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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ABSTRACT |
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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 >>
,
-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
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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INTRODUCTION |
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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 -adrenergic receptor (26). The mechanisms of desensitization of the
-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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MATERIALS AND METHODS |
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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 ClAfter 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 asWhere 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
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 ![]() |
RESULTS |
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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
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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 ofSimilar 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
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
Isc, unlike
the response in physiological KRB medium. In contrast, the
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,
Isc = 35.1 ± 8.2 µA/cm2; for UTP alone,
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
Isc (for
bumetanide plus UTP,
Isc = 30.2 ± 7.7 µA/cm2; for UTP alone,
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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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
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 × 106 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
,
-methylene-ATP (
,
-methATP) stimulated the
Isc only at very
high agonist concentrations from the luminal side
(
Isc at
10
4 M = 2.5 ± 1.4 µA/cm2;
Isc at
10
3 M = 19.1 ± 7.7 µA/cm2;
n = 3).
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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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
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Time course.
The time course of homologous desensitization in MGEP cell monolayers
was investigated by measuring the maximal
Isc response to
luminal 104 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
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 × 106 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
106 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 105 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
[Ca2+]i
response (for UTP preincubation,
Isc = 11.0 ± 0.9 µA/cm2; for no
preincubation,
Isc = 20.5 ± 2.9 µA/cm2,
n = 3).
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Ca2+
mobilization.
As shown in Fig. 5B,
inset, UTP increased
Ca2+ mobilization in MGEP cells,
with an EC50 (1.1 × 106 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
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 × 106 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
[Ca2+]i
and
Isc after
10
4 M UTP rechallenge.
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DISCUSSION |
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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
108 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 -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
[Ca2+]i
and
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.
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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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