Cl⁻secretion in ATP-treated renal epithelial C7–MDCK cells is mediated by activation of P_2Y1 receptors, phospholipase A₂ and protein kinase A

Methods

Cell culture

C7–MDCK cells, obtained as previously described (Gekle et al. 1994), were cultured in Hepes-NaHCO₃-buffered Dulbecco's modified Eagle's medium (DMEM, pH 7.4) supplemented with 2.5 g l⁻¹ sodium bicarbonate, 2 g l⁻¹ Hepes, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin and 10% fetal bovine serum. Upon reaching subconfluency, they were passaged 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 × 10³ cells cm⁻² in coverslips, 35 mm Petri dishes, 12-well plates or 1 cm² permeable inserts (Corning Brand Transwell plate inserts, Fisher Scientific, Montreal, PQ, USA).

Adenovirus-mediated gene transduction

Cells seeded in permeable inserts or 12-well plates were subjected to 24 h serum deprivation and incubated for the next 24 h in DMEM containing 0.1% bovine serum albumin (BSA) and 3 × 10⁹ viral particles (v.p.) ml⁻¹ of E1⁻, E3⁻ replication-deficient adenovirus (Ad5) encoding the cDNA for the PKA inhibitor, PKI (AdPKI). We have previously confirmed the efficiency of AdPKI transduction in a specific inhibition of PKA in intact cells (Hogarth et al. 2004). Adenovirus encoding the CMV-driven LacZ (AdLacZ) gene served as a control.

Electrical measurements

After 3–4 days of seeding on Transwell inserts, R_te values were measured with EVOM^. (World Precision Instruments, Sarasota, FL, USA). For I_SC measurements, the Transwell inserts containing monolayers with R_te in the range from 1500 to 3500 Ω × cm² were subjected to 24 h serum deprivation and mounted between halves of an Ussing chamber (Warner Instrument Corp., Hamden, CT, USA). Fluid in each half of the chamber 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 Instrument Corp.). Basolateral and apical solutions, 16 and 14 ml, respectively, containing DMEM with 0.1% BSA were circulated by airlifting with 5% CO₂/95% air and kept at 37°C in a water jacket. For more details, see Bourcier et al. (2002).

[Ca²⁺]_i, measurement

C7–MDCK cells grown on glass coverslips were incubated for 30–40 min in medium B containing 140 mm NaCl, 5 mm KCl, 1 mm MgCl₂, 1 mm CaCl₂, 5 mm glucose, 20 mm Hepes-Tris buffer (pH 7.4) and supplied with 5 μm fura 2-AM. Then, they were washed twice with medium B and kept for up to 30 min at room temperature before the experiments. Coverslips with fura 2-loaded cells were placed in the bottom of a laminar flow-through chamber mounted on the stage of a Nikon inverted microscope equipped for epifluorescence (Eclipse TE300, Nikon, Tokyo, Japan). The cells were illuminated at 340 and 380 nm with a 100 W mercury lamp and interference filters (Chroma Technology Corp., Brattleboro, VT, USA) mounted on a filter wheel (Sutter Lambda 10-C, Sutter Instruments, MA, USA) and a dichroic mirror (510/40 nm, Chroma Technology Corp.). Images of single cells at 510 nm-emitted light were acquired via a 40× objective (CFI PL FLUOR, Nikon) and a Princeton T57 Micromax CCD camera at the rate of one ratio image per 4 s. In this imaging system (Canbara Packard Canada, Mississauga, ON, USA), cell illumination and fluorescence image acquisition hardware were run by MetaFluor software (Universal Imaging Corp., West Chester, PA, USA).

cAMP production

Cells seeded in 12-well plates were washed twice with medium A and incubated for 1 h in 1 ml of medium B with or without ATP and indomethacin. After aspiration of this medium, the cells were treated with 1 ml of 1 m perchloric acid, and cAMP production was quantified as previously described (Orlov et al. 1999).

PLA2 assay

PLA₂ assay was performed in accordance with a slightly modified method (Xing et al. 1997). Briefly, after 15 min stimulation with agonists in 35 cm² flasks, the medium was aspirated and the cells were washed with ice-cold medium B containing 0.2% BSA, then scraped in 1 ml of medium containing (mm): 20 NaCl, 25 Hepes (pH 7.5), 2 EDTA, 2 EGTA, 200 μm Na₃VO₄, 1 μg ml⁻¹ leupeptin, 1 μg ml⁻¹ aprotinin and 1 mm PMSF. After 2 × 10 s sonication, the cell lysate was centrifuged (1000 g, 10 min), and 200 ml of supernatant was added to 0.8 ml of medium containing (mm): 25 Hepes (pH 7.5), 1 EDTA, 4 CaCl₂, 1 DTT, 200 μm Na₃VO₄, 1 μg ml⁻¹ leupeptin, 1 μg ml⁻¹ aprotinin, 1 mm PMSF and 10 μm 1-stearoyl-2-[1-¹⁴C]-arachidonyl-sn-glycerol. After 1 h incubation at 37°C, the reaction was stopped by the addition of 1.5 ml of 1: 2 (v/v) chloroform–methanol. Arachidonic acid was separated from total lipid extracts by thin layer chromatography and quantified in a liquid scintillation counter.

Western blot analysis

After stimulation with the desired agonists in 12-well plates, the cells were lysed on ice in 0.125 ml of buffer containing 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 2 mm EDTA, 2 mm EGTA, 25 mm Hepes (pH 7.5), 10% glycerol, 1 mm NaF, 200 μm Na₃VO₄, and protease inhibitors (1 μg ml⁻¹ leupeptin, 1 μg ml⁻¹ aprotinin and 1 mm PMSF). The lysates were cleared from insoluble material by centrifugation at 20 000 g for 10 min, boiled in Laemmli buffer, subjected to polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA, USA). The membranes were treated with primary antibodies followed by horseradish peroxide-conjugated secondary antibodies (Calbiochem, San Diego, CA, USA), and developed by enhanced chemiluminescence reaction (Pierce).

Total RNA

Total RNA was isolated from cells seeded in 75 cm² flasks by Trizol reagent (Invitrogen Burlington, ON, Canada). First strand cDNA synthesis was carried out with 2 mg of total RNA and random primers using SuprScript First-Strand Synthesis 11904–018 (Invitrogen) as recommended by the manufacturer.

Real-time PCR

Real-time PCR was performed on 2 ng reverse-transcribed RNA using a QuantiTect SYBR kit (Qiagen, Valencia, CA, USA) in conjunction with primers specific for canine P_2Y receptor subtypes shown in Brindikova et al. (2003), i.e. P_2Y4, P_2Y6 and P_2Y11, and Table 1 and manufactured by Integrated DNA Technologies (Coralville, IA, USA). Thermocycling and detection was performed using a Opticon Monitor system (MJ Research, Hercules, CA, USA). Melting curve analysis, gel electrophoresis of PCR products and sequencing were used to confirm primer specificity.

Reagents

The previously described (Lum et al. 1999) replication-deficient adenovirus vector encoding PKI gene (AdPKI) was kindly provided by Dr R. Green (University of Illinois at Chicago, Chicago, IL, USA). The control adenovirus (AdLacZ) gene was a gift from Dr M. Dunn (Medical College of Wisconsin, Milwaukee, WI, USA). Phospho-PKA substrate (RRXS*/T*) and antivasodilator-stimulated phosphoprotein (VASP) rabbit monoclonal antibodies were obtained from Cell Signalling (Hornby, ON, Canada) and Calbiochem (San Diego, CA, USA), respectively. DMEM, calf serum and other ingredients for cell culture were purchased from Gibco Laboratories (Burlington, ON, Canada). 1-Stearoyl-2-[1-¹⁴C]-arachidonyl-sn-glycerol was procured from Amersham (Baie d'Urfé, PQ, Canada), fura 2-AM from Molecular Probes (Eugene, OR, USA), ATP, UTP, ADP, UDP, 2MeSATP, BAPTA-AM from Sigma (St. Louis, MO, USA), U73122 and U73343 from Calbiochem, and arachidonyltrifluoromethyl ketone (AACOCF3) and arachidonylmethyl ketone (AACOCH3) from BIOMOL (Plymouth Meeting, PA, USA). Salts and buffers were obtained from Sigma and Anachemia Science (Montreal, PQ, Canada).

Results

Role of PKA in agonist-stimulated I_SC

Both basal and apical applications of ATP resulted in rapid development of I_SC, reaching maximal values 30 s after ATP addition, which was followed by attenuation with a decline of ∼50% in 10 min (Fig. 1). As shown in our previous studies, positive deflection of I_SC in ATP-treated C7–MDCK monolayers indicates Cl⁻ movement from the basolateral to the apical surface (Bourcier et al. 2002). Basolateral application of PGE₁ or the β-adrenergic receptor agonist isoproterenol resulted in I_SC with approximately similar maximal values (I_SC-MAX) but more long-lasting kinetics compared to ATP-treated cells. Unlike ATP, isoproterenol was inactive when added to the apical surface. The transepithelial ion current triggered by apical PGE₁ displayed ∼5-fold less I_SC-MAX compared to basolateral application of this compound (Fig. 1). The asymmetrical action of isoproterenol and PGE₁ on I_SC is consistent with previous data obtained in ATCC–MDCK cells (Brown & Simmons, 1981; Simmons, 1991b). Application of a permeable cAMP analogue (8-Br-cAMP) led to slowly developing I_SC compared to ATP, isoproterenol and PGE₁, whereas 8-Br-cGMP was inactive (Fig. 1). Negative results were also obtained with 10 μm adenosine (data not shown), indicating a lack of contribution of the ecto-ATPase/P1-receptor-coupled pathway in ATP-induced transcellular ion current. In subsequent experiments, we added isoproterenol, PGE₁ and ATP to the serosal solution, whereas other compounds were applied to both surfaces of the cell monolayers.

Figure 2 illustrates the effect of ATP, isoproterenol and PGE₁ on PKA activity, estimated by western blotting with antibodies against the phosphorylated PKA substrates containing the RRXS*/T* PKA motif and by electrophoretic shift of phosphorylated VASP – an established substrate of PKA. Both methodological approaches revealed that all tested compounds rapidly activated PKA with more than 20-fold elevation of the VASP∼P/VASP ratio within 1 min of stimulation (Fig. 3). After 10 min of incubation with ATP, this ratio was decreased by ∼2-fold but it did not significantly change at that time in cells treated with isoproterenol or PGE₁.

To further examine the role of PKA in Cl⁻ secretion, we took two different approaches. First, we treated cells with H-89, a potent and selective cell-permeable inhibitor of PKA (Davies et al. 2000). Second, we overexpressed the PKA-inhibitor protein, PKI, by adenovirus-mediated gene transduction approach (AdPKI). In previous studies, we and others demonstrated that this approach provides PKI delivery to vascular endothelial and smooth muscle cells with about 100% efficiency (Lum et al. 1999; Hogarth et al. 2004). Figure 4A shows that both H-89 and AdPKI differentially suppressed VASP phosphorylation triggered by ATP, isoproterenol or PGE₁. Importantly, the inhibitory action of PKA inhibitors on I_SC-MAX (Fig. 4C) was in proportion to attenuation of the VASP∼P/VASP ratio (Fig. 4B). Neither agonist-induced I_SC nor VASP phosphorylation was significantly affected by adenovirus-mediated transduction of the LacZ gene used as a control (data not presented).

Role of PLA₂ in agonist-stimulated I_SC

In ATCC–MDCK (Post et al. 1996, 1998; Xing et al. 1999) and C7–MDCK cells (Orlov et al. 1999), ATP sharply augments cAMP production in a manner partially inhibited by indomethacin, suggesting an autocrine PLA₂/AA/COX/PGE-mediated pathway of PKA activation. This hypothesis is consistent with data showing activation of PLA₂ (Post et al. 1996; Xing et al. 1999) and augmented production of AA (Firestein et al. 1996; Xing et al. 1997, 1999) and PGE₂ (Post et al. 1998) in ATP-treated ATCC–MDCK cells. To examine the role of PLA₂ in P_2Y-induced Cl⁻ secretion, we treated the cells with an inhibitor of this enzyme, AACOCF₃, or with its inactive structural analogue AACOCH₃. Both ATP-induced [¹⁴C]-AA release from 1-Stearoyl-2-[1-¹⁴C]-arachidonyl-sn-glycerol and ATP-induced I_SC-MAX across C7–MDCK monolayers were almost completely abolished by AACOCF₃ but not AACOCH₃ (Fig. 5).

We next tested for the role of COX by use of the inhibitor indomethacin. Figure 6 illustrates that indomethacin inhibited ATP-induced cAMP production in C7–MDCK cells with an IC₅₀ of 0.3 μm, a concentration similar to that producing half-maximal inhibition of ATP-induced I_SC. Moreover, indomethacin almost completely abolished VASP phosphorylation triggered by ATP (Fig. 7A). In contrast, neither VASP phosphorylation (Fig. 7A) nor elevation of transepithelial ion current produced by isoproterenol or PGE₁ (Fig. 7B) was affected by COX inhibition.

Both basal and apical application of 10 μm AA (C_20:4) transiently increased I_SC in monolayers of C7–MDCK cells (Fig. 8A). This effect was not reproduced by additions of linoleic (C_18:2) and oleic (C_18:1) acids derived by PLA₂ activation but unable to generate prostanoids. At the same concentration, AA caused marked activation of PKA, as indicated by VASP phosphorylation. Both VASP phosphorylation and AA-induced I_SC were partially suppressed by AdPKI and almost completely abolished by additions of H-89 and indomethacin (Fig. 8B and C).

Role of [Ca²⁺]_i and other intermediates of P_2Y-induced signalling

ATP rapidly augmented [Ca²⁺]_i, as indicated by more than two-fold elevation of the fura-2 F₃₄₀/F₃₈₀ fluorescence ratio in 10–20 s of cell stimulation (Fig. 9). [Ca²⁺]_i was almost completely normalized in 3–4 min of ATP addition, a result consistent with our previous findings (Orlov et al. 1999).

We failed to detect any effect of indomethacin (Fig. 9), isoproterenol or PGE₁ on baseline [Ca²⁺]_i and its modulation by ATP. Transient elevation of [Ca²⁺]_i was also produced by inhibition of endoplasmic reticulum Ca²⁺-ATPase with thapsigargin. Pretreatment with thapsigargin completely abolished ATP-induced Ca_i²⁺ signalling. ATP-induced rise of [Ca²⁺]_i was also sharply diminished in cells loaded with the intracellular Ca²⁺ chelator BAPTA. Neither thapsigargin nor BAPTA affected the sustained elevation of [Ca²⁺]_i triggered by the Ca²⁺-ionophore ionomycin (Fig. 9).

The sharp increase of [Ca²⁺]_i detected in thapsigargin-treated cells (Fig. 9) was not accompanied by any transcellular ion current (Fig. 10). The addition of ionomycin resulted in short-lasting I_SC with 3–5-fold lower maximal values than in response to ATP. Neither thapsigargin nor ionomycin significantly affected I_SC-MAX in ATP-treated cells, but each produced a more rapid decline of transcellular charge movement. I_SC-MAX in ATP-treated cells was also insensitive to the presence of BAPTA. We noted, however, that in contrast to thapsigargin and ionomycin, BAPTA attenuated the normalization of ATP-induced I_SC (Fig. 10).

In previous studies, it was shown that activation of P_2Y-purinoceptors in C7–MDCK cells enhanced the production of IP₃ and phosphorylation of ERK1/2 that was abolished by U73122 and PD98059, inhibitors of phospholipase C (PLC) and ERK1/2, respectively (Orlov et al. 1999). Increased PLC activity in ATP-treated cells strongly suggests activation of diacylglycerol- and 14β-phorbol 12-myristate 13-acetate (PMA)-sensitive isoforms of PKC. The presence of these isoforms in C7–MDCK cells is supported by data showing that 30 min incubation with 0.1 μm 4β-PMA almost completely blocked Na⁺,K⁺,Cl⁻ cotransport (Orlov et al. 1999). Neither PD98059 nor 4β-PMA affected baseline and ATP-induced I_SC in C7–MDCK monolayers (Table 2). U73122 decreased ATP-induced I_SC by 20–30%. However, a similar effect was also detected with its structural non-active analogue U73343 (Table 2).

Identification of P_2Y receptor subtypes

Using northern blot analysis, we detected the presence of P_2Y1, P_2Y2 and P_2Y11 mRNA species in C7 cells (Brindikova et al. 2003). Real-time PCR shows that these cells express abundant levels of mRNA for P_2Y2 and P_2Y1 receptor subtypes whereas the content of P_2Y11, P_2Y12 and P_2Y14 mRNA is 5–6 orders of magnitude lower than that of P_2Y2 (Fig. 11). Expression of mRNA for P_2Y4, P_2Y6 and P_2Y13 receptors was below the limit of detection.

We observed that half-maximal VASP phosphorylation on C7 cells occurs at concentrations of 2MeSATP, ATP, ADP and UTP of ∼0.3, 5 30 and >100 μm, respectively (Fig. 12A and B). These data are consistent with a rank order of potency for activation of cloned P_2Y1 (2MeSATP > ATP > ADP ≫ UTP) rather than P_2Y2 (ATP = UTP ≫ 2MeSATP) and P_2Y11 (ATP > 2MeSATP ≫ ADP) receptors (Kunapuli & Daniel, 1998; Vassort, 2001;). 2MeSATP was much more potent than UTP in activating of I_SC in the C7 monolayer (Fig. 12C) whereas half-maximal elevation of [Ca²⁺]_i was detected at UTP and 2MeSATP concentrations of ∼1 and 10 μm, respectively (Fig. 12D).

Discussion

We previously reported that in C7–MDCK monolayers, a widely used principal cell model, ATP transient elevates I_SC, whose positive deflection and dependence on the presence of Na⁺, K⁺ and Cl⁻ in serosal and mucosal solutions indicate basolateral-to-apical Cl⁻ secretion (Bourcier et al. 2002). In these cells, P_2Y agonists trigger diverse signalling, including activation of PLC, transient elevation of [Ca²⁺]_i and enhanced cAMP production that was partially abolished by COX inhibition with indomethacin (Orlov et al. 1999). The crosstalk of these signalling systems in the activation of Cl⁻ secretion is poorly understood. Here, we report that P2Y-induced Cl⁻ secretion in monolayers of C7–MDCK cells is triggered by activation of PKA via a PLA₂/COX-mediated signalling pathway (Fig. 13). This conclusion is strongly supported by the following observations: (i) both ATP-induced increment of AA synthesis and I_SC were abolished by the PLA₂ inhibitor AACOCF₃ but were insensitive to its non-active structural analogue AACOCH₃ (Fig. 5); (ii) the COX inhibitor indomethacin demonstrated similar affinity in its ability to decrease I_SC and cAMP production triggered by ATP (Fig. 6); (iii) Cl⁻ secretion in C7–MDCK monolayers was triggered by other cAMP-increasing agonists, including isoproterenol and PGE₁ (Fig. 1). These compounds, as well as ATP, led to rapid activation of PKA, assayed by phosphorylation of VASP and other proteins containing the PKA phosphorylation motif. The kinetics of ATP-, isoproterenol- and PGE₁-induced PKA activation correlated with the kinetics of I_SC triggered by these stimuli (Figs 2 and 3); (iv) PKA activity stimulated by ATP, isoproterenol and PGE₁ was diminished by a cell-permeable inhibitor of this enzyme H-89 or by use of an adenovirus vector encoding the inhibitory subunit of PKA (AdPKI). Both approaches used for PKA inhibition sharply reduced I_SC caused by the addition of ATP, isoproterenol and PGE₁ (Fig. 4); (v) the addition of AA was sufficient to activate PKA and transepithelial ion current (Fig. 8). Indomethacin completely blocked the increment of PKA and I_SC triggered by ATP and AA (Figs 7 and 8) but did not affect the activation of PKA and I_SC by isoproterenol or PGE₁ (Fig. 7). Previous data have indicated a key role for ATP release and P_2Y receptor- and COX-promoted generation of AA release in establishing basal and nucleotide receptor-stimulated levels of cAMP in MDCK cells (Ostrom et al. 2000, 2001).

In parallel with increase in activity of PLA₂ and PKA, activation of P_2Y receptors in C7–MDCK cells leads to transient elevation of [Ca²⁺]_i (Gordjani et al. 1997; Orlov et al. 1999). Using renal epithelial cells of different origins, including ATCC–MDCK, several laboratories have documented the presence of Ca²⁺-activated Cl⁻ and K⁺ conductances sensitive to NPPB and charybdotoxin, respectively (Steidl et al. 1991; Tauc et al. 1993; Gandhi et al. 1998; Boese et al. 2000). Previously, we reported that NPPB, applied to both surfaces of C7–MDCK monolayers, and charybdotoxin, added to the serosal side, inhibited ATP-induced I_SC (Bourcier et al. 2002). Viewed collectively, such data implicate Ca²⁺_i-mediated signalling in nucleotide receptor-induced Cl⁻ secretion. However, our results do not support this hypothesis. Indeed, the kinetics of [Ca²⁺]_i elevation triggered by thapsigargin were approximately similar to those observed in ATP-treated cells, whereas BAPTA strongly suppressed ATP-induced Ca²⁺_i signalling (Fig. 9). Despite the sharp modulation of Ca²⁺_i-induced signalling, neither baseline nor ATP-induced I_SC-MAX was affected by thapsigargin or BAPTA (Fig. 10). We also did not observe any impact of PLC, ERK1/2 MAPK and PKC (other intermediates of intracellular signalling activated in ATP-treated C7–MDCK cells (Orlov et al. 1999)) on P_2Y-stimulated Cl⁻ secretion (Table 2).

The opposite effects of thapsigargin and ionomycin versus BAPTA on the kinetics of attenuation of ATP-induced I_SC (Fig. 10) indicate that transient elevation of [Ca²⁺]_i contributes to rapid inactivation of ATP-induced I_SC, which contrasts with the relatively sustained I_SC observed for cells treated with PGE₁ and other cAMP-increasing stimuli (Fig. 1). The role of PLA₂ in downregulation of cAMP production detected in forskolin-treated ATCC–MDCK cells (Ostrom et al. 2001) and delayed activation of Na⁺ channels seen in PGE₂-treated C7–MDCK cells (Wegmann & Nüsing, 2003) should be also considered as possible mechanisms underlying the distinct kinetics observed for modulation of I_SC by these stimuli.

Real-time PCR data show that the level of P_2Y1 and P_2Y2 expression in C7–MDCK cells is 5–6 orders of magnitude higher than that of other P_2Y receptor subtypes (Fig. 11). The comparison of rank order of potency of nucleotides detected in our study (Fig. 12) with data published for cloned P_2Y receptors (Kunapuli & Daniel, 1998; Vassort, 2001) strongly suggests that activation of PKA and I_SC triggered by basolateral application of agonists is mediated by P_2Y1 receptors with low sensitivity to UTP, whereas UTP-sensitive P2Y₂ receptors contribute to Ca²⁺ signalling (Fig. 12D). It was recently shown that haemagglutinin-tagged P_2Y1 and P_2Y2 receptors mainly reside on basolateral and apical membranes of MDCK cells, respectively (Wolff et al. 2005). Considering this, the role of P_2Y2 receptor-coupled signalling cascade in Cl⁻ secretion seen under apical application of ATP (Fig. 1) should be examined further.

In conclusion, our results show that Cl⁻ secretion in monolayers of C7–MDCK cells subjected to basolateral application of ATP is triggered by P_2Y1 receptors and involves a Ca²⁺_i-independent PLA₂/COX/PKA-mediated signalling pathway (Fig. 13), but leave numerous questions unresolved: Which G proteins mediate upstream signalling triggered by P_2Y1 receptors? Does PLA₂-mediated AA production contribute to Cl⁻ secretion via prostanoid synthesis, or does AA also directly affect other targets (Mignen & Shuttleworth, 2000; Carattino et al. 2003)? Is PKA in ATP-treated cells activated exclusively via cAMP production or is the activity of this enzyme also affected by cAMP-independent signalling (Zhong et al. 1997; Dulin et al. 2001; Niu et al. 2001)? Nucleotide receptor-induced transcellular ion fluxes are not a unique feature of MDCK cell monolayers. Indeed, augmented Cl⁻ secretion and attenuated Na⁺ reabsorption were detected in ATP-treated cells derived from mouse and rabbit inner medullary and cortical collecting ducts (McCoy et al. 1999; Boese et al. 2000; Cuffe et al. 2000; Unwin et al. 2003). What is the contribution of P_2Y-mediated Cl⁻ secretion to the regulation of kidney function under normal and pathophysiological conditions? Such questions will be the focus of our forthcoming studies.