HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 35, 32925-32932, August 31, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/35/32925    most recent M103313200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sørensen, C. E. Articles by Novak, I. Search for Related Content PubMed PubMed Citation Articles by Sørensen, C. E. Articles by Novak, I. Social Bookmarking                      What's this?

Visualization of ATP Release in Pancreatic Acini in Response to Cholinergic Stimulus

USE OF FLUORESCENT PROBES AND CONFOCAL MICROSCOPY^*

Christiane E. Sørensen and Ivana Novak

From the August Krogh Institute, Universitetsparken 13, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark

Received for publication, April 13, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The energy providing substrate ATP can be released from various cells and act extracellularly to regulate the same cells or neighboring cells. However, the pathway for ATP release and the eliciting physiological stimulus are unclear. Recently, we showed that ATP activates P2X and P2Y purinergic receptors on pancreatic ducts. Thus, it was relevant to ask whether the upstream acini could be the source of releasable ATP and what the stimulus might be. We used freshly prepared rat pancreatic acini and applied conventional luminescence measurements of luciferin/luciferase reaction. As a new application of this reaction in confocal microscopy, we monitored luciferin fluorescence as a sign of ATP release by single acini. In addition we used quinacrine to mark ATP stores, which were similar to those marked with fluorescent ATP, 2'-(or-3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate, but only partially overlapping with those marked by acridine orange and LysoTracker Red. In functional studies we show that native pancreatic acini release ATP in response to various stimuli but most importantly to cholinergic stimulation, a very likely physiological stimulus in this epithelium. In a close vicinity of acini we detect about 9 µM ATP after cholinergic stimulation. Thus, ATP is poised as the paracrine mediator between pancreatic acini and ducts.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A variety of purinergic receptors and ecto-nucleotidases indicate that extracellular ATP is of importance as an autocrine or paracrine mediator. ATP and other nucleotides can be released from various cells by several mechanisms including a nonselective release from cytoplasm, transport via a plasma membrane transporter, or a release from exocytotic granules where ATP is compartmentalized. The latter type of ATP release is found in neuronal, neuro-endocrine, and endocrine cells (1-3). In epithelia many stimuli can elicit release of ATP by yet unresolved mechanisms, and in many cases it is not clear to what extent these are physiological rather than experimental stimuli. For example, in epithelia ATP release has been shown in response to stretch, stress, and cell volume changes (4-8). As one of the possible mechanisms, it has been discussed whether the intracellular ATP is transported by ATP-binding cassette proteins, including the cystic fibrosis transmembrane conductance regulator (7, 8). Pancreatic ducts contain a number of luminal and also basolateral purinergic receptors from both P2X and P2Y families (P2X4, P2X7, P2Y2, and P2Y4) (9). Thus, it is important to find where ATP is released and under what conditions. Pancreatic acini form the bulk of the pancreatic tissue, they surround pancreatic ducts, and empty their enzyme-rich secretion into the ductal lumen. The major aim of the present study was to find whether acini are the source of releasable ATP and to find the possible physiological stimulus.

One hurdle in ATP research field is the difficulty of monitoring ATP release directly from living cells. Presently, the most commonly used method is the luminometer measurement of photon generation from the reaction of luciferin and ATP that is catalyzed by luciferase. When applied to a group of cells, mostly cultured cells, however, it is not possible to distinguish between ATP releases from healthy cells compared with release from lysed or dead cells (see below). Consequently, a lot of effort has been put into developing new methods to monitor ATP release (10-12). In the present study, in an extension of the conventional luminescence methods on pancreatic acini, we developed new methods of monitoring of ATP release in single acini utilizing luciferin fluorescence, as well as quinacrine and MANT-ATP¹ fluorescence detected by the confocal laser scanning microscopy.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- All standard chemicals, Collagenase V, and quinacrine dihydrochloride were obtained from Sigma. Fluorophores, H₂DIDS, luciferin, and luciferase were obtained from Molecular Probes (Leiden, The Netherlands). The ATP bioluminescence kit HS II was from (Roche Molecular Biochemicals). Tissue culture media were from Life Technologies, Inc.

Pancreas Tissue Preparation and Measurement of ATP in Cell Suspensions-- Pancreas was obtained from female Wistar rats and cut into small pieces. For some experiments pancreatic lobules were dissected with sharpened forceps and used without further treatments. Acinar preparation was based on our earlier published method of collagenase digestion with some modifications (13). Tissue was incubated with 1.3 mg/ml collagenase V for 40 min and finely dispersed by passages through glass pipettes. Homogenized suspension was passed through a 150-µm nylon filter, and acini were washed with Dulbecco's modified Eagle's medium 1000/Ham's F-12 medium. Finally, acini were suspended in 3 ml of bicarbonate-free Ringer (BIC) containing 145 mmol/liter Na⁺, 3.6 mmol/liter K⁺, 1.5 mmol/liter Ca²⁺, 1 mmol/liter Mg²⁺, 122 mmol/liter Cl, 25 mmol/liter gluconate, 2 mmol/liter phosphate, 5 mmol/liter glucose, 2 mg/ml bovine serum albumin, and 0.25 mg/ml trypsin inhibitor (pH 7.4). After equilibration for 15-20 min at 37 °C, the acinar suspension was gently rotated to ensure proper mixing of the cells, except for experiments shown in Fig. 1. A hypotonic shock was induced by reducing NaCl from 122 mmol/liter to 72 mmol/liter by exchanging half of the BIC medium with a medium containing 30 mmol/liter NaCl. In one series of experiments acini were stimulated with carbachol (CCH). Samples of supernatants were taken at timed intervals, and each sample was immediately centrifuged (15 s at 1000 × g) to remove cells possibly present in the supernatant and heated to 98 °C for 1 min to eliminate ecto-nucleotidase activity. The amount of ATP in the samples was measured using the high sensitivity ATP bioluminescence kit HS II following the instructions of the manufacturer. ATP standards were dissolved in the same medium used for cell suspensions. For ATP measurements 100 µl of luciferin-luciferase reagent was injected into a 100-µl sample or an ATP standard. The resulting light emission was measured in our laboratory-made luminometer. Standard curves were generated for each series of measurements. ATP concentrations in the supernatant were normalized to 10% cytocrit.

Measurements with Calcein, Quinacrine, and Vesicular Markers using Confocal Laser Scanning Microscope-- A confocal spectral laser scanning microscope (Leica SP CLSM, Leica Microsystems Heidelberg GmbH, Germany) equipped with ArKr and UV lasers and 63× 1.2NA PL APO and 20× 1.7 NA HC PL APO objectives was used to monitor fluorescent signals. The real frame scanning time for time series was usually set at 0.88 s for 512 × 512 pixels. Emission intensities of various fluorophores were selected with the CLSM spectrophotometer unit and collected with a photomultiplier. Analysis of the data was performed with Leica's physiology package software and MetaMorph 4.0 software (Universal Imaging Corporation, West Chester, PA). The tissues were held in the experimental chamber by means of holding pipettes and/or attached to a coverslip with Cell-Tak (Becton Dickinson Labware, Bedford, MA). The tissues/cells were suspended in the control BIC, and the experiments were performed at 25 °C. For volume measurements, acini were loaded with 5 µM calcein acetoxymethyl ester for 30 min (14). The fluorescence signal was monitored at 500-540 nm upon 488 nm excitation. Putative ATP stores in pancreatic cells were monitored with quinacrine. The cells were incubated with 1-5 µM quinacrine dihydrochloride for 5-15 min, and fluorescence was detected at 490-540 nm with 476 nm excitation. For detection of acidic organelles, pancreatic tissues were incubated with 5 µM acridine orange for 15-20 min or with 25-50 nM LysoTracker Red for 40-50 min. Acridine orange was excited with 476 and 488 nm, and the fluorescence was monitored at 510-550 and 620-680 nm. LysoTracker Red was excited with 568 nm, and the fluorescence signal was detected at 580-640 nm. In several experiments acini were incubated with 25-50 µM MANT-ATP for 1-5 h in a Dulbecco's modified Eagle's medium 100/F12 medium supplemented with 10% fetal calf serum. Upon excitation with 364 nm, the fluorescence signal was detected at 430-480 nm. Unlike other fluorophores used in this study, MANT-ATP bleached relatively fast.

Measurement of ATP Using Luciferin and Confocal Laser Scanning Microscope-- Luciferin-luciferase reagent (HSII) was added to pancreatic cells (about 10% cytocrit) suspended in BIC solution in a 1:5 dilution, and the luciferin fluorescence measurements were begun within 10 min. In some experiments D-luciferin (0-1.67 mM) and luciferase (25-188 µg/ml) were added separately. All of the experiments were performed at 25 °C. Luciferin was excited with 364 nm, and the emission fluorescence was monitored at 510-550 nm (see "Results"). In measurements on pancreatic cells fluorescence signals were collected from regions around and inside of acini. Pancreatic cells were stimulated with carbachol and as a control with BIC blanks. ATP concentration was determined indirectly as a disappearance of the substrate luciferin, utilizing the luciferin fluorescence. Subsequently, one or more concentrations of ATP were used for in situ calibrations. Agonists dissolved in small volumes were added directly to the bath, and only experiments where acini did not move or were not affected by mechanical stimulation were evaluated. In some experiments 0.5 mM H₂DIDS was added to the bathing medium.

The results are presented as single experiments; summaries are given with the mean values ± S.E., where n refers to a number of independent preparations; fluorescence and transmission images and as time sequences of fluorescence intensities in regions of interests.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ATP Release in Acinar Cell Suspension-- Presently, the most commonly used method for measurement of ATP release in cultured epithelia is the luminometer detection of photon generation from reaction of luciferin and ATP that is catalyzed by luciferase. Initially, we applied this method to measure ATP release from freshly prepared pancreatic acini obtained from rat pancreas. Acini were gently shaken to ensure proper mixing with the medium. This mechanical stimulation caused a clear transient increase in ATP in the supernatant collected from acinar suspension immediately following the mechanical stress (Fig. 1). Subsequent decrease in ATP was most likely due to hydrolysis of ATP by ecto-nucleotidases, including CD39 present in pancreatic acini (15-17). We presume that in pancreatic acini, as in many other tissues, mechanical disturbances and stretch may be more relevant as the experimental tool rather than physiological stimulus. It is important to take account for this background mechanical disturbance. Acinar suspensions were shaken throughout the course of subsequent experiments, and extra care had to be taken in experiments with single acini.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   ATP release in pancreatic acini suspension with mechanical stimulation. ATP release was monitored as luminescence of the luciferin/luciferase reaction. Mechanical stress was induced by 1 min of gentle shaking.

In exocrine glands secretory cells undergo volume changes during secretion (decrease and increase). Because cell swelling (caused by hypotonic stress) is known to release ATP in several epithelial cells (4, 5, 18), we asked whether this also happens in pancreatic acini. Fig. 2a shows experiments where first a control solution and then a hypotonic solution was added to cell suspensions, such that the final ionic strength was 60% of the control. Although there was an indication that ATP was released in the hypotonic medium, there were large variations in responses. We considered whether these variations had something to do with cell swelling. Hence, in separate experiments using CLSM we monitored volume changes in single acinar cells loaded with calcein (Fig. 2, b and c), similar to the published study on airway epithelial cells (14). Immediately after the hypotonic shock as the cells swelled, concentration of the fluorophore within the single cells decreased as seen by a steeper slope in the intensity curve (Fig. 2c). Acinar cells swelled within about 60 s, but not in a synchrony. Because ATP release from acini may only be lasting during this short period of swelling and because ATP is hydrolyzed by ecto-nucleotidases, monitoring of small amounts of ATP released by sampling of supernatant is problematical.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Effect of hypotonic solution on ATP release and acinar cell volume. a, ATP release was measured in a pancreatic acini suspension as in Fig. 1. b, two small acini consisting of three or four cells show calcein fluorescence in a CLSM. Cell volume was estimated indirectly by changes in calcein fluorescence within marked regions in four cells. The bar indicates 20 µm. c, immediately after the hypotonic shock (NaCl was decreased from 122 to 72 mmol/l), an abrupt decrease in the fluorescence indicates a decrease in the calcein concentration caused by a cell swelling. A steady, smaller fall in the fluorescence before and after the hypotonic shock is due to bleaching and/or efflux of calcein. Similar results were obtained in three experiments.

Pancreatic enzyme secretion is elicited by cholinergic stimulation normally mediated by parasympathetic nerves. Carbachol, a nondegradable cholinergic agonist, was used to stimulate pancreatic acini in a suspension. There was a transient increase in ATP in response to 10 and 100 µM carbachol, as shown in the depicted experiment (Fig. 3). In another five experiments we could observe that ATP increased transiently in some experiments, whereas in others the increase was more sustained. The peak ATP released after carbachol stimulation (100 µM) was 22.3 ± 2.8 nmol/liter and the prestimulation level was 7.1 ± 0.6 nmol/liter (n = 5). ATP detected in the supernatant is the balance between ATP released and ATP hydrolyzed. It is also clear that ATP released could be due to carbachol-stimulated release, but also, as in the above experiments (Figs. 1 and 2), it could be due to cell stress or even cell lysis. We calculated that it takes a lysis of 1 in 100,000 cells in 10% cytocrit to free about 4 nmol/liter of ATP in the suspension. The message from these experiments is that measurement of ATP release from a group of cells is difficult, if there is no control of cell viability at the time of the measurement. Therefore, we set out to develop other methods, and in the following paragraphs we will outline our new approach to monitor release of ATP in single pancreatic acini.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   The cholinergic agonist carbachol stimulates ATP release in pancreatic acini. ATP release in acinar suspension was monitored as in Fig. 1. The suspension was gently shaken throughout the experiment. Similar results were obtained in another five experiments.

Distribution of Quinacrine, Acridine Orange, LysoTracker Red, and MANT-ATP Fluorescence in Pancreas-- Quinacrine, an acridine compound, binds to nucleic acids and therefore has been used as a marker of ATP stores in histological preparations of various cells (19). In a couple of preparations it has been used as a marker of granular release (20, 21). We applied quinacrine to freshly dissected pancreatic lobules and pancreatic tissues prepared from suspensions (Fig. 4a). Pancreatic acini, even intact lobules, quickly accumulated quinacrine. In contrast, pancreatic ducts labeled only very weakly with this fluorophore. Using high magnifications and difference interference contrast optics, we could localize the predominant part of quinacrine in zymogen granules (Fig. 4a) and in addition a very weak and diffuse fluorescence underneath the basolateral membrane (Fig. 4e). In unstimulated pancreatic acini the quinacrine fluorescence was stable. However, after carbachol addition the fluorescence intensity in the apical and also basal poles of acini decreased (Fig. 4e), possibly as ATP was released. This was the case in both isolated acini and pancreatic lobules.

View larger version (76K): [in this window] [in a new window]   Fig. 4.   a, labeling of pancreatic cells with quinacrine. Quinacrine images and the corresponding transmission images of a dissected pancreatic lobule (first and second images), an isolated acinus (third and fourth images), and a duct (fifth and sixth images). Quinacrine fluorescence is concentrated evenly in zymogen granules. Pancreatic ducts show very weak quinacrine fluorescence. b, labeling of pancreatic cells with acridine orange. Overlay images of the green and red-orange fluorescence in a dissected pancreatic lobule (first image), isolated acini (second, third, and fourth images). The fifth image is a transmission image corresponding to the acinus in the fourth image. The sixth image is an overlay image of the acridine orange fluorescence in a pancreatic duct. Acridine orange was not compartmentalized in dissected lobules. Isolated acini accumulated acridine orange, and the red-orange fluorescence was observed in regions adjacent to the nucleus and in small organelles close to the basal poles of the acini (see text). c, labeling of pancreatic acini with LysoTracker Red. Fluorescence images and simultaneously obtained transmission images of three pancreatic acini labeled with LysoTracker Red. Images are shown in pairs. This acid store marker partitioned to organelles in the perinuclear regions, some zymogen granules and into vesicles close to the basolateral membrane. d, accumulation of fluorescent MANT-ATP in pancreatic acini. Fluorescence images are shown in pseudo-colors as indicated by the intensity bar. Pancreatic acini were incubated with MANT-ATP. The first and second images show acini incubated with MANT-ATP for 1 and 2 h, respectively. The fluorescence was confined to the cytosol and regions adjacent to the nucleus. The third image is the transmission image corresponding to the acinus in the second image. The fourth, fifth, and sixth images show that after 3-5 h of incubation many acinar cells exhibit fluorescence in the zymogen granular regions. e, release of quinacrine by carbachol. The fluorescence and transmission images of a pancreatic acinus used for quantitative measurements of the quinacrine release. The isolated acinus was stimulated with CCH (10 µM), and the intensity of quinacrine was monitored under apical and basolateral membrane regions with time. In all panels, the bars indicate 20 µm, and the results are typical images of 20-57 acini obtained in four to eight independent preparations for each fluorophore.

Because quinacrine is a weak base, it could become accumulated in cellular acidic compartments. The following experiments were carried out to test this theory. Acridine orange, another marker of acidic stores, undergoes concentration and pH-dependent shift from the green to the red-orange emission. A similar shift is also seen when acridine orange binds to double- and single-stranded nucleic acids. In our experiments, acridine orange, unlike quinacrine, did not easily compartmentalize in intact pancreatic lobules, unless the tissue was enzymatically prepared (Fig. 4b). Acridine orange showed an even green fluorescence in pancreatic ducts with a few red fluorescent vesicles (Fig. 4b). In pancreatic acini the acridine orange distribution was pronounced in perinuclear regions, presumably trans-Golgi condensing vacuoles, lysosomes, and some early zymogen granules. The red-orange fluorescence was also detected in small vesicles close to the basolateral membrane, presumably endosomes. Another acid store indicator, LysoTracker Red, had a distribution in pancreatic acini similar to that of acridine orange (Fig. 4c).

In several experiments pancreatic acini were incubated with the fluorescent ATP indicator MANT-ATP. Initially (1 h), the probe was distributed evenly in the cytoplasm, and after some time it became more concentrated in the peri-nuclear regions (2 h), presumably in the Golgi region. In acini that survived 3-5 h of incubation, and in which most likely de novo protein synthesis has taken place, MANT-ATP was accumulated in zymogen granules (Fig. 4d).

ATP Release Detected by Luciferin Fluorescence-- The oxidation of luciferin, catalyzed by luciferase and consuming ATP, results in a light emission that is detected by a luminometer. The design of the conventional confocal microscope is not suitable for luminescence detection. Therefore, we utilized another part of this reaction to monitor ATP in the extracellular medium. Namely, we monitored the disappearance of the substrate luciferin as a decrease in the luciferin fluorescence in the medium surrounding cells (see Figs. 6-8). Luciferin was excited at 364 nm, and Fig. 5 (a and b) shows the emission spectrum for luciferin alone and for luciferin plus luciferase mix before and after addition of ATP. Utilizing the CLSM spectrophotometer unit, the emission spectra were collected in xz scans using the settings and the chamber similar to those in experiments with pancreatic cells. Fig. 5c shows the relation between the luciferin concentration and the fluorescence intensity monitored at 510-550 nm. During the course of the experiment luciferin accumulated within acinar cells (see below). Nevertheless, it was the extracellular solution surrounding pancreatic cells that was most responsive to additions of small amounts of exogenous ATP in the presence of luciferase (Figs. 6-8). Fig. 6 shows a control experiment with luciferin, initially in the absence of luciferase. Here the luciferin intensity in acinar cells and the surrounding medium remained constant, even at high ATP or carbachol concentrations. Only after luciferase was added did the fluorescence intensity in the medium decrease, and it further decreased with an extra addition of ATP. In experiments where both luciferin and luciferase were present in the bath, the background luciferin intensity decreased depending on the added exogenous ATP concentrations (Figs. 7 and 8). We do not expect that the time course of the luciferin disappearance would be the same as the bioluminescence signal, where the flash or the sustained light generation measured as the photon flux depends on release of the inhibitory product oxyluciferin and a large number of factors affecting this process (22). Nevertheless, one would expect that the time course of the luciferin concentration response is also complex, depending on the rate of luciferase-mediated ATP breakdown, composition of the physiological medium containing cells and cell products, ecto-nucleotidase activity of cells, diffusion of used up media and fresh media, and the mixing of added samples. In the present experimental system, some transient responses were observed with exogenous ATP (e.g. 16 µM ATP in Fig. 7). We estimated that the maximum decrease in the luciferin fluorescence immediately after each ATP addition was the most reliable indication of how much luciferin and therefore ATP was present in the test region. Standard curves of the exogenous ATP concentration versus the changes in luciferin fluorescence were made for each experiment (Figs. 7 and 8).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Fluorescence emission spectrum of luciferin and dependence of fluorescence on luciferin concentration. a, D-luciferin was excited with 364 nm, and the emission fluorescence intensity of a solution in xz scans was monitored with the spectrophotometer unit of the confocal microscope. b, the same spectrum was obtained with the luciferin and luciferase mix (HSII). The addition of ATP (~1 mM) only reduced the intensity of the fluorescence. c, the emission intensity (510-550 nm) was proportional to the luciferin concentration.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   ATP and carbachol do not affect luciferin fluorescence unless luciferase is present. a, the fluorescence of luciferin with time within some cells in the acinus (i) and in the surrounding bath solution (o). Note the high luciferin fluorescence in the bath solution. The intensity bar is shown below the image. b, effect of ATP, CCH, and luciferase. No changes were detected in the luciferin fluorescence until luciferase (4.8 µg/100 µl) was added and ATP in the medium was used up. The arrows indicate concentrations of the agonists in the bath at the single addition.

View larger version (44K): [in this window] [in a new window]   Fig. 7.   Carbachol decreases luciferin intensity around pancreatic acini indicating ATP release. a, the transmission image of isolated acini. b-d, the luciferin fluorescence of the same acini at 30, 210, and 350 s. The bars indicate 20 µm. e, CCH and then exogenous ATP was added at the indicated times, and the fluorescence intensity of regions adjacent to acini (marked in b) was monitored. f, simultaneously, the fluorescence intensity within 5 cells (marked in c) was also monitored. The arrows indicate concentrations of the agonists in the bath at the single addition. g, in situ calibration curve of the intensity changes with exogenous ATP. In this experiment CCH caused a release of about 25 µM ATP.

View larger version (53K): [in this window] [in a new window]   Fig. 8.   Carbachol decreases the luciferin intensity in the acinar lumen. a, images of a pancreatic acinus where the fluorescence intensity (in marked regions) was measured with time. The scale bar indicates 20 µm. The luciferin fluorescence intensity is represented in pseudo-colors, as indicated by the bar. b, effect of CCH and exogenous ATP on the luciferin intensity adjacent to the acinus (upper part) and in three luminal regions within the acinus (lower part). The decreases in the fluorescence intensity with CCH indicate appearance of released ATP in the bath and possibly the lumen. c, in situ calibration curve of intensity changes of luciferin in the bath with various exogenous ATP concentrations. In this experiment CCH caused release of about 20 µM ATP into the bath. A larger decrease in intensity in the lumen would correspond to a release of about 70 µM ATP. Similar calibration curves of bath ATP concentrations versus changes in bath luciferin fluorescence were made for 11 experiments where CCH was tested and in seven further experiments where only exogenous ATP was tested.

The decisive experiment was that addition of carbachol to acini caused a decrease in the luciferin intensity around acini (Figs. 7 and 8). Addition of the blank solution (BIC) without carbachol had no effect on the luciferin fluorescence (Fig. 8) and was used as a control. Furthermore, effects of the agonist or ATP on luciferin were dependent on luciferase (Fig. 6). The carbachol effect on the luciferin fluorescence indicated that there was a release of ATP from pancreatic acini (Figs. 7 and 8). In each experiment with pancreatic acini or in the subsequent cell free preparation under the same conditions, calibration curves with exogenous ATP were made (Figs. 7g and 8c). From these it was determined how much ATP acini released in response to carbachol in that particular experiment. From 11 independent experiments we estimated that cholinergic stimulation caused a release of 9 ± 3 µM ATP from acini (about 10% cytocrit) to the bath. Although the ATP release was most easily detected in the medium surrounding acini (Figs. 7 and 8), in some small acini it was also possible to detect a fall in the fluorescence in the acinar lumen following the carbachol stimulation (Fig. 8). This effect could indeed be due to ATP release from granules, if we assume that luciferin and luciferase could diffuse into the lumen during the incubation. However, we cannot exclude the possibility that a part of the fluorescence fall was due to a dilution of luciferin by the secreted fluid or due to quenching of the signal by the secreted protein. Nevertheless, in cell-free experiments, albumin (1 mg/ml) quenched the luciferin signal by less than 10%. Possibly then, these experiments indicate that some ATP is released into the lumen of acini. Lack of the effect of exogenous ATP on the luminal fluorescence is puzzling, but because the first generation of ducts that would be present in acinar clusters are lined with CD39,² exogenous ATP at the given concentrations might have difficulties reaching the lumen.

Luciferin Uptake-- During the course of experiments, luciferin often accumulated in acinar cells, but not in duct cells (Fig. 9, a-d) or blood vessels. In acinar cells luciferin accumulated in the cytoplasm and not in zymogen granules. One would not expect luciferin to accumulate passively, but because it has a carboxyl group, it is possible that one of the organic anion transporters might transport it into acinar cells. To test this theory we used H₂DIDS as a broadly acting inhibitor. Fig. 9 (e-h) shows that with H₂DIDS in the medium, the luciferin uptake was markedly delayed. In another set of experiments, we have tested whether the luciferin uptake could be induced. Fig. 10 shows that at relatively high concentrations of extracellular ATP, luciferin is taken up by a few cells, presumably those that have P2 receptors.³ After some time the neighboring acini also fluoresce with luciferin, probably as luciferin spreads to other cells via gap junction.

View larger version (84K): [in this window] [in a new window]   Fig. 9.   Luciferin accumulation in pancreatic cells. a-d, luciferin often accumulates in pancreatic acini (a, b, and d) but not in pancreatic ducts (c and d). Acini and ducts were incubated with luciferin (1 mM) for 15-30 min, and images were accumulated in xy or xz scans. Luciferin appears as light regions within the cytoplasm of acini and as light gray bathing solution (see the intensity bar). The images were taken from different preparations. e-h, preincubation with H2DIDS (0.5 mM) delayed or abolished accumulation of luciferin in acini, which remained dark (nonfluorescent) over 30 min. These are the consecutive images of the same acinus taken in the xy plane (e-g) and the xz plane (h). These results are representative of eight experiments. In all of the panels the bars indicate 20 µm.

View larger version (80K): [in this window] [in a new window]   Fig. 10.   Stimulation of luciferin uptake with high concentrations of ATP. The incubation medium contained luciferin (1.67 mM) and H2DIDS (0.5 mM). The following images were recorded: the differential interference contrast image (DIC); the luciferin fluorescence shown as accumulated scans (at 0 and 600 s) and single scans during the time series (at 10, 60, and 120 s). The addition of ATP evoked an increase in the luciferin fluorescence in two cells marked 1 and 3. Image taken at 600 s indicates that the fluorescence spread to other cells within the acinus. The lower panel shows the intensity changes with ATP in six cells (regions 1-6) and the surrounding medium (regions 7-9). These results are representative of nine similar experiments. The scale bars indicate 20 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Using the conventional luminescence measurements of luciferin/luciferase reaction, the present study on acinar suspensions shows that acini release ATP in response to a mechanical stress, cholinergic stimulation, and possibly volume changes. Clearly, the most physiologically relevant stimulus for this tissue would be the cholinergic stimulation. We verified this finding at a single cell/acinus level by using the luciferin fluorescence, the quinacrine release, and the fluorescent ATP marker and monitored signals in a confocal laser scanning microscope.

Quinacrine is a putative marker of ATP and adenine nucleotide stores and as shown in histological preparations it is accumulated in intestinal nerves, marginal cells of cochlea, and adrenal chromaffin cells (19, 23). In pancreatic acini our study shows that it is accumulated in secretory vesicles, which release their contents upon stimulation (Fig. 4). Similar quinacrine release was reported for insulin secreting cells and acini, although the precise localization of the fluorophore to secretory granules was not possible in these imaging studies (20, 21). Because quinacrine is a weak base, it is possible that it would accumulate in acidic stores. However, other acid store markers, acridine orange and LysoTracker Red, accumulated in stores that only partially overlapped with quinacrine stores (Fig. 4). Granules closest to the apical membrane were only marked with quinacrine. Interestingly, there are only a few contradicting reports about pH values in zymogen granules. Although some studies on isolated acinar cells and isolated zymogen granules propose that the granular pH is acidic, because they accumulate acridine orange (24, 25), other reports indicate that pH of pancreatic and parotid gland granules are only slightly acidic and that their pH may depend on their maturity, with those most mature being least acidic (26, 27). Qualitatively, our results with acridine orange and LysoTracker Red indicate that only some granules are acidic, whereas the apically positioned granules are least acidic. In addition, the zymogen granule pH may depend on the functional state of acini. Returning back to ATP stores, it seems that MANT-ATP also accumulates in zymogen granules after a prolonged incubation. Other fluorescent ATP derivatives, -ATP, have been used to study transport of ATP into isolated brain synaptic vesicles (28). The advantage of MANT-ATP is that it has a more favorable excitation wavelength, making it more suitable for visualization in CLSM and intact preparations. If we assume that quinacrine and MANT-ATP mark ATP stores, this part of our study indicates that some ATP is stored in zymogen granules. In that respect the exocrine pancreas is similar to some endocrine, neuro-endocrine cells, and neurons in the way that they compartmentalize ATP.

The method of luciferin fluorescence as a detector of extracellular ATP released from cells is a new application of the old well known reaction. In comparison with the sensitive luminometric detection of a few photons on the product side, use of the confocal microscope for detection of the fluorescence disappearance is not as sensitive. In addition the present system with acinar cells is not ideal to study the kinetics of the reaction (see "Results"). Nevertheless, the advantage of the method is that one can visualize single intact acini and cells and monitor their release of ATP into the medium semi-quantitatively (Figs. 7 and 8). Our estimate is that acini (about 10% cytocrit) release around 9 µM ATP to the surrounding medium after the carbachol application. Similar local ATP concentrations are suggested for activated platelets as estimated by surface-attached firefly luciferase, 14-20 µM (11), and for pancreatic  cells as estimated by a biosensor technique, 25 µM (10). In acini the ATP release is most easily detected in the medium surrounding acini (Figs. 7 and 8), but some release might occur into the acinar lumen (Fig. 8). This agrees with a detection of ATP release using the quinacrine method and using MANT-ATP as the ATP store marker (Fig. 4). Taking these findings together, it is likely that zymogen granules contain releasable ATP stores. Nevertheless, we cannot exclude the possibility that ATP is also released or secreted from cytoplasm via an apical or basolateral transporter. The ATP release in acini is relatively slow (in the range of minutes) (Figs. 3, 7, and 8) and intact with fluid and enzyme secretion. Nevertheless, one would expect ATP to be degraded by various ecto-nucleotidases, including the demonstrated CD39 (17). This would explain the fact that the ATP detected in the supernatant of the acini suspension was some 100 times lower (Fig. 3).

Interestingly, pancreatic acini take up luciferin by an H₂DIDS-sensitive transport. Possibly this could be one of the carriers capable of transporting anions, such as the DIDS-sensitive kidney organic anion transporter, or hepatocyte multidrug resistance protein MRP3 that is found with Northern blotting also in pancreas (29, 30). Luciferin transport is triggered by high ATP concentrations through a small number of cells possessing P2 receptors and may spread to other cells via gap junctions. Our recent study on ATP-mediated Ca²⁺ signals in pancreatic acini shows indeed that only about 10% of acinar cells have functional P2 receptors.³

Taken together, our experiments show that pancreatic acini release ATP in response to several stimuli. Clearly the most physiological relevant stimulus is the cholinergic stimulation. This conclusion is supported by three independent series of experiments: luminescence measurements on cells suspension, ATP markers (MANT-ATP, quinacrine), and on a cell level as luciferin consumption. Interestingly, our latest study shows that pancreatic acini have very few functional P2 receptors.³ We postulate that it might be a functional necessity for acini to avoid being stimulated by their own ATP, thus preventing autocrine initiation of autodigestive processes in pancreas. Instead, released ATP is "reserved" for the downstream ducts. Thus, ATP might act distally on pancreatic ducts possessing functional purinergic receptors. Luminal ATP acts via P2X4/P2X7 purinoceptors to increase Ca²⁺ and Na⁺ influx (9). ATP is broken down by luminal CD39 or other ecto-nucleotidases, and adenosine acts via P1 receptors to open luminal Cl channels (17). ATP (or its breakdown products) can thus affect ductal secretion and potentiate secretion evoked by secretin (31). Hence, ATP released from acini may explain the long standing dilemma in pancreatic physiology, namely that cholinergic stimulation of acinar secretion potentiates ductal secretion evoked by secretin (32, 33).

	ACKNOWLEDGEMENTS

We are grateful to H. N. Rasmussen and J. Amstrup for critical discussion of the work. The technical assistance of A. Nielsen, A. V. Olsen, and B. Petersen is greatly acknowledged.

	FOOTNOTES

^* This work was supported by the Danish Medical and Science Research Councils.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.

To whom correspondence should be addressed. Tel.: 45-3532-1645; Fax: 45-3532-1567; E-mail: inovak@aki.ku.dk.

Published, JBC Papers in Press, May 31, 2001, DOI 10.1074/jbc.M103313200

² C. E. Sørensen, J. Amstrup, and I. Novak, manuscript in preparation.

³ I. Novak, R. Nitschke, and J. Amstrup, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: MANT-ATP, 2'-(or-3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate; BIC, a bicarbonate-free Ringer; CCH, carbachol; CLSM, confocal laser scanning microscope; H₂DIDS, 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				T. Nakamoto, D. A. Brown, M. A. Catalan, M. Gonzalez-Begne, V. G. Romanenko, and J. E. Melvin Purinergic P2X7 Receptors Mediate ATP-induced Saliva Secretion by the Mouse Submandibular Gland J. Biol. Chem., February 20, 2009; 284(8): 4815 - 4822. [Abstract] [Full Text] [PDF]

				D. Heitzmann and R. Warth Physiology and Pathophysiology of Potassium Channels in Gastrointestinal Epithelia Physiol Rev, July 1, 2008; 88(3): 1119 - 1182. [Abstract] [Full Text] [PDF]

				L. Tourneur, S. Mistou, A. Schmitt, and G. Chiocchia Adenosine Receptors Control a New Pathway of Fas-associated Death Domain Protein Expression Regulation by Secretion J. Biol. Chem., June 27, 2008; 283(26): 17929 - 17938. [Abstract] [Full Text] [PDF]

				E. C. Thrower, S. Osgood, C. A. Shugrue, T. R. Kolodecik, A. M. Chaudhuri, J. R. Reeve Jr, S. J. Pandol, and F. S. Gorelick The novel protein kinase C isoforms -{delta} and -{varepsilon} modulate caerulein-induced zymogen activation in pancreatic acinar cells Am J Physiol Gastrointest Liver Physiol, June 1, 2008; 294(6): G1344 - G1353. [Abstract] [Full Text] [PDF]

				K. Ishibashi, K. Okamura, and J. Yamazaki Involvement of apical P2Y2 receptor-regulated CFTR activity in muscarinic stimulation of Cl- reabsorption in rat submandibular gland Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2008; 294(5): R1729 - R1736. [Abstract] [Full Text] [PDF]

				C. Hille and B. Walz Characterisation of neurotransmitter-induced electrolyte transport in cockroach salivary glands by intracellular Ca2+, Na+ and pH measurements in duct cells J. Exp. Biol., February 15, 2008; 211(4): 568 - 576. [Abstract] [Full Text] [PDF]

				S. M. Kreda, S. F. Okada, C. A. van Heusden, W. O'Neal, S. Gabriel, L. Abdullah, C. W. Davis, R. C. Boucher, and E. R. Lazarowski Coordinated release of nucleotides and mucin from human airway epithelial Calu-3 cells J. Physiol., October 1, 2007; 584(1): 245 - 259. [Abstract] [Full Text] [PDF]

				R. Corriden, P. A. Insel, and W. G. Junger A novel method using fluorescence microscopy for real-time assessment of ATP release from individual cells Am J Physiol Cell Physiol, October 1, 2007; 293(4): C1420 - C1425. [Abstract] [Full Text] [PDF]

				G. Burnstock Physiology and Pathophysiology of Purinergic Neurotransmission Physiol Rev, April 1, 2007; 87(2): 659 - 797. [Abstract] [Full Text] [PDF]

				G. G. Yegutkin, S. S. Samburski, S. Jalkanen, and I. Novak ATP-consuming and ATP-generating Enzymes Secreted by Pancreas J. Biol. Chem., October 6, 2006; 281(40): 29441 - 29447. [Abstract] [Full Text] [PDF]

				S.-R. Jung, K. Kim, B. Hille, T. D. Nguyen, and D.-S. Koh Pattern of Ca2+ increase determines the type of secretory mechanism activated in dog pancreatic duct epithelial cells J. Physiol., October 1, 2006; 576(1): 163 - 178. [Abstract] [Full Text] [PDF]

				M. P. Abbracchio, G. Burnstock, J.-M. Boeynaems, E. A. Barnard, J. L. Boyer, C. Kennedy, G. E. Knight, M. Fumagalli, C. Gachet, K. A. Jacobson, et al. International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy Pharmacol. Rev., September 1, 2006; 58(3): 281 - 341. [Abstract] [Full Text] [PDF]

				S. F. Okada, R. A. Nicholas, S. M. Kreda, E. R. Lazarowski, and R. C. Boucher Physiological Regulation of ATP Release at the Apical Surface of Human Airway Epithelia J. Biol. Chem., August 11, 2006; 281(32): 22992 - 23002. [Abstract] [Full Text] [PDF]

				G. G. Yegutkin, A. Mikhailov, S. S. Samburski, and S. Jalkanen The Detection of Micromolar Pericellular ATP Pool on Lymphocyte Surface by Using Lymphoid Ecto-Adenylate Kinase as Intrinsic ATP Sensor Mol. Biol. Cell, August 1, 2006; 17(8): 3378 - 3385. [Abstract] [Full Text] [PDF]

				N. Ullrich, A. Caplanusi, B. Brone, D. Hermans, E. Lariviere, B. Nilius, W. Van Driessche, and J. Eggermont Stimulation by caveolin-1 of the hypotonicity-induced release of taurine and ATP at basolateral, but not apical, membrane of Caco-2 cells Am J Physiol Cell Physiol, May 1, 2006; 290(5): C1287 - C1296. [Abstract] [Full Text] [PDF]

				S. Chivasa, B. K. Ndimba, W. J. Simon, K. Lindsey, and A. R. Slabas Extracellular ATP Functions as an Endogenous External Metabolite Regulating Plant Cell Viability PLANT CELL, November 1, 2005; 17(11): 3019 - 3034. [Abstract] [Full Text] [PDF]

				D. A. Brown, J. I. E. Bruce, S. V. Straub, and D. I. Yule cAMP Potentiates ATP-evoked Calcium Signaling in Human Parotid Acinar Cells J. Biol. Chem., September 17, 2004; 279(38): 39485 - 39494. [Abstract] [Full Text] [PDF]

				J. Peti-Peterdi, A. Fintha, A. L. Fuson, A. Tousson, and R. H. Chow Real-time imaging of renin release in vitro Am J Physiol Renal Physiol, August 1, 2004; 287(2): F329 - F335. [Abstract] [Full Text] [PDF]

				S.-R. Jung, M.-H. Kim, B. Hille, T. D. Nguyen, and D.-S. Koh Regulation of exocytosis by purinergic receptors in pancreatic duct epithelial cells Am J Physiol Cell Physiol, March 1, 2004; 286(3): C573 - C579. [Abstract] [Full Text]

				M. Yamasaki, R. Masgrau, A. J. Morgan, G. C. Churchill, S. Patel, S. J. H. Ashcroft, and A. Galione Organelle Selection Determines Agonist-specific Ca2+ Signals in Pancreatic Acinar and {beta} Cells J. Biol. Chem., February 20, 2004; 279(8): 7234 - 7240. [Abstract] [Full Text] [PDF]

				Q. Li, X. Luo, W. Zeng, and S. Muallem Cell-specific Behavior of P2X7 Receptors in Mouse Parotid Acinar and Duct Cells J. Biol. Chem., November 28, 2003; 278(48): 47554 - 47561. [Abstract] [Full Text] [PDF]

				E. R. Lazarowski, R. C. Boucher, and T. K. Harden Mechanisms of Release of Nucleotides and Integration of Their Action as P2X- and P2Y-Receptor Activating Molecules Mol. Pharmacol., October 1, 2003; 64(4): 785 - 795. [Full Text] [PDF]

				C. E Sorensen, J. Amstrup, H. N Rasmussen, I. Ankorina-Stark, and I. Novak Rat pancreas secretes particulate ecto-nucleotidase CD39 J. Physiol., September 15, 2003; 551(3): 881 - 892. [Abstract] [Full Text] [PDF]

				T. Henttinen, S. Jalkanen, and G. G. Yegutkin Adherent Leukocytes Prevent Adenosine Formation and Impair Endothelial Barrier Function by Ecto-5'-nucleotidase/CD73-dependent Mechanism J. Biol. Chem., June 27, 2003; 278(27): 24888 - 24895. [Abstract] [Full Text] [PDF]

				J. Leipziger Control of epithelial transport via luminal P2 receptors Am J Physiol Renal Physiol, March 1, 2003; 284(3): F419 - F432. [Abstract] [Full Text] [PDF]

				J. Arreola and J. E Melvin A novel chloride conductance activated by extracellular ATP in mouse parotid acinar cells J. Physiol., February 15, 2003; 547(1): 197 - 208. [Abstract] [Full Text] [PDF]

				I. Novak ATP as a Signaling Molecule: the Exocrine Focus Physiology, February 1, 2003; 18(1): 12 - 17. [Abstract] [Full Text] [PDF]

				W. Namkung, J. A. Lee, W. Ahn, W. Han, S. W. Kwon, D. S. Ahn, K. H. Kim, and M. G. Lee Ca2+ Activates Cystic Fibrosis Transmembrane Conductance Regulator- and Cl--dependent HCO3 Transport in Pancreatic Duct Cells J. Biol. Chem., January 3, 2003; 278(1): 200 - 207. [Abstract] [Full Text] [PDF]

				R. A. North Molecular Physiology of P2X Receptors Physiol Rev, October 1, 2002; 82(4): 1013 - 1067. [Abstract] [Full Text] [PDF]

				F. Thevenod Ion channels in secretory granules of the pancreas and their role in exocytosis and release of secretory proteins Am J Physiol Cell Physiol, September 1, 2002; 283(3): C651 - C672. [Abstract] [Full Text] [PDF]

				M. Idzko, S. Dichmann, D. Ferrari, F. Di Virgilio, A. la Sala, G. Girolomoni, E. Panther, and J. Norgauer Nucleotides induce chemotaxis and actin polymerization in immature but not mature human dendritic cells via activation of pertussis toxin-sensitive P2y receptors Blood, July 18, 2002; 100(3): 925 - 932. [Abstract] [Full Text] [PDF]

				A. la Sala, S. Sebastiani, D. Ferrari, F. Di Virgilio, M. Idzko, J. Norgauer, and G. Girolomoni Dendritic cells exposed to extracellular adenosine triphosphate acquire the migratory properties of mature cells and show a reduced capacity to attract type 1 T lymphocytes Blood, March 1, 2002; 99(5): 1715 - 1722. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 276/35/32925    most recent M103313200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sørensen, C. E. Articles by Novak, I. Search for Related Content PubMed PubMed Citation Articles by Sørensen, C. E. Articles by Novak, I. Social Bookmarking                      What's this?