Research ArticleExperimental Studies

Extracellular ATP Induces Cytoplasmic and Nuclear Ca2+ Transients via P2Y2 Receptor in Human Biliary Epithelial Cancer Cells (Mz-Cha-1)

CHRISTOPH ELSING, TIHOMIR GEORGIEV, CHRISTIAN A. HÜBNER, REGINA BOGER, WOLFGANG STREMMEL and THORSTEN SCHLENKER
Anticancer Research September 2012, 32 (9) 3759-3767;

Abstract

Extracellular nucleotides such as adenosine triphosphate (ATP) play a role in biliary epithelial cell function. Since nucleotide receptors are potential targets for various diseases related to epithelial cell dysfunction and cancer, the purpose of this study was to investigate the expression and to functionally characterize the nucleotide receptor subtypes in biliary epithelial cancer cells (Mz-Cha-1). Extracellular ATP dose-dependently resulted in an intracellular Ca2+ increase (mean effective concentration (EC50) 40 μM). Uridine triphosphate (UTP) produced a similar Ca2+ response and cross-desensitation was observed. The rank order of tested agonists was ATP=UTP>> adenosine>ADP=AMP>α,β-methylene-ATP. This confirms the functional expression of purinoceptor P2Y2 and P2Y4 in biliary epithelial cancer cell membranes. mRNAs for P2Y1, P2Y2, P2Y4 and P2Y6 purinergic receptor subtypes were found, whereas western blot analysis suggested only the expression of P2Y2 receptors. Confocal imaging and nuclear staining was used to compartmentalize ATP-induced cytosolic and nuclear Ca2+-transients, indicating a role for secretory ATP in regulating nuclear function, by increasing nuclear Ca2+ concentrations. These data define the expression profile of P2Y receptors on human biliary epithelial cancer cells and indicate P2Y2 receptors as being potential targets in new treatment strategies for biliary cancer.

Biliary epithelial cells contribute significantly to the production and the final bile composition, by secretion and absorption of fluids and electrolytes mediated by ion channels, transporters and ectoenzymes (1). Extracellular ATP and its derivatives have been demonstrated to be present in bile and it is thought that they play a central role in autocrine and paracrine regulation of biliary epithelial cell function (2-6). Extracellular ATP elicits its effect on biliary epithelial cells (7) via the activation of purinergic receptors P2X and P2Y. P2X receptors and their role in cholangiocyte secretion have been recently demonstrated (5). Several data have demonstrated a role for P2Y2 receptor subtypes (8) in different models of biliary epithelial cells (2, 9, 10). ATP binding to these receptors stimulates phospholipase C, which generates inositol-1,4,5-triphosphate (IP3). IP3 then triggers calcium release from intracellular stores via its receptors (IP3Rs). Although IP3R is also found on nuclear envelopes of different cell types (11), the regulation of extracellular ATP-induced nuclear Ca2+ signalling in cholangiocytes is poorly understood. Confocal microscopy and adequate dyes are now able to compartmentalize nuclear [Ca2+]i in intact cells (12) and to detect rapid changes upon agonist stimulation.

Cholangiocellular carcinoma (CCC) is the second most common type of primary liver cancer with increasing incidence (13). Chronic inflammation and hereditary biliary malformations are established risk factors (14).

Extracellular ATP is thought to be a signalling molecule that may serve as a danger signal to alert the immune system of tissue damage and therewith inflammation (15) and possibly takes part in promoting or preventing malignant transformation (16). Although purinergic-signalling-related proteins have been used in diagnostics as tumor markers and the therapeutic potential of purines or purine analogues is exploited (16), study of the precise role of purinergic receptor signalling in biliary epithelial cancer cells is warranted. We therefore investigated the purinergic receptors, the receptor-mediated calcium liberation process and the involved calcium stores in a biliary cancer cell line, Mz-Cha-1.

Materials and Methods

Materials. Adenosine-5’-triphosphate (ATP), uridine-5’-triphosphate (UTP) and trypsin were obtained from Boehringer Mannheim, (Mannheim, Germany). The calcium chelating agent ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetra acetic acid (EGTA), NiCl2, verapamil hydrochloride, nifedipine, ryanodine, glycyl-L-phenylalanine-β-naphthylamide (GPN), p-trifluoromethoxy-phenylhydrazone (FCCP), oligomycin, thapsigargin, 2,5-di-(t-butyl)-hydroquinone (tBuBHQ), adenosine-5’-diphosphate (ADP), the P2X preferring agonist α,β-methylene-ATP, adenosine-5’-monophosphate (AMP), adenosine, N6,2’-O-dibutyryladenosine-3’,5’-cyclic-monophosphate (db-cAMP), the Ca2+ ionophore ionomycin, and pertussis toxin (PTX) were purchased form Sigma Chemical Co. (Munich, Germany). Fluo-3 and Fluo-4 acetoxymethyl ester and Pluronic F 127 were purchased from Molecular Probes Inc. (Eugene, OR, USA). All other chemicals used were from different sources and of the highest available grade.

Cell culture. The human biliary epithelial cancer cell line Mz-ChA-1 was used for the experiments. Mz-ChA-1 cells are derived from a human adenocarcinoma of the biliary tree and were kindly provided by Dr. Knuth (17). Cells were maintained in culture at 37°C in a 5% carbon dioxide incubator in hydrogen carbonate-containing Dulbecco's modified Eagle's medium (DMEM; Gibco, BRL, Berlin, Germany) supplemented with 10% heat-inactivated fetal calf serum (FCS; Boehringer Mannheim), 100 IU/ml penicillin and 100 μg/ml streptomycin (Biochrom KG, Berlin, Germany).

At the time of the experiments (50-60% confluent cell monolayer), the medium was removed and the cells were washed with incubation buffer containing 135 mmol/l NaCl, 5 mmol/l KCl, 0.8 mmol/l MgSO4, 1.2 mmol/l CaCl2, 0.8 mmol/l Na2HPO4, 10 mmol/l HEPES (pH 7.4), and 5 mmol/l glucose.

Measurement of cellular [Ca2+]i. For intracellular [Ca2+]i measurement, cells attached on glass coverslips were loaded with the Ca2+-sensitive fluorescent dye fluo-3 acetoxymethyl ester for 60 min at 37°C in DMEM, containing 10% FCS and 0.05% Pluronic F-127 to enhance loading efficiency. As Fluo-3 cannot be ratio imaged, it does not allow the absolute [Ca2+]i concentration to be determined. Coverslips were placed in culture dishes and mounted on the stage of a confocal laser scanning microscope (Zeiss, LSM-310, Carl Zeiss, Jena, Germany)). Cells were superfused continuously with a HEPES buffer balanced salt solution at pH 7.4 at 37°C, containing 135 mM NaCl, 5 mM KCl, 0.8 mM Mg2SO4, 1.2 mM CaCl2, 0.8 mM Na2HPO4, 10 mM HEPES and 5 mM glucose. Cells were excited with the internal 488 nm argon laser. Emitted light was recorded using 525-565 nm band pass filter. Images were obtained at 1 or 5 s intervals using the frame mode of the system. Data from subsequent scans were expressed as changes in fluorescence intensity over time.

To characterise [Ca2+]i transients after stimulation of purinergic receptors, different concentrations of various agonists were added to the superfusion buffer. The role of extracellular Ca2+ was assessed by exposing Mz-ChA-1 cells to ATP in Ca2+-free buffer supplemented with the calcium chelator EGTA 2 mM. Physiological extracellular Ca2+ concentrations were subsequently reconstituted by the addition of 2 mM CaCl2.

The effect of inhibition of voltage-dependent Ca2+ channels on [Ca2+]i transients was investigated after preincubation of cells with verapamil (10 μM) and nifedipine (20 μM). Possibly involved 2Na+/Ca2+ exchange was inhibited by NiCl2 (5 mM). The Ca2+ ionophor ionomycin was solubilized in ethanol and subsequently used at 1 μM for depletion of intracellular Ca2+ stores in Ca2+-free buffer.

Detection of simultaneous nuclear and cytosolic calcium transients. Cells grown on coverslips were loaded with the calcium-sensitive dye fluo-4 AM (2 μM; 7-10 min) and the cell-permeable nuclear indicator ethidium bromide (Vol% 1/500; 50 min), as described above. The coverslips were placed on the stage of a laser scanning microscope (Olympus I×70, Hamburg, Germany) and were continuously superfused with the extracellular buffer (described above). Cells were excited at 488 nm with an internal laser (argon/krypton laser, Omnichrome ©643R-OLYM-AO3; Melles Griot, Inc., Helium Neon Lasers, Carlsbad, CA, USA). Emission was recorded using a 510-550 nm band pass filter. Ethidium bromide was detected on a second photomultiplier using a high pass filter (>585 nm) and data were transferred to a computer. Images were obtained every 2.5 s using the frame mode of the system. Data were analyzed using ImageJ (NIH, Bethesda, Maryland, USA). A small part of the ethidium bromide signal passed the 510-550 band pass filter. Since this signal was however unaffected by Ca2+, changes in Ca2+ concentrations were therefore not recorded.

RNA isolation. RNA was isolated from confluent MZ-ChA1 cultures the with TRIZOL reagent (Life Technologies Inc., Grand Island, N.Y., USA), according to the manufacturer's instructions.

Reverse transcriptase polymerase chain reaction (RT-PCR) for evaluation of P2Y subtype mRNA expression. For cDNA synthesis 2 μg of total RNA were used as a template for first-strand synthesis in a 20-μl reaction volume containing 5 nmol dNTP, 10 pmol random hexamers, 200 units SUPERSCRIPT TMII (Life Technologies Inc., Grand Island, NY, USA), 50 mM Tris HCl (pH 8.3), 75 mM KCl, 3 mM MGCl2 and 10 mM dithiothreitol in diethyl pyrocarbonate-treated distilled and deionized water. The reaction was incubated for 50 min at 42°C and stopped by heating to 70°C for 15 min. Using a Perkin-Elmer GeneAmp PCR System 2400 (Perkin-Elmer Corporation, CT, USA.) the following PCR was carried out: 2 μl of the cDNA preparation, 10 μM NTP, 20 mM Tris HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 2 units rTaq DNA polymerase (LifeTechnologies Inc.) and 10 pM of sense and antisense primer were used in a 50-μl reaction mix. The conditions for denaturation, annealing and extension were as follows: after 94°C for 3 min, 3 cycles at 94°C for 30 s, 62°C for 30 s, 72°C for 2 min and subsequently 35 cycles at 94°C for 30 s, 60°C for 30 s, 72°C for 1 min 30 s, followed by a 10-min incubation at 72°C. The PCR products were seperated by electrophoresis on a 1.5% agarose gel, stained with ethidium bromide. The specifity of the amplified products was tested by sequencing.

To verify that the amplified products were from mRNA, controls with total RNA were also performed. The primer oligonucleotides of the different P2Y genes were selected and used as previously described (18).

The primers for P2Y1 were: sense 5’-TGTGGTGTACCCCCTCAAGTCCC-3’, antisense 5’-ATCCGTAACAGCCCAGAATCAGCA-3’ (260 bp); for P2Y2: sense 5’-CCAGGCCCCCGTGCTCTACTTTG-3’, antisense 5’-CATGTTGATGGCGTTGAGGGTGTG-3’ (367 bp); for P2Y4: sense 5’-CGTCTTCTCGCCTCCGCTCTCT-3’, antisense 5’-GCCCTGCACTCATCCCCTTTTCT-3’ (433 bp); for P2Y6: sense 5’-CCGCTGAACATCTGTGTC-3’ antisense 5’-AGAGCCATGCCATAGGGGC-3’ (464 bp). Glyceraldehyde-3-phosphate dehydrogenase was used as a housekeping gene (sense 5’-GGTCGGAGTCAACGGATTTGGTCG-3’: antisense 5’-CCTCCGACGCCTGCTTCACCAC-3’), yielding a 782-bp product.

Western blot analysis. Protein was extracted from cultured Mz-Cha-1 cells using Daub lysis buffer: 50 mM Hepes (pH 7.5), 150 mM NaCl, 1% Triton ×100, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml aprotinin (19). Cell lysates were centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was collected and the protein concentration was determined by the Bradford Protein Assay (Bio-Rad, Munich, Germany). Equal amounts of protein (40 μg) were then separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and were transferred to nitrocellulose membranes. Immunodetection was performed using primary rabbit antibodies against P2Y1, P2Y2, P2Y4, P2Y6 (Alomone Laboratories, Jerusalem, Israel). Rat brain was used as a positive control. Peroxidase-conjugated species-specific secondary antibodies (Santa Cruz Biotechnology, Heidelberg, Germany) were used at a dilution of 1:10,000. Bound antibodies were visualized using chemiluminiscent substrate (Perkin Elmer, Zaventem, Belgium) and exposure to Amersham Hyperfilm ECL.

Results

[Ca2+]i transients in biliary epithelial cells stimulated by ATP. Compared to the total cell number, the proportion of responding biliary epithelial cancer cells increased with ATP concentrations between 1 and 200 μM. The mean effective concentration (EC50) was observed at 40 μM (Figure 1). Several different reaction patterns were noted (Figure 2). After stimulation by 100 μM ATP, most cells displayed a high initial elevation of [Ca2+]i, followed by oscillations of [Ca2+]i at a rate of 1 * 10−2 Hz (Figure 2A). In some cells, the [Ca2+]i transient terminated after one oscillation (Figure 2B). Further patterns were predominantly observed during lower ATP concentrations (10 μM) (Figure 2 C and D). In spontaneously oscillating cells, extracellular ATP increased the oscillation frequency from 5.7±1.5 * 10−3 Hz to 13.7±3.5 * 10−3 Hz.

Characterization of purinergic receptors on biliary epithelial cancer cells. Subsequently, we compared the efficiency of various purinergic agonists (100 μM) to induce a [Ca2+]i response (Figure 3). The proportion of reacting cells after stimulation by UTP was almost comparable to ATP and cross-desensitization was noticed. Pre-treatment of cells with ATP or UTP abolished the Ca2+ response to supramaximal concentrations of ATP or UTP (200 μM) (data not shown). The rank order of tested agonists was ATP=UTP>> adenosine>ADP=AMP>α,β-meATP. This profile is in line with the functional expression of purinergic receptors of the P1 and P2Y2 subtype (6, 9, 20, 21).

Figure 1.
Figure 1.

Dose-response curve of the human biliary epithelial cancer cell line (Mz-Cha-1). Extracellular ATP produces a concentration-dependent increase (EC50 40 μM) in intracellular Ca2+ levels. Data represent the mean±SD from 3-14 experiments of 2-3 different preparations.

Importance of extracellular Ca2+. To evaluate the [Ca2+]i signal in biliary epithelial cells in more detail, we investigated the role of intra- and extracellular Ca2+ for ATP-induced [Ca2+]i transients. The overall proportion of reacting cells did not differ significantly for media with or without Ca2+ (80% compared to 84%, p> 0.05). However, the shape of the signaling pattern for ATP-induced [Ca2+]i transients was different. Spike-like responses predominated and oscillatory or plateau-like [Ca2+]i transients terminated prematurely when Ca2+ was withdrawn from the extracellular medium, indicating a pivotal role for extracellular Ca2+ in maintaining the [Ca2+]i signal (Figure 4A). To characterize Ca2+ entry mechanisms from the extracellular medium, cells were preincubated with verapamil (10 μM) or nifedipine (20 μM) to inhibit voltage-dependent Ca2+ channels. However, neither the signal shape nor the duration of plateau-like [Ca2+]i transients differed significantly in the presence of either substance, indicating that mechanisms other than voltage-dependant Ca2+ channels are important for the influx of Ca2+ during ATP-induced calcium oscillations. NiCl2 (1mM) is known to inhibit 2Na+/Ca2+exchange, but did not influence the ATP-mediated Ca2+ response in our experiments (data not shown).

Figure 2.
Figure 2.

Ca2+ signalling patterns in individual biliary epithelial cancer cells (Mz-Cha-1). At concentrations of 100 μM ATP, most cells react with oscillations (A and B). Patterns C and D were observed at lower (10 μM) ATP concentration predominantly. Representative confocal measurements of single cells are shown.

Importance of intracellular Ca2+ stores. Thapsigargin depletes IP3-sensitive Ca2+ stores by inhibition of smooth endoplasmatic reticulum Ca2+ (SERCA) pumps. In the presence of extracellular Ca2+, thapsigargin (2 or 5 μM) induced a gradual increase of [Ca2+]i and a subsequent plateau-like elevation of [Ca2+]i in the used cell line. This plateau-like elevation of [Ca2+]i was terminated by removal of extracellular Ca2+ and was not observed for cells in Ca2+-free medium. This may suggest that plateau-like Ca2+ elevations depend on Ca2+ influx from the extracellular medium.

In the presence of extracellular Ca2+ thapsigargin was not able to inhibit a subsequent ATP response. Interestingly, ATP did not induce [Ca2+]i oscillations after application of thapsigargin, indicating a central role of IP3-sensitive Ca2+ stores for oscillatory Ca2+ responses (Figure 4B).

In the absence of extracellular Ca2+, depletion of IP3-sensitive Ca2+ stores by thapsigargin should prevent a further response to ATP, as ATP is thought to act via production of IP3. However, even then, neither thapsigargin nor tBuBHQ, another inhibitor of SERCA pumps, nor their combination was able to prevent a further increase of [Ca2+]i by ATP (Figure 4C). To compare this effect with that of other mediators acting via IP3, we tested the effect of carbachol in this setting, but carbachol did not further increase [Ca2+]i. These observations suggest that ATP not only releases a thapsigargin-sensitive intracellular Ca2+ pool but also a second pool, which can not be mobilized by thapsigargin or carbachol (Figure 4D).

In excitable and some non-excitable cells, ryanodine leads to liberation of Ca2+ from ryanodine-sensitive intracellular Ca2+ stores. As ryanodine did not release intracellular [Ca2+]i, nor inhibit the thapsigargin-resistant ATP response in our experiments, we found no evidence for the involvement of ryanodine-sensitive Ca2+ stores in the purinergic response.

As lysosomes often contain high Ca2+ concentrations, we mobilized lysosomal Ca2+ by GPN, which is known to permeabilize these organelles very specifically by osmotic swelling (22). Although GPN (0.2 mM) induced Ca2+ release from intracellular Ca2+ stores, it did not inhibit the thapsigargin-resistant ATP response in cells in Ca2+-free medium, thus excluding a lysosomal origin of the ATP-sensitive Ca2+ pool.

To investigate the role of the mitochondrial Ca2+ pool, we incubated Mz-ChA-1 cells with oligomycin, or FCCP. Both substances are known to inhibit oxidative phosphorylation and thereby lead to accumulation of high intramitochondrial Ca2+ concentrations. After a 5-min incubation with oligomycin 5 μM or FCCP 2 μM, the proportion of viable cells did not decrease significantly, as determined by trypan blue exclusion (viability 95% vs. 92 %, p>0.05). But neither uncoupler affected the thapsigargin-resistant ATP response, thus excluding a mitochondrial localization of the carbachol-resistant, but ATP-sensitive Ca2+ pool.

The Ca2+ ionophore ionomycin, which caused a marked increase of [Ca2+]i even in Ca2+-free medium, was more effective at releasing intracellularly stored Ca2+ than was ATP and was able to prevent a subsequent ATP-triggered [Ca2+]i transient.

Nuclear Ca2+ transients induced by extracellular ATP in biliary epithelial cancer cells. Since nuclei contain Ca2+, we investigated whether extracellular ATP could also increase nuclear [Ca2+]i. With confocal microscopy, the compartmentalization of the intracellular Ca2+ signal is possible. Stimulation of Mz-Cha-1 cells with extracellular ATP in Ca2+ containing media induced nuclear and cytosoloic [Ca2+]i transients in almost all investigated cells (Figure 5A). When cells were pre-incubated with thapsigargin in Ca2+-free and EGTA-containing medium, a Ca2+ transient in the nucleus and cytoplsma was still observed (Figure 5B). These data suggest that secretory extracellular ATP might regulate nuclear function by increasing nuclear [Ca2+]i concentrations. Profiles generated from line scan images in representative cells (Figure 5C) suggest that nuclear Ca2+ increase precedes the cytosolic response (Figure 5D).

Gene and protein expression of P2Y receptor subtypes in Mz-ChA-1 cells. To verify the pharmacological data, the P2Y receptor expression was determined at the molecular level in cultured Mz-Cha-1 cells. Gene expression for P2Y receptor subtypes was investigated using PCR methods. P2Y1, P2Y2, P2Y4, and P2Y6 mRNA was expressed in the human biliary epithelial cancer cell line (Figure 6A).

Figure 3.
Figure 3.

Ca2+ response of the Mz-Cha-1 biliary epithelial cancer cell line after stimulation with different purinergic agonists (ATP=adenosine triphosphate; UTP=uridine triphosphate; ADP=adenosine diphosphate; AMP=adenosine monophosphate; α, β-me-ATP=α, β-methyleneadenosine triphosphate; Me-S-ATP=methylthioadenosine triphosphate; Adenosine (100 μM)). Data indicate the P1, P2Y2 and P2Y4 purinergic receptor subtype expression. Data represent the mean±SD from 8 experiments.

At the protein level, immunoblots showed only the presence of P2Y2 in the human biliary epithelial cancer cell line (60 kDa) (Figure 6B). The 120 kDa line in rat brain probably represents receptor oligomerization in whole organ lysate (23). The P2Y1,-4 and -6 antibodies revealed no significant bands in Mz-Cha-1 cells, but strong signals in rat brain.

Discussion

Biliary epithelial cells are involved in hepatic secretory processes and constitute targets for injury, inflammatory and neoplastic biliary diseases, i.e. cholangiocellular carcinoma. Small ligands, such as ATP, are present in bile and are secreted by hepatocytes and biliary epithelial cells (3, 24) and play a role in autocrine and paracrine regulation of the epithelial cell function.

Our data confirm previous data obtained in isolated rat cells, that human biliary epithelial cancer cells also express purinergic receptors. Purinoceptors are a family of membrane-bound receptors that bind extracellular nucleotides. P2 is the term used for receptors which are mainly activated by ATP, whereas P1 receptors respond to adenosine preferentially (25). Both subtypes have been shown to be expressed in biliary epithelial cells (9, 21). As UTP and ATP were equipotent in eliciting a Ca2+ response and cross-desensitization occurred between both agonists, we conclude that P2 receptors in the human biliary epithelial cell line are of the P2Y subtype. This binding characteristic is typical for P2Y2 receptors and might be explained by a model with a common binding site for the triphosphate tail and two distinct ribose and an adenine or uracil binding site, respectively (25).

Figure 4.
Figure 4.

Ca2+ signalling patterns in individual biliary epithelial cancer cells (Mz-Cha-1) under different experimental conditions to clarify the role of extra- and intracellular Ca2+ stores. Representative confocal recordings of at least five different experiments are shown.

The presence of P2Y2 receptors on biliary epithelial cells has been suggested from pharmacological and genetical studies. In the present study, to our knowledge for the first time, we investigated the expressions of P2Y receptor subtypes on a genetic and protein level in a human biliary epithelial cancer cell line. As recent studies have suggested that the P2Y receptors designated as P2Y5 and P2Y7 do not belong to the family of nucleotide receptors (8), we used primer pairs for the human P2Y1, P2Y2, P2Y4, and P2Y6 receptors. In accordance with the pharmacological characterization at the functional level, P2Y2 receptor-specific mRNA was detected in the human biliary epithelial cell line. However, specific mRNA for the other three P2Y receptor subtypes was also found in the Mz-Cha-1 cells. Since ADP and UDP, which are the most potent agonists for P2Y1 and P2Y6 receptors, respectively, only caused changes in [Ca2+]i in about 5% of cells, this strongly suggests that these receptor subtypes are only expressed in few cells at a functional level. Furthermore, it is possible that the [Ca2+]i reaction, after stimulation with these agonists was due to contamination of the commercial ADP and UDP preparations with the respective nucleotide triphosphates. The existence of different receptor subtype mRNA species points to post-transcriptional processes as being important regulators for functional P2Y protein expression. Because P2Y2 and P2Y4 receptors are only sensitive to ATP and UTP, albeit with different sensitivites, demonstration of functional co-expression in the same cells is almost impossible. Our western blot analysis, however, indicates the presence of P2Y2 receptors solely in the biliary epithelial cancer cell line.

Figure 5.
Figure 5.

Ca2+ signalling in individual biliary epithelial cells. Confocal imaging was used to differentiate between nuclear and cytosolic signals after the addition of 100 μM ATP (arrow). Representative recordings in calcium-containing medium (A) and in calcium-free medium after thapsigargin pre-treatment (B) are shown, taken from at least five different experiments. A representative line scan image (XT plot) of nuclear (nu) and cytosolic (cy) Ca2+ transients (C) in Ca2+-containing media after addition of 100 μM ATP. The profiles (D) were taken from the line scan image averaging 20 pixel of the X direction.

We have demonstrated for the first time the existence of nuclear [Ca2+]i transients in a biliary epithelial cancer cell line. The nuclear calcium transient was not abolished by SERCA inhibitors. This is in line with previous studies performed in pancreatic β cells (11), where the nuclear [Ca2+]i transient was independent of endoplasmatic Ca2+ stores. One limitation of our study might be that there are differences in the distribution and the behaviour of the Ca2+-sensitive dyes in different cellular compartments (26). We therefore used only cells with evenly distributed fluo-4 fluorescence in the experiments. Nuclear Ca2+ increase preceded the cytosolic response. We did not determine the reason for the difference in nuclear and cytosolic Ca2+ changes in the biliary epithelial line, however, it can be speculated that different IP3 receptors may be expressed in different compartments of the cells (27).

Figure 6.
Figure 6.

A: Identification of P2Y receptors in the Mz-Cha-1 biliary epithelial cell line. RT-PCR was utilized to probe cDNA from Mz-Cha-1 cells for P2Y1 (lane 1), P2Y2 (lane 3), P2Y4 (lane 5) and P2Y6 (lane 7). Lanes 2, 4, 6, and 8 represent total RNA as appropriate controls. Results are consistent with the genetical expression of the P2Y1, -2,-4 and -6 receptors cDNA in Mz-Cha-1 cells. Together with the agonist data, it is assumed that only P2Y2 and P2Y4 receptors are functionally expressed in this biliary epithelial cell line. B: Western blot analysis of P2Y receptor protein expression in the Mz-Cha-1 (Mz) biliary epithelial cancer cell line. On the protein level, only P2Y2 receptors are expressed in the cell line. RB: Rat brain.

Purinoceptors have been implicated in the pathophysiology of a number of diseases, including diabetes, thrombosis and cancer. In gastrointestinal tract tumours, the expression of P2Y2 receptor subtype has been found to inhibit proliferation (28-30). Taking into account that extracellular ATP modulates gene expression (31), this molecule might have distinct physiological relevance other than influencing secretory processes in biliary epithelial cells and might also play a role in inflammation and carcinogenesis, thus offering putative chances for the development of highly selective therapies.

  • Received April 14, 2012.
  • Revision received July 10, 2012.
  • Accepted July 11, 2012.

References

  1. ↵
    1. Elsing C,
    2. Kassner A,
    3. Hübner C,
    4. Böhli H,
    5. Stremmel W
    : Absorptive and secretory mechanisms in biliary epithelial cell. J Hepatol 24(S1): 121-127, 1996.
    OpenUrlPubMed
  2. ↵
    1. Schlenker T,
    2. Romac JM,
    3. Sharara AI,
    4. Roman RM,
    5. Kim SJ,
    6. LaRusso N,
    7. Liddle RA,
    8. Fitz JG
    : Regulation of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes. Am J Physiol 273: G1108-1117, 1997.
    OpenUrlPubMed
  3. ↵
    1. Roman RM,
    2. Fitz JG
    : Emerging roles of purinergic signalling in gastrointestinal epithelial secretion and hepatobiliary function. Gastroenterology 116: 964-979, 1999.
    OpenUrlPubMed
    1. Dranoff JA,
    2. Masyuk AI,
    3. Kruglov EA,
    4. LaRusso NF,
    5. Nathanson MH
    : Polarized expression and functional P2Y ATP receptors in rat bile duct epithelia. Am J Gastrointest Liver Physiol 281: 1059-1067, 2001.
    OpenUrl
  5. ↵
    1. Doctor RB,
    2. Matzakos T,
    3. McWilliams R,
    4. Johnson S,
    5. Feranchak AP,
    6. Fitz JG
    : Purinergic regulation of cholangiocyte secretion: identification of a novel role for P2X receptors. Am J Physiol 288: G779-786, 2005.
    OpenUrl
  6. ↵
    1. Feranchak AP,
    2. Lewis MA,
    3. Kresge C,
    4. Sathe M,
    5. Bugde A,
    6. Luby-Phelps K,
    7. Antich PP,
    8. Fitz JG
    : Initiation of purinergic signalling by exocytosis of ATP-containing vesicles in liver epithelium. J Biol Chem 285: 8138-8147, 2010.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Minagawa N,
    2. Ehrlich BE,
    3. Nathanson MH
    : Calcium signaling in cholangiocytes. World J Gastroenterol 12: 3466-3470, 2006.
    OpenUrlPubMed
  8. ↵
    1. Burnstock G
    : Purine and pyrimidine receptors. Cell Mol Life Sci 64: 1471-1483, 2007.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Elsing C,
    2. Kassner A,
    3. Stremmel W
    : Sodium, hydrogen antiporter activation by extracellular adenosine triphosphate in biliary epithelial cells. Gastroenterology 111: 1321-1332, 1996.
    OpenUrlPubMed
  10. ↵
    1. Dranoff JA,
    2. Masyuk AI,
    3. Kruglov EA,
    4. LaRusso NF,
    5. Nathanson MH
    : Polarized expression and function of P2Y ATP receptors in rat bile duct epithelia. Am J Physiol 281: G1059-1067, 2001.
    OpenUrl
  11. ↵
    1. Chen Z,
    2. Li Z,
    3. Peng G,
    4. Chen X,
    5. Yin W,
    6. Kotlikoff MI,
    7. Yuan Z,
    8. Ji G
    : Extracellular ATP-induced nuclear Ca2+ transient is mediated by inositol 1,4,5-triphosphate receptors in mouse pancreatic β-cells. Biochem Biophys Res Commun 382: 381-384, 2009.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Burnier M,
    2. Centeno G,
    3. Burki E,
    4. Brunner HR
    : Confocal microscopy to analyse cytosolic and nuclear calcium in cultured vascular cells. Am J Physiol 266: C1118-1127, 1994.
    OpenUrlPubMed
  13. ↵
    1. Ustundag Y,
    2. Bayraktar Y
    : Cholangiocarcinoma: a compact review of the literature. World J Gastroenterol 14: 6458-6466, 2008.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Malhi H,
    2. Gores GJ
    : Cholangiocarcinoma: Modern advances in understanding a deadly old disease J Hepatol 45: 856-867, 2006.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Mortaz E,
    2. Braber S,
    3. Nazary M,
    4. Givi ME,
    5. Nijkamp FP,
    6. Folkerts G
    : ATP in the pathogenesis of lung emphysema. Eur J Pharmacol 619: 92-96, 2009.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Deli T,
    2. Csernoch L
    : Extracellular ATP and cancer: An overview with special reference to P2 purinergic receptors. Pathol Oncol Res 14: 219-231, 2008.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Knuth A,
    2. Gabbert H,
    3. Dippold W,
    4. Klein O,
    5. Sachse W,
    6. Bitter-Suermann D,
    7. Prellwitz W,
    8. Meyer zum Büschenfelde KH
    : Biliary adenocarcinoma. Characterization of three new tumor cell lines. J Hepatol 1: 579-596, 1985.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Schöfl C,
    2. Ponczek M,
    3. Mader T,
    4. Warning M,
    5. Benecke H,
    6. von zur Mühlen A,
    7. Mix H,
    8. Cornberg M,
    9. Böker KH,
    10. Manns MP,
    11. Wagner S
    : Regulation of cytosolic free calcium concentration by extracellular nucleotides in human hepatocytes. Am J Physiol 276: G164-172, 1999.
    OpenUrlPubMed
  19. ↵
    1. Tovell VE,
    2. Sanderson J
    : Distinct P2Y receptor subtypes regulate calcium signalling in human retinal pigmentepithelial cells. Invest Ophtalmol Vis Sci 49: 350-357, 2008.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. McGill JM,
    2. Basavappa S,
    3. Mangel AW,
    4. Shimokura GH,
    5. Middleton JP,
    6. Fitz JG
    : Adenosine triphosphate activates ion permeabilities in biliary epthelial cells. Gastroenterology 107: 236-243, 1994.
    OpenUrlPubMed
  21. ↵
    1. Wolkoff LI,
    2. Perrone RD,
    3. Grubman SA,
    4. Lee DW,
    5. Soltoff SP,
    6. Rogers LC,
    7. Beinborn M,
    8. Fang SL,
    9. Cheng SH,
    10. Jefferson DM
    : Purinoceptor P2u identification and function in human intrahepatic biliary epithelial cells. Cell Calcium 17: 375-383, 1995.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Berg TO,
    2. Stromhaug PE,
    3. Berg T,
    4. Seglen PO
    : Separation of lysosomes and autophagosomes by means of glycyl-phenylalanine-naphthylamide, a lysosome-disrupting cathepsin-C substrate. Eur J Biochem 221: 595-602, 1994.
    OpenUrlPubMed
  23. ↵
    1. Yoshioka K,
    2. Nakata H
    : ATP- and adenosine-mediated signaling in the central nervous system: purinergic receptor complex: generating adenine nucleotide-sensitive adenosine receptors. J Pharmacol Sci 94: 88-94, 2004.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Gatof D,
    2. Kilic G,
    3. Fitz JG
    : Vesicular exocytosis contributes to volume sensitive ATP-release in biliary cells. Am J Physiol 286: G538-546, 2004.
    OpenUrlCrossRef
  25. ↵
    1. Conigrave AD,
    2. Jiang L
    : Review: Ca2+-mobilizing receptors for ATP and UTP. Cell Calcium 17: 111-119, 1995.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Gerasimenco O,
    2. Gerasimenko J
    : New aspects of nuclear calcium signalling. J Cell Sci 117: 3087-3094, 2004.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Kim JC,
    2. Son MJ,
    3. Subedi KP,
    4. Kim do H,
    5. Woo SH
    : IP3-induced cytosolic and nuclear Ca2+ signals in HL-1 atrial myocytes: possible role of IP3 receptor subtypes. Mol Cells 29: 387-395, 2010.
    OpenUrlPubMed
  28. ↵
    1. Höpfner M,
    2. Lemmer K,
    3. Jansen A,
    4. Hanski C,
    5. Riecken EO,
    6. Gavish M,
    7. Mann B,
    8. Buhr H,
    9. Glassmeier G,
    10. Scherübl H
    : Expression of functional P2-purinergic receptors in primary cultures of human colorectal carcinoma cells. Biochem Biophys Res Commun 251: 811-817, 1998.
    OpenUrlCrossRefPubMed
    1. Maaser K,
    2. Höpfner M,
    3. Kap H,
    4. Sutter AP,
    5. Barthel B,
    6. von Lampe B,
    7. Zeitz M,
    8. Scherübl H
    : Extracellular nucleotides inhibit growth of human oesophageal cancer cells via P2Y(2)-receptors. Br J Cancer 86: 636-644, 2002.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Wang MX,
    2. Ren LM,
    3. Shan BE
    : Inhibitory effects of extracellular adenosine triphosphate on growth of esophageal carcinoma cells. World J Gastroenterol 11: 5915-5919, 2005.
    OpenUrlPubMed
  31. ↵
    1. Janssen LJ,
    2. Farkas L,
    3. Rahman T,
    4. Kolb MR
    : ATP stimulates (Ca2+) waves and gene expression in cultured human fibroblasts. Int J Biochem Cell Biol 41: 2477-2484, 2009.
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
Extracellular ATP Induces Cytoplasmic and Nuclear Ca2+ Transients via P2Y2 Receptor in Human Biliary Epithelial Cancer Cells (Mz-Cha-1)
CHRISTOPH ELSING, TIHOMIR GEORGIEV, CHRISTIAN A. HÜBNER, REGINA BOGER, WOLFGANG STREMMEL, THORSTEN SCHLENKER
Anticancer Research Sep 2012, 32 (9) 3759-3767;
Twitter logo Facebook logo Mendeley logo

Jump to section