Desensitization of P2Y2 receptor-activated transepithelial anion secretion

Lane L. Clarke1, Matthew C. Harline1, Miguel A. Otero2, Geraldine G. Glover3, Richard C. Garrad3, Brent Krugh3, Nancy M. Walker1, Fernando A. González2, John T. Turner4, and Gary A. Weisman3

1 Dalton Cardiovascular Research Center and Department of Veterinary Biomedical Sciences and Departments of 3 Biochemistry and 4 Pharmacology, University of Missouri-Columbia, Columbia, Missouri 65211; and 2 Department of Chemistry, University of Puerto Rico, Río Piedras, Puerto Rico 00931


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Desensitization of P2Y2 receptor-activated anion secretion may limit the usefulness of extracellular nucleotides in secretagogue therapy of epithelial diseases, e.g., cystic fibrosis (CF). To investigate the desensitization process for endogenous P2Y2 receptors, freshly excised or cultured murine gallbladder epithelia (MGEP) were mounted in Ussing chambers to measure short-circuit current (Isc), an index of electrogenic anion secretion. Luminal treatment with nucleotide receptor agonists increased the Isc with a potency profile of ATP = UTP > 2-methylthioATP >> alpha ,beta -methylene-ATP. RT-PCR revealed the expression of P2Y2 receptor mRNA in the MGEP cells. The desensitization of anion secretion required a 10-min preincubation with the P2Y2 receptor agonist UTP and increased in a concentration-dependent manner (IC50 approx  10-6 M). Approximately 40% of the anion secretory response was unaffected by maximal desensitizing concentrations of UTP. Recovery from UTP-induced desensitization was rapid (<10 min) at preincubation concentrations less than the EC50 (1.9 × 10-6 M) but required progressively longer time periods at greater concentrations. UTP-induced total inositol phosphate production and intracellular Ca2+ mobilization desensitized with a concentration dependence similar to that of anion secretion. In contrast, maximal anion secretion induced by Ca2+ ionophore ionomycin was unaffected by preincubation with a desensitizing concentration of UTP. It was concluded that 1) desensitization of transepithelial anion secretion stimulated by the P2Y2 receptor agonist UTP is time and concentration dependent; 2) recovery from desensitization is prolonged (>90 min) at UTP concentrations >10-5 M; and 3) UTP-induced desensitization occurs before the operation of the anion secretory mechanism.

receptor regulation; chloride secretion; bicarbonate secretion; cystic fibrosis; mouse; gallbladder; epithelium


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CYSTIC FIBROSIS (CF) is the most common lethal genetic disease of the Caucasian population (12). The disease is caused by mutations in the CF transmembrane conductance regulator (cftr) gene, which encodes a cAMP-activated anion channel (CFTR) (1, 43). CFTR is primarily expressed in epithelial tissues and normally functions in the secretion of salt and water onto the epithelial surface (29, 50). Loss of this function leads to blockage in the lumens of glands and ducts by inspissated mucus and debris, resulting in the common manifestations of CF, such as pancreatic insufficiency, obstructive bowel disease, cholecystitis/gallstones, and failure of the airway mucociliary apparatus (33, 57). In the search for therapies to correct the deficiency of transepithelial fluid secretion, previous studies have demonstrated that CF epithelia express an alternative apical membrane anion conductance (GCla) that is activated by intracellular Ca2+ mobilization and, therefore, that can compensate for the loss of CFTR (59). The relevance of targeting the GCla for CF pharmacotherapy has been strengthened by the observation that the GCla is the dominant anion conductive pathway in the airway and pancreatic duct epithelia of CFTR knockout mice that have a relatively mild disease phenotype in these organs (10).

Previous studies have shown that the GCla of CF epithelia can be regulated by the activity of a luminal membrane receptor, the P2Y2 (formerly P2u) receptor for extracellular nucleotides (7, 30, 35, 40). The P2Y2 receptor is a G protein-coupled receptor that activates the phospholipase C-dependent generation of inositol 1,4,5-trisphosphate and intracellular Ca2+ mobilization (5, 35). The P2Y2 receptor has an agonist profile that is characterized by the equal potencies and efficacies of ATP and UTP (16, 40). Although ATP is considered to be the endogenous ligand for the P2Y2 receptor, its usefulness in an aerosolized therapy for CF lung disease is limited by the potential for its rapid metabolism to the bronchoconstrictive agent adenosine (14). In contrast, UTP applied to the surfaces of airway epithelia would be metabolized to the relatively inactive base uridine (38). Therefore, UTP has been advocated as the prototypical compound for aerosol therapy in CF disease (4, 30, 37).

A potential limitation of nucleotide therapy for CF relates to the propensity of G protein-coupled receptors to undergo desensitization upon repeated agonist exposure, i.e., homologous desensitization (45). The phenomenon of homologous desensitization by G protein-coupled receptors has been particularly well studied for the beta -adrenergic receptor (26). The mechanisms of desensitization of the beta -adrenergic receptor have been categorized into 1) a rapid phase that is dependent on receptor phosphorylation and that involves the uncoupling of the receptor from the Gs protein, 2) a secondary phase that involves receptor sequestration from the cell surface and that requires protein dephosphorylation for reversal, and 3) a long-term phase involving receptor downregulation and degradation that requires de novo protein synthesis for reversal (3, 41, 47, 62). Little is known about the pathway for the desensitization of endogenous P2Y2 receptors. In a study of airway epithelia, Brown et al. (5) have shown that total inositol phosphate (IP) generation activated by the P2Y2 receptor desensitizes in response to repeated application of UTP. However, it has not been determined whether the subsequent events, including intracellular Ca2+ mobilization and stimulation of channel activity, also undergo desensitization during repeated P2Y2 receptor stimulation. Therefore, in the present study, we investigated the process of P2Y2 receptor desensitization as it relates to the clinically relevant function of modulating transepithelial anion secretion. The murine gallbladder was used as a model epithelium because we have previously shown that this tissue responds to UTP stimulation with Ca2+-activated anion secretion (24).


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Animals

C57Blk/6 mice (2-4 mo of age) were obtained from Laboratory Animal Resources, Dalton Cardiovascular Research Center, University of Missouri-Columbia (Columbia, MO). The mice were maintained on standard laboratory mouse chow and water ad libitum. Before each experiment, the mice were fasted overnight but were provided drinking water. All experiments involving the animals were approved by the University of Missouri-Columbia Institutional Animal Care and Use Committee.

Cell Culture

Murine gallbladder epithelial (MGEP) cells were isolated from surgically excised gallbladders with Hanks' balanced salt solution containing 0.25% (wt/vol) trypsin and 1 mM EDTA (37°C for 30 min). After vigorous shaking to free the epithelial cells from the underlying stroma, the remnant of the gallbladder wall (i.e., muscle layers) was removed and the cell suspension was treated 1:1 (vol:vol) with soybean trypsin inhibitor (1 mg/ml in Ham's F-12 medium). After centrifugation (500 g for 5 min), the cells were resuspended and then plated on Transwell-COL permeable supports (Corning Costar, Cambridge, MA) in Ham's F-12 medium containing 1 µg/ml insulin, 7.5 µg/ml transferrin, 1 µM hydrocortisone, 30 nM triiodothyronine, 2.5 ng/ml epidermal growth factor (EGF), and 10 ng/ml endothelial cell growth substance and supplemented (1:1) with 3T3 fibroblast-conditioned DMEM containing 2% (vol/vol) calf serum (CS), as previously described for the murine epithelium. After two passages, the cells were replated in the same medium onto a Transwell-COL support. After 1 day in culture, the support was suspended over a confluent monolayer of TEX S-6 fibroblast cells, a packaging cell line producing a retrovirus harboring the simian virus 40 (SV40) large T (LT) antigen and the neomycin resistance gene (a gift from Dr. Ian Summerhayes, New England Deaconess Hospital, Boston, MA) (42). The coculture was maintained for 7 days in DMEM supplemented with 2 mM glutamine, 0.5 µg/ml insulin, 0.125 µg/ml transferrin, 5 nM hydrocortisone, 67.5 µg/ml glucose, and 10% (vol/vol) CS. The luminal medium contained 1 µg/ml Polybrene for the first 3 days of coculture.

Transformation of the gallbladder cells was confirmed by selection in growth medium containing 400 µg/ml Geneticin and by immunohistochemical staining with a monoclonal antibody to the SV40 LT antigen (clone OH1; 1:100 dilution; Biogenesis) and a secondary FITC-conjugated anti-mouse IgG polyclonal antibody (DAKO, High Wycombe, UK) (20). The immortalized MGEP cells were continuously passaged onto Transwell-COL permeable supports as described above. Passages 32-88 were used in the present experiments. For bioelectric measurements, MGEP cells were cultured on permeable collagen supports affixed across a 4.5-mm aperture in polycarbonate cups, as previously described (7). The cell monolayers were used 6-9 days after being cultured on the collagen supports to coincide with the maximal baseline transepithelial potential difference of the preparations.

Bioelectric Measurements

Freshly excised gallbladders were opened longitudinally and mounted in standard Ussing chambers (exposed tissue surface area = 0.125 cm2), as previously described (11). The serosal surface of the gallbladder preparation was supported by a large-gauge nylon mesh. For studies using the MGEP cell line, the cultured preparations were mounted in Ussing chambers specifically designed to fit the polycarbonate culture cups (35). The luminal and basolateral surfaces of the gallbladders or cell cultures were bathed independently with 4 ml of a standard Krebs-Ringer bicarbonate (KRB) solution, which consisted of (in mM) 115 NaCl, 25 NaHCO3, 2.4 K2HPO4, 0.4 KH2PO4, 1.2 CaCl2, 1.2 MgCl2, and 10 glucose. In ion-substitution studies, a nominally Cl-- and HCO-3-free Ringer solution was formulated by replacing Cl- with gluconate- and HCO-3 with TES- (3 mM CaSO4 was added to compensate for Ca2+ chelation by gluconate-). For a nominally Na+-free Ringer solution, NaCl was replaced with N-methyl-D-glucamine chloride and NaHCO3 was replaced with choline bicarbonate. All Ringer solutions were recirculated with a 95% O2-5% CO2 gas lift and warmed to 37°C.

After being mounted in the modified Ussing chambers, the epithelial preparations were continuously voltage-clamped to zero transepithelial potential difference (i.e., short-circuited) with an automatic voltage clamp (VCC-600; Physiologic Instruments, San Diego, CA) and calomel electrodes connected to the chamber baths with 4% agar-KRB bridges. The short-circuit current (Isc) and the automatic fluid resistance compensation current were applied through Ag-AgCl electrodes that were in contact with the chamber baths via 4% agar-KRB bridges. Every 5 min during the experiment, the transepithelial resistance (Rt) values of the preparations were monitored by measuring the current deflections of the preparations in response to 2-mV voltage pulses and applying Ohm's law. Before each experiment, the offset of the transepithelial calomel electrodes and the compensation of the fluid resistance between the ends of the potential-sensing bridges were carefully performed by using an Ussing chamber with only nylon mesh (for gallbladder) or a culture cup without a monolayer (for cell preparations). Sign conventions are reported such that the Isc of the epithelial preparation is referenced to the serosal bath.

Intracellular Ca2+ Measurements

The MGEP cells were grown in tissue culture flasks, washed with HEPES-buffered saline (HBS; in mM: 120 NaCl, 4.0 KCl, 1.2 KH2PO4, 1.0 MgSO4, 15.0 Tris-HEPES, pH 7.4), and detached by incubation with 10 ml of HBS containing 1.0 mM EDTA. Cells were pelleted and resuspended in HBS containing 10.0 mM glucose, 0.1% (wt/vol) BSA, and 1.0 mM CaCl2 (HBS+). Fura 2-AM (2 µM) was added for 30 min at 37°C, and the cells were pelleted, resuspended in HBS+ at a density of 0.5-1.0 × 106 cells/ml, and equilibrated for 20 min at 37°C. Two-milliliter aliquots of cell suspension were pelleted and resuspended in 2.0 ml of HBS+. Changes in the concentration of intracellular free Ca2+ ([Ca2+]i) in response to UTP and/or other compounds were determined as previously described (21, 51).

Measurement of Total IP Production

Agonist-induced IP production in MGEP cells grown to confluence in 24-well plates was determined. The cells were incubated overnight in inositol-free DMEM (Sigma, St. Louis, MO) containing 5.0 µCi/ml myo-[2-3H(N)]inositol (22.3 Ci/mmol; Dupont-NEN, Boston, MA). The cells were washed twice with HBS and incubated for 5 min at 37°C in 0.5 ml of HBS in the presence or absence of UTP. The cells were washed once and incubated for 5 min at 37°C in 0.5 ml of HBS containing 30 mM LiCl in the presence or absence of UTP. The reactions were terminated by aspirating the medium; aspiration was followed by the addition of 0.3 ml of ice-cold 10% (vol/vol) perchloric acid. The perchloric acid extract was transferred to a test tube, the wells were rinsed with 0.3 ml of ice-cold water, and the wash water was combined with the extract. HEPES (225 mM, 0.3 ml) and KOH (2.0 M, 0.3 ml) were added to neutralize the perchloric acid. The separation of IP from inositol and glycerophosphoinositol in the neutralized extract and the measurement of 3H-labeled IP in disintegrations per minute were performed as described previously (61).

RT-PCR

P2Y1, P2Y2, P2Y4, and P2Y6 receptors were expressed in human 1321N1 astrocytoma cells as described previously (40). Total RNA was isolated from MGEP and 1321N1 cell transfectants with an RNeasy kit (Qiagen, Chatsworth, CA). The removal of deoxynucleic acids was accomplished through treatment with ribonuclease-free deoxyribonuclease followed by a clean-up step with the RNeasy kit. The quantity, purity, and integrity of the RNA were determined by ultraviolet (UV) spectroscopy and agarose gel electrophoresis. Total RNA (1 µg) was reverse transcribed with 1.6 µg of oligopoly(dT)15 primer and a 1st Strand cDNA synthesis kit for RT-PCR (Boehringer-Mannheim, Indianapolis, IN). Duplicate cDNA synthesis reactions were carried out for each RNA isolate in the presence or absence (control) of RT. Ten percent of the resulting cDNA was used as a template in the PCR performed with the Expand high-fidelity PCR system (Boehringer Mannheim). The P2Y1 primers (upstream: 5'-AAGACCGGCTTCCAGTTCTACTAC-3', downstream: 5'-CACATTTCTGGGGTCTGGAAATCC-3'), P2Y2 primers (upstream: 5'-CTTCAACGAGGACTTCAAGTACGTCG-3', downstream: 5'-CATGTTGATGGCGTTGAGGGTGTGG-3'), P2Y4 primers (upstream: 5'-ATGAGGATTTCAAGTTCATCCTGC-3', downstream: 5'-TAGACCACGTTGACAATGTTCAGT-3'), and P2Y6 primers (upstream: 5'-TGCCACCCACAACCTGTGTCTACCG-3', downstream: 5'-AGTAGAAGAGGATGGGGTCCAGCAC-3') were designed to be receptor subtype specific but to recognize regions of high homology among species (based on the rat, human, and mouse sequences available from GenBank). Primers specific for the amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Clontech, Palo Alto, CA) were used as controls for the presence and integrity of the cDNA. Reaction mixtures contained 20 pmol of primer and 3.5 units of the high-fidelity polymerase mixture in a 50-µl final reaction volume. The PCR was carried out for 30 cycles as follows: denaturation at 95°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 2 min, with a 5-min extension at 72°C after the final cycle. The resulting PCR products were resolved on a 1.0% (wt/vol) agarose gel containing 10 µg/ml ethidium bromide and detected under UV illumination. The PCR products were excised from the agarose gels, and the DNA was purified with the GFX PCR DNA and gel band purification kit (Pharmacia Biotech, Piscataway, NJ). After purification, the PCR products were sequenced at the University of Missouri-Columbia Molecular Biology DNA Core Facility.

Data Analysis

Differences between the basal Isc before treatment and the maximal values of Isc induced by specific agonists are reported as Delta Isc (i.e., Delta Isc = basal Isc - treatment Isc). In desensitization studies, variations in the Isc responses to identical concentrations of agonist between different passages of the cells were found (for example, responses to 10-4 M UTP varied from 18 to 30 µA/cm2). Therefore, the Delta Isc response of an individual cell preparation to a second exposure of agonist was normalized as a percentage of the preincubation Delta Isc response [i.e., (Delta Isc for treatment/Delta Isc for preincubation) × 100%].

Where appropriate, statistical analysis was by Student's t-test for paired and unpaired observations. If more than two groups were compared, the means were analyzed by a one-way ANOVA to test for statistically significant differences. If significant differences existed, Dunnett's t-test was used post hoc to compare each treatment mean to the control mean. Data in Fig. 4D were not normally distributed, and a Kruskal-Wallis one-way ANOVA on ranks was used to test for statistically significant differences. If significant differences existed within a group, a Dunn's test was used post hoc to compare each treatment mean to the preincubation Delta Isc mean. A probability value of P < 0.05 was considered statistically significant. Unless otherwise stated, all values are given as means ± SE.

Materials

In all studies, ATP, UTP, and alpha ,beta -methylene-ATP (Boehringer Mannheim) were added from fresh stocks prepared in the appropriate Ringer solution. When used, 2-methylthio-ATP (Boehringer Mannheim) was added from a stock aliquot prepared in the appropriate Ringer solution and frozen at -80°C. Ionomycin (Calbiochem, San Diego, CA) was prepared as a stock in ethanol. All other reagents were obtained from either Fischer Scientific (Springfield, NJ), Sigma, or Aldrich (Milwaukee, WI).


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Desensitization of the Anion Secretory Response to UTP in Freshly Excised Murine Gallbladder Mucosa

Freshly excised gallbladder mucosa responds to luminal UTP (10-4 M) with an abrupt increase in the Isc, which rapidly reaches a maximum and then decreases to a sustained level that is approximately twofold above baseline (Fig. 1A). After the UTP was washed from the luminal bath, the addition of a second equimolar concentration of UTP resulted in a peak Isc response that was reduced by 55% relative to the initial Isc response (cumulative data in Fig. 1, inset). The plateau phase of the Isc response measured at 1 min after the peak response was also reduced by 53.5% (initial UTP Delta Isc = 74.0 ± 7.8 µA/cm2; rechallenge UTP Delta Isc = 39.6 ± 6.9 µA/cm2). Thus, simulating a topical treatment regimen by repeated exposure of the epithelium to an equimolar concentration of UTP resulted in the desensitization of the Isc response. Ion substitution studies were used to investigate the ionic basis of the inward Isc stimulated by UTP. As shown in Fig. 1B, the removal of either HCO-3 or Cl- from the KRB solutions significantly reduced the UTP-induced Isc response. The removal of both anions decreased the UTP-induced Isc by 96%, indicating that the major fraction of the response represents anion secretion.


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Fig. 1.   Desensitization of UTP-stimulated anion secretion in freshly excised murine gallbladder mucosa. Gallbladders were mounted in standard Ussing chambers and bathed in physiological Krebs-Ringer bicarbonate (KRB) solution. A: desensitization. UTP (10-4 M) was added to the luminal bathing solution (arrows) for 10 min and then was washed from the luminal bath by repeated solution changes before rechallenge with UTP. Upward deflections in the short-circuit current (Isc) trace (spikes) result from repetitive 2-mV pulses used to estimate transepithelial resistance. Inset: mean Delta Isc (basal Isc - maximal stimulated Isc) ± SE from 7 experiments. * P < 0.05 vs. Delta Isc for initial UTP addition (paired t-test). B: ion substitution. Ion substitution studies were used to determine ionic basis of Isc response. Gallbladders were bathed bilaterally with Ringer solution containing standard KRB (Cl- + HCO-3), Cl- alone, HCO-3 alone, or neither (Cl- and HCO-3 free) and treated with 10-4 M UTP in the luminal bath. Bars, mean Delta Isc values ± SE (n = 6-9 experiments). * P < 0.05 vs. KRB with UTP (ANOVA-protected Dunnett's t-test).

Anion Secretory Response to UTP in Isolated Murine Gallbladder Epithelial Cells

The number of epithelial cells in the murine gallbladder is limiting, and Isc responses in native mucosa may be complicated by the effects of submucosal elements (immune cells, neural tissue, etc.). Therefore, we developed an MGEP cell line by transformation of isolated gallbladder epithelial cells with SV40 LT (42). When grown on permeable collagen matrix supports, the MGEP cells form confluent monolayers that typically have a baseline Isc of -6.3 ± 0.6 µA/cm2 and a transepithelial resistance of 621 ± 67 Omega  · cm2 (n = 30; >70 passages).

Similar to the freshly excised gallbladder preparations, MGEP cell monolayers responded to UTP treatment with an abrupt increase in Isc followed by a decrease to a sustained level above baseline (for an example, see Fig. 4A). To investigate the ionic basis of the UTP-stimulated Delta Isc response in the MGEP cells, ion substitution and inhibitor studies were performed with 10-4 M UTP as the agonist. As shown in Fig. 2A, removal of Cl- and HCO-3 from the bathing medium abolished the UTP-stimulated Delta Isc, unlike the response in physiological KRB medium. In contrast, the Delta Isc response to UTP was not diminished by removal of Na+ from the luminal bath (Fig. 2A) or by treatment of the luminal membrane with Na+ channel blocker amiloride (10 µM; for amiloride plus UTP, Delta Isc = 35.1 ± 8.2 µA/cm2; for UTP alone, Delta Isc = 32.9 ± 6.5 µA/cm2; n = 3). Recent studies of the murine gallbladder have shown that most of the response to cAMP stimulation results from electrogenic bicarbonate secretion (34). To investigate whether the UTP-induced anion current is largely carried by HCO-3 rather than Cl-, we pretreated MGEP monolayers with bumetanide, an inhibitor of Na+-K+-2Cl- cotransport (22), for 5 min before the addition of 10-4 M UTP. Bumetanide (10-4 M) did not significantly reduce the UTP-induced Delta Isc (for bumetanide plus UTP, Delta Isc = 30.2 ± 7.7 µA/cm2; for UTP alone, Delta Isc = 32.6 ± 4.6 µA/cm2, n = 3). This finding is consistent with a recent report that UTP stimulates electrogenic bicarbonate secretion across the gallbladder from CFTR knockout mice (25).


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Fig. 2.   Nucleotide agonist-Isc response potency profiles for murine gallbladder epithelial (MGEP) cells. Confluent monolayers were mounted in Ussing chambers and, unless otherwise noted, were bathed in standard KRB solution. A: ion substitution and inhibitor studies. MGEP cell monolayers were bathed with either KRB (n = 7), Ringer solution without permeant anions (Cl- + HCO-3 free; n = 5), KRB without Na+ in the luminal bath [Na+ free (L); n = 5], KRB containing 1 mM DIDS in the luminal bath [KRB + DIDS (L); n = 5], or KRB containing 200 µM glibenclamide in the luminal bath [KRB + Glib (L); n = 3] and then treated with 10-4 M UTP in the luminal solution. Bars, mean Delta Isc values ± SE. * P < 0.05 vs. KRB with UTP (ANOVA-protected Dunnett's t-test). B: potency profile for agonists added to the luminal membrane. Symbols, mean Delta Isc values ± SE for 4-7 monolayers at each concentration. 2-MeSATP, 2-methylthio-ATP. C: potency profile for agonists added to the basolateral membrane. Symbols, mean Delta Isc values ± SE for 4-7 monolayers at each concentration. Dotted lines, EC50 estimates for agonists (see text).

Previous studies of murine airway epithelia (i.e., nasal and tracheal) have shown that ATP or UTP activates a luminal membrane (Ca2+ mediated) anion conductance that is distinct from CFTR and that is inhibited by the anion channel blocker DIDS (2, 9). To examine the possibility that this conductance mediates anion secretion across MGEP cell monolayers, the Isc response to UTP was measured after the addition of DIDS (300 µM) to the luminal solution. As shown in Fig. 2A, treatment with DIDS significantly inhibited the UTP-stimulated Isc response by ~60% in the MGEP cell monolayers. In contrast, when the monolayers were pretreated with glibenclamide (200 µM), a compound that blocks CFTR channel activity (46), the UTP-stimulated Delta Isc was not affected. This finding is consistent with a recent report that the anion secretory response to UTP is not decreased in the CFTR knockout murine gallbladder epithelium (23).

P2 receptor agonist potency profiles in MGEP cells. Concentration-effect studies with nucleotide receptor agonists were used to investigate the location of P2 receptor subtypes on the luminal and basolateral membranes of polarized MGEP cell monolayers. As shown in Fig. 2B, the concentration-effect curve for luminal membrane treatment with UTP was similar to the curve generated with the presumed endogenous ligand, ATP. The EC50 values were 1.9 × 10-6 M for UTP and 1.3 × 10-6 M for ATP. The equal potencies and efficacies of UTP and ATP are unique to the P2Y2 receptor among the cloned mammalian P2 receptors (16, 56). The ATP analog 2-methylthio-ATP (2-MeSATP) also caused a significant increase in the Isc response from the luminal side but was less potent (EC50 = 5.1 × 10-5 M) and less efficacious than either ATP or UTP. The analog alpha ,beta -methylene-ATP (alpha ,beta -methATP) stimulated the Isc only at very high agonist concentrations from the luminal side (Delta Isc at 10-4 M = 2.5 ± 1.4 µA/cm2; Delta Isc at 10-3 M = 19.1 ± 7.7 µA/cm2; n = 3).

The concentration-effect curves for P2 receptor agonists added to the basolateral bath of MGEP cell cultures are shown in Fig. 2C. Basolateral ATP was significantly less potent (EC50 = 2.1 × 10-5 M) and efficacious than luminal ATP for generation of an Isc response. Basolateral 2-MeSATP had a potency (EC50 = 3.9 × 10-5 M) similar to that of basolateral ATP but was less efficacious. In contrast to luminal treatment, basolateral UTP addition stimulated very little change in the Isc response except at 1 mM concentration. Basolateral treatment with alpha ,beta -methATP did not produce a change in the Isc response at concentrations as high as 10-3 M.

RT-PCR of P2 receptor mRNA. RT-PCR was used to identify P2Y receptor subtype-specific mRNA in MGEP cells; 1321N1 astrocytoma cells expressing recombinant P2Y receptors served as positive controls. Primers were designed according to the sequences of cDNA for the P2Y receptor subtypes: P2Y1, P2Y2, P2Y4, and P2Y6 (13, 16, 17, 36, 40, 56). As shown in Fig. 3, primers specific for the P2Y2 receptor amplified PCR products whose identities were verified by DNA sequencing and comparison to the sequence of the cloned murine P2Y2 receptor cDNA (n = 3). Although not apparent in the gel shown in Fig. 3, faint bands of approximately the correct size for P2Y1 and P2Y6 were occasionally detected in the MGEP cells. However, these bands could not be consistently reproduced from RNA taken at successive passages of the MGEP cell line, and the quantity of PCR product was insufficient for sequence analysis.


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Fig. 3.   Detection of P2Y receptor mRNA in MGEP cells by RT-PCR. Total RNA (1 µg) from MGEP cells was reverse transcribed, and resulting cDNA was used to detect expression of mRNA for the P2Y1, P2Y2, P2Y4, and P2Y6 receptors by PCR. Human 1321N1 astrocytoma cells expressing the recombinant P2 receptors (turkey P2Y1, mouse P2Y2, human P2Y4, and rat P2Y6) were used as positive controls. PCR products were resolved on 1.0% (wt/vol) agarose gels containing 10 µg/ml ethidium bromide and photographed under ultraviolet illumination. The identities of the PCR products were verified by DNA sequencing. Primers specific for glyceraldehyde-3-phosphate dehydrogenase were used as reaction controls. Reactions without RT were used to control for DNA contamination of the samples.

Desensitization of the Anion Secretory Response to UTP in MGEP Cells

The preceding studies suggest that the P2Y2 receptor activity in MGEP cell monolayers is predominantly associated with the luminal membrane. To examine whether the Isc response of MGEP cell monolayers also desensitizes with UTP treatment, we performed Ussing chamber studies on polarized MGEP cells grown on permeable collagen supports. As shown by the experiment depicted in Fig. 4A, 10-4 M UTP stimulated an increase in Isc, which rapidly attained a maximal value and then decreased toward baseline over 10 min. Subsequent washout of the UTP followed by a second addition of 10-4 M UTP resulted in a peak Isc response that was reduced by 43% relative to the initial Isc response (n = 7). The plateau phase of the Isc response measured at 1 min after the peak Isc response was also reduced by 44% (initial UTP Delta Isc = 6.3 ± 0.8 µA/cm2; rechallenge UTP Delta Isc = 2.8 ± 0.6 µA/cm2). Note, however, that the Isc profile of the UTP response differs from that for the freshly excised mucosal preparations (compare with Fig. 1). Compared with results for the excised mucosa, the UTP-induced Isc response in the cultured preparations is rapid and the magnitude of the plateau phase is reduced. These differences may be due to the release of paracrine mediators from nonepithelial cells in the mucosal preparations, which could prolong the Isc response. However, it is unlikely that these mediators would act by stimulating cAMP-dependent anion secretion (i.e., CFTR) because we have observed similar UTP-induced Isc profiles in CFTR knockout murine gallbladders (unpublished observation). Alternatively, the presence of EGF in the culture medium may suppress the Isc by increasing the intracellular concentration of the D-inositol 3,4,5,6-tetrakisphosphate, an endogenous inhibitor of Ca2+-mediated Cl- secretion (54).


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Fig. 4.   Desensitization of UTP-stimulated anion secretion in MGEP cells. MGEP cell monolayers were mounted in Ussing chambers and bathed with a standard KRB solution. A: continuous Isc trace demonstrating desensitization of Isc response to UTP. UTP (10-4 M) was added to luminal bathing solution (arrows). UTP was removed from luminal bath by repeated solution changes (wash). Upward deflections in Isc trace (spikes) result from repetitive 2-mV pulses used to estimate transepithelial resistance. B: time course of Isc response desensitization. MGEP cell monolayers were exposed to UTP (10-4 M) in the luminal bath for times indicated, then washed and re-treated with UTP (10-4 M) after 5 min. Responses are mean percent of the preincubation Delta Isc values ± SE; n = 5. Mean preincubation Delta Isc values: 26.5 ± 1.9 µA/cm2 at 5 min; 27.8 ± 2.6 µA/cm2 at 10 min; 33.4 ± 3.2 µA/cm2 at 30 min; and 30.6 ± 1.7 µA/cm2 at 60 min. * P < 0.05 vs. time 0 (ANOVA-protected Dunnett's t-test). C: noncumulative concentration-effect curve for UTP-induced desensitization of Isc response. MGEP cells were preincubated with indicated concentrations of UTP in luminal bath for 10 min. UTP was removed by multiple solution changes, and MGEP cells were reexposed to equimolar concentration of UTP after 5 min. Dotted line, IC50 for desensitization (0.8 × 10-6 M). Responses are mean percent of the preincubation Delta Isc values ± SE; n = 3-7 at each concentration. Mean preincubation Delta Isc values for UTP concentrations were 0.9 ± 0.6 µA/cm2 at 10-8 M (n = 4), 6.3 ± 1.1 µA/cm2 at 10-7 M (n = 4), 15.6 ± 1.8 µA/cm2 at 10-6 M (n = 4), 30.0 ± 3.1 µA/cm2 at 10-5 M (n = 6), 35.9 ± 4.7 µA/cm2 at 10-4 M (n = 6), and 32.7 ± 2.9 µA/cm2 at 10-3 M (n = 7). D: effect of UTP concentration on recovery of UTP-stimulated Isc response. MGEP cells were preincubated with indicated concentrations of UTP in luminal bath for 10 min. UTP was removed by multiple solution changes, and monolayers were allowed to recover for times indicated before reexposure to equimolar concentration of UTP. Responses are mean percent of the preincubation Delta Isc values ± SE; n = 3-7 at each concentration. Mean preincubation Delta Isc values for UTP concentrations were 16.7 ± 1.5 µA/cm2 at 10-6 M (n = 11), 27.0 ± 2.4 µA/cm2 at 10-5 M (n = 14), 32.7 ± 2.2 µA/cm2 at 10-4 M (n = 16), and 30.3 ± 3.1 µA/cm2 at 10-3 M (n = 17). * P < 0.05 vs. preincubation Delta Isc (ANOVA-protected Dunn's test).

Time course. The time course of homologous desensitization in MGEP cell monolayers was investigated by measuring the maximal Isc response to luminal 10-4 M UTP after the preincubation of the cells with 10-4 M UTP for 5, 10, 30, or 60 min. As shown in Fig. 4B, maximal desensitization of UTP-stimulated Delta Isc occurred after a 10-min preincubation with 10-4 M UTP. Preincubation periods longer than 10 min did not further alter the magnitude of desensitization.

Concentration dependence. The effect of UTP preincubation concentration on the magnitude of Isc desensitization was examined by exposing the cell monolayers to various concentrations of UTP for 10 min and, after washing the luminal surface with several volumes of KRB, measuring the Isc response to an equimolar concentration of UTP. As shown in Fig. 4C, homologous desensitization of the Isc response to UTP occurred in a concentration-dependent manner with an IC50 (0.8 × 10-6 M) that was approximately equal to the EC50 for activation of the Isc response. Maximal desensitization occurred at preincubation concentrations of UTP >10-4 M, but approximately 40% of the total Isc response to UTP remained, even at the highest UTP concentrations.

Recovery. It was investigated whether the preincubation concentration of UTP affected the time required for recovery of the Isc response from desensitization. MGEP cell monolayers were preincubated with various concentrations of UTP for 10 min, then allowed 10, 90, or 180 min to recover before treatment with an equimolar concentration of UTP. As shown in Fig. 4D, the UTP-induced Isc response completely recovered within 90 min of pretreatment with 10-6 M and 10-5 M UTP. However, a recovery time of >90 min was required with higher UTP concentrations (10-4 and 10-3 M).

Desensitization of the Effector and Signaling Pathway for UTP-Induced Anion Secretion in MGEP Cells

In epithelial tissues, anion secretion is stimulated by increasing the activity of plasma membrane ion transporters, e.g., Ca2+-activated apical membrane anion channels and basolateral K+ channels (2, 15). The signaling pathway from the P2Y receptor to the Ca2+-activated ion channels involves the generation of the second-messenger inositol 1,4,5-trisphosphate and the mobilization of intracellular free Ca2+ (5, 35). To determine whether UTP-induced desensitization of anion secretion involves desensitization of both the transport mechanism and the P2Y2 receptor signaling pathway, we investigated the effect of UTP pretreatment on three steps in the pathway, i.e., the Ca2+-activated anion conductance/transport mechanism, Ca2+ mobilization, and IP generation.

Ca2+-mediated anion secretion. To investigate the possibility that homologous desensitization of the UTP-induced Isc response occurs at the level of the Ca2+-activated anion transport mechanism, UTP-pretreated MGEP cells were exposed to the Ca2+ ionophore ionomycin to increase [Ca2+]i independent of receptor activation. A concentration-response study indicated that 10-5 M ionomycin produced a maximal Isc response in MGEP cells (10-8-10-4 M ionomycin was tested). As shown in Fig. 5A, preincubation of MGEP cells with 10-4 M UTP had no effect on the Isc response to 10-5 M ionomycin. In contrast, the UTP preincubation desensitized the subsequent Isc response to 10-4 M UTP. Although these experiments demonstrated that the anion transport mechanism per se was fully functional, we also tested a lower concentration of ionomycin (10-8 M) to assess whether desensitization occurs between [Ca2+]i mobilization and activation of anion secretion. Interestingly, preincubation with 10-4 M UTP reduced the subsequent Isc response to the 10-8 M ionomycin, indicating that a component of desensitization occurs distal to the Delta [Ca2+]i response (for UTP preincubation, Delta Isc = 11.0 ± 0.9 µA/cm2; for no preincubation, Delta Isc = 20.5 ± 2.9 µA/cm2, n = 3).


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Fig. 5.   UTP-induced desensitization of effector and signaling pathways in MGEP cells. A: desensitization of Ca2+-mediated anion secretion. MGEP cells were mounted in Ussing chambers and bathed with KRB solution. Monolayers were preincubated with UTP (10-4 M) or vehicle (Veh, KRB) in luminal bath for 10 min, washed, and treated with either UTP (10-4 M) or ionomycin (Iono, 10-5 M) after 5 min. Responses are mean Delta Isc values ± SE after UTP or ionomycin treatment; n = 6. Bars, mean Delta Isc values ± SE. * P < 0.05 vs. preincubation Delta Isc (paired t-test). B: noncumulative concentration-effect curve for UTP-induced desensitization of intracellular Ca2+ mobilization. MGEP cells were preincubated with indicated concentrations of UTP in luminal bath for 10 min before measurement of intracellular Ca2+ concentration ([Ca2+]i) as described in MATERIALS AND METHODS. UTP was removed and washed cells were exposed to equimolar concentration of UTP after 5 min. Responses are values of mean % of the Delta [Ca2+]i induced by UTP preincubation ± SE; n = 3. Dotted line, IC50 for desensitization (0.7 × 10-6 M UTP). Mean preincubation Delta [Ca2+]i values for UTP concentrations were 8.7 ± 4.3 nM at 10-8 M, 26.7 ± 0.7 nM at 10-7 M, 371.0 ± 29.1 nM at 10-6 M, 588.7 ± 64.3 nM at 10-5 M, 679.1 ± 25.0 nM at 10-4 M, and 745.3 ± 58.5 nM at 10-3 M. Inset, noncumulative concentration-effect curve for UTP-induced Ca2+ mobilization. Responses are means ± SE (n = 6) for indicated concentrations. EC50 was estimated at 1.1 × 10-6 M UTP. C: UTP-induced desensitization of total inositol phosphate (IP) production. MGEP cells were labeled overnight with [3H]inositol in inositol-free DMEM. Preincubation conditions for [3H]inositol-treated MGEP cells: a and b, medium alone; c and d, 10-4 M UTP in the luminal bath for 15 min. UTP was removed, and washed cells in a and c were not treated; cells in b and d were exposed after 5 min in medium to 2 × 10-6 M UTP for 15 min. Responses are means of total IP released (in dpm) ± SE; n = 3. Inset, noncumulative concentration-effect curve for UTP-induced production of total IP. Responses are mean dpm ± SE (n = 3) for indicated concentrations. EC50 was estimated at 2 × 10-6 M UTP.

Ca2+ mobilization. As shown in Fig. 5B, inset, UTP increased Ca2+ mobilization in MGEP cells, with an EC50 (1.1 × 10-6 M) similar to the effective concentration of UTP for induction of the Isc response (see Fig. 2B). To determine whether the UTP-stimulated increase of [Ca2+]i in MGEP cells desensitizes similarly to the Isc response, [Ca2+]i was measured during a desensitization protocol similar to the one used in the Isc studies, i.e., preincubation with various concentrations of UTP for 10 min, a washing of the cells, and rechallenge with an equimolar UTP concentration. As shown in Fig. 5B, homologous desensitization of the calcium response to UTP occurred in a concentration-dependent manner with an IC50 (0.7 × 10-6 M UTP) nearly identical to the IC50 for homologous desensitization of the Isc response (0.8 × 10-6 M). Note also that approximately 35% of the [Ca2+]i response remained at the highest UTP concentrations, which compares favorably with the residual Delta Isc response at the maximal UTP doses (~40%; see Fig. 4C).

IP production. As shown in Fig. 5C, inset, UTP stimulates total IP production in a concentration-dependent manner (EC50 = 2.1 × 10-6 M). To determine whether desensitization occurs at the level of IP generation, MGEP cells were preincubated with 10-4 M UTP (a concentration shown to induce maximal desensitization of UTP-stimulated anion secretion; see Fig. 4C), then rechallenged with 2 × 10-6 M UTP. As shown in Fig. 5C, stimulation of total IP production by UTP in MGEP cells was greatly attenuated (89.4%) by prior exposure to 10-4 M UTP. In separate experiments when MGEP cells were rechallenged with 10-4 M UTP, stimulation of total IP production was reduced 52.8% by preincubation with 10-4 M UTP (for preincubation, stimulation of IP production was 1,201 ± 112 dpm; for rechallenge, it was 567 ± 214 dpm, n = 3). The residual total IP production (47.2%) after rechallenge with 10-4 M UTP also compares favorably with the residual Delta [Ca2+]i and Delta Isc after 10-4 M UTP rechallenge.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The regulation of anion secretion by P2Y2 receptors is particularly relevant to secretagogue therapy of CF epithelial disease. The loss of CFTR Cl- channels in CF patients results in altered ion composition and reduced volume of epithelial salt and water secretion (8, 29). The discovery that an alternative anion conductance, i.e., the Ca2+-activated anion conductance, is expressed in some epithelial tissues and can be stimulated by topical application of the P2Y2 receptor agonist UTP has provided a therapeutic strategy to overcome the CF-related deficiency in transepithelial anion secretion (4, 30). However, one shortcoming of this approach is that anion secretion stimulated by UTP is transient (<1 h) (7, 30) and may require repeated application of the agonist to be therapeutically useful. It is apparent from this study that homologous desensitization of the anion secretory response occurs with repeated exposure of an epithelial tissue to UTP. Therefore, an understanding of the P2Y2 receptor desensitization process will be necessary to advance the use of extracellular UTP for the treatment of CF.

The anion secretory responses elicited by UTP in freshly excised murine gallbladder were reproduced in cell lines developed by transformation of primary gallbladder cell isolates. After transformation, the MGEP cells were continuously cultured on permeable collagen filters in an effort to maintain the phenotypic characteristics of a polarized gallbladder epithelium. We have subsequently found in >90 passages that the basal Isc and Rt of MGEP cell monolayers fall within a narrow range. Similar to what occurs with a freshly excised gallbladder epithelium, UTP stimulated an anion secretory response in MGEP cells that desensitized with repeated addition of the agonist. Furthermore, the MGEP cells demonstrated a P2 receptor agonist potency profile consistent with the agonist profile in primary cultures of human airway epithelia (35). Thus the MGEP cell line appears to be a useful model for investigating P2 receptor regulation of epithelial ion transport.

The present study specifically investigated the desensitization of UTP-stimulated transepithelial anion secretion. In polarized epithelia, transepithelial anion secretion stimulated by luminal UTP typically involves the activation of a non-CFTR anion conductance in the luminal membrane and a K+ conductance in the basolateral membrane (which maintains membrane potential) (7). These events are dependent on intracellular Ca2+ mobilization, which may result in covalent modification of the transporter proteins, e.g., via phosphorylation (44), and which may contribute to the desensitization process. However, the transport mechanism mediating anion secretion in MGEP cells responded fully to ionophore-induced increases in [Ca2+]i after preincubation with a desensitizing concentration of UTP. This observation suggests that the transport proteins per se stimulated by UTP-induced Ca2+ mobilization either do not desensitize or undergo rapid resensitization. In contrast, UTP pretreatment caused desensitization of subsequent UTP-stimulated anion secretion with a concentration dependence that mirrored the desensitization of UTP-stimulated Ca2+ mobilization. The desensitization process in MGEP cells was further correlated with the desensitization of UTP-stimulated total IP generation (Fig. 5C). In previous studies of airway epithelial cells, Brown et al. (5) have also shown that total IP generation desensitizes with repeated UTP exposure. The time course and IC50 for UTP desensitization reported in that study agree remarkably well with the desensitization parameters for UTP-stimulated Isc responses measured in the MGEP cells. Together, these findings indicate that a major component of the desensitization of UTP-stimulated transepithelial anion secretion occurs at the level of receptor activation. Recent studies of the recombinant P2Y2 receptor have shown that desensitization and sequestration of the receptor are likely due to phosphorylation of the COOH terminus by G protein-coupled receptor kinases (19). In addition to detecting receptor desensitization, experiments using 10-8 M ionomycin after UTP preincubation detected another component of desensitization that occurs between the increase in Ca2+ mobilization and activation of the anion transport proteins. This finding is consistent with recent studies by Ho et al. (27) and others, which have shown that D-inositol 3,4,5,6-tetrakisphosphate generated during receptor-mediated Ca2+ mobilization produces long-term inhibition of Ca2+-activated Cl- secretion through a pathway independent and probably downstream of changes in [Ca2+]i (27, 54).

Similar to findings with other polarized epithelia (35, 52), the agonist potency profiles of the Isc response in MGEP cells indicated that UTP was equipotent to ATP when added to the luminal solution. This characteristic potency profile has been a principal means of P2Y2 receptor identification (16, 40) because radioligand binding assays, selective antagonists, and specific antibodies for P2Y receptor subtypes have been difficult to develop (58). In the present study, receptor subtype identification was facilitated by RT-PCR-based detection of P2 receptor mRNA (39, 53). This assay detected the presence of P2Y2 receptor mRNA, which was subsequently verified by sequencing the PCR product. Thus the evidence favors the conclusion that luminal UTP activates the P2Y2 receptor to stimulate net anion secretion across the MGEP epithelium. These results are in agreement with findings obtained with polarized epithelial cell lines from other tissues, including lung (35), kidney (18, 63), pancreatic duct (6), sweat gland (31), and salivary gland (52, 53).

Regulation of the epithelial P2Y2 receptor appears to be consistent with that for other G protein-coupled receptors linked to phospholipase C (32, 49, 51, 55). The desensitization process for these receptors is modeled after that of the beta -adrenergic receptor (26, 45) and involves acute desensitization, sequestration, and the longer-term process of downregulation (19). Although the present study focused on acute agonist desensitization, it was found that the recovery of UTP-stimulated anion secretion at high UTP pretreatment concentrations (>10-5 M) was slow, being incomplete after 90 min. A similar phenomenon has been previously noted in studies of anion secretion across airway epithelia (28) and Madin-Darby canine kidney cells (60) (the latter study did not involve UTP activation of a Ca2+-activated anion conductance). These observations suggest that a significant fraction of epithelial cell P2Y2 receptors are internalized when the apical membrane is exposed to high UTP concentrations. Therefore, optimal secretagogue therapy for CF may require either a restricted concentration range of UTP or adjunct treatment targeting the desensitization pathway. However, 35-40% of the UTP-stimulated Delta [Ca2+]i and Delta Isc remained even at high UTP concentrations. Thus either a subset of P2Y2 receptors or a different P2 receptor subtype in MGEP cells is apparently refractory to UTP-induced desensitization. This fraction of the epithelial cell P2 receptor complement may be sufficient to yield therapeutic benefit in the nucleotide treatment of CF disease.


    ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Michael Lethem (Brighton Univ., Brighton, UK) for the immunohistochemical detection of the SV40-LT antigen in the MGEP cell line and Dr. T. Kendall Harden for the provision of the turkey P2Y1 and rat P2Y6 receptor cDNAs. We also acknowledge the expert technical assistance of Amy Norcom, Jean M. Camden, and Charles Jorgensen.


    FOOTNOTES

Financial support for this work was provided by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48816 (L. L. Clarke), National Institute of Dental Research Grant DE-07389 (J. T. Turner and G. A. Weisman), National Institute of General Medical Sciences Grant GM-36887 (G. A. Weisman), the Minority Biomedical Research Support-National Institutes of Health Program (F. A. González), the University of Missouri-Columbia Food for the 21st Century Program (G. A. Weisman), and the Cystic Fibrosis Foundation (G. A. Weisman, L. L. Clarke, and J. T. Turner).

R. C. Garrad is a Cystic Fibrosis Foundation Postdoctoral Fellow. F. A. González is an Alfred P. Sloan Research Fellow.

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

Address for reprint requests and other correspondence: L. L. Clarke, 324D Dalton Cardiovascular Research Center, Research Park, Univ. of Missouri-Columbia, Columbia, MO 65211 (E-mail: clarkel{at}missouri.edu).

Received 1 April 1998; accepted in final form 17 December 1998.


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DISCUSSION
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