Pflugers Arch - Eur J Physiol (2004) 449: 66–75 DOI 10.1007/s00424-004-1293-2
ION CHANNEL S, TRANSPORTERS
L. Guerra . M. Favia . T. Fanelli . G. Calamita . M. Svelto . A. Bagorda . K. A. Jacobson . S. J. Reshkin . V. Casavola
Stimulation of Xenopus P2Y1 receptor activates CFTR in A6 cells
Received: 13 April 2004 / Accepted: 26 April 2004 / Published online: 3 July 2004 # Springer-Verlag 2004
Abstract Nucleotide binding to purinergic P2Y receptors contributes to the regulation of a variety of physiological functions in renal epithelial cells. Here, we investigate the regulatory mechanism of the P2Y1 receptor agonist 2methylthioadenosine diphosphate (2-MeSADP) on Cl− transport in A6 cells, a commonly used model of the distal section of the Xenopus laevis nephron. Protein and mRNA expression analysis together with functional measurements demonstrated the basolateral location of the Xenopus P2Y1 receptor. 2-MeSADP increased intracellular [Ca2+] and cAMP and Cl− efflux, responses that were all inhibited by the specific P2Y1 receptor antagonist MRS 2179. Cl−efflux was also inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide. Inhibition of either protein kinase A (PKA) or the binding between A-kinase-anchoring proteins (AKAPs) and the regulatory PKA RII subunit blocked the 2MeSADP-induced activation of CFTR, suggesting that PKA mediates P2Y1 receptor regulation of CFTR through one or more AKAPs. Further, the truncation of the PDZ1 domain of the scaffolding protein Na+/H+ exchanger regulatory factor-2 (NHERF-2) inhibited 2-MeSADPdependent stimulation of Cl− efflux, suggesting the involvement of this scaffolding protein. Activation or inhibition of PKC had no effect per se on basal Cl− efflux but potentiated or reduced the 2-MeSADP-dependent stimulation of Cl− efflux, respectively. These data suggest that the X. laevis P2Y1 receptor in A6 cells can increase
L. Guerra . M. Favia . T. Fanelli . G. Calamita . M. Svelto . A. Bagorda . S. J. Reshkin . V. Casavola (*) Department of General and Environmental Physiology, University of Bari, Via Amendola 165/A, 70126 Bari, Italy e-mail:
[email protected] K. A. Jacobson Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Md., USA
both cAMP/PKA and Ca2+/PKC intracellular levels and that the PKC pathway is involved in CFTR activation via potentiation of the PKA pathway. Keywords Xenopus P2Y1 . CFTR . NHERF-2 . A6
Introduction In recent years, extracellular nucleotides have been identified as an important class of signal molecules that mediate different biological effects in a variety of cells and tissues. The biological actions of these extracellular nucleotides result from binding to and activation of their respective transmembrane cell surface purinergic (P2) receptors. P2 receptors are subdivided into two families: the ionotropic (P2X) receptors that act as ion channels and the G protein-coupled, metabotropic (P2Y) receptors [1, 6, 18]. At present, at least eight distinct mammalian P2Y receptor subtypes have been identified. These can be subdivided further on the basis of their specific pharmacological selectivity for different nucleotides: the adeninenucleotide-preferring receptors responding mainly to ADP and ATP (P2Y1, P2Y11, P2Y12, P2Y13) and the uracilnucleotide-preferring receptors (P2Y2, P2Y4, P2Y6). Binding of ATP to the G protein-coupled P2Y receptor can activate phospholipase C (PLC), leading to inositol1,4,5-trisphosphate (IP3) production and mobilization of internal Ca2+ stores and can either increase [3, 17, 44] or diminish [1, 18, 25] cAMP production, depending on the tissue or cell type. One prominent feature of P2 receptors is their extraordinarily wide-spread tissue expression, and the kidney in particular expresses purinoceptors abundantly. The study of the physiological role of purinergic signalling in different cell types in specific nephron segments is complex, due primarily to (a) the diversity of renal cell types in which extracellular nucleotides behave as important regulators of epithelial ion transport; (b) the diversity of purinoceptors expressed along the nephron; and (c) the different behaviour evinced by the same
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receptor in different species [52]. Moreover, different subtypes of purinoceptors, in particular P2Y receptors, can be co-expressed in the same cell and often in the same membrane domain of the epithelial cell [27]. Renal cell lines derived from different species have been employed to elucidate the physiological role of extracellular ATP in the kidney and have proven a useful tool for studying the regulation of electrolyte transport such as that of Na+, K+ and Cl− [3, 22, 36, 37, 38]. In A6 cells, an amphibian distal cell line, P2Y receptor stimulation leads to Cl−secretion mediated by a calciumdependent Cl−channel [5, 38]. In a recent study, we demonstrated that in a cell line derived from A6 cells, A6NHE3, the activation of a P2Y1-like receptor by the specific agonist 2-methylthioadenosine diphosphate (2MeSADP) induced both cAMP production and an increase in cytosolic [Ca2+] ([Ca2+]i) [3]. The aim of this study was to characterize further, in A6 cells, the P2Y receptor and to study the mechanism of its regulation of Cl− secretion. Using a PCR probe designed specifically for the recently described Xenopus laevis P2Y1 receptor [16], this study demonstrated that A6 cells express the same receptor and that its activation increases both cAMP and [Ca2+]i. Electrophysiological data demonstrate further that the Xenopus P2Y1 receptor is localized mainly on the basolateral side of the polarized A6 monolayer and that its activation induces Cl−secretion by stimulation of cystic fibrosis transmembrane conductance regulator (CFTR) channels.
using a pair of β-actin primers, BAF (5′-CAGATCATGTTTGAGACCTT-3′) and BAR (5′-CGGATGTCMACGTCACACTT-3′; M=A+C), which yield a 509-bp DNA fragment (33 cycles of PCR). For the Northern blotting studies, RNA samples (10 µg per lane) were electrophoresed through formaldehyde-1% agarose gels and transferred to nylon membranes and hybridized with the above RT-PCR fragment of the X. laevis P2Y cDNA as a probe labelled with a-[32P]deoxycytidine 5′-triphosphate. Hybridization was performed in a solution of 50% formamide, 5×saline-sodium citrate (SSC), 5×Denhardt’s solution, 1% SDS and 100 µg/ml denatured salmon sperm DNA at 42 °C for 20 h. The membranes were washed twice in 2×SSC, 0.1% SDS at room temperature for 15 min each and once in 0.1×SSC, 0.1% SDS at 42 °C for 30 min. Membranes were autoradiographed with intensifying screens for 3 days. Protein extraction and Western blot analysis A total cellular lysate was prepared according to [4]. An 60-µg protein aliquot was separated in 7.5% SDS-PAGE. The separated proteins were transferred to Immobilon P (Millipore) in a Trans-Blot semidry electrophoretic transfer cell (Bio-Rad) for immunoblotting. Immunocomplexes were detected with enhanced chemiluminescence (ECL) reagent (Amersham). The following antibodies were used: anti P2Y1 polyclonal antibody against the human P2Y1 peptide (Sigma-RBI, Milan, Italy, dilution 1:400).
Materials and methods Cell culture Experiments were performed on cells of the with A6/C1 subclone of the A6-2F3 line (passage 114–128) selected functionally on the basis of high transepithelial resistance and responsiveness to aldosterone [50]. A6/C1 cell lines were grown in 0.8×concentrated DMEM containing 25 mM NaHCO3, 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin solution with a final osmolarity of 240–250 mOsmol. Cells generally reached confluency 7–8 days after seeding when the culture medium was changed 3 times a week. RT-PCR and Northern blot analysis Total RNA from A6 cells was isolated using TRIzol reagent (Invitrogen, San Diego, Calif., USA), following the manufacture’s protocol. RT-PCR was carried out using a kit (GeneAmp RNA PCR Core, Perkin-Elmer, Branchburg, N.J., USA) with the X. laevisP2Y-specific primers XLP2Y1 upstream (5′-ACAGAAGTCTTTCTCTCAGC3′) and XLP2Y1 downstream (5′-TCACAAGCTTGTGTCCCCAT-3′), which yield a 1,082-bp fragment of the X. laevis P2Y coding region. RT-PCR was verified and normalized against the β-actin expression
Measurements of transepithelial short-circuit current and Cl− transport Transepithelial potential difference (ΔV) and short-circuit current (Isc) were measured in a modified Ussing chamber employing standard methods [12]. The electrical parameters were measured at room temperature in the following Cl− medium (in mM): NaCl 110, MgSO4 0.5, KCl 3, KH2PO4 1, HEPES 10, glucose 5, CaCl2 1 (pH 7.5). Measurement of Cl− efflux and apical CFTR function Cl− efflux was measured with the aid of the Cl− sensitive dyeN-ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE) according to [4, 8]. In brief, A6 cells were seeded onto collagen-coated cell culture inserts having polyethylene terephthalate (PET) filters (Falcon Becton-Dickinson Labware, USA) as described above. Monolayers were loaded overnight in culture medium containing 5 mM MQAE at 28 °C in a CO2 incubator. Fluorescence was recorded with a Varian Cary Eclipse spectrofluorometer using 360 nm (bandwidth 10 nm) for excitation and measuring emission at 450 nm (bandwidth 10 nm). All experiments were performed at room temperature in HEPES-buffered bicarbonate-free media (Cl−
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medium, as above and Cl−-free medium: NaNO3 105, KNO3 3.2, MgSO4 0.8, NaH2PO4 1, HEPES 10, Ca(NO3)2 5, glucose 5). At the start of the experiment, the monolayers were perfused at a constant rate of 2 ml/min in Cl− medium. To measure the Cl− efflux across the apical membrane, the apical Cl− perfusion medium was changed to a Cl−-free medium and MQAE fluorescence intensity followed. At the end of each experiment a two-point calibration was made: the maximal intensity of fluorescence (F0) was determined by perfusing the cells with a Cl−-free medium (nitrate substituted) on both sides of the monolayer and the minimum fluorescence (FKSCN) was obtained by then exposing the cells to a solution containing KSCN (in mM: KSCN 110, MgSO4 1, HEPES 10, CaSO4 1, glucose 5 and 5 µM valinomycin). For data analysisFKSCN was subtracted from the experimentally measured fluorescence (F) and the resulting fluorescence divided byF0, with efflux being expressed in arbitrary slope changes [Δ(F/F0)/minute]. Measurements of [Ca2+]i Cells were seeded at low density on glass cover-slips and used the following day for microspectrofluorimetric measurements of [Ca2+]i with the dye Fura-2-AM [47]. Samples were excited at 340 nm and 380 nm and emission monitored at 510 nm. The 340/380 ratio was converted to [Ca2+]i according to [23]. The composition of the Ringer solution used in these experiments was (in mM): NaCl 101.4, MgSO4 0.5, CaCl2 1.4, KCl 5.4, NaHCO3 8, NaH2PO4 0.9, HEPES 1, glucose 5 (pH 7.5).
Fig. 1 A Electrophoretic analysis of the RT-PCR product amplified from A6 total RNA using specific primers for the Xenopus laevis purinoreceptor (XP2Y1) and for β-actin as positive control. B Northern blot analysis of the amplified RTPCR product after separation on a 1% agarose gel and analysed using a probe for X. laevis P2Y1. C Western blot analysis using a specific rabbit antibody directed against a peptide of human P2Y1. The P2Y1 receptor migrated as a 65 kDa band. D Electrophoretic analysis of the RT-PCR product amplified from A6 and MDCK total RNA using specific primers for the P2Y11 and for β-actin as positive control
Cyclic AMP determination Cells were plated in a 12-well microculture plates and grown to approximately 70% confluency. Prior to treatment of cells, growth medium was removed and cells were equilibrated for 30 min in Ringer. Unless otherwise indicated the incubations with agonists were conducted for 10 min at 28 °C in presence of the phosphodiesterase inhibitors 10 µM rolipram plus 1 mM 3-isobutyl-1methylxanthine (IBMX). After incubation the cells were washed twice with a Ringer solution and 0.25 ml 5% trichloroacetic acid solution was added to extract cAMP. Intracellular cAMP was then determined using the cAMP [3H] assay system (Amersham TRK 432). Transient transfection Briefly, 2.5 µg of construct containing Na+/H+ exchanger regulatory factor-2 (NHERF-2) PDZ1 domain-truncated cDNA was diluted in 250 µl DMEM (without FBS or antibiotics), to which 2 µl Escort IV (Sigma) was added and incubated for 30 min. A6 cells (~70% confluent; culture inserts 0.9 cm2 diameter) were washed once with DMEM and incubated with the DNA-lipid mix for 6 h, at the end of which 250 µl FBS-containing media was added and the cells incubated for a further 48 h before use. To monitor the level of transfection of cells with plasmids containing the NHERF-2 construct and the X-press tag, cells were seeded after Cl− transport measurements on collagen-coated Teflon filters, fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, incubated with fluores-
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cein isothiocyanate (FITC)-conjugated anti X-press antibody for 2 h and observed with a Nikon microscope.
can not exclude this possibility since as a positive control for the P2Y11 receptor we used the MDCK cell line and the human sequence for the primers [44].
Materials Fura-2-AM and MQAE were purchased from Molecular Probes (Eugene, Ore., USA); 12-o-tetradecanoylphorbol13-acetate (TPA), glibenclamide, DIDS, H89 and 2MeSADP were purchased from Sigma RBI; Ro-31-8220 and BAPTA-AM were purchased from Calbiochem. Data presentation Results are expressed as mean±SE. The significance of differences between means was established using Student’s t-test for paired or unpaired data as appropriate. P<0.05 was considered significant.
Results A6 epithelia express the Xenopus P2Y1 receptor Recently, Cheng et al. [16] have provided evidence of the expression of the transcript for the P2Y1 receptor in X. laevis (Xenopus P2Y1), the translated amino acid sequence of which shares 83% similarity with its human counterpart. This evidence for the P2Y1-like receptor in X. laevis led us to assess its expression in the A6 renal cell line, which is derived from the distal part of the X. laevis nephron. Figure 1A shows a representative electrophoretic analysis of the RT-PCR product amplified from A6 total RNA using primers specific for the Xenopus P2Y1. A strong band of the expected size (1,000 bp) suggested the presence of the Xenopus P2Y1 mRNA and its expression in A6 cells was further demonstrated by Northern blots, which detected a transcript of about 3.0 kb (Fig. 1B). These RT-PCR and Northern blot results were confirmed by Western blot analysis employing specific rabbit antibodies directed against a sequence of the human P2Y1 receptor (amino acid residues 242–258) that is conserved in Xenopus P2Y1. As shown in Fig. 1C, a band of the expected Mr (65 kDa) was detected in the A6 cell homogenate. Recently, we have demonstrated that 2-MeSADP increases both cAMP/PKA and Ca2+/PKC intracellular levels in A6-NHE3 cells, a cell line obtained by stable transfection of A6 cells with the cDNA encoding the rat isoform of NHE3 [3]. Until now the only P2Y receptor subtype reported to activate both the phosphoinositide and the cAMP pathways when specifically activated by adenine nucleotides is the P2Y11 receptor [17, 52]. To explore the possible involvement of the P2Y11 receptor, we performed RT-PCR using the same conditions and the same primers (those described in [44]) for the P2Y11 receptor. The results reported in Fig. 1D suggest that the P2Y11 receptor is not expressed in A6 cells, even if we
Localization of purinoceptor P2Y1-like receptors in A6 monolayers To localize the Xenopus P2Y1 receptor, we measured the effect of the P2Y1 synthetic agonist, 2-MeSADP, on shortcircuit current (Isc) in confluent A6 cell monolayers. Figure 2A shows a typical experiment in which only the basolateral application of 100 nM 2-MeSADP elicited a rapid transient increase of Isc and the transepithelial potential difference ΔV. The peak amplitude, reached after about 2 min, was calculated as the difference between the basal and maximal Isc (ΔIsc=6.82±0.80 µA/cm2, n=14). Apical application of 2-MeSADP at the physiological concentration of 100 nM had no effect on Isc. A small, but not significant increase of Isc was observed with apical treatment only with a much higher, non-physiological concentration (50 µM 2-MeSADP, ΔIsc=1.33±0.48 µA/ cm2, n=4), probably due either to an non-specific interaction, or the involvement of receptors such as the P2Y2like receptors that are characterized by a low affinity for 2MeSADP [5]. To establish whether the basolateral, 2-MeSADP-dependent stimulation of Isc involved the P2Y1 receptor directly, the cells were pre-treated on the basolateral side with the selective P2Y1 antagonist MRS 2179 (1 µM) [10], which reduced the basolateral 2-MeSADP-induced Isc peak by ~80% (Fig. 2B). We have reported previously the expression of the A2a adenosine receptor in the basolateral membrane of these cells [13]. The lack of any effect of pretreatment with the A2a-antagonist 8-(3chlorostyryl)caffeine (CSC, 1 µM) on the stimulatory action of 2-MeSADP (Fig. 2B) confirmed the involvement of only Xenopus P2Y1 receptors in the 2-MeSADPdependent increase of Isc. 2-MeSADP-induced increases in apical Cl− permeability In monolayers exposed to amiloride, basolateral application of 2-MeSADP (100 nM) induced a transient increase in Isc (ΔIsc) that was not significantly different from the ΔIsc obtained in monolayers untreated with amiloride (6.41±0.77, n=24 vs. 6.82±0.80 µA/cm2, n=14 for amiloride treated and untreated monolayers, respectively). This finding suggests that basolateral 2-MeSADP stimulates exclusively an amiloride-insensitiveIsc component, probably apical Cl− channels. In A6 cells, patch-clamp experiments have demonstrated two types of apical Cl− channels that are regulated by either calcium and/or cAMP [35]. The CFTR Cl−channel is also found on the apical membrane of A6 cells [33]. We therefore investigated the effects of apically added Cl− channel inhibitors on the peak response of Isc to 2-MeSADP. DIDS, a blocker of
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Fig. 2 A Typical time course of the short-circuit current (Isc) and transepithelial potential difference (ΔV) during apical and basolateral stimulation of confluent A6 monolayers with the potent P2Y1 synthetic agonist 2-methylthioadenosine diphosphate (2-MeSADP, 100 nM). After an initial period in which Isc was allowed to stabilize, 2-MeSADP was added first to the apical side and then, after a 10 min wash-out period, to the basolateral side of the monolayer. B Effect of the P2Y1-selective antagonist MRS 2179 (1 µM, solid bar) and of the adenosine A2a receptor antagonist 8-(3chlorostyryl)caffeine (CSC, 1 µM, cross-hatched bar) on the 2MeSADP dependent increase of transepithelial short-circuit current. Means±SE, n=8 independent experiments
Ca2+-activated Cl− channels [2], had no significant effect on the stimulatory 2-MeSADP response, while a 5 min preincubation of the monolayer with 100 µM glibenclamide, an inhibitor of cAMP-stimulated Cl−currents [43, 48] almost completely abolished the response to basolateral 2-MeSDAP (Fig. 3). These data suggest that the 2MeSADP-mediated increase in Isc is due to electrogenic Cl− secretion via apical CFTR Cl− channels. These data were confirmed further by fluorimetric measurements using a Cl− sensitive dye, MQAE, in which Cl− transport rates across apical A6 cell membranes were measured as the initial rate of change in F/F0 after replacement of Cl− in the medium by nitrate (see Materials and methods). Figure 4A illustrates a typical experiment. Under non-stimulated conditions, the A6 monolayer has a very low apical Cl− permeability [0.0126±0.0016 Δ(F/ F0)/min, n=23] as shown earlier [5]. Figure 4A shows that 2-MeSADP increased the Cl− efflux rate, a response inhibited significantly by preincubation of the apical side with glibenclamide. In control experiments, a second 2MeSADP addition in the absence of glibenclamide induced a Cl− efflux comparable to the first 2-MeSADP stimulation (data not shown). The results of ten experi-
Fig. 3 Effects of glibenclamide, a specific inhibitor of cAMPstimulated Cl− currents (100 µM, hatched bar) and DIDS, a blocker of Ca2+-activated Cl−channels (100 µM, cross-hatched bar) on the 2-MeSADP-induced increase of apical, amiloride-independent Isc in A6 monolayers. Control: cells not pretreated with inhibitors. Means ±SE, n=5 independent experiments
ments are reported in Fig. 4B; the glibenclamide-sensitive component of Cl− efflux (i.e. CFTR-mediated Cl− transport) calculated as the difference between 2-MeSADPstimulated fluorescence in the absence and presence of glibenclamide was 0.0176±0.0033 Δ(F/F0)/min, n=10. A similar increase in the glibenclamide-sensitive component of Cl− efflux was obtained when A6 cells were treated with 10 µM forskolin (FSK), an activator of adenylate cyclase [0.0169±0.002 Δ(F/F0)/min, n=9]. 2-MeSADP increases both [Ca2+]i and cAMP levels In Fura-2-AM loaded A6 cells, 100 nM 2-MeSADP increased [Ca2+]i, from 35.9±5.6 to 124.6±11.3 nM (n=7). This increase was prevented completely by preincubation with 1 µM MRS 2179 (36.52±7.83 nM, n=4). We next measured the accumulation of intracellular cAMP to evaluate directly the involvement of adenylate cyclase in the action of 2-MeSADP. As shown in Fig. 5, addition of 100 nM 2-MeSADP to the basolateral side of the monolayer resulted in a significant increase of cAMP that was prevented by the P2Y1 receptor antagonist, MRS 2179. In MDCK cells, P2Y2 receptor activation can stimulate cAMP formation in a indomethacin-sensitive manner [44]. As preincubation with indomethacin had no effect on 2-MeSADP-dependent cAMP production (Fig. 5), we can exclude a role for both phospholipase A2-dependent release of arachidonic acid and P2Y2 receptor activation in its action.
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Fig. 5 Effect of 2-MeSADP in the absence or presence of the P2Y1 antagonist MRS 2179 and indomethacin on cAMP production. The level of intracellular cAMP in A6 cells was measured after cells had been incubated for 10 min with 100 nM 2-MeSADP or a preincubation with 1 µM MRS 2179 for 5 min or 10 µM indomethacin for 10 min before the addition of 2-MeSADP. Incubation with forskolin (FSK) was used as positive control. P values are in relation to the control. Means±SE, n=6 independent experiments Fig. 4 A Typical recording showing changes in intracellular Cl−dependentN-ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE) fluorescence (expressed as the F/F0 ratio) when the A6 cell monolayer was treated with 100 nM 2-MeSADP following replacement of apical chloride by nitrate in the absence or presence of 100 µM glibenclamide. Glibenclamide was applied for 5 min before apical anion substitution. B Summary of experiments as inA. The glibenclamide-sensitive Cl− efflux rates across the apical membrane (open bar) were calculated as the difference in rate of change of the F/F0 ratio [Δ(F/F0)/min] in the absence of (stippled bar) and presence of (hatched bar) glibenclamide. In the same monolayer, we first followed the rate of Cl− efflux after 2-MeSADP treatment and then, always in the same monolayer, analysed the effect of glibenclamide; this permitted the use of two-tailed, paired Student’s t-test for determination of significance. Means±SE, n=10 independent experiments
These data demonstrate that the same low concentration of 2-MeSADP that stimulated CFTR also induced both the cAMP and [Ca2+]i signal transduction systems simultaneously in these cells. Cross-talk between the PKA and PKC signalling systems has been demonstrated in many cellular systems. To test this hypothesis, intracellular cAMP was measured in presence of TPA or BAPTA-AM under control conditions and after 2-MeSADP application. Neither 100 nM TPA nor 20 µM BAPTA-AM significantly influenced the basal cAMP levels (105±4.3%, n=3; 99±6.5%, n=3, of control after TPA or BAPTA-AM treatment, respectively, n=3) or the 2-MeSADP-dependent cAMP production (151±6.7%, n=3; 141±4.7%, n=3; of control values after TPA or BAPTA-AM treatment, respectively).
Signal transduction systems involved in 2-MeSADP stimulated Cl− efflux We next analysed the involvement of the cAMP/PKA and/ or Ca2+/PKC signal transduction pathways in the 2MeSADP-dependent stimulation of Cl− transport. The involvement of PKA was analysed by preincubating MQAE-loaded A6 monolayers with 1 µM H89, a PKA inhibitor. As seen in Fig. 6, H89 strongly inhibited (−89.2 ±10.8%, n=5) the glibenclamide-sensitive Cl− efflux across the apical membrane while having no effect on basal Cl− efflux. This result was confirmed by Iscmeasurements in which H89 pre-treatment inhibited the 2-MeSADP-dependent transient increase in Isc to the same extent (−82.4±5.1%, P<0.001, n=4). Endogenous PKC-mediated phosphorylation of CFTR can have a permissive role in priming the CFTR channel for acute activation by PKA [28] or can inhibit or stimulate CFTR activity depending on the sequence phosphorylated [15]. In the present study, 50 nM Ro-318220, a PKC inhibitor [7], significantly inhibited the 2MeSADP-dependent transient peak of Isc by −36.2±8.3% (n=10, P<0.02) while pretreatment with the PKC activator TPA (100 nM) potentiated the 2-MeSADP-dependent transient peak of Isc by +51.6±6.8%, (n=10, P<0.02). This potentiation by TPA was attenuated strongly by apical glibenclamide (−86.8±8.6%, n=6). Preincubation of the A6 monolayers for 5 min with Ro-31-8220 prevented the subsequent TPA-dependent potentiation of the 2-MeSADP increase in CFTR activity, demonstrating that TPA was functioning by activation of PKC (−26.5±6.7%,n=4, n.s. vs. pretreatment with Ro-31-8220 alone). Pretreatment with TPA alone (100 nM) induced no significant response but, interestingly, converted cell monolayers that were
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Fig. 6 Effect of pre-incubation with H89 (1 µM, cross-hatched bar), an inhibitor of PKA, the synthetic peptide S-Ht31 (100 µM, solid bar) or its inactive control S-Ht31-P (100 µM, hatched bar) on 2-MeSADP-dependent Cl− efflux. Means±SE,n=5 independent experiments
unresponsive to 2-MeSADP into 2-MeSADP-responding monolayers. Preincubation with 20 µM BAPTA-AM for 30 min prior to 2-MeSADP addition had no effect on the stimulatory action of 2-MeSADP (+15.3±19.6%, n=4, n. s.).
protein-protein association, we preincubated A6 cell monolayers for 30 min with an amphipathic membranepermeable peptide coupled to stearate residues, S-Ht31 (100 µM), corresponding to the RII binding motif of a human thyroid AKAP that blocks the binding of RII to AKAPs [11]. As shown in Fig. 6, preincubation with SHt31 completely prevented the stimulatory effect of 2MeSADP on Cl− secretion, while the inactive control peptide S-Ht31-P had no effect. These data suggest that one or more AKAPs mediate the association of PKA with CFTR and this association is necessary for the stimulation of CFTR by 2-MeSADP. Moreover, since the PDZ1 domain of NHERF-2 is known to interact with high affinity with the C-terminal domain of CFTR [45], we determined whether the PDZ1 domain of NHERF-2 was involved in the regulation of the 2-MeSADP-dependent increase of CFTR mediated Cl− efflux in A6 cells. We therefore measured Cl− efflux from A6 monolayers that had been transfected with plasmids containing the cDNA encoding for NHERF-2 truncated in the PDZ1 domain (NHERF-2-ΔPDZ1). As can be seen in Fig. 7, in A6 cells transfected with NHERF-2-ΔPDZ1 the increase of CFTRmediated Cl−efflux elicited by either 2-MeSADP or FSK treatment was abolished completely, while the treatment with liposome complex only had no effect. These results demonstrate clearly that the PDZ1 domain of NHERF-2 is essential for the 2-MeSADP-dependent and PKA-dependent regulation of CFTR channel activity.
Role of the A-kinase anchoring protein (AKAP) and NHERF-2 in the 2-MeSADP-dependent regulation of CFTR activity
Discussion
We have reported recently that, in A6 cells, CFTR interacts with NHERF-2 and that NHERF-2, in turn, associates with ezrin [4], which functions as an AKAP [30]. To determine whether the PKA-dependent CFTR regulation induced by 2-MeSADP is mediated by this
Stimulation of Cl− secretion by extracellular nucleotides has been reported in a number of secretory epithelia including the airway [28, 43, 49], colonic cells [31], epididymal cells [14], pancreatic duct cells [39, 41] and the kidney [5, 19, 36, 51]. In this study we used A6 cells, a
Fig. 7 Effect of truncation of the PDZ1 domain of the scaffolding protein Na+/H+ exchanger regulatory factor-2 (NHERF-2) on both 2MeSADP and FSK-mediated regulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity. CFTR-mediated Cl− efflux was measured in A6 cells 48 h after transfection with
2.5 µg of NHERF-2-ΔPDZ1 cDNA or with only Escort IV. Means ±SE, n=4 experiments per treatment. Inset: level of transfection (as revealed by immunofluorescence microscopy using FITC-conjugated anti X-press antibody) in a non-transfected and a transfected A6 monolayer on the filter after Cl− efflux measurements
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renal cell line derived from X. laevis, to examine the location of the P2Y1 receptor and the signal transduction pathways involved in P2Y1 receptor-mediated Cl− secretion. Although purinergic receptors are expressed in A6 cells [3, 5, 38, 40], the specific receptor subtypes and their mechanisms of action are still undefined. Recently, a single transcript of RNA of 3.2 kb encoding the P2Y1 receptor in the brain, spinal cord, liver and muscle of adult Xenopus has been isolated [16]. The following findings from the present study show that A6 cells express basolateral X. laevis P2Y1 receptors: (a) both RT-PCR using primers and Northern blot analysis with a probe specific for the X. laevis P2Y1 receptor demonstrated the expression of its mRNA, while Western blot analysis revealed the expression of the protein for the X. laevis P2Y1 receptor; (b) only basolateral treatment with physiological concentrations (nanomolar) of the specific P2Y1 receptor agonist 2-MeSADP induced a rapid, transient increase in Isc and an increase of Cl− efflux; (c) this stimulatory effect of 2-MeSADP on Cl− efflux was almost completely reversed by pretreatment with MRS 2179, a selective antagonist at human P2Y1 receptors [10]. The presence of cAMP- and Ca2+-activated Cl−channels displaying single-channel conductances of 8 and 3 pS, respectively, has been demonstrated in the apical membranes of A6 cells grown on permeable filters [33, 35]. Here, we observed a strong inhibition of the 2-MeSADP stimulated Cl− efflux when the apical side of the monolayers were preincubated with glibenclamide, a well-known inhibitor of cAMP-stimulated Cl− currents [20, 48], while apical preincubation with DIDS, a blocker of Ca2+-activated Cl− channels [2], had no significant effect on the stimulatory 2-MeSADP response. This result suggests that in A6 cells the channel most likely mediating the Cl− secretory response induced by 2-MeSADP is the cAMP-activated Cl− channel CFTR. P2Y receptors function via two signal transduction pathways: receptor activation can increase [Ca2+]i by stimulating PLC activity [21] or it can either increase [26, 31, 44] or decrease [18, 25] cAMP production, depending on the tissue or cell type examined. In the present study in A6 cells that endogenously express Xenopus P2Y1 receptors, 2-MeSADP stimulated both cAMP production (Fig. 5) and [Ca2+]i at the same concentration (100 nM) at which it stimulated Cl−efflux (Figs. 2 and 4). Cheng et al. [16] have reported that 2-MeSADP activation of the Xenopus P2Y1 receptor transfected into a heterologous system (Cos-7 cells) mobilized intracellular calcium with a higher EC50 than for mammalian or avian receptors and, even at 50 µM, did not increase cAMP. Those authors suggested that this difference could be due to an incorrect protein folding of the amphibian receptor when expressed in mammalian cells. In support of this hypothesis, in our cellular system, which expresses endogenous XenopusP2Y1 receptors on the basolateral membrane, we observed second messenger production even at a 500-fold lower concentration of 2-MeSADP (100 nM). Our observation regarding the involvement of the X. laevis P2Y1 receptor in CFTR activation differs from that obtained in another
cellular system [34] in which P2Y1 receptor activation does not regulate CFTR activity in CHO cells expressing an endogenous P2Y1 receptor and a stably transfected CFTR. A possible explanation for this discrepancy in receptor signalling mechanisms may be that the X. laevis P2Y1 receptor is a more archaic form, leading to the induction of cAMP in addition to calcium mobilization in the amphibian cells by activating two signal transduction systems (Fig. 5). This is in line with the known high tissue and/or species plasticity of receptor down-stream mechanisms. Recently, there has been a paradigm change in the understanding of PKA-dependent regulation in which specificity is achieved by its tight localization at the functional subcellular compartment via binding to AKAPs and the subsequent targeting of this complex to specific proteins by binding to scaffolding proteins such as NHERF-2. A6 cells express the NHERF-2 isoform endogenously and this forms a complex with CFTR via the AKAP ezrin and PKA that permits the PKA-dependent regulation of CFTR [4]. In the present study, incubation with S-Ht31, a peptide that blocks the interaction of PKA with AKAPs [30, 32], blocked the 2-MeSADP-dependent stimulation of CFTR activity, suggesting that an AKAP is also involved in the PKA-mediated regulation of Cl− efflux by 2-MeSADP. Moreover, the fact that the truncation of the NHERF-2 PDZ1 domain, which interacts with CFTR [24], abrogated the 2-MeSADP-dependent regulation of CFTR activity, demonstrates that the CFTR association with the NHERF-2 PDZ1 domain is critical for its regulation by 2-MeSADP. In addition to PKA-dependent activation, CFTR activity is also modulated by PKA-independent mechanisms. Initial PKC-mediated phosphorylation may be required for the acute activation of CFTR channel activity by PKA [29]. Interestingly, the direct phosphorylation of CFTR by PKC has been suggested to either stimulate or inhibit directly depending on the consensus sequence of CFTR that is phosphorylated [15]. While the PKC-phosphorylated region is highly conserved among species, the inhibitory T682 site is not present in the amphibian CFTR and its absence may have contributed to the strong potentiation of the 2-MeSADP activation observed in the present study. Here, we found that: (a) pretreatment with the PKC inhibitor Ro-31-8220 inhibited the 2-MeSADPdependent Cl− efflux significantly; (b) pretreatment with TPA, an activator of PKC, while having no effect per se on 2-MeSADP-stimulated Cl− efflux, potentiated the Cl− transport response to 2-MeSADP. This last observation is reinforced by experiments in which TPA pretreatment transformed non 2-MeSADP responding A6 monolayers into responders. (c) The potentiating effect of TPA was prevented completely by preincubation with Ro-31-8220. Altogether, these data suggest that in A6 cells expressing endogenous Xenopus P2Y1 receptor, the latter’s activation stimulates CFTR by a mechanism involving mainly the PKA-dependent signal transduction pathway, but which can be amplified in a PKC-dependent manner. That neither TPA nor BAPTA-AM had any
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significant effect on either basal or 2-MeSADP-induced cAMP imply that PKC acts directly on CFTR [9, 29] and not via the involvement of Ca2+-activated isoforms of adenylate cyclase [42]. On the basis of our results we cannot formulate the precise mechanism by which PKC can potentiate the effect of PKA and we cannot distinguish between a direct PKC effect on CFTR protein or an indirect effect by phosphorylation of NHERF-2. Indeed, PKC-mediated phosphorylation of serine 162 of NHERF1 interferes with its ability to interact with and stimulate CFTR in respiratory Calu-3 cells [46]. However, in the NHERF-2 isoform expressed in A6 cells the serine 162 is replaced by glutamine, which could also explain the strong potentiation of the 2-MeSADP activation by PKC observed in the present study. In conclusion, all these data suggest that NHERF may play an important role in facilitating activation of CFTR at the cell membrane by promoting its phosphorylation via the anchoring of the catalytic subunit of PKA to the vicinity of the channel by the NHERF-2-ezrin macromolecular complex. Acknowledgements This work was supported by grants from: Telethon, Italy, grant E.1125; the Italian Cystic Fibrosis Research Foundation and CEGBA (Centro di Eccellenza di Genomica in Campo Biomedico ed Agrario). We thank Dr. Pann-Ghill Suh of Life Science and School of Environmental Engineering, Pohang University of Science and Tecnology, Pohang 790-784, South Korea, for the kind gift of NHERF-2-ΔPDZ1.
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