomena.

	FOOTNOTES

 
^* This work was supported by grants from the Kidney Foundation of Canada (to S. N. O.) and National Institutes of Health Grant GM66232 (to P. A. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Centre de Recherche, CHUM-Technopôle Angus, 2901 Rachel Es Rm. 313, Montreal, Quebec H1W 4A4, Canada. Tel.: 514-890-8000 (ext. 23615); Fax: 514-412-7638; E-mail: sergei.n.orlov{at}umontreal.ca.

² The abbreviations and trivial names used are: NKCC, Na⁺,K⁺,Cl^– cotransporter(s); ADPS, adenosine 5'-[-thio]diphosphate; 2MeSATP, 2-(methylthio)-ATP; MRS2179, 2'-deoxy-N⁶-methyladenosine-3',5'-biphosphate; MRS2500, 2'-iodo-N⁶-methyl-(N)-metanocarba-2'-deoxyadenosine-3',5'-biphosphate; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; AM, acetoxymethyl ester; MDCK, Madin-Darby canine kidney; ARL 67156, 6-N,N'-diethyl-,-dibromomethylene-D-adenosine-5-triphosphate.

	ACKNOWLEDGMENTS

 
The technical assistance of Monique Poirier and the editorial help of Ovid Da Silva (Research Support Office, Centre de Recherche, CHUM) are appreciated.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Russell, J. M. (2000) Physiol. Rev. 80, 212–276
2. Delpire, E., Rauchman, M. I., Beier, D. R., Hebert, S. C., and Gullans, S. R. (1994) J. Biol. Chem. 269, 25677–25682[Abstract/Free Full Text]
3. Orlov, S. N., Tremblay, J., and Hamet, P. (1996) Am. J. Physiol. 270, C1388–C1397
4. Orlov, S. N., Tremblay, J., and Hamet, P. (1996) J. Membr. Biol. 153, 125–135[CrossRef][Medline] [Order article via Infotrieve]
5. Delpire, E. (2000) News Physiol. Sci. 15, 309–312[Abstract/Free Full Text]
6. Akar, F., Jiang, G., Paul, R. J., and O'Neill, W. C. (2001) Am. J. Physiol. 281, C579–C584
7. Anfinogenova, Y. J., Baskakov, M. B., Kovalev, I. V., Kilin, A. A., Dulin, N. O., and Orlov, S. N. (2004) Pflügers Arch. 449, 42–55[CrossRef][Medline] [Order article via Infotrieve]
8. Greger, R. (2000) Am. J. Med. Sci. 319, 51–62[CrossRef][Medline] [Order article via Infotrieve]
9. Gamba, G. (2005) Physiol. Rev. 85, 423–493[Abstract/Free Full Text]
10. Breyer, M. D., and Ando, Y. (1994) Annu. Rev. Physiol. 56, 711–739[CrossRef][Medline] [Order article via Infotrieve]
11. Bailey, M. A., Hillman, K. A., and Unwin, R. J. (2000) J. Auton. Nerv. Syst. 81, 264–270[CrossRef][Medline] [Order article via Infotrieve]
12. Chan, C. M., Unwin, R. J., and Burnstock, G. (2001) Exp. Nephrol. 6, 200–207
13. Fredholm, B. B., Abbracchio, M. P., Burnstock, G., Daly, J. W., Harden, T. K., Jackobson, K. A., Leff, P., and Williams, M. (1994) Pharmacol. Rev. 46, 143–156[Medline] [Order article via Infotrieve]
14. Williams, M., and Jarvis, M. F. (2000) Biochem. Pharmacol. 59, 1173–1185[CrossRef][Medline] [Order article via Infotrieve]
15. Bailey, M. A., Imbert-Teboul, M., Turner, C., Marsy, S., Strai, K., Burnstock, G., and Unwin, R. J. (2000) Kidney Int. 58, 1893–1901[CrossRef][Medline] [Order article via Infotrieve]
16. Rice, W. R., Burton, F. M., and Fiedeldey, D. T. (1995) Am. J. Respir. Cell Mol. Biol. 12, 27–32[Abstract]
17. Schwiebert, E. M., Wallace, D. P., Braunstein, C. M., King, S. R., Peti-Peterdi, J., Hanaoka, K., Guggino, W. B., Guay-Woodford, L. M., Bell, P. D., Sullivan, L. P., Grantham, J. J., and Taylor, A. L. (2002) Am. J. Physiol. 282, F763–F775
18. Takeda, M., Kobayashi, M., and Endou, H. (1998) Biochem. Mol. Biol. Int. 44, 657–664[Medline] [Order article via Infotrieve]
19. Tokuyama, Y., Hara, M., Jones, E. M., Fan, Z., and Bell, G. I. (1995) Biochem. Biophys. Res. Commun. 211, 211–218[CrossRef][Medline] [Order article via Infotrieve]
20. Gagnon, F., Dulin, N. O., Tremblay, J., Hamet, P., and Orlov, S. N. (1999) J. Membr. Biol. 167, 193–204[CrossRef][Medline] [Order article via Infotrieve]
21. Gagnon, F., Orlov, S. N., Tremblay, J., and Hamet, P. (1998) Biochim. Biophys. Acta 1369, 233–239[Medline] [Order article via Infotrieve]
22. Brindikova, T. A., Bourcier, N., Torres, B., Pchejetski, D., Gekle, M., Maximov, G. V., Montminy, V., Insel, P. A., Orlov, S. N., and Isenring, P. (2003) Am. J. Physiol. 285, C1445–C1453
23. Gekle, M., Wunsch, S., Oberleithner, H., and Silbernagl, S. (1994) Pflugers Arch. 428, 157–162[CrossRef][Medline] [Order article via Infotrieve]
24. Orlov, S. N., Dulin, N. O., Gagnon, F., Gekle, M., Douglas, J. G., Schwartz, J. H., and Hamet, P. (1999) J. Membr. Biol. 172, 225–234[CrossRef][Medline] [Order article via Infotrieve]
25. Bourcier, N., Grygorczyk, R., Gekle, M., Berthiaume, Y., and Orlov, S. N. (2002) J. Membr. Biol. 186, 131–143[CrossRef][Medline] [Order article via Infotrieve]
26. Taurin, S., Dulin, N. O., Pchejetski, D., Grygorczyk, R., Tremblay, J., Hamet, P., and Orlov, S. N. (2002) J. Physiol. 543, 835–847[Abstract/Free Full Text]
27. Akimova, O. A., Bourcier, N., Taurin, S., Bundey, R. A., Grygorczyk, K., Gekle, M., Insel, P. A., Dulin, N. O., and Orlov, S. N. (2005) J. Physiol. 568, 789–801[Abstract/Free Full Text]
28. von Kügelgen, I. (2006) Pharmacol. Ther. 110, 415–432[CrossRef][Medline] [Order article via Infotrieve]
29. Burnstock, G., and Knight, G. E. (2004) Int. Rev. Cytol. 240, 31–304[Medline] [Order article via Infotrieve]
30. Zambon, A. C., Hughes, R. J., Meszaros, J. G., Wu, J. J., Torres, B., Brunton, L. L., and Insel, P. A. (2000) Am. J. Physiol. 279, F1045–F1052
31. Berra-Romani, R., Rinaldi, C., Rageeb, A., Castelli, L., Magistretti, J., Taglietti, V., and Tanzi, F. (2004) J. Vasc. Res. 41, 166–173[CrossRef][Medline] [Order article via Infotrieve]
32. Drakulish, D. A., Spellmon, C., and Hexum, T. D. (2004) Eur. J. Pharmacol. 485, 137–140[CrossRef][Medline] [Order article via Infotrieve]
33. Cattaneo, M., Lecchi, A., Ohno, M., Joshi, B. V., Besada, P., Tchilibon, S., Lombardi, R., Bischofberger, N., Harden, T. K., and Lacobson, K. A. (2004) Biochem. Pharmacol. 68, 1995–2002[CrossRef][Medline] [Order article via Infotrieve]
34. Paulmichl, M., Pfleilshifter, J., Woll, E., and Lang, F. (1991) J. Cell. Physiol. 147, 68–75[CrossRef][Medline] [Order article via Infotrieve]
35. Delles, C., Haller, T., and Dietl, P. (1995) J. Physiol. 486, 557–569[Abstract/Free Full Text]
36. Anfinogenova, Y. J., Rodriguez, X., Grygorczyk, R., Adragna, N., Lauf, P. K., Hamet, P., and Orlov, S. N. (2001) Cell Physiol. Biochem. 11, 295–310[CrossRef][Medline] [Order article via Infotrieve]
37. Hanson, P. I., and Schulman, H. (1992) Annu. Rev. Biochem. 61, 559–601[CrossRef][Medline] [Order article via Infotrieve]
38. Vassort, G. (2001) Physiol. Rev. 81, 767–806[Abstract/Free Full Text]
39. Kunapuli, S. P., and Daniel, J. D. (1998) Biochem. J. 336, 513–523
40. Wolff, S. C., Qi, A.-D., Harden, T. K., and Nicholas, R. A. (2005) Am. J. Physiol. 288, C624–C632
41. Qi, A.-D., Wolff, S. C., and Nicholas, R. A. (2005) J. Biol. Chem. 280, 29169–29175[Abstract/Free Full Text]
42. Haas, M., and Forbush, B., III (2000) Annu. Rev. Physiol. 62, 515–534[CrossRef][Medline] [Order article via Infotrieve]
43. Mount, D. B., Delpire, E., Gamba, G., Hall, A. E., Poch, E., Hoover, R. S., and Hebert, S. C. (1999) J. Exp. Biol. 201, 2091–2102
44. Gagnon, K. B., England, R., and Delpire, E. (2006) Am. J. Physiol. 290, C134–C142
45. Gagnon, K. B. E., England, R., and Delpire, E. (2006) Mol. Cell. Biol. 26, 689–698[Abstract/Free Full Text]
46. Gamba, G. (2004) Am. J. Physiol. 288, F245–F252
47. Piechotta, K., Lu, J., and Delpire, E. (2002) J. Biol. Chem. 277, 50812–50819[Abstract/Free Full Text]
48. Lazarosski, E. L., and Boucher, R. C. (2001) News Physiol. Sci. 16, 1–5[Abstract/Free Full Text]
49. Joseph, S. M., Buchakjian, M. R., and Dubyak, G. R. (2003) J. Biol. Chem. 278, 23331–23342[Abstract/Free Full Text]
50. Ostrom, R. S., Gregorian, C., and Insel, P. A. (2000) J. Biol. Chem. 275, 11735–11739[Abstract/Free Full Text]
51. Schwiebert, E. M., and Zsembery, A. (2003) Biochim. Biophys. Acta 1615, 7–32[Medline] [Order article via Infotrieve]

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