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Am J Physiol Gastrointest Liver Physiol 288: G779-G786, 2005. First published November 4, 2004; doi:10.1152/ajpgi.00325.2004
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LIVER AND BILIARY TRACT

Purinergic regulation of cholangiocyte secretion: identification of a novel role for P2X receptors

R. Brian Doctor,1 Thomas Matzakos,1 Ryan McWilliams,1 Sylene Johnson,1 Andrew P. Feranchak,2 and J. Gregory Fitz2

1University of Colorado Health Sciences Center, Denver, CO 80262 and 2University of Texas Southwestern Medical Center, Dallas, Texas

Submitted 21 July 2004 ; accepted in final form 28 October 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The P2X family of ligand-gated cation channels is comprised of seven distinct isoforms activated by binding of extracellular purines. Although originally identified in neurons, there is increasing evidence for expression of P2X receptors in epithelia as well. Because ATP is released by both hepatocytes and cholangiocytes, these studies were performed to evaluate whether P2X receptors are present in cholangiocytes and contribute to local regulation of biliary secretion and bile formation. RT-PCR of cDNA from cultured normal rat cholangiocytes detected transcripts for P2X receptors 2, 3, 4, and 6; products from P2X3 and P2X4 were robust and always detectable. In cholangiocyte lysates, P2X4 protein was readily detected, and immunohistochemical staining of intact rat liver revealed P2X4 protein concentrated in intrahepatic bile ducts. To assess the functional significance of P2X4, isolated Mz-ChA-1 cells were exposed to the P2X4-preferring agonist 2',3'-O-(4-benzoyl-benzoyl)-ATP (BzATP), which activated inward currents of –18.2 + 3.0 pA/pF. In cholangiocyte monolayers, BzATP but not P2X3 agonists elicited robust Cl– secretory responses (short-circuit current) when applied to either the apical ({Delta}Isc 22.1 ± 3.3 µA) or basolateral (18.5 ± 1.6 µA) chamber, with half-maximal stimulation at ~10 µM and ~1 µM, respectively. The response to BzATP was unaffected by suramin (not significant) and was inhibited by Cu2+ (P < 0.01). These studies provide molecular and biochemical evidence for the presence of P2X receptors in cholangiocytes. Functional studies indicate that P2X4 is likely the primary isoform involved, representing a novel and functionally important component of the purinergic signaling complex modulating biliary secretion.

liver; bile formation; adenosine 5'-triphosphate; cholangiocyte

ATP IS RELEASED INTO BILE by both hepatocytes and cholangiocytes where it functions as a potent autocrine/paracrine stimulus for cholangiocyte secretion (18, 19). These effects are mediated by activation of purinergic receptors in the plasma membrane. Under basal conditions, extracellular ATP concentrations are low. However, increases in cell volume, activation of protein kinase C, and stimulation of exocytosis increase membrane ATP permeability (10). Once in bile, ATP has direct access to the cholangiocyte apical membrane, and even low concentrations of ATP (half-maximal stimulation near 300 nM) are sufficient to increase intracellular Ca2+ concentration [Ca2+]i and activate membrane Cl– and K+ channels (22). The resulting transepithelial transport of Cl– contributes importantly to alkalinization and dilution of bile (7). Thus definition of the proteins involved in purinergic signaling offers attractive options for development of new therapeutic agents capable of modifying the volume and composition of bile.

Most attention has been focused on P2Y receptors as the prototype effector pathway responsible for Ca2+-dependent secretory responses (7, 22). Agonist (ATP) binding to these G protein-coupled receptors stimulates phospholipase C, generates inositol-1,4,5-trisphosphate, and releases calcium from intracellular stores. In liver, P2Y2 receptors represent the best characterized member of this family. cDNAs, mRNAs and protein corresponding to P2Y2 receptors are readily detectable in cholangiocytes, and the P2Y2-preferring agonist UTP effectively stimulates cholangiocyte secretion, consistent with an important physiological role for these receptors (7, 22).

Several observations suggest that additional P2 receptor types also contribute to the cholangiocyte response to ATP. Notably, ATP is detectable in human bile in concentrations of ~1.5 µM, substantially above those required for half-maximal activation of P2Y receptors (4). Furthermore, in intact tissue, the secretory response to ATP is sustained, whereas P2Y2 receptors typically show rapid desensitization in the continued presence of agonist (21). Finally, the observed pharmacological properties of the apical vs. basolateral domains differ, and are not readily explained by a single dominate population of P2Y2 receptors (22).

The concept that there may be coexpression of multiple purinergic receptors within a single cell type is supported by the identification of multiple P2 receptor transcripts in other cholangiocyte models, including the novel finding of partial transcripts corresponding to P2X receptors (7, 25). P2X receptors are molecularly distinct from the P2Y family and function as ligand-gated, calcium-permeable cation channels. cDNAs encoding seven members of this family (referred to as P2X1–7) have been identified in neuronal cells, and their properties have been reviewed in detail (17). P2X receptors have homologies to the epithelial sodium channel and encode a protein containing only two transmembrane domains. The amino- and COOH termini are thought to be located intracellularly, and a larger extracellular loop comprises an ATP binding domain. P2X receptors function in vivo as homo- or heterotrimers or hexamers, and activation by higher concentrations of ATP leads to opening of a calcium-permeable nonselective cation pore and depolarization of postsynaptic membranes (17).

The expression and potential physiological roles of the different P2X receptor subtypes in epithelia is not clear. Using 2',3'-O-(4-benzoyl-benzoyl)-ATP (BzATP) as a P2X-preferring agonist, Taylor et al. (25) demonstrated in intestinal T84 cells that receptor activation is associated with Cl– secretion. Transcripts encoding different P2X receptor types are also detectable in other epithelial models, supporting the concept that P2X receptors might function in parallel with P2Y2 receptors to mediate more sustained responses. Because P2X4 appears to be present in ductular epithelia (2), we evaluated the hypothesis that P2X receptors are present in cholangiocytes and function in parallel with P2Y2 receptors as a pathway for ATP-dependent regulation of cholangiocyte secretion and ductular bile formation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell models. Patch-clamp studies were performed by using isolated Mz-Cha-1 cells, originally isolated from human adenocarcinoma of the gallbladder (14). These exhibit phenotypic features of differentiated biliary epithelium and are amenable to single cell recording techniques. In addition, when cultured as described, they have been utilized as models for biliary ATP release, ATP degradation, and purinergic signaling (9, 18).

Because Mz-ChA-1 cells do not form high-resistance monolayers in culture, additional molecular and Ussing chamber studies were performed utilizing normal rat cholangiocytes (NRC) isolated from intrahepatic bile ducts (26). These cells express phenotypic features of differentiated biliary epithelium including receptors, signaling pathways, and ion channels similar to those found in primary cells (18, 21). Moreover, increases in [Ca2+]i are followed by opening of membrane K+ and Cl– channels, producing an increase in short-circuit current (Isc) (see below). NRC monolayers were cultured on rat-tail collagen slabs as previously described (21) and passaged onto collagen-coated semipermeable (24-mm diameter, 0.4-µm pore) Costar transwell supports (Corning) 7–10 days before all electrophysiological and molecular studies. This protocol permits highly polarized cells, the development of a high transepithelial resistance (Rt > 1,000 {Omega}·cm2), and net apical Cl– secretion (18, 21).

Reagents. ATP, suramin, BzATP, and other ATP analogs were obtained from Sigma (St. Louis, MO). ATP at a concentration of 100 µM was utilized as a nonselective agonist to maximally activate both P2X and P2Y receptors. BzATP (0.1–100 µM) was utilized as a P2X-selective agonist (25, 27). In heterologous expression studies, BzATP causes half-maximal activation of P2X4 and P2X7 receptors at concentrations of 1–10 µM (12, 17, 25); it is a weak agonist for other P2X and P2Y receptors. {alpha}{beta}-Methylene-ATP is an agonist for P2X3 but not P2X4 receptors (17).

Detection of P2X receptor RNAs by RT-PCR. Total RNA was extracted from homogenized NRC (26), isolated Rattus norvegicus hepatocytes, and whole kidney and liver preparations using RNeasy mini kits (Qiagen, Valencia, CA). Independent sense and antisense primers were designed from known cloned receptor sequences, available in GenBank, for the P2X1 and -2 receptors. Previously published (23) primer sequences were used for the remaining receptors. Primer sequences and lengths of the expected PCR products are shown in Table 1. PCR conditions were 94°C for 2 min, 35 cycles (94°C for 30 s, 58°C for 30 s, 72°C for 60 s) and 72°C for 7 min. Amplification products were separated by gel electrophoresis (1.5% agarose) and visualized with ethidium bromide staining. PCR products were extracted by using a QIAEX II Gel Extaction Kit (Qiagen) and sequenced by using an automated sequencer to confirm their identities.


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  		Table 1. P2X receptor primers for RT-PCR

 
Immunoblotting. Cells and tissues were lysed in 5x PAGE buffer [5% (wt/vol) SDS, 25% (wt/vol) sucrose, 5 mM EDTA, 50 mM Tris·HCl, at pH 8.0] with 5% {beta}-mercaptoethanol and the resultant proteins were run on 3.5–14.5% SDS-PAGE gels and transferred onto nitrocellulose paper (6). Briefly, after blots were blocked with 5% nonfat milk, they were labeled with primary antibody (P2X1–2, -4, -6, -7 from Alomone Labs, Jerusalem, Israel; P2X3 from Chemicon International, Temecula, CA), washed, probed with species-specific horseradish peroxidase-conjugated anti-IgG, and developed with SuperSignal West Pico Luminol/Enhancer solution (Pierce Chemical, Rockford, IL). Developed blots were visualized by using an Epichemi3 Darkroom system (UVP Bioimaging Systems, Upland, CA).

Immunohistochemistry. Rat liver sections were permeabilized with 0.1% Tween in PBS and blocked (10% fetal calf serum, 0.25% glycine in PBS) for 30 min. Blocked samples were incubated in primary antibody (1:100, prepared in blocking solution) for 60 min. After washing, samples were incubated in Alexa Flour 546 goat anti-rabbit IgG (Molecular Probes, Eugene, OR) for 30 min, followed by 4,6-diamidino-2-phenylindole for nuclear staining. Sections were viewed by using an inverted Olympus IX70 microscope. Images were refined by using Deltavision digital deconvolution.

Whole cell currents. Whole cell currents were measured by using patch-clamp recording techniques (11) as previously described (9). Studies were performed at room temperature (22–25°C) 24–48 h after plating of cells on 35-mm collagen-covered plates. The standard extracellular solution contained (in mM): 140 NaCl, 4 KCl, 1 KH2PO4, 2 MgCl2, 1 CaCl2, 5 glucose, and 10 HEPES/NaOH (pH 7.3). The standard pipette (intracellular) solution contained (in mM) 130 KCl, 10 NaCl, 2 MgCl2, 10 HEPES/KOH (pH 7.3) and ~100 nM calculated free [Ca2+]. The low divalent cation extracellular solution was similar to the standard solution except 0 mM MgCl2 and 0.5 mM CaCl2. With these solutions, activation of a cation conductance results in inward currents at the holding potential of –40 mV (cation equilibrium potential +1 mV). Cells were viewed through an inverted phase contrast microscope using Hoffman optics at a magnification of x600 (Olympus IMT-2). Patch pipettes were pulled from Corning 7052 glass and had resistances of 3–6 M{Omega}. Recordings were made with an Axopatch IC amplifier (Axon Instruments, Foster City, CA), and signals were filtered at 2 kHz bandwidth using a four-pole low-pass Butterworth filter. Currents were analyzed by using pClamp software (version 6.0 Axon Instruments, Foster City, CA). Results are compared with control studies measured on the same day to minimize any effects of day-to-day variability and reported as current density (pA/pF) to normalize for differences in cell size.

Ussing chamber analysis. NRC cells were utilized to study the role of P2X receptors in regulation of transepithelial Cl– secretion (18, 20). Cells were grown to confluence on collagen-treated polycarbonate filters with a pore size of 0.4 µm (Costar, Cambridge, MA) until resistance was >1,000 {Omega}·cm2 (EVOHM; World Precision Instruments, Sarasota, FL) (21). Cells were mounted in a Trans-24 mini-perfusion system for tissue culture cups (Jim's Instrument Manufacturing, Iowa City, Iowa). All experiments were carried out at 37°C, and basolateral and apical (luminal) sides were bubbled with O2 through air-lift circulators. The standard extracellular buffer solution contained (in mM) 140 NaCl, 4 KCl, 1 KH2PO4, 2 MgCl2, 1 CaCl2, 5 glucose, and 10 HEPES/NaOH (pH 7.3). Transepithelial voltage was clamped to 0 mV, and Isc was recorded through agar bridges (3% agar in 1 M KCl) connected to Ag-AgCl electrodes (cartridge electrodes, World Precision Instruments). The Isc is the net sum of electrogenic ion movement from the basolateral to the apical chamber, and hence, a reflection of transepithelial secretion (18). Experimental results were compared with control studies performed on the same day to minimize any potential effects of day-to-day variability in current amplitude.

Statistics. Results are presented as means ± SE, with n representing the number of culture plates or repetitions for each assay as indicated. Student's paired or unpaired t-test was used to assess statistical significance as indicated, and P values < 0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cholangiocytes express multiple P2X receptor isoform transcripts. To evaluate whether transcripts for P2X receptors are present in cholangiocytes, cDNA from NRC cells was probed by RT-PCR using primers specific for P2X1 through P2X7 (Table 1). All studies were performed under identical culture conditions using cells from passage 15–25 and capable of forming differentiated, polarized monolayers. In Fig. 1, the positive controls from renal and brain tissues (bottom) show amplified products of appropriate size. In a representative study of NRC cell RNA, clear bands corresponding to P2X3 and P2X4 are present, as well as a faint band corresponding to P2X6. In seven separate studies, P2X3 and P2X4 were always present (7 of 7) and produced robust RT-PCR bands. P2X1 was identified as a faint band in 1 of 7 studies, P2X2 was detected in 4 of 7 studies, and P2X6 was detected in 2 of 7 studies. P2X5 and P2X7 were not detected over a broad range of conditions in the presence of positive controls.



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  		Fig. 1. Identification of P2X receptor transcripts in normal rat cholangiocytes (NRC). RT-PCR was utilized to probe cDNA from normal rat cholangiocytes. Controls are from rat kidney (P2X1) and rat brain (P2X2–7). In the example shown, representative products of the appropriate size were detected for P2X3 and P2X4 receptors, and a faint band corresponding to the P2X6 receptor was also detected.

 
Identification of P2X4 receptor protein in cholangiocytes. Expression of P2X receptor proteins in NRC cells was performed by Western blot analysis using isoform-specific antibodies. Rat brain lysates served as a positive control; and rat liver lysates (~80% composed of hepatocytes) were evaluated for comparison (Fig. 2). P2X4 was readily detected in NRC cell lysates and showed a higher level of expression per milligram protein than either the brain or liver samples. The predicted molecular mass of P2X4 is ~46 kDa, but it is detected biochemically as a broader band or doublet of 60–70 kDa due to glycosylation (27). P2X2 and P2X6 also were observed over multiple studies. P2X7 was never detected despite positive controls. The antibody for P2X3 was uninformative (failure to specifically detect protein in positive controls) over a range of conditions.



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  		Fig. 2. Immunoblotting of P2X receptor protein in liver cells. Rabbit polyclonal antibodies to P2X receptors were utilized to probe proteins from rat brain, intact rat liver, and NRC. In NRC, P2X4 protein was detected as a band of 60–70 kDa; P2X2 and P2X6 were also detectable with lower signals. P2X7 was not detected despite positive controls. Note the comparatively high levels of P2X4 protein in cholangiocytes compared with whole brain and liver tissues.

 
Immunolocalization of P2X4. On the basis of the presence of P2X4 RNA and protein in cholangiocytes, rat liver sections were evaluated by immunohistochemical staining with the same P2X4 antibody to evaluate expression and localization of P2X4 in native bile duct epithelial cells. Within the intact liver, P2X4 was observed in liver parenchymal cells but had markedly higher signals within ductular epithelial cells (Fig. 3, top, arrows). Higher magnification showed that P2X4 was distributed along both the apical and basolateral membrane domains (Fig. 3, bottom, left). These findings confirm that P2X4 receptor protein is present in native cholangiocytes, and suggest that it is distributed in both the apical and basolateral domains of intrahepatic bile duct epithelial cells.



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  		Fig. 3. Immunolocalization of P2X4 receptors to intrahepatic bile ducts. Immunohistochemical staining of sections from intact rat liver are shown; green corresponds to P2X4 and blue corresponds to nuclear staining with 4,6-diamidino-2-phenylindole. Top: image at low magnification (bar = 10 µ) represents a liver lobule from the central vein (CV) to portal tract (PT). P2X4 was most prominent in bile duct cells (arrow). Bottom: higher magnification views of the same duct (bar = 5 µ) are shown with (left) and without (right) the P2X4 antibody. P2X4 was distributed along both the apical and basolateral membrane domains.

 
Functional role for P2X receptors in regulation of cholangiocyte secretion. Among the different P2X receptors detected by RT-PCR, BzATP is a weak agonist for P2X1, P2X2, P2X3, and P2X6, but a potent agonist for P2X4 (12, 17). It is also an agonist for P2X7, but these receptors were not identifiable in cholangiocytes by either RT-PCR or Western blot analysis. In contrast, {alpha}{beta}-methylene-ATP is a potent agonist for P2X3 but not P2X4 receptors (12, 17). To evaluate the potential functional role of P2X receptors, in initial studies whole cell recordings were performed by using isolated Mz-ChA-1 cells to evaluate the biophysical response to BzATP. This approach permits detection of the initial transient inward currents through the cation pore too small for detection in intact monolayers. Under basal conditions, resting current density was –1.4 ± 0.3 pA/pF at a holding potential of –40 mV (n = 8). Exposure to BzATP (10 µM) stimulated an inward current that activated nearly instantaneously, peaked at –18.2 ± 3.0 pA/pF (n = 4, P < 0.01), and then decreased within seconds to a lower, more sustained plateau (Fig. 4). The amplitude of the peak response increased to –28.8 ± 5.6 pA/pF (n = 4) in low divalent cation solution, features consistent with opening by BzATP of a nonselective cation pore.



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  		Fig. 4. Activation of inward currents by 2',3'-O-(4-benzoyl-benzoyl)-ATP (BzATP). A: imunoblotting of proteins with antibodies to P2X4 shows that Mz-ChA-1 cell lysates also contain P2X4 immunoreactivity. The smaller band of the doublet predominated. B: whole cell currents were measured in Mz-ChA-1 cells using patch-clamp techniques at a holding potential of –40 mV. Exposure to BzATP activated inward currents that peaked nearly instantaneously and then decreased to a lower plateau. Peak response was increased in solutions with a low divalent cation concentration. C: average current density (pA/pF) under basal conditions (n = 8) and after exposure to BzATP (10 µM) in control (n = 4) and low divalent cation (n = 4) solutions.

 
The sustained increase in conductance caused by BzATP in single cells had properties of Ca2+-activated Cl– channels as recently identified in biliary cells (8). In cholangiocyte monolayers, activation of these channels leads to transepithelial Cl– secretion as measured by an increase in Isc (18). Because Mz-ChA-1 cells do not form differentiated monolayers, additional studies were performed by using NRCs to assess whether P2X receptors might be functionally important in cholangiocyte secretion. In NRC cells in monolayer culture, basal transepithelial resistance averaged 2,264 ± 189 {Omega}·cm2; transepithelial voltage was –14.8 ± 1.4 mV (lumen negative); and basal Isc at a voltage clamp potential of 0 mV was 33.3 ± 1.6 µA (n = 23). Effects of secretory agonists are reported as {Delta}Isc, reflecting changes from basal values. The P2X4-preferring agonist BzATP was a potent agonist of secretion, whether applied to the apical or basolateral domain (Fig. 5, top). In the example shown, basal Isc was 28 µA. Selective exposure of the basolateral membrane to BzATP (100 µM) caused a rapid initial {Delta}Isc of ~22 µA, followed by a sustained secretory response of ~10 µA. The sustained response in the continued presence of agonist (e.g., lack of desensitization) persisted for >10 min in all studies. Subsequent exposure to the nonselective purinergic agonist ATP (100 µM) caused an additional increase with a transient peak and a sustained increase in {Delta}Isc to ~15 µA above initial levels.



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  		Fig. 5. Activation of Cl– secretion by apical and basolateral P2X4 agonists. Top: representative recording of short-circuit current (Isc) from NRC cells mounted in an Ussing chamber. Addition of BzATP to the basolateral chamber caused a rapid increase in Isc, followed by a longer sustained plateau above basal levels. Subsequent exposure to the nonselective P2 agonist ATP caused a further increase in Isc. Concentration dependence of the peak response ({Delta}Isc) to BzATP when added selectively to the apical (middle) or basolateral (lower) chamber are shown [n ≥ 4 at each concentration (M)].

 
The peak secretory responses to BzATP applied to the apical or basolateral chambers are shown in Fig. 5, middle and bottom, respectively (n = 5–14 at each concentration). The peak {Delta}Isc responses were produced at BzATP concentrations of ≥10–4 M (100 µM), and values were similar when applied selectively to either the apical (22.1 ± 3.3 µA, n = 14, P < 0.001) or the basolateral membrane (18.5 ± 1.6 µA, n = 5, P < 0.001). In each case, peak values were not markedly different from those produced by subsequent exposure to 100 µM ATP. The concentration of BzATP required for half-maximal responses was ~10 µM when applied to the apical domain and ~1 µM when applied to the basolateral domain.

The secretory response exhibited several features anticipated for activation of P2X4 receptors. First, BzATP is a potent agonist for P2X4 but not other receptors detected in these cells. Second, suramin is an effective inhibitor of P2X3 and other family members but not P2X4 (17). In the presence of high concentrations of suramin (100 µM), BzATP (100 µM) still elicited a robust secretory response. In the experiments shown in Fig. 6A, the response to BzATP (100 µM) was measured in the absence (n = 5) vs. the presence of suramin (n = 7) on the same study days and no inhibition was observed (not significant). Third, the response to BzATP was inhibited by Cu2+, which has been shown to inhibit P2X4 receptors with an IC50 of ~9 µM (5). After activation by BzATP, exposure to Cu2+ (100 µM) inhibited currents by >90% within 3 min (n = 6, P < 0.01; Fig. 6B). Similar results were obtained when Cu2+ was added before exposure to BzATP (data not shown). Finally, exposure to the P2X3-preferring agonist {alpha}{beta}-methylene-ATP elicited comparatively small responses (Fig. 7). After initial activation of currents by BzATP, subsequent exposure to {alpha}{beta}-methylene-ATP (100 µM) had no additional effect (Fig. 7A). Alternatively, initial exposure to high concentrations of {alpha}{beta}-methylene-ATP caused a comparatively small increase in Isc, but failed to influence the response to subsequent BzATP. A representative recording and averaged data are shown in Fig. 7B (n = 5–7). Collectively, these biophysical features including activation by BzATP, slow desensitization, insensitivity to suramin, and inhibition by Cu2+ strongly support the concept that P2X4 receptors are prominent contributors to purinergic regulation of Cl– secretion in bile duct epithelia.



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  		Fig. 6. Properties of BzATP-induced secretory response. The change in Isc was measured as described in Fig. 5. A: response to BzATP (100 µM) was measured in the absence ({bullet}) vs. presence ({circ}) of suramin, which failed to inhibit the response (not significant). B: Isc was first stimulated by exposure to BzATP (100 µM) in all studies. After 10 min, exposure to CuCl2 ({circ}, 100 µM) inhibited the response compared with control monolayers ({bullet}, P < 0.01). Data presented as means ± SE for 5–7 monolayers.

 


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  		Fig. 7. Properties of BzATP-induced secretory response. To evaluate the potential role of P2X3 receptors, the relative effects of the P2X3-preferring agonist {alpha}{beta}-methylene-ATP were compared with those of the P2X4-preferring agonist BzATP at equal concentrations of 100 µM to maximally stimulate secretory responses. A: after activation of Isc by BzATP, subsequent exposure to {alpha}{beta}-methylene-ATP after 10 min failed to cause any further increase in Isc. B: representative recording and summary data are shown. Initial exposure to high concentrations of {alpha}{beta}-methylene-ATP produced a reproducible but comparatively small increase in Isc compared with that produced by subsequent exposure to BzATP. Data presented as means ± SE for 5–7 monolayers.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
ATP is present in bile and functions as an autocrine/paracrine factor that contributes to regulation of biliary cell secretion and volume. Previous studies (7, 18, 22) indicate that these effects are mediated, in part, by activation of P2Y2 receptors. The present studies provide molecular and biophysical evidence that P2X receptors are also present and are functionally important in cholangiocytes, and indicate that P2X4 is one of the major isoforms involved.

P2X4 receptors were originally identified in brain tissues where they were found to have a unique pharmacological profile including activation by ATP and ATP-{gamma}-S; little or no affinity for 2-methylthio-ATP, ADP, and {alpha}{beta}-methylene-ATP; and relative insensitivity to the purinoceptor antagonist suramin (3). Unlike P2X3 and other P2X family members, P2X4 receptors comparatively slow desensitization in the continued presence of agonist (27). With the use of a monoclonal antibody to the ectodomain of P2X4, monomers have a molecular mass of ~60 kDa (2). In addition to the widespread distribution of receptors in brain tissues, immunoreactivity is also detectable more broadly in tracheal, parotid, and intestinal epithelial cells; and in the epithelium lining salivary, pancreatic, and intrahepatic bile ducts (2). Despite this broad distribution, there is less evidence of a clear functional role for P2X4 receptor proteins in epithelia.

In previous studies, exposure of cholangiocytes to ATP has been shown to release calcium from intracellular stores. This response is mediated, in part, by P2Y2 receptors through a G protein-dependent mechanism and is reproduced by UTP in concentrations that fail to activate P2X receptors (7, 18). However, multiple additional P2Y family members including P2Y1, P2Y2, and P2Y6 also appear to be present, leading to a complex pharmacological signature in intact tissues that is not easily reconciled with a single dominant pathway. Recently, mRNAs corresponding to P2X receptors were identified in biliary cells, suggesting an alternative pathway for purinergic regulation of cholangiocyte secretion (7, 25). However, exposure of cholangiocytes to P2X-preferring agonists 2-methylthio-ATP, {alpha}{beta}-methylene-ATP, and BzATP failed to produce measurable increases in [Ca2+]i (7). Notably, in many other cell types, agonist-induced calcium influx through P2X4 is only detectable in Na+-free medium due to competition for Na+ and Ca2+ through the P2X pore (1, 10); and the secretory response to BzATP is sustained even in the absence of detectable increases in [Ca2+]i (27). Thus the lack of a detectable increase in bulk cytosolic [Ca2+] does not exclude a contribution of local increases in calcium concentration through the P2X4 pore.

In the present studies, several observations provide support for the concept that P2X4 receptors are present, are functionally important, and represent the dominant P2X isoform responsible for regulation of secretion and ductular bile formation. First, P2X4 RNA was detected by RT-PCR in 7 of 7 studies. Second, a protein of appropriate molecular mass was identified in cholangiocyte lysates. Second, the same protein in lesser amounts was also detected in whole liver and isolated hepatocytes (data not shown), suggesting a broader role for these receptors in other hepatic functions as well. Third, immunohistochemical studies identified P2X4-specific protein in intrahepatic bile ducts of normal rat liver. Finally, the P2X4 receptor agonist BzATP stimulated inward currents in isolated cells and a robust secretory response if applied to the apical or the basolateral surface of cells in monolayer culture. The properties of this response, including half-maximal stimulation at concentrations of 1–10 µM, slow desensitization, insensitivity to suramin, and inhibition by Cu2+, are consistent with both endogenous P2X4 receptors and heterologous expression models (12, 25, 27). Collectively, these findings provide the first evidence for a functional role for P2X receptors in bile formation, localize P2X4 receptors to both the apical and basolateral membrane domains, and suggest that P2X4 is the primary isoform involved.

Several potential limitations and qualifications of these studies merit emphasis. First, it should be emphasized that other P2X isoforms are also expressed, but their functional roles are difficult to ascertain, due to the lack of highly specific agonists and antagonists. At the RNA level, transcripts for P2X3 and P2X4 were present in all studies; P2X1, P2X2, and P2X6 were present in some studies; and P2X5 and P2X7 were never detected. At the protein level, however, only P2X4 and P2X6 were reliably detected. This is relevant to the interpretation of the response to BzATP, because it is also an agonist for P2X7 receptors (12). In the absence of detectable P2X7 expression, however, the simplest explanation is that the response is mediated by P2X4 proteins. Similarly, despite positive RT-PCR results, Western blot analysis for P2X3 protein was not informative (failure to detect positive controls). However, BzATP is a weak agonist for P2X3 receptors (17), these receptors typically desensitize over milliseconds in the continued presence of agonist, and the P2X3-preferring agonist {alpha}{beta}-methylene-ATP elicited comparatively small secretory responses (13). It is important to emphasize that even lower levels of expression of other P2X isoforms could have important biological effects. There is considerable diversity in the ionic currents attributable to P2X receptors, presumably due to both intrinsic differences in the different isoforms but also to their ability to associate with other P2X receptor isoforms (2). P2X4 and P2X6 subunits, for example, can form heterooligomers resulting in different functional properties (13, 15). Thus even low levels of coexpression of P2X6 or other isoforms could modify the regulatory or conductive responses to agonists.

Second, the present studies utilized electrophysiological measurement of Isc as a surrogate measure of transepithelial secretion. Although this approach is consistent with the prevailing concept that secretion is driven by transepithelial Cl– transport (24), fluid secretion was not directly assessed, and confirmation will require more direct measurements in isolated bile duct units or other models as they become available.

Third, there is increasing evidence for considerable heterogeneity between small and large cholangiocytes and between different regions within the intrahepatic biliary tree (16). Given that these cells appear to express P2Y2, P2X4, and other purinergic receptors, it will be important to define the cellular strategies behind the presence of multiple ATP effector pathways. The distinctive functional properties of P2X4 vs. P2Y2 receptors provide certain clues, such as, higher agonist concentrations are required for P2X4, they exhibit slower desensitization, and they mediate calcium influx and not intracellular release. However, a more complete understanding will require definition of whether there is heterogeneity among cells in different regions of the biliary tree, and whether cholangiocytes have the capacity to regulate the type and number of receptors in response to changing physiological demands. In neurons, for example, P2X4 is rapidly internalized after agonist binding (17).

Taken together, these findings indicate that P2X4 receptors represent a novel and functionally important component of the purinergic signaling complex in cholangiocytes. On the basis of the sustained secretory response to BzATP, it is attractive to speculate that the P2X4 receptors in the apical membrane, which are accessible through bile, are well suited as targets for pharmacological therapy aiming to modulate the volume and composition of bile. Thus it will be important to clarify the distinct roles of P2X and P2Y2 receptors, and to determine the cellular strategies responsible for trafficking, localization, and plasma membrane retention of these receptors under physiological conditions and during cholestasis, gallstone formation, and ischemia where ductular function is likely to be impaired.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported, in part, by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46082 (to J. G. Fitz), DK-43278 (to J. G. Fitz), DK-34039 (to R. B. Doctor), DK-57729 (to R. B. Doctor), and DK-61480 (to A. P. Feranchak).


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Fitz, UT-Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390 (E-mail:greg.fitz{at}utsouthwestern.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Baricordi OR, Ferrari D, Melchiorri L, Chiozzi P, Hanau S, Chiari E, Rubini M, and Di Virgilio F. An ATP-activated channel is involved in mitogenic stimulation of human T lymphocytes. Blood 87: 682–690, 1996.[Abstract/Free Full Text]
  2. Bo X, Kim M, Nori SL, Schoepfer R, Burnstock G, and North RA. Tissue distribution of P2X4 receptors studied with an ectodomain antibody. Cell Tissue Res 313: 159–165, 2003.[CrossRef][ISI][Medline]
  3. Bo X, Zhang Y, Nassar M, Burnstock G, and Schoepfer R. A P2X purinoceptor cDNA conferring a novel pharmacological profile. FEBS Lett 375: 129–133, 1995.[CrossRef][ISI][Medline]
  4. Chari RS, Schutz SM, Haebig JA, Shimokura GH, Cotton PB, Fitz JG, and Meyers WC. Adenosine nucleotides in bile. Am J Physiol Gastrointest Liver Physiol 270: G246–G252, 1996.[Abstract/Free Full Text]
  5. Coddou C, Morales B, Gonzalez J, Grauso M, Gordillo F, Bull P, Rassendren F, and Huidobro-Toro JP. Histidine 140 plays a key role in the inhibitory modulation of the P2X4 nucleotide receptor by copper but not zinc. J Biol Chem 278: 36777–36785, 2003.[Abstract/Free Full Text]
  6. Doctor RB, Bennett V, and Mandel LJ. Degradation of spectrin and ankyrin in the ischemic rat kidney. Am J Physiol Cell Physiol 264: C1003–C1013, 1993.[Abstract/Free Full Text]
  7. Dranoff JA, Masyuk AI, Kruglov EA, LaRusso NF, and Nathanson MH. Polarized expression and function of P2Y ATP receptors in rat bile duct epithelia. Am J Physiol Gastrointest Liver Physiol 281: G1059–G1067, 2001.[Abstract/Free Full Text]
  8. Feranchak AP, Doctor RB, Troetsch M, Brookman K, Johnson SM, and Fitz JG. Calcium-dependent regulation of secretion in biliary epithelial cells: the role of apamin-sensitive SK channels. Gastroenterology 127: 903–913, 2004.
  9. Feranchak AP, Roman RM, Doctor RB, Salter KD, Toker A, and Fitz JG. The lipid products of phosphoinositide 3-kinase contribute to regulation of cholangiocyte ATP and chloride transport. J Biol Chem 274: 30979–30986, 1999.[Abstract/Free Full Text]
  10. Gatof D, Kilic G, and Fitz JG. Vesicular exocytosis contributes to volume-sensitive ATP release in biliary cells. Am J Physiol Gastrointest Liver Physiol 286: G538–G546, 2004.[Abstract/Free Full Text]
  11. Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch clamp techniques for high resolution recording from cells and cell-free membrane patches. Pflügers Arch 391: 85–100, 1981.[CrossRef][ISI][Medline]
  12. He ML, Gonzalez-Iglesias AE, and Stojilkovic SS. Role of nucleotide P2 receptors in calcium signaling and prolactin release in pituitary lactotrophs. J Biol Chem 278: 46270–46277, 2003.[Abstract/Free Full Text]
  13. Khakh BS, Bao XR, Labarca C, and Lester HA. Neuronal P2X transmitter-gated cation channels change their ion selectivity in seconds. Nat Neurosci 2: 322–330, 1999.[CrossRef][ISI][Medline]
  14. Knuth A, Gabbert H, Dippold W, Klein O, Sachsse W, Bitter-Suermann D, Prellwitz W, and Meyer zum Buschenfelde KH. Biliary adenocarcinoma characterization of three new human tumor cell lines. J Hepatol 1: 579–596, 1985.[CrossRef][ISI][Medline]
  15. Le KT, Babinski K, and Seguela P. Central P2X4 and P2X6 channel subunits coassemble into a novel heteromeric ATP receptor. J Neurosci 18: 7152–7159, 1998.[Abstract/Free Full Text]
  16. Marzioni M, Glaser SS, Francis H, Phinizy JL, LeSage G, and Alpini G. Functional heterogeneity of cholangiocytes. Semin Liver Dis 22: 227–240, 2002.[CrossRef][ISI][Medline]
  17. North RA. Molecular physiology of P2X receptors. Physiol Rev 82: 1013–1067, 2002.[Abstract/Free Full Text]
  18. Roman RM, Feranchak AP, Salter KD, Wang Y, and Fitz JG. Endogenous ATP release regulates Cl– secretion in cultured human and rat biliary epithelial cells. Am J Physiol Gastrointest Liver Physiol 276: G1391–G1400, 1999.[Abstract/Free Full Text]
  19. Roman RM and Fitz JG. Emerging roles of purinergic signaling in gastrointestinal epithelial secretion and hepatobiliary function. Gastroenterology 116: 964–979, 1999.[CrossRef][ISI][Medline]
  20. Salter KD, Fitz JG, and Roman RM. Domain-specific purinergic signaling in polarized rat cholangiocytes. Am J Physiol Gastrointest Liver Physiol 278: G492–G500, 2000.[Abstract/Free Full Text]
  21. Salter KD, Roman RM, LaRusso NR, Fitz JG, and Doctor RB. Modified culture conditions enhance expression of differentiated phenotypic properties of normal rat cholangiocytes. Lab Invest 80: 1775–1778, 2000.
  22. Schlenker T, Romac JMJ, Sharara A, Roman RM, Kim S, LaRusso N, Liddle R, and Fitz JG. Regulation of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes. Am J Physiol Gastrointest Liver Physiol 273: G1108–G1117, 1997.[Abstract/Free Full Text]
  23. Shibuya I, Tanaka K, Hattori Y, Uezono Y, Harayama N, Noguchi J, Ueta Y, Izumi F, and Yamashita H. Evidence that multiple P2X purinoceptors are functionally expressed in rat supraoptic neurones. J Physiol 51: 351–367, 1999.
  24. Singh SK, Mennone A, Gigliozzi A, Fraioli F, and Boyer JL. Cl–-dependent secretory mechanisms in isolated rat bile duct epithelial units. Am J Physiol Gastrointest Liver Physiol 281: G438–G446, 2001.[Abstract/Free Full Text]
  25. Taylor AL, Schwiebert LM, Smith JJ, King C, Jones JR, Sorscher EJ, and Schwiebert EM. Epithelial P2X purinergic receptor channel expression and function. J Clin Invest 104: 875–884, 1999.[ISI][Medline]
  26. Vroman B and LaRusso N. Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells. Lab Invest 74: 303–313, 1996.[ISI]
  27. Zsembery A, Boyce AT, Liang L, Peti-Peterdi J, Bell PD, and Schwiebert EM. Sustained calcium entry through P2X nucleotide receptor channels in human airway epithelial cells. J Biol Chem 278: 133398–133408, 2003.



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