Cl–secretion in ATP-treated renal epithelial C7–MDCK cells is mediated by activation of P2Y1 receptors, phospholipase A2 and protein kinase
1 Centre de recherche, Centre hospitalier de l'Université de Montréal (CHUM-Htel-Dieu), Montreal, PQ, Canada
2 Department of Medicine, University of Chicago, Chicago, IL, USA
3 Department of Pharmacology, University of California, San Diego, CA, USA
4 Department of Physiology, University of Wurzburg, Germany
Abstract
This study examines the mechanism of P2Y-induced Cl– secretion in monolayers of C7–Madin–Darby canine kidney (MDCK) cells triggered by basolateral application of ATP and measured as transcellular short current (ISC). Both ATP-induced arachidonic acid (AA) synthesis and ISC in ATP-treated cells were abolished by the phosholipase A2 (PLA2) inhibitor, AACOCF3. The cyclo-oxygenase inhibitor indomethacin decreased ISC and cAMP production in ATP-treated cells with an IC50 of 0.3 μM. ATP led to rapid activation of cAMP-dependent protein kinase A (PKA), as estimated by phosphorylation of a vasodilator-stimulated phosphoprotein. PKA activity and ISC evoked by ATP, as well as by prostaglandin E1 (PGE1), were diminished in the presence of the PKA inhibitor H-89 or an adenovirus-mediated expression of PKA-inhibitor protein, PKI. In contrast, indomethacin completely blocked the increment of PKA and ISC triggered by ATP and AA, but did not affect PKA activation and ISC detected with PGE1. The kinetics of [Ca2+]i elevation in ATP- and thapsigargin-treated cells were similar and suppressed by the Cai2+ chelator BAPTA. Neither baseline nor maximal increment of ATP-induced ISC was affected by thapsigargin and BAPTA. Real-time PCR showed that C7 cells express more mRNA for P2Y1 and P2Y2 than for other P2Y receptor subtypes. The rank order of potency (2MeSATP > ATP > ADP >> UTP) indicates that P2Y1 rather than P2Y2 receptors contribute to PKA and ISC activation. Viewed collectively, these data show that Cl– secretion in C7–MDCK monolayers treated with basolateral ATP is triggered by P2Y1 receptors and is mediated by subsequent [Ca2+]i-independent activation of PLA2 and PKA.
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Introduction
Because of compensatory reactions occurring in proximal tubular segments via tubuloglomerular feedback, renal collecting ducts are considered a major target for the regulation of water/salt homeostasis by hormones and neurotransmitters, such as arginine vasopressin, atrial natriuretic peptide, prostanoids, mineralocorticoids and catecholamines, providing electrogenic Na+ absorption or Cl– secretion (Breyer & Ando, 1994). Evidence for the involvement in the regulation of renal salt and water homeostasis of heterotrimeric G-protein-coupled P2Y receptors affected by ATP, UTP, and ADP comes from data showing the presence of mRNA encoding these receptors, the major sources of extracellular nucleosides and modulation of renal function in vitro and in situ by nucleotide receptor agonists and antagonists (for recent review, see Chan et al. 2001; Vallon, 2003; Leipziger, 2003; Komlosi et al. 2005).
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The pathophysiological roles of nucleotide-mediated signalling in the kidney are only beginning to emerge. Thus, Rost et al. (2002) reported that in the rat model of mesangial proliferative glomerulonephritis, the P2 receptor antagonist PPADS dose-dependently reduced early glomerular mesangial cell proliferation and increased serum creatine and urea. In cysts excised from the kidneys of patients suffering from autosomal dominant polycystic kidney disease, the concentration of ATP is increased up to 10 μM (Wilson et al. 1999). Based on these results, Schwiebert et al. (2002) proposed that P2Y receptors contribute to enhanced Cl– secretion, i.e. a key cell physiological marker of this disease.
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Data on ion fluxes regulation by nucleotides in the distal tubule have mainly been obtained with Madin–Darby canine kidney (MDCK) cells. In these renal epithelial cells, ATP-induced transcellular short-circuit current (ISC) (Simmons, 1981b) is accompanied by diverse signalling triggered by activation of P2Y receptors that results in transient elevation of [Ca2+]i; opening of Ca2+-sensitive K+ channels and hyperpolarization, production of cAMP, inositol 1,4,5-triphospate (IP3), arachidonic acid (AA) and prostaglandin E2 (PGE2); activation of protein kinase C (PKC), extracellular signal-related kinase (ERK1/2) mitogen-activated protein kinase (MAPK), etc. (Paulmichl & Lang, 1988; Friedrich et al. 1989; Paulmichl et al. 1991; Insel et al. 1996, 2001). Along with ATP, ISC in MDCK cells can also be triggered by cAMP-increasing compounds, such as adrenaline, isoproterenol, forskolin, 8-Br-cAMP, PGE1 and PGE2 (Brown & Simmons, 1981; Simmons, 1991a; Woo et al. 1998), and suppressed by the intracellular Ca2+ chelator BAPTA as well as by inhibitors of cyclo-oxygenase (COX) such as indomethacin (Simmons, 1981a; Zegarra-Moran et al. 1995).
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It should be emphasized that MDCK cells available from the American Type Culture Collection (ATCC–MDCK) are heterogeneous and develop different levels of transepithelial electrical resistance (Rte). By seeding ATCC–MDCK cells at low density, Gekle et al. (1994) isolated C7 and C11 subclones possessing high and low Rte and resembling principal and intercalated cells, respectively, from the collecting duct. Importantly, P2Y-induced signalling shows striking differences in ATCC–MDCK and C7–MDCK cells: inhibition of Na+,K+,Cl– cotransport and activation of ERK1/2 MAPK and capacitive calcium entry seen in ATP-treated ATCC–MDCK cells (Gagnon et al. 1999) are absent in C7–MDCK cells (Orlov et al. 1999).
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We have reported that ATP triggers ISC in C7– but not in C11–MDCK cells (Bourcier et al. 2002). We also documented that in C7–MDCK cells P2Y-induced ISC is mediated by activation of apical and basolateral anion channels that have different sensitivities to benzoic acid derivatives and that results in transcellular Cl– movement from the serosal to the mucosal compartment (Bourcier et al. 2002; Brindikova et al. 2003). In the present study, we employed several independent tools to activate and inhibit cAMP-, phospholipase A2 (PLA2)- and Ca2+-mediated signalling, and compared their action on the kinetics of P2Y-driven Cl– secretion in C7–MDCK monolayers. Data obtained in these studies allowed us to conclude that Cl– secretion in ATP-treated C7 cells is triggered by P2Y1 receptors and is mediated by subsequent activation of PLA2 and PKA independently of the sharp elevation of [Ca2+]i.
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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 x 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).
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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 x 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.
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Electrical measurements
After 3–4 days of seeding on Transwell inserts, Rte values were measured with EVOM. (World Precision Instruments, Sarasota, FL, USA). For ISC measurements, the Transwell inserts containing monolayers with Rte in the range from 1500 to 3500 x 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).
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[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 40x 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).
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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 x 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.
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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).
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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 P2Y receptor subtypes shown in Brindikova et al. (2003), i.e. P2Y4, P2Y6 and P2Y11, 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.
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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).
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Results
Role of PKA in agonist-stimulated ISC
Both basal and apical applications of ATP resulted in rapid development of ISC, 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 ISC 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 -adrenergic receptor agonist isoproterenol resulted in ISC with approximately similar maximal values (ISC-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 ISC-MAX compared to basolateral application of this compound (Fig. 1). The asymmetrical action of isoproterenol and PGE1 on ISC 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 ISC 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.
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Typical records of ISC modulation by 100 μM ATP, 10 μM isoproterenol (ISO), 1 μM PGE1, 500 μM 8-Br-cAMP or 500 μM 8-Br-cGMP applied to the basal (bas) and the apical (ap) surface of C7–MDCK 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 VASPP/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.
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Western blotting was carried out with antibodies against phosphorylated PKA-substrates containing PKA phosphorylation motif (RRXS*/T*) (A) and anti-VASP (B) antibodies in C7–MDCK cells treated with 10 μM isoproterenol (ISO), 1 μM PGE1 and 100 μM ATP.
Mean values obtained from three experiments are shown. The VASPP/VASP ratio in the absence of any stimuli was taken as 1.0.
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 ISC-MAX (Fig. 4C) was in proportion to attenuation of the VASPP/VASP ratio (Fig. 4B). Neither agonist-induced ISC nor VASP phosphorylation was significantly affected by adenovirus-mediated transduction of the LacZ gene used as a control (data not presented).
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A, representative blot showing the effects of AdPKI (3 x 109 v.p. ml–1) and H-89 (10 μm) on VASP phosphorylation in control cells (CON) and cells treated for 2 min with 100 μm ATP, 10 μm isoproterenol (ISO) or 1 μm PGE1. B, the effect of AdPKI and H-89 on the VASPP/VASP ratio and C, maximal values of ISC (ISC-MAX) triggered by ATP, isoproterenol and PGE1. AdPKI and H-89 were applied, respectively, 24 h and 20 min before addition of the above-listed stimuli. Means ± S.E.M. from three (VASP phosphorylation) and four (ISC) experiments are shown. The VASPP/VASP ratio and ISC-MAX values obtained with ATP-, isoproterenol- or PGE1-treated cells in the absence of AdPKI and H-89 were taken as 100%.
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Role of PLA2 in agonist-stimulated ISC
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 P2Y-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 ISC-MAX across C7–MDCK monolayers were almost completely abolished by AACOCF3 but not AACOCH3 (Fig. 5).
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AACOCF3 and AACOCH3 were added at concentrations of 200 μM 30 min before stimulation of cells with 100 μM ATP. Radioactivity of AA spots in the absence of any additions (dpm (mg protein)-1) was taken as 100%. The maximal values of ISC developed in ATP-treated cells are shown. Means ± S.E.M. from experiments performed in guadruplicate (PLA2 activity) or from three experiments (ISC) are given.
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 ISC. 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.
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Cells were preincubated with indomethacin 20 min before ATP addition. In the absence of ATP, cAMP production varied in the range from 6–10 pmol (mg protein)–1 h–1 and was not affected by indomethacin. triggered by values in the absence of indomethacin were taken as 100%. Means ± S.E.M. from experiments performed in quadruplicate (cAMP) or from three experiments (ISC) are given.
Indomethacin (10 μM) was added 20 min before 100 μM ATP; 10 μM isoproterenol and 1 μM PGE1 were applied for the next 2 (A) or 10 (B) min. Means ± S.E.M. from three experiments are shown.
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Both basal and apical application of 10 μM AA (C20:4) transiently increased ISC in monolayers of C7–MDCK cells (Fig. 8A). This effect was not reproduced by additions of linoleic (C18:2) and oleic (C18:1) 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 ISC were partially suppressed by AdPKI and almost completely abolished by additions of H-89 and indomethacin (Fig. 8B and C).
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A, typical record of ISC triggered by basal (bas) and apical (ap) application of AA, linoleic acid (LA) and oleic acid (OA) at a concentration of 10 μM. B, effect of indomethacin (IND, 10 μM), AdPKI (30 v.p. ml–1) and H-89 (10 μM) on VASP phosphorylation in the absence and presence of 10 μM AA. Cells were pretreated for 24 h with AdPKI and for 20 min with indomethacin or H-89 and then AA was applied for 2 min. C, effect of indomethacin, AdPKI and H-89 on maximal ISC values triggered by the addition of 10 mM AA. ISC-MAX values obtained in AA-treated cells in the absence of the above-listed inhibitors were taken as 100%. Means ± S.E.M. from three experiments are shown.
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Role of [Ca2+]i and other intermediates of P2Y-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).
BAPTA-AM was added at a concentration of 25 μM simultaneously with fura-2AM. For more details, see Methods.
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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 Cai2+ 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).
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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 ISC with 3–5-fold lower maximal values than in response to ATP. Neither thapsigargin nor ionomycin significantly affected ISC-MAX in ATP-treated cells, but each produced a more rapid decline of transcellular charge movement. ISC-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 ISC (Fig. 10).
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Thapsigargin, ionomycin and BAPTA were added to both serosal and mucosal medium, whereas ATP was applied to the basolateral surface of C7–MDCK monolayers. BAPTA-AM was added 30 min before stimulation with ATP.
In previous studies, it was shown that activation of P2Y-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 14-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 4-PMA almost completely blocked Na+,K+,Cl– cotransport (Orlov et al. 1999). Neither PD98059 nor 4-PMA affected baseline and ATP-induced ISC in C7–MDCK monolayers (Table 2). U73122 decreased ATP-induced ISC by 20–30%. However, a similar effect was also detected with its structural non-active analogue U73343 (Table 2).
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Identification of P2Y receptor subtypes
Using northern blot analysis, we detected the presence of P2Y1, P2Y2 and P2Y11 mRNA species in C7 cells (Brindikova et al. 2003). Real-time PCR shows that these cells express abundant levels of mRNA for P2Y2 and P2Y1 receptor subtypes whereas the content of P2Y11, P2Y12 and P2Y14 mRNA is 5–6 orders of magnitude lower than that of P2Y2 (Fig. 11). Expression of mRNA for P2Y4, P2Y6 and P2Y13 receptors was below the limit of detection.
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Real-time PCR was performed using P2Y subtype-specific primer pairs in conjunction with SYBR Green dye I. mRNA abundance was normalized to GADPH expression and presented relative to P2Y2 receptors. Means ± S.E.M. from experiments performed in triplicate are shown.
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 P2Y1 (2MeSATP > ATP > ADP >> UTP) rather than P2Y2 (ATP = UTP >> 2MeSATP) and P2Y11 (ATP > 2MeSATP >> ADP) receptors (Kunapuli & Daniel, 1998; Vassort, 2001;). 2MeSATP was much more potent than UTP in activating of ISC 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).
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The VASPP/VASP ratio in the absence of any stimuli was taken as 1.0. The maximal increments of ISC and [Ca2+]i triggered by 2MeSATP and UTP, respectively, were taken as 100%. Mean values obtained from three experiments are shown.
Discussion
We previously reported that in C7–MDCK monolayers, a widely used principal cell model, ATP transient elevates ISC, 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, P2Y 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 ISC 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 ISC 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 ISC 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 ISC 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 ISC triggered by ATP and AA (Figs 7 and 8) but did not affect the activation of PKA and ISC by isoproterenol or PGE1 (Fig. 7). Previous data have indicated a key role for ATP release and P2Y 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).
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1, Cl– channels; AC, adenylyl cyclase; EP, PGE receptors; PL, AA-containing phospholipids; INDO, indomethacin; a.m and b.m., apical and basolateral membrane, respectively; , unknown steps or intermediates; and —|, activatory and inhibitory stimuli, respectively. Compounds used to verify this model appear in italics. For other abbreviations and details, see text.
In parallel with increase in activity of PLA2 and PKA, activation of P2Y 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 ISC (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 ISC-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 P2Y-stimulated Cl– secretion (Table 2).
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The opposite effects of thapsigargin and ionomycin versus BAPTA on the kinetics of attenuation of ATP-induced ISC (Fig. 10) indicate that transient elevation of [Ca2+]i contributes to rapid inactivation of ATP-induced ISC, which contrasts with the relatively sustained ISC 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 ISC by these stimuli.
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Real-time PCR data show that the level of P2Y1 and P2Y2 expression in C7–MDCK cells is 5–6 orders of magnitude higher than that of other P2Y receptor subtypes (Fig. 11). The comparison of rank order of potency of nucleotides detected in our study (Fig. 12) with data published for cloned P2Y receptors (Kunapuli & Daniel, 1998; Vassort, 2001) strongly suggests that activation of PKA and ISC triggered by basolateral application of agonists is mediated by P2Y1 receptors with low sensitivity to UTP, whereas UTP-sensitive P2Y2 receptors contribute to Ca2+ signalling (Fig. 12D). It was recently shown that haemagglutinin-tagged P2Y1 and P2Y2 receptors mainly reside on basolateral and apical membranes of MDCK cells, respectively (Wolff et al. 2005). Considering this, the role of P2Y2 receptor-coupled signalling cascade in Cl– secretion seen under apical application of ATP (Fig. 1) should be examined further.
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In conclusion, our results show that Cl– secretion in monolayers of C7–MDCK cells subjected to basolateral application of ATP is triggered by P2Y1 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 P2Y1 receptorsDoes 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 P2Y-mediated Cl– secretion to the regulation of kidney function under normal and pathophysiological conditionsSuch questions will be the focus of our forthcoming studies.
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References
Boese SH, Glanville M, Aziz O, Gray MA & Simmons NL (2000). Ca2+ and cAMP-activated Cl– conductance mediate Cl– secretion in a mouse renal inner collecting duct cell line. J Physiol 523, 325–338.
Bourcier N, Grygorczyk R, Gekle M, Berthiaume Y & Orlov SN (2002). Purinergic-induced ion current in monolayers of C7–MDCK cells: role of basolateral and apical ion transporters. J Membr Biol 186, 131–143.
, 百拇医药
Breyer MD & Ando Y (1994). Hormonal signaling and regulation of salt and water transport in the collecting duct. Annu Rev Physiol 56, 711–739.
Brindikova TA, Bourcier N, Torres B, Pchejetski D, Gekle M, Maximov GV, Montminy V, Insel PA, Orlov SN & Isenring P (2003). Purinergic-induced signaling in C11-MDCK cells inhibits the secretory Na-K-Cl cotransporter. Am J Physiol Cell Physiol 285, C1445–C1453.
Brown CDA & Simmons NL (1981). Catecholamine-stimulation of Cl– secretion in MDCK cell epithelium. Biochim Biophys Acta 649, 427–435.
, 百拇医药
Carattino MD, Hill WG & Kleyman TR (2003). Arachidonic acid regulates surface expression of epithelial sodium channels. J Biol Chem 278, 36202–36213.
Chan CM, Unwin RJ & Burnstock G (2001). Potential functional roles of extracellular ATP in kidney and urinary tract. Exp Nephrol 6, 200–207.
Cuffe JE, Biefeld-Ackermann A, Thomas J, Leipziger J & Korbmacher C (2000). ATP stimulates Cl– secretion and reduces amiloride-sensitive Na+ absorbtion on M-1 mouse cortical collecting duct. J Physiol 524, 77–90.
, 百拇医药
Davies SP, Reddy H, Caivano M & Cohen P (2000). Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351, 95–105.
Dulin NO, Niu J, Browning DD, Ye RD & Voyno-Yasenetskaya T (2001). Cyclic AMP-independent activation of protein kinase A by vasoactive peptides. J Biol Chem 276, 20827–20830.
Firestein BL, Xing M, Hughes RJ, Corvera CU & Insel PA (1996). Heterogeneity of P2U- and P2Y-purinergic receptor regulation of phospholipases in MDCK cells. Am J Physiol 271, F610–F618.
, 百拇医药
Friedrich F, Weiss H, Paulmichl M & Lang F (1989). Activation of potassium channels in renal epithelioid cells (MDCK) by external ATP. Am J Physiol 256, C1016–C1021.
Gagnon F, Dulin NO, Tremblay J, Hamet P & Orlov SN (1999). ATP-induced inhibition of Na+,K+,Cl– cotransport in Madin–Darby canine kidney cells: lack of involvement of known purinoceptor-coupled signaling pathways. J Membr Biol 167, 193–204.
Gandhi R, Elble RC, Gruber AD, Schreur KD, Fuller CM & Paulli BU (1998). Molecular and functional characterisation of a calcium-sensitive chloride channel from mouse lung. J Biol Chem 273, 31096–31101.
, 百拇医药
Gekle M, Wunsch S, Oberleithner H & Silbernagl S (1994). Characterization of two MDCK-cell subtypes as a model system to study principal cell and intercalated cell properties. Pflugers Arch 428, 157–162.
Gordjani N, Nitschke R, Greger R & Leipziger J (1997). Capacitive Ca2+ entry (CCE) induced by luminal and basolateral ATP in polarised MDCK–C7 cells is restricted to the basolateral membrane. Cell Calcium 22, 121–128.
, 百拇医药 Hogarth K, Sandbo N, Taurin S, Kolenko V, Miano JM & Dulin NO (2004). Dual role of PKA in phenotypic modulation of vascular smooth muscle cells by extracellular ATP. Am J Physiol Cell Physiol 287, C449–C456.
Insel PA, Fierstein BL, Xing M, Post SR, Jacobson JP, Balboa MA & Hughes RJ (1996). P2-purinoceptors utilize multiple signalling pathways in MDCK–D1 cells. J Auton Pharmacol 16, 311–313.
Insel PA, Ostrom RS, Zambon AC, Hughes RJ, Balboa MA, Shehnaz D, Gregorian C, Torres B, Firestein BL, Xing M & Post SR (2001). P2Y receptors of MDCK cells: epithelial cell regulation by extracellular nucleotides. Clin Exp Pharmacol Physiol 28, 351–354.
, 百拇医药
Komlosi P, Fintha A & Bell PD (2005). Renal cell-to-cell communication via extracellular ATP. Physiology 20, 86–90.
Kunapuli SP & Daniel JD (1998). P2 receptor subtypes in the cardiovascular system. Biochem J 336, 513–523.
Leipziger J (2003). Control of epithelial transportt via luminal P2 receptors. Am J Physiol Renal Physiol 284, F419–F432.
Lum H, Jaffe HA, Schulz IT, Masood A, RayChaudhury A & Green RD (1999). Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction. Am J Physiol 277, C580–C588.
, 百拇医药
McCoy DE, Taylor AL, Kudlow BA, Karlson K, Slattery MJ, Schwiebert LM, Schwiebert EM & Stanton BA (1999). Nucleotides regulate NaCl transport in mIMCD-K2 cells via P2X and P2Y purinergic receptors. Am J Physiol 277, F552–F559.
Mignen O & Shuttleworth TJ (2000). I (ARC), a novel arachidonate-regulated, noncapacitative Ca2+ entry channel. J Biol Chem 275, 9114–9119.
Niu J, Vaiskunaite R, Suzuki N, Kozasa T, Carr DW, Dulin N & Voyno-Yasenetskaya TA (2001). Interaction of heterotrimeric G13 protein with an A-kinase-anchoring protein 110 (AKAP110) mediates cAMP-independent PKA activation. Curr Biol 11, 1688–1690.
, http://www.100md.com
Orlov SN, Dulin NO, Gagnon F, Gekle M, Douglas JG, Schwartz JH & Hamet P (1999). Purinergic regulation of Na+,K+,Cl– cotransport and MAP kinases is limited to C11-MDCK cells resembling intercalated cells from collecting ducts. J Membr Biol 172, 225–234.
Ostrom RS, Gregorian C, Drenan RM, Gabot K, Rana BK & Insel PA (2001). Key role for constitutive cyclooxygenase-2 of MDCK cells in basal signaling and response to released ATP. Am J Physiol Cell Physiol 281, C524–C531.
, 百拇医药
Ostrom RS, Gregorian C & Insel PA (2000). Cellular release of and response to ATP as key determinants of the set-point of signal transduction pathways. J Biol Chem 275, 11735–11739.
Paulmichl M & Lang F (1988). Enhancement of intracellular calcium concentration by extracellular ATP and UTP in Madin Darby canine kidney cells. Biochem Biophys Res Comm 156, 1139–1143.
Paulmichl M, Pfleilshifter J, Woll E & Lang F (1991). Cellular mechanisms of ATP-induced hyperpolarization in renal epitheloid MDCK-cells. J Cell Physiol 147, 68–75.
, 百拇医药
Post SR, Jacobson JP & Insel PA (1996). P2 purinergic receptor agonists enhance cAMP production in Madin-Darby canine kidney epithelial cells via an autocrine/paracrine mechanism. J Biol Chem 271, 2029–2032.
Post SR, Rump C, Zambon A, Hughes RJ, Buda MD, Jacobson JP, Kao CC & Insel PA (1998). ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells. J Biol Chem 273, 23093–23097.
Rost S, Daniel S, Schulze-Lohoff E, Baumert HG, Lambrecht G & Huge C (2002). P2 receptor antagonist PPADS inhibits mesangial cell proliferation in experimental mesangial proliferative glomerulonephritis. Kidney Int 62, 1659–1671.
, 百拇医药
Schwiebert EM, Wallace DP, Braunstein CM, King SR, Peti-Peterdi J, Hanaoka K, Guggino WB, Guay-Woodford LM, Bell PD, Sullivan LP, Grantham JJ & Taylor AL (2002). Autocrine extracellular purinergic signaling in epithelial cells derived from polycystic kidneys. Am J Physiol 282, F763–F775.
Simmons NL (1981a). Identification of a purine (P2) receptor linked to ion transport in a cultured renal (MDCK) epithelium. Br J Pharmacol 73, 379–384.
, http://www.100md.com
Simmons NL (1981b). Stimulation of Cl– secretion by exogenous ATP in cultured epithelial monolayers. Biochim Biophys Acta 646, 231–242.
Simmons NL (1991a). Chloride secretion stimulated by prostaglandin E1 and by forskolin in a canine renal epithelial cell line. J Physiol 432, 459–472.
Simmons NL (1991b). The effect of hypo-osmolarity upon transepithelial ion transport in cultured renal epithelial layers (MDCK). Pflugers Arch 419, 572–578.
, 百拇医药
Steidl M, Ritter M & Lang F (1991). Regulation of potassium conductance by prostaglandins in cultured renal epitheloid (Madin–Darby canine kidney) cells. Pflugers Arch 418, 431–436.
Tauc M, Gastineau M & Poujeol P (1993). Toxin sensitivity of the calcium-dependent rubidium efflux in Madin–Darby canine kidney cells. Biochem Biophys Res Comm 190, 596–601.
Unwin RJ, Bailey MA & Burnstock G (2003). Purinergic signaling along the renal tubule: the current state of play. News Physiol Sci 18, 237–241.
, 百拇医药
Vallon V (2003). Tubuloglomerular feedback and the control of glomerular filtration rate. News Physiol Sci 18, 169–174.
Vassort G (2001). Adenosine 5'-triphosphate: a P2-purinergic agonist in the myocardium. Physiol Rev 81, 767–806.
Wegmann M & Nüsing RM (2003). Prostaglandin E2 stimulates sodium reabsorption in MDCK cells, a renal collecting duct principal cell model. Prostagland Leukotr Essent Fatty Acids 69, 315–322.
, 百拇医药
Wilson PD, Hovater JS, Casey CC, Fortenberry JA & Schwiebert EM (1999). ATP release mechanisms in primary cultures of epithelia derived from cysts of polycystic kidneys. J Am Soc Nephrol 10, 218–229.
Wolff SC, Qi AD, Harden TK & Nicholas RA (2005). Polarized expression of human P2Y receptors in epithelial cells from kidney, lung, and colon. Am J Physiol Cell Physiol 288, C624–C632.
Woo JS, Inoue CN, Hanaoka K, Schwiebert EM, Guggino SE & Guggino WB (1998). Adenyl cyclase is involved in desensitization and recovery of ATP-stimulated Cl– secretion in MDCK cells. Am J Physiol 274, C371–C378.
, 百拇医药
Xing M, Firestein BL, Shen GH & Insel PA (1997). Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P2U receptors in MDCK-D1 cells: involvement of MAP kinase-dependent and – independent pathways. J Clin Invest 99, 805–814.
Xing M, Post S, Ostrom RS, Samardzija M & Insel PA (1999). Inhibition of phospholipase A2-mediated arachidonic acid release by cyclic AMP defines a negative feedback loop for P2Y receptor activation in Madin-Darby canine kidney D1 cells. J Biol Chem 274, 10035–10038.
, 百拇医药
Zegarra-Moran O, Rommeo G & Galietta LJV (1995). Regulation of transepithelial ion transport by two different purinoceptors in the apical membrane of canine kidney (MDCK) cells. Br J Pharmacol 114, 1052–1056.
Zhong H, SuYang H, Erdjument-Bromage H, Tempst P & Ghosh S (1997). The transcriptional activity of NF-B is regulated by the IB-associated PKAc subunit through a cyclic AMP-independent mechanism. Cell 89, 413–424., http://www.100md.com(A. Olga Akimova, Nathalie)
2 Department of Medicine, University of Chicago, Chicago, IL, USA
3 Department of Pharmacology, University of California, San Diego, CA, USA
4 Department of Physiology, University of Wurzburg, Germany
Abstract
This study examines the mechanism of P2Y-induced Cl– secretion in monolayers of C7–Madin–Darby canine kidney (MDCK) cells triggered by basolateral application of ATP and measured as transcellular short current (ISC). Both ATP-induced arachidonic acid (AA) synthesis and ISC in ATP-treated cells were abolished by the phosholipase A2 (PLA2) inhibitor, AACOCF3. The cyclo-oxygenase inhibitor indomethacin decreased ISC and cAMP production in ATP-treated cells with an IC50 of 0.3 μM. ATP led to rapid activation of cAMP-dependent protein kinase A (PKA), as estimated by phosphorylation of a vasodilator-stimulated phosphoprotein. PKA activity and ISC evoked by ATP, as well as by prostaglandin E1 (PGE1), were diminished in the presence of the PKA inhibitor H-89 or an adenovirus-mediated expression of PKA-inhibitor protein, PKI. In contrast, indomethacin completely blocked the increment of PKA and ISC triggered by ATP and AA, but did not affect PKA activation and ISC detected with PGE1. The kinetics of [Ca2+]i elevation in ATP- and thapsigargin-treated cells were similar and suppressed by the Cai2+ chelator BAPTA. Neither baseline nor maximal increment of ATP-induced ISC was affected by thapsigargin and BAPTA. Real-time PCR showed that C7 cells express more mRNA for P2Y1 and P2Y2 than for other P2Y receptor subtypes. The rank order of potency (2MeSATP > ATP > ADP >> UTP) indicates that P2Y1 rather than P2Y2 receptors contribute to PKA and ISC activation. Viewed collectively, these data show that Cl– secretion in C7–MDCK monolayers treated with basolateral ATP is triggered by P2Y1 receptors and is mediated by subsequent [Ca2+]i-independent activation of PLA2 and PKA.
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Introduction
Because of compensatory reactions occurring in proximal tubular segments via tubuloglomerular feedback, renal collecting ducts are considered a major target for the regulation of water/salt homeostasis by hormones and neurotransmitters, such as arginine vasopressin, atrial natriuretic peptide, prostanoids, mineralocorticoids and catecholamines, providing electrogenic Na+ absorption or Cl– secretion (Breyer & Ando, 1994). Evidence for the involvement in the regulation of renal salt and water homeostasis of heterotrimeric G-protein-coupled P2Y receptors affected by ATP, UTP, and ADP comes from data showing the presence of mRNA encoding these receptors, the major sources of extracellular nucleosides and modulation of renal function in vitro and in situ by nucleotide receptor agonists and antagonists (for recent review, see Chan et al. 2001; Vallon, 2003; Leipziger, 2003; Komlosi et al. 2005).
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The pathophysiological roles of nucleotide-mediated signalling in the kidney are only beginning to emerge. Thus, Rost et al. (2002) reported that in the rat model of mesangial proliferative glomerulonephritis, the P2 receptor antagonist PPADS dose-dependently reduced early glomerular mesangial cell proliferation and increased serum creatine and urea. In cysts excised from the kidneys of patients suffering from autosomal dominant polycystic kidney disease, the concentration of ATP is increased up to 10 μM (Wilson et al. 1999). Based on these results, Schwiebert et al. (2002) proposed that P2Y receptors contribute to enhanced Cl– secretion, i.e. a key cell physiological marker of this disease.
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Data on ion fluxes regulation by nucleotides in the distal tubule have mainly been obtained with Madin–Darby canine kidney (MDCK) cells. In these renal epithelial cells, ATP-induced transcellular short-circuit current (ISC) (Simmons, 1981b) is accompanied by diverse signalling triggered by activation of P2Y receptors that results in transient elevation of [Ca2+]i; opening of Ca2+-sensitive K+ channels and hyperpolarization, production of cAMP, inositol 1,4,5-triphospate (IP3), arachidonic acid (AA) and prostaglandin E2 (PGE2); activation of protein kinase C (PKC), extracellular signal-related kinase (ERK1/2) mitogen-activated protein kinase (MAPK), etc. (Paulmichl & Lang, 1988; Friedrich et al. 1989; Paulmichl et al. 1991; Insel et al. 1996, 2001). Along with ATP, ISC in MDCK cells can also be triggered by cAMP-increasing compounds, such as adrenaline, isoproterenol, forskolin, 8-Br-cAMP, PGE1 and PGE2 (Brown & Simmons, 1981; Simmons, 1991a; Woo et al. 1998), and suppressed by the intracellular Ca2+ chelator BAPTA as well as by inhibitors of cyclo-oxygenase (COX) such as indomethacin (Simmons, 1981a; Zegarra-Moran et al. 1995).
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It should be emphasized that MDCK cells available from the American Type Culture Collection (ATCC–MDCK) are heterogeneous and develop different levels of transepithelial electrical resistance (Rte). By seeding ATCC–MDCK cells at low density, Gekle et al. (1994) isolated C7 and C11 subclones possessing high and low Rte and resembling principal and intercalated cells, respectively, from the collecting duct. Importantly, P2Y-induced signalling shows striking differences in ATCC–MDCK and C7–MDCK cells: inhibition of Na+,K+,Cl– cotransport and activation of ERK1/2 MAPK and capacitive calcium entry seen in ATP-treated ATCC–MDCK cells (Gagnon et al. 1999) are absent in C7–MDCK cells (Orlov et al. 1999).
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We have reported that ATP triggers ISC in C7– but not in C11–MDCK cells (Bourcier et al. 2002). We also documented that in C7–MDCK cells P2Y-induced ISC is mediated by activation of apical and basolateral anion channels that have different sensitivities to benzoic acid derivatives and that results in transcellular Cl– movement from the serosal to the mucosal compartment (Bourcier et al. 2002; Brindikova et al. 2003). In the present study, we employed several independent tools to activate and inhibit cAMP-, phospholipase A2 (PLA2)- and Ca2+-mediated signalling, and compared their action on the kinetics of P2Y-driven Cl– secretion in C7–MDCK monolayers. Data obtained in these studies allowed us to conclude that Cl– secretion in ATP-treated C7 cells is triggered by P2Y1 receptors and is mediated by subsequent activation of PLA2 and PKA independently of the sharp elevation of [Ca2+]i.
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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 x 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).
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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 x 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.
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Electrical measurements
After 3–4 days of seeding on Transwell inserts, Rte values were measured with EVOM. (World Precision Instruments, Sarasota, FL, USA). For ISC measurements, the Transwell inserts containing monolayers with Rte in the range from 1500 to 3500 x 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).
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[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 40x 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).
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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 x 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.
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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).
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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 P2Y receptor subtypes shown in Brindikova et al. (2003), i.e. P2Y4, P2Y6 and P2Y11, 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.
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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).
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Results
Role of PKA in agonist-stimulated ISC
Both basal and apical applications of ATP resulted in rapid development of ISC, 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 ISC 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 -adrenergic receptor agonist isoproterenol resulted in ISC with approximately similar maximal values (ISC-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 ISC-MAX compared to basolateral application of this compound (Fig. 1). The asymmetrical action of isoproterenol and PGE1 on ISC 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 ISC 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.
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Typical records of ISC modulation by 100 μM ATP, 10 μM isoproterenol (ISO), 1 μM PGE1, 500 μM 8-Br-cAMP or 500 μM 8-Br-cGMP applied to the basal (bas) and the apical (ap) surface of C7–MDCK 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 VASPP/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.
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Western blotting was carried out with antibodies against phosphorylated PKA-substrates containing PKA phosphorylation motif (RRXS*/T*) (A) and anti-VASP (B) antibodies in C7–MDCK cells treated with 10 μM isoproterenol (ISO), 1 μM PGE1 and 100 μM ATP.
Mean values obtained from three experiments are shown. The VASPP/VASP ratio in the absence of any stimuli was taken as 1.0.
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 ISC-MAX (Fig. 4C) was in proportion to attenuation of the VASPP/VASP ratio (Fig. 4B). Neither agonist-induced ISC nor VASP phosphorylation was significantly affected by adenovirus-mediated transduction of the LacZ gene used as a control (data not presented).
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A, representative blot showing the effects of AdPKI (3 x 109 v.p. ml–1) and H-89 (10 μm) on VASP phosphorylation in control cells (CON) and cells treated for 2 min with 100 μm ATP, 10 μm isoproterenol (ISO) or 1 μm PGE1. B, the effect of AdPKI and H-89 on the VASPP/VASP ratio and C, maximal values of ISC (ISC-MAX) triggered by ATP, isoproterenol and PGE1. AdPKI and H-89 were applied, respectively, 24 h and 20 min before addition of the above-listed stimuli. Means ± S.E.M. from three (VASP phosphorylation) and four (ISC) experiments are shown. The VASPP/VASP ratio and ISC-MAX values obtained with ATP-, isoproterenol- or PGE1-treated cells in the absence of AdPKI and H-89 were taken as 100%.
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Role of PLA2 in agonist-stimulated ISC
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 P2Y-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 ISC-MAX across C7–MDCK monolayers were almost completely abolished by AACOCF3 but not AACOCH3 (Fig. 5).
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AACOCF3 and AACOCH3 were added at concentrations of 200 μM 30 min before stimulation of cells with 100 μM ATP. Radioactivity of AA spots in the absence of any additions (dpm (mg protein)-1) was taken as 100%. The maximal values of ISC developed in ATP-treated cells are shown. Means ± S.E.M. from experiments performed in guadruplicate (PLA2 activity) or from three experiments (ISC) are given.
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 ISC. 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.
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Cells were preincubated with indomethacin 20 min before ATP addition. In the absence of ATP, cAMP production varied in the range from 6–10 pmol (mg protein)–1 h–1 and was not affected by indomethacin. triggered by values in the absence of indomethacin were taken as 100%. Means ± S.E.M. from experiments performed in quadruplicate (cAMP) or from three experiments (ISC) are given.
Indomethacin (10 μM) was added 20 min before 100 μM ATP; 10 μM isoproterenol and 1 μM PGE1 were applied for the next 2 (A) or 10 (B) min. Means ± S.E.M. from three experiments are shown.
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Both basal and apical application of 10 μM AA (C20:4) transiently increased ISC in monolayers of C7–MDCK cells (Fig. 8A). This effect was not reproduced by additions of linoleic (C18:2) and oleic (C18:1) 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 ISC were partially suppressed by AdPKI and almost completely abolished by additions of H-89 and indomethacin (Fig. 8B and C).
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A, typical record of ISC triggered by basal (bas) and apical (ap) application of AA, linoleic acid (LA) and oleic acid (OA) at a concentration of 10 μM. B, effect of indomethacin (IND, 10 μM), AdPKI (30 v.p. ml–1) and H-89 (10 μM) on VASP phosphorylation in the absence and presence of 10 μM AA. Cells were pretreated for 24 h with AdPKI and for 20 min with indomethacin or H-89 and then AA was applied for 2 min. C, effect of indomethacin, AdPKI and H-89 on maximal ISC values triggered by the addition of 10 mM AA. ISC-MAX values obtained in AA-treated cells in the absence of the above-listed inhibitors were taken as 100%. Means ± S.E.M. from three experiments are shown.
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Role of [Ca2+]i and other intermediates of P2Y-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).
BAPTA-AM was added at a concentration of 25 μM simultaneously with fura-2AM. For more details, see Methods.
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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 Cai2+ 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).
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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 ISC with 3–5-fold lower maximal values than in response to ATP. Neither thapsigargin nor ionomycin significantly affected ISC-MAX in ATP-treated cells, but each produced a more rapid decline of transcellular charge movement. ISC-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 ISC (Fig. 10).
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Thapsigargin, ionomycin and BAPTA were added to both serosal and mucosal medium, whereas ATP was applied to the basolateral surface of C7–MDCK monolayers. BAPTA-AM was added 30 min before stimulation with ATP.
In previous studies, it was shown that activation of P2Y-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 14-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 4-PMA almost completely blocked Na+,K+,Cl– cotransport (Orlov et al. 1999). Neither PD98059 nor 4-PMA affected baseline and ATP-induced ISC in C7–MDCK monolayers (Table 2). U73122 decreased ATP-induced ISC by 20–30%. However, a similar effect was also detected with its structural non-active analogue U73343 (Table 2).
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Identification of P2Y receptor subtypes
Using northern blot analysis, we detected the presence of P2Y1, P2Y2 and P2Y11 mRNA species in C7 cells (Brindikova et al. 2003). Real-time PCR shows that these cells express abundant levels of mRNA for P2Y2 and P2Y1 receptor subtypes whereas the content of P2Y11, P2Y12 and P2Y14 mRNA is 5–6 orders of magnitude lower than that of P2Y2 (Fig. 11). Expression of mRNA for P2Y4, P2Y6 and P2Y13 receptors was below the limit of detection.
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Real-time PCR was performed using P2Y subtype-specific primer pairs in conjunction with SYBR Green dye I. mRNA abundance was normalized to GADPH expression and presented relative to P2Y2 receptors. Means ± S.E.M. from experiments performed in triplicate are shown.
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 P2Y1 (2MeSATP > ATP > ADP >> UTP) rather than P2Y2 (ATP = UTP >> 2MeSATP) and P2Y11 (ATP > 2MeSATP >> ADP) receptors (Kunapuli & Daniel, 1998; Vassort, 2001;). 2MeSATP was much more potent than UTP in activating of ISC 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).
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The VASPP/VASP ratio in the absence of any stimuli was taken as 1.0. The maximal increments of ISC and [Ca2+]i triggered by 2MeSATP and UTP, respectively, were taken as 100%. Mean values obtained from three experiments are shown.
Discussion
We previously reported that in C7–MDCK monolayers, a widely used principal cell model, ATP transient elevates ISC, 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, P2Y 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 ISC 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 ISC 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 ISC 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 ISC 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 ISC triggered by ATP and AA (Figs 7 and 8) but did not affect the activation of PKA and ISC by isoproterenol or PGE1 (Fig. 7). Previous data have indicated a key role for ATP release and P2Y 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).
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1, Cl– channels; AC, adenylyl cyclase; EP, PGE receptors; PL, AA-containing phospholipids; INDO, indomethacin; a.m and b.m., apical and basolateral membrane, respectively; , unknown steps or intermediates; and —|, activatory and inhibitory stimuli, respectively. Compounds used to verify this model appear in italics. For other abbreviations and details, see text.
In parallel with increase in activity of PLA2 and PKA, activation of P2Y 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 ISC (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 ISC-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 P2Y-stimulated Cl– secretion (Table 2).
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The opposite effects of thapsigargin and ionomycin versus BAPTA on the kinetics of attenuation of ATP-induced ISC (Fig. 10) indicate that transient elevation of [Ca2+]i contributes to rapid inactivation of ATP-induced ISC, which contrasts with the relatively sustained ISC 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 ISC by these stimuli.
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Real-time PCR data show that the level of P2Y1 and P2Y2 expression in C7–MDCK cells is 5–6 orders of magnitude higher than that of other P2Y receptor subtypes (Fig. 11). The comparison of rank order of potency of nucleotides detected in our study (Fig. 12) with data published for cloned P2Y receptors (Kunapuli & Daniel, 1998; Vassort, 2001) strongly suggests that activation of PKA and ISC triggered by basolateral application of agonists is mediated by P2Y1 receptors with low sensitivity to UTP, whereas UTP-sensitive P2Y2 receptors contribute to Ca2+ signalling (Fig. 12D). It was recently shown that haemagglutinin-tagged P2Y1 and P2Y2 receptors mainly reside on basolateral and apical membranes of MDCK cells, respectively (Wolff et al. 2005). Considering this, the role of P2Y2 receptor-coupled signalling cascade in Cl– secretion seen under apical application of ATP (Fig. 1) should be examined further.
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In conclusion, our results show that Cl– secretion in monolayers of C7–MDCK cells subjected to basolateral application of ATP is triggered by P2Y1 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 P2Y1 receptorsDoes 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 P2Y-mediated Cl– secretion to the regulation of kidney function under normal and pathophysiological conditionsSuch questions will be the focus of our forthcoming studies.
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References
Boese SH, Glanville M, Aziz O, Gray MA & Simmons NL (2000). Ca2+ and cAMP-activated Cl– conductance mediate Cl– secretion in a mouse renal inner collecting duct cell line. J Physiol 523, 325–338.
Bourcier N, Grygorczyk R, Gekle M, Berthiaume Y & Orlov SN (2002). Purinergic-induced ion current in monolayers of C7–MDCK cells: role of basolateral and apical ion transporters. J Membr Biol 186, 131–143.
, 百拇医药
Breyer MD & Ando Y (1994). Hormonal signaling and regulation of salt and water transport in the collecting duct. Annu Rev Physiol 56, 711–739.
Brindikova TA, Bourcier N, Torres B, Pchejetski D, Gekle M, Maximov GV, Montminy V, Insel PA, Orlov SN & Isenring P (2003). Purinergic-induced signaling in C11-MDCK cells inhibits the secretory Na-K-Cl cotransporter. Am J Physiol Cell Physiol 285, C1445–C1453.
Brown CDA & Simmons NL (1981). Catecholamine-stimulation of Cl– secretion in MDCK cell epithelium. Biochim Biophys Acta 649, 427–435.
, 百拇医药
Carattino MD, Hill WG & Kleyman TR (2003). Arachidonic acid regulates surface expression of epithelial sodium channels. J Biol Chem 278, 36202–36213.
Chan CM, Unwin RJ & Burnstock G (2001). Potential functional roles of extracellular ATP in kidney and urinary tract. Exp Nephrol 6, 200–207.
Cuffe JE, Biefeld-Ackermann A, Thomas J, Leipziger J & Korbmacher C (2000). ATP stimulates Cl– secretion and reduces amiloride-sensitive Na+ absorbtion on M-1 mouse cortical collecting duct. J Physiol 524, 77–90.
, 百拇医药
Davies SP, Reddy H, Caivano M & Cohen P (2000). Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351, 95–105.
Dulin NO, Niu J, Browning DD, Ye RD & Voyno-Yasenetskaya T (2001). Cyclic AMP-independent activation of protein kinase A by vasoactive peptides. J Biol Chem 276, 20827–20830.
Firestein BL, Xing M, Hughes RJ, Corvera CU & Insel PA (1996). Heterogeneity of P2U- and P2Y-purinergic receptor regulation of phospholipases in MDCK cells. Am J Physiol 271, F610–F618.
, 百拇医药
Friedrich F, Weiss H, Paulmichl M & Lang F (1989). Activation of potassium channels in renal epithelioid cells (MDCK) by external ATP. Am J Physiol 256, C1016–C1021.
Gagnon F, Dulin NO, Tremblay J, Hamet P & Orlov SN (1999). ATP-induced inhibition of Na+,K+,Cl– cotransport in Madin–Darby canine kidney cells: lack of involvement of known purinoceptor-coupled signaling pathways. J Membr Biol 167, 193–204.
Gandhi R, Elble RC, Gruber AD, Schreur KD, Fuller CM & Paulli BU (1998). Molecular and functional characterisation of a calcium-sensitive chloride channel from mouse lung. J Biol Chem 273, 31096–31101.
, 百拇医药
Gekle M, Wunsch S, Oberleithner H & Silbernagl S (1994). Characterization of two MDCK-cell subtypes as a model system to study principal cell and intercalated cell properties. Pflugers Arch 428, 157–162.
Gordjani N, Nitschke R, Greger R & Leipziger J (1997). Capacitive Ca2+ entry (CCE) induced by luminal and basolateral ATP in polarised MDCK–C7 cells is restricted to the basolateral membrane. Cell Calcium 22, 121–128.
, 百拇医药 Hogarth K, Sandbo N, Taurin S, Kolenko V, Miano JM & Dulin NO (2004). Dual role of PKA in phenotypic modulation of vascular smooth muscle cells by extracellular ATP. Am J Physiol Cell Physiol 287, C449–C456.
Insel PA, Fierstein BL, Xing M, Post SR, Jacobson JP, Balboa MA & Hughes RJ (1996). P2-purinoceptors utilize multiple signalling pathways in MDCK–D1 cells. J Auton Pharmacol 16, 311–313.
Insel PA, Ostrom RS, Zambon AC, Hughes RJ, Balboa MA, Shehnaz D, Gregorian C, Torres B, Firestein BL, Xing M & Post SR (2001). P2Y receptors of MDCK cells: epithelial cell regulation by extracellular nucleotides. Clin Exp Pharmacol Physiol 28, 351–354.
, 百拇医药
Komlosi P, Fintha A & Bell PD (2005). Renal cell-to-cell communication via extracellular ATP. Physiology 20, 86–90.
Kunapuli SP & Daniel JD (1998). P2 receptor subtypes in the cardiovascular system. Biochem J 336, 513–523.
Leipziger J (2003). Control of epithelial transportt via luminal P2 receptors. Am J Physiol Renal Physiol 284, F419–F432.
Lum H, Jaffe HA, Schulz IT, Masood A, RayChaudhury A & Green RD (1999). Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction. Am J Physiol 277, C580–C588.
, 百拇医药
McCoy DE, Taylor AL, Kudlow BA, Karlson K, Slattery MJ, Schwiebert LM, Schwiebert EM & Stanton BA (1999). Nucleotides regulate NaCl transport in mIMCD-K2 cells via P2X and P2Y purinergic receptors. Am J Physiol 277, F552–F559.
Mignen O & Shuttleworth TJ (2000). I (ARC), a novel arachidonate-regulated, noncapacitative Ca2+ entry channel. J Biol Chem 275, 9114–9119.
Niu J, Vaiskunaite R, Suzuki N, Kozasa T, Carr DW, Dulin N & Voyno-Yasenetskaya TA (2001). Interaction of heterotrimeric G13 protein with an A-kinase-anchoring protein 110 (AKAP110) mediates cAMP-independent PKA activation. Curr Biol 11, 1688–1690.
, http://www.100md.com
Orlov SN, Dulin NO, Gagnon F, Gekle M, Douglas JG, Schwartz JH & Hamet P (1999). Purinergic regulation of Na+,K+,Cl– cotransport and MAP kinases is limited to C11-MDCK cells resembling intercalated cells from collecting ducts. J Membr Biol 172, 225–234.
Ostrom RS, Gregorian C, Drenan RM, Gabot K, Rana BK & Insel PA (2001). Key role for constitutive cyclooxygenase-2 of MDCK cells in basal signaling and response to released ATP. Am J Physiol Cell Physiol 281, C524–C531.
, 百拇医药
Ostrom RS, Gregorian C & Insel PA (2000). Cellular release of and response to ATP as key determinants of the set-point of signal transduction pathways. J Biol Chem 275, 11735–11739.
Paulmichl M & Lang F (1988). Enhancement of intracellular calcium concentration by extracellular ATP and UTP in Madin Darby canine kidney cells. Biochem Biophys Res Comm 156, 1139–1143.
Paulmichl M, Pfleilshifter J, Woll E & Lang F (1991). Cellular mechanisms of ATP-induced hyperpolarization in renal epitheloid MDCK-cells. J Cell Physiol 147, 68–75.
, 百拇医药
Post SR, Jacobson JP & Insel PA (1996). P2 purinergic receptor agonists enhance cAMP production in Madin-Darby canine kidney epithelial cells via an autocrine/paracrine mechanism. J Biol Chem 271, 2029–2032.
Post SR, Rump C, Zambon A, Hughes RJ, Buda MD, Jacobson JP, Kao CC & Insel PA (1998). ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells. J Biol Chem 273, 23093–23097.
Rost S, Daniel S, Schulze-Lohoff E, Baumert HG, Lambrecht G & Huge C (2002). P2 receptor antagonist PPADS inhibits mesangial cell proliferation in experimental mesangial proliferative glomerulonephritis. Kidney Int 62, 1659–1671.
, 百拇医药
Schwiebert EM, Wallace DP, Braunstein CM, King SR, Peti-Peterdi J, Hanaoka K, Guggino WB, Guay-Woodford LM, Bell PD, Sullivan LP, Grantham JJ & Taylor AL (2002). Autocrine extracellular purinergic signaling in epithelial cells derived from polycystic kidneys. Am J Physiol 282, F763–F775.
Simmons NL (1981a). Identification of a purine (P2) receptor linked to ion transport in a cultured renal (MDCK) epithelium. Br J Pharmacol 73, 379–384.
, http://www.100md.com
Simmons NL (1981b). Stimulation of Cl– secretion by exogenous ATP in cultured epithelial monolayers. Biochim Biophys Acta 646, 231–242.
Simmons NL (1991a). Chloride secretion stimulated by prostaglandin E1 and by forskolin in a canine renal epithelial cell line. J Physiol 432, 459–472.
Simmons NL (1991b). The effect of hypo-osmolarity upon transepithelial ion transport in cultured renal epithelial layers (MDCK). Pflugers Arch 419, 572–578.
, 百拇医药
Steidl M, Ritter M & Lang F (1991). Regulation of potassium conductance by prostaglandins in cultured renal epitheloid (Madin–Darby canine kidney) cells. Pflugers Arch 418, 431–436.
Tauc M, Gastineau M & Poujeol P (1993). Toxin sensitivity of the calcium-dependent rubidium efflux in Madin–Darby canine kidney cells. Biochem Biophys Res Comm 190, 596–601.
Unwin RJ, Bailey MA & Burnstock G (2003). Purinergic signaling along the renal tubule: the current state of play. News Physiol Sci 18, 237–241.
, 百拇医药
Vallon V (2003). Tubuloglomerular feedback and the control of glomerular filtration rate. News Physiol Sci 18, 169–174.
Vassort G (2001). Adenosine 5'-triphosphate: a P2-purinergic agonist in the myocardium. Physiol Rev 81, 767–806.
Wegmann M & Nüsing RM (2003). Prostaglandin E2 stimulates sodium reabsorption in MDCK cells, a renal collecting duct principal cell model. Prostagland Leukotr Essent Fatty Acids 69, 315–322.
, 百拇医药
Wilson PD, Hovater JS, Casey CC, Fortenberry JA & Schwiebert EM (1999). ATP release mechanisms in primary cultures of epithelia derived from cysts of polycystic kidneys. J Am Soc Nephrol 10, 218–229.
Wolff SC, Qi AD, Harden TK & Nicholas RA (2005). Polarized expression of human P2Y receptors in epithelial cells from kidney, lung, and colon. Am J Physiol Cell Physiol 288, C624–C632.
Woo JS, Inoue CN, Hanaoka K, Schwiebert EM, Guggino SE & Guggino WB (1998). Adenyl cyclase is involved in desensitization and recovery of ATP-stimulated Cl– secretion in MDCK cells. Am J Physiol 274, C371–C378.
, 百拇医药
Xing M, Firestein BL, Shen GH & Insel PA (1997). Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P2U receptors in MDCK-D1 cells: involvement of MAP kinase-dependent and – independent pathways. J Clin Invest 99, 805–814.
Xing M, Post S, Ostrom RS, Samardzija M & Insel PA (1999). Inhibition of phospholipase A2-mediated arachidonic acid release by cyclic AMP defines a negative feedback loop for P2Y receptor activation in Madin-Darby canine kidney D1 cells. J Biol Chem 274, 10035–10038.
, 百拇医药
Zegarra-Moran O, Rommeo G & Galietta LJV (1995). Regulation of transepithelial ion transport by two different purinoceptors in the apical membrane of canine kidney (MDCK) cells. Br J Pharmacol 114, 1052–1056.
Zhong H, SuYang H, Erdjument-Bromage H, Tempst P & Ghosh S (1997). The transcriptional activity of NF-B is regulated by the IB-associated PKAc subunit through a cyclic AMP-independent mechanism. Cell 89, 413–424., http://www.100md.com(A. Olga Akimova, Nathalie)