1 INSERM U442, Université de Paris-Sud, bât 443, 91405 Orsay, France
2 INSERM U356, IFR 58, Institut des Cordeliers, 15 rue de lEcole de Médecine, 75270 Paris, France
3 Université Libre de Bruxelles, IRIBHN, Faculté de Médecine, Campus Erasme, Route de Lennik 808, B-1070 Brussels, Belgium
4 Université Libre de Bruxelles, Faculté des Sciences CP231, Boulevard du Triomphe, B-1050 Brussels, Belgium
Author for correspondence (e-mail: laurent.combettes{at}ibaic.u-psud.fr)
Accepted February 27, 2001
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SUMMARY |
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Key words: Calcium oscillations, Waves, Liver, Gap junctions
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INTRODUCTION |
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In the liver, coordinated intercellular Ca2+ waves were first observed in doublets or triplets of freshly isolated hepatocytes (Nathanson and Burgstahler, 1992; Combettes et al., 1994), and have more recently been demonstrated in the isolated intact perfused liver (Nathanson et al., 1995; Robbgaspers and Thomas 1995; Motoyama et al., 1999; Patel et al., 1999). It is well known that cells in a tissue may communicate directly via gap junctions (the junctional coupling pathway) or indirectly via a chemical messenger that is released by the cell into the extracellular medium, where it stimulates a target cell (paracrine pathway). Intercellular Ca2+ waves may be propagated via one or both of these pathways. For example, hepatocytes can communicate both through gap junctions (Saez et al., 1989) and via paracrine factors such as ATP (Schlosser et al., 1996). However, numerous results obtained on isolated multicellular systems of rat hepatocytes (doublets or triplets of cells tightly connected by gap junctions) argue against a role for the paracrine pathway in the propagation and the coordination of the intercellular Ca2+ waves. Indeed, propagation of Ca2+ waves between isolated hepatocytes or among connected hepatocytes within multiplets following mechanical stimulation of one cell disappears as soon as the preparation is perfused (Schlosser et al., 1996; Tordjmann et al., 1997). By contrast, perfusion of InsP3-dependent agonists such as vasopressin or noradrenaline induces coordinated intercellular Ca2+ waves that are inhibited by selectively blocking gap-junction coupling (Nathanson and Burgstahler, 1992; Tordjmann et al., 1997). Thus, it is clear that functional gap junctions are fundamental to ensure the coordination of these Ca2+ signals. However, the mechanisms ensuring this coordination are not precisely known and are still a matter of debate (Höfer, 1999; Dupont et al., 2000).
Ca2+ and InsP3 are the two most likely candidates for the intercellular messenger involved in the propagation and coordination of intercellular Ca2+ waves in connected hepatocytes. It has been shown, especially in doublets of hepatocytes, that Ca2+ ions and InsP3 can spread from one cell to another through gap junctions (Saez et al., 1989; Niessen and Willecke, 2000; reviewed by Sanderson et al., 1994). Moreover, both second messengers are thought to induce an autocatalytic increase in [Ca2+]i (reviewed by Taylor, 1998). In fact, in a large number of different cultured cell types, it has been shown that propagation of Ca2+ waves induced by mechanical stimulation of one cell probably relies on the progressive diffusion of the InsP3, massively synthesized in the stimulated cell, to its neighbors (Sanderson, 1995; Sneyd et al., 1995). However, experimental results obtained in multiplets of connected hepatocytes suggest that such a mechanism cannot account for the coordination of apparent intercellular Ca2+ waves in these cells. The main difference between hepatocytes and the other tissues is that, although gap-junction permeability is essential for coordinating Ca2+ oscillations in the coupled cells, each hepatocyte needs to be stimulated to display Ca2+ oscillations. Focal stimulation of one cell within a doublet by submaximal concentrations of InsP3-dependent agonists, such as vasopressin or noradrenaline, does not induce a Ca2+ increase in the adjacent cells (Tordjmann et al., 1997). Two recent independent studies using different mathematical models propose that intercellular Ca2+ waves arise because each individual hepatocyte in multiplets displays repetitive Ca2+ spikes with a slight phase-shift with respect to neighboring cells; both studies emphasize the crucial role of gap-junction coupling to coordinate these Ca2+ spikes (Höfer, 1999; Dupont et al., 2000). However, whereas it is suggested in the first model that synchronization requires gap-junctional diffusion of cytosolic Ca2+ (Höfer, 1999), it is proposed in the other model that Ca2+ spikes are coordinated by the junctional diffusion of small amounts of InsP3 (Dupont et al., 2000). In the present experimental study, the purpose was to determine the respective roles of Ca2+ and InsP3 in the coordination of Ca2+ oscillations in hepatocytes. Experiments were performed on doublets and triplets of hepatocytes, which are real fragments of the liver cell plate. Our results indicate that gap-junctional diffusion of InsP3 plays a key role in the coordination of Ca2+ signals among connected hepatocytes.
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MATERIALS AND METHODS |
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Preparation of hepatocytes
Isolated rat hepatocytes were prepared from fed female Wistar rats by limited collagenase digestion of rat liver, as previously described (Combettes et al., 1994). In these conditions, about 20% cells were associated by two (doublet) or three (triplet) and were distinguished from aggregates of non-connected cells in conventional light microscopy by screening for dilated bile canaliculi, indicators of maintained functional polarity (Gautam et al., 1987). After isolation, rat hepatocytes were maintained (5x105 cells/ml) at 4°C in Williams medium E supplemented with 10% foetal calf serum, penicillin (100,000 units/ml) and streptomycin (100 µg/ml). Cell viability, assessed by trypan blue exclusion, remained greater than 96% for 4-5 hours.
Measurement of intracellular Ca2+ in individual cells
Loading of hepatocytes with fura2
Hepatocytes were loaded with fura2, as previously described (Tordjmann et al., 1997), either by injection (see below) or by incubation with the dye. Small aliquots of the suspended hepatocytes (5x105 cells) were diluted in 2 ml of Williams medium E modified as described above, then plated onto dish glass coverslips coated with collagen I, and incubated for 60 minutes at 37°C under an atmosphere containing 5% CO2. After cell plating, the medium was removed and replaced with a medium containing 3 µM fura2/AM or fura2PE3/AM for 30 minutes at 37°C under an atmosphere containing 5% CO2. The coverslips were then washed twice with a saline solution (20 mM HEPES, 116 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 0.96 mM NaH2PO4, 5 mM NaHCO3, and glucose 1 g/l, pH 7.4). Dish coverslips were put onto a thermostatted holder (34°C) on the stage of a Zeiss Axiovert 35 microscope set up for epifluorescence microscopy.
Microinjection
Microinjection was performed using an Eppendorf microinjector (5242), as described previously (Tordjmann et al., 1997). Micropipettes with an internal tip diameter of 0.5 µm (Femtotips, Eppendorf) were filled with test agents together with 5 mM fura2 in a buffer solution containing 100 mM KCl, 20 mM NaCl, 10 mM Hepes adjusted to pH 7.1. After microinjection, cells were allowed to recover for at least 10 minutes. The success of microinjection was assessed by monitoring the morphology of cells before and after manipulation and checking the ability of the cell to retain injected fura2 and low [Ca2+]i. Purification and determination of activity of the recombinant type I InsP3 5-phosphatase (19 µmol/minute/ml in this study) was performed as described previously (Communi et al., 1996; De Smedt et al., 1997). InsP3 5-phosphatase was inactivated at 90°C for 20 minutes. Cells were microinjected either with inactivated InsP3 5-phosphatase or with InsP3 5-phosphatase (activity: 120 nmol/min/ml in the pipette) as described above.
Determination of [Ca2+]i changes in hepatocytes
Ca2+ imaging was as described previously (Tordjmann et al., 1997). Briefly, the excitation light was supplied by a high pressure xenon arc lamp (75 watt), and the excitation wavelengths were selected by 340 and 380 nm filters (10 nm bandwidth) mounted in a processor-controlled rotating filter wheel (Sutter, Novato, CA) between the UV lamp and the microscope. Fluorescence images were collected either by a low-light level ISIT camera (Lhesa Électronique, Cergy Pontoise, France) or a CCD camera (Princeton Instruments, Evry, France), digitized and integrated in real time by an image processor (Metafluor, Princeton Instruments).
Simultaneous determination of gap-junction permeability and [Ca2+]i changes in hepatocytes multiplets
Gap-junction permeability was determined in hepatocyte multiplets using fluorescence recovery after photobleaching (FRAP). These experiments were performed on an inverted confocal microscope (Zeiss, LSM510) using the fluorescent tracer calcein, a low molecular weight (623 Da) fluorescent dye that has been previously reported to be an effective marker for gap-junction communication (Tomasetto et al., 1993; Jacobi et al., 1998). To avoid cell injury, this fluorescent compound can be loaded into cultured cells in its esterified form, calcein/AM. [Ca2+]i changes were followed with the fluorescent Ca2+-sensitive dye Ca2+-orange. Because the excitation and emission characteristics of calcein (ex=494 nm;
em=520 nm) do not significantly overlap with those of Ca2+-orange (
ex=546 nm;
em=580 nm), both the [Ca2+]i and the spread of calcein can be simultaneously evaluated. Thus, hepatocytes were incubated with calcein/AM (3 µM) and Ca2+-orange/AM (5 µM) for 20 minutes at 37°C. Cells were then washed three times and incubated for 10-20 minutes to allow complete ester hydrolysis. Calcein and Ca2+-orange fluorescence were then observed at 520 nm and 580 nm, respectively, after excitation was provided by the 488 nm and the 546 nm lines of a krypton/argon laser. The AOTF system of the Zeiss confocal microscope allows use of the same Argon-Krypton laser to both image and bleach the fluorescence in numerous cells of interest. After baseline fluorescence images were obtained at each wavelength, agents were added and change in [Ca2+]i was monitored on line by following the change in Ca2+-orange fluorescence (see Fig. 3). Then, the fluorescence emitted by calcein was bleached by exposure of a given cell to high (100%) intensity laser light (488 nm) close to the calcein excitation maximum (494 nm). Complete or almost complete photobleaching was achieved after 30 scans for a total duration of 15 seconds (Fig. 3). Recovery of fluorescence in the bleached cell was monitored for 8 minutes. After correction and normalization, fluorescence was plotted over time to generate fluorescence recovery curves which were fitted to an exponential function using a scientific plotting program (Origin, MicroCal). Analysis allow us to obtain two values: the degree of recovery (%) and the rate of recovery.
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RESULTS AND DISCUSSION |
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Focal application of ionomycin
Focal application of ionomycin was achieved by positioning a glass micropipette close to the cell of interest and applying a constant pressure via the Eppendorf injector, delivering picoliter quantities of ionomycin-containing solution (500 nM). As shown in Fig. 1 (left panel), the ionomycin-microperfused cell exhibited a rapid and high [Ca2+]i rise; by contrast, [Ca2+]i remained at a basal low level in the non-microperfused cell. However, after ionomycin was washed away, global perfusion of noradrenaline (1 µM) induced well coordinated [Ca2+]i oscillations within the two cells (right panel).
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To investigate this possibility, gap-junction permeability was quantified in dying hepatocyte doublets using FRAP in the presence or absence of high agonist concentrations. This technique has the advantage over other methods to be relatively non-invasive and quantitative. Indeed, when cells are loaded with a low molecular weight fluorescent probe (1 kDa), the recovery of fluorescence in the bleached target cell will reflect the influx of dye from the connected unbleached cells (Wade et al., 1986; Meyvis et al., 1999). Then, the rate of refill is a measure of gap-junction permeability. In the present study, hepatocytes were loaded with calcein, a low molecular weight (623 Da) fluorescent dye that has been previously reported to be an effective marker for gap-junction communication (Tomasetto et al., 1993; Jacobi et al., 1998) and Ca2+-orange as an intracellular Ca2+ indicator. A representative confocal image of an hepatocyte doublet loaded with calcein and Ca2+-orange is shown in Fig. 3C.
First, the baseline fluorescence of a doublet was obtained simultaneously for each dye by confocal microscopy (see Materials and Methods). After about 30 seconds, a maximal concentration of agonist was perfused. As revealed by the fluorescence increase of Ca2+-orange, perfusion of vasopressin (10 nM) induced, as usual, a sustained [Ca2+]i increase in the two cells (Fig. 3A). Then, as described in Materials and Methods, the fluorescence emitted by calcein was bleached in one cell (indicated as cell 2 in Fig. 3) by selectively exposing the latter cell to the full laser power at 488 nm. This resulted in the nearly complete ablation of the cells calcein fluorescence (Fig. 3C,b; Fig. 3B, trace 2). Note that, in accordance with its excitation wavelength characteristic, Ca2+-orange was not greatly affected by this strong illumination at 488 nm (Fig. 3C; Fig. 3A, trace 2). Subsequently, the microscope settings were returned to the recording configuration, and the refill was monitored (Fig. 3B). The fluorescence recovery was then plotted and fitted to an exponential function (Fig. 4), yielding two characteristic values: the degree (%) and the rate (seconds) of recovery. As shown in Fig. 4 and summarized in Table 1, hepatocyte doublets from control experiments were extensively interconnected by gap junctions, as they showed a rapid and high degree of recovery. As expected for a gap-junction-dependent effect, octanol (0.5 mM) nearly totally blocked this refill (Fig. 4; Table 1). At this concentration of octanol, the effect of gap-junction cell coupling was reversible as octanol could be washed out and coupling restored (data not shown). By contrast, in vasopressin (10 nM)- or noradrenaline (10 µM)-treated hepatocytes, we found that coupling was similar to control experiments, suggesting that stimulation with these maximal concentrations of agonists had no effect on gap-junction permeability (Table 1). Thus, it can be concluded that the passage of small signaling molecules is possible under conditions of physiological stimulation.
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Nevertheless, it should be remembered that coordination could extend across an intermediate cell in which the release of intracellular Ca2+ was inhibited by heparin, at least for the first few spikes following stimulation (Tordjmann et al., 1997). This suggests that an increase in Ca2+ is not crucial for the coordination of Ca2+ oscillations.
Investigation of the possible role of InsP3 as a coordinating messenger for Ca2+ spiking among connected hepatocytes
Ca2+ signals in response to increasing agonist concentrations
The other likely candidate that could be responsible for the coordination of Ca2+ spiking among connected hepatocytes is InsP3. If junctional InsP3 diffusion is involved, it can be expected that the degree of coupling increases with the level of InsP3, and thus also with the concentration of the agonist. To test this hypothesis, hepatocyte multiplets were perfused with increasing concentrations of InsP3-dependent agonists (Fig. 5). Note that the proportion of responsive cells in these conditions was low (30-50%). Nevertheless, application of very low concentrations of noradrenaline (0.02 µM), to doublet or triplet of hepatocytes sometimes induced Ca2+ oscillations (Fig. 5 and data not shown). However, these oscillations were not coordinated, as if the cells were not functionally coupled by gap junctions (each cell having its own oscillation frequency) even though gap junctions efficiently connected these cells as they were loaded by cell to cell diffusion of microinjected fura2. Raising the concentration of noradrenaline (0.05 µM) not only increased the frequency of the Ca2+ oscillations as expected (see blue and green traces in Fig. 5 for example), but also led to coordinated oscillations (Fig. 5, middle panel). Finally, perfusion of a higher concentration of noradrenaline (0.1 µM) induced a good coordination of the Ca2+ oscillations among the connected hepatocytes. Because, in contrast with the amplitude of Ca2+ oscillations, the InsP3 level increases with increasing concentrations of agonists (Thomas et al., 1991; Thomas et al., 1996), these results suggest that InsP3, rather than Ca2+, is responsible for the coordination of Ca2+ oscillations between connected hepatocytes. At low stimulation, production of InsP3 is indeed too small to allow for an efficient junctional InsP3 diffusion, and the cells thus oscillate at their own intrinsic frequencies; raising the level of InsP3, via the increase of agonist concentrations, allows the diffusion of significant amounts of InsP3 through gap junctions, a phenomenon that can account for the observed coordination in Ca2+ oscillations (Dupont et al., 2000).
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As described previously, InsP3-dependent agonists at low concentration induced uncoordinated Ca2+ oscillations among connected hepatocytes (Fig. 6, left panels). However, when cells were incubated with 8Bromo-cAMP (10 µM) for 5 minutes we observed that, in 70% of all the multiplets analyzed (15 doublets and 8 triplets), re-addition of the same low concentration of vasopressin or noradrenaline, induced well-coordinated [Ca2+]i oscillations (Fig. 6A and data not shown). This suggest that in these conditions, the small quantity of InsP3 diffusing through gap junctions was sufficient to coordinate Ca2+ oscillations. Alternatively, it has been shown that cAMP can increase the permeability of gap junctions (Loewenstein, 1985; Burghardt, 1995), an effect that could account for coordination of Ca2+ signals. Although the concentration of 8Bromo-cAMP required to increase gap-junction permeability is usually higher (0.1-1 mM) than that used in this study (10 µM), this hypothesis has been checked. Gap-junction permeability was quantified using FRAP in dying hepatocytes doublets incubated for 5 minutes with or without 8Bromo-cAMP (10 µM). Results are summarized in Table 1 and show that gap-junction permeability was not significantly affected by 8Bromo-cAMP. Thus, the cAMP-induced increase in the degree of coordination of Ca2+ oscillations observed at low levels of stimulation might result from the shift in the sensitivity of the InsP3R.
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Thus, both cAMP and thimerosal significantly increased the level of coordination of Ca2+ spiking between connected hepatocytes at low agonist concentrations. Our interpretation of the latter results is that a shift in the affinity of the InsP3Rs for InsP3 potentiates the coordinating effect of the small amounts of InsP3 flowing through gap junctions. It should be emphasized that the increase in coordination by cAMP and thimerosal would not be predicted by a model based on Ca2+ diffusion through gap junctions, as the amplitude of Ca2+ oscillations does not depend on the presence of cAMP or thimerosal.
Effect of the increase of InsP3 5-phosphatase activity in hepatocytes
Finally, we have studied the role of a putative InsP3 intercellular diffusion in synchronization of Ca2+ oscillations by microinjection of type I InsP3 5-phosphatase in the intermediate cell of a triplet. Type I InsP3 5-phosphatase is the most widespread InsP3 5-phosphatase and efficiently metabolizes InsP3 to produce inositol 1,4-bisphosphate, which does not mobilize Ca2+ (Putney et al., 1989; Erneux et al., 1989; Verjans et al., 1994). It has been shown recently that over-expression of this enzyme in CHO cells deeply affected the pattern of Ca2+ oscillations; in some cases it even abolished the stimulus-induced Ca2+ signal (De Smedt et al., 1997). In the present study, we thus used microinjected InsP3 5-phosphatase to specifically decrease the level of InsP3 in a particular hepatocyte. Triplets were first loaded with fura2 and treated with noradrenaline (0.1 µM), which elicited trains of coordinated Ca2+ oscillations in the three connected cells (Fig. 7, left panel). After washing out noradrenaline, the intermediate cells of the triplets were microinjected with InsP3 5-phosphatase, as described in Materials and Methods. In the vast majority of injected multiplets (9/13), the renewed superfusion of noradrenaline (0.1 µM) elicited Ca2+ oscillations in the non-injected cells only, whereas Ca2+ remained at a low basal level in the InsP3 5-phosphatase-injected cell. In the remaining injected cells (4 out of 13), the frequency of oscillations was strongly reduced (data not shown). In all cases, Ca2+ oscillations in the two remaining responding cells appear uncoordinated (n=13 triplets). It is worth noting that InsP3 5-phosphatase did not inhibit gap-junction permeability because, when fura2 was co-injected with 5-phosphatase into the intermediate cells of the triplets, the dye diffused into the two adjacent connected cells and again, no coordination was observed in the two remaining responding cells (n=3, data not shown).
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To summarize, there is no coordination of Ca2+ oscillations when the production of InsP3 is too low, that is, in the presence of low concentrations of agonist or when InsP3 is rapidly metabolized (by InsP3 5-phosphatase). By contrast, coordination can be achieved either by increasing the InsP3 concentration (via a rise in agonist concentration) or by increasing the affinity of the InsP3 receptor for InsP3, even at a very low levels of stimulation.
In conclusion, we have shown that in doublets of hepatocytes connected by gap junctions, a high and sustained increase in [Ca2+]i does not induce any detectable [Ca2+]i rise in the adjacent cells, although gap junctions are fully operable. Similar results have been obtained in other cell types (Demer et al., 1993; Yule et al., 1996; reviewed by Sanderson et al., 1994) and are in agreement with the poor ability of Ca2+ to diffuse over a long distance (Albritton et al., 1992). However, because Ca2+ is known to be the principal regulator of its own release through an increase in the effect of InsP3 on its receptor (reviewed by Taylor, 1998), it has been suggested in other cell types, such as aortic endothelial cells, pancreatic acinar cells or articular chondrocytes, that the diffusion of small amounts of Ca2+ between adjacent cells could serve to sensitize InsP3R in the neighboring cells and thus allow for the intercellular propagation of the signal (Yule et al., 1996; DAndrea and Vittur, 1997; reviewed by Tordjmann et al., 2000).
Our results in hepatocytes cannot exclude any role for Ca2+ in the coordination of Ca2+ spiking in this cell type. It remains plausible that diffusion of Ca2+ might play a role when the Ca2+ sequestration mechanism is partly impaired (i.e. at high levels of InsP3). In this case, intercellular diffusion of Ca2+ could smooth out the differences in Ca2+ signals among connected cells caused by possible heterogeneities in cell shape or in the diverse InsP3-independent fluxes (Höfer, 1999). Our results show however, that even if such Ca2+-activated Ca2+ release through InsP3R takes place in hepatocytes (Taylor, 1998), it is not crucial for the coordination of Ca2+ oscillations induced by InsP3-dependent agonists among connected cells. By contrast, coordination of Ca2+ oscillations among connected hepatocytes is fully dependent on the level of InsP3 diffusing from cell to cell.
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ACKNOWLEDGMENTS |
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