Investigation of the roles of Ca2+ and InsP3 diffusion in the coordination of Ca2+ signals between connected hepatocytes
Caroline Clair1,
Cécile Chalumeau2,
Thierry Tordjmann1,
Josiane Poggioli2,
Christophe Erneux3,
Geneviève Dupont4 and
Laurent Combettes1
1 INSERM U442, Université de Paris-Sud, bât 443, 91405 Orsay, France
2 INSERM U356, IFR 58, Institut des Cordeliers, 15 rue de l’Ecole 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
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Fig. 1. Focal application of ionomycin to connected rat hepatocytes. Hepatocytes were injected with fura2. The two parts of the figure show successive measures of [Ca2+]i in the same hepatocyte doublet and are representative of those obtained using 4 doublets in 3 independent experiments. The first part of the figure shows the Ca2+ response when one cell within the doublet was focally microperfused with ionomycin (500 nM in the micropipette) for the time shown by the upper horizontal bars. In these conditions, only the stimulated cell (indicated by arrow) within the doublet responded. Following ionomycin wash out, global superfusion of the doublet with noradrenaline (1 µM) induced tightly coordinated [Ca2+]i oscillations in both cells. For technical convenience, tracings were interrupted (the gap represents 3 minutes).
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Fig. 2. Absence of apparent Ca2+ diffusion between connected rat hepatocytes. Hepatocyte doublets were injected with fura2 and heparin (10 mg/ml in the pipette) as described in methods section. After injection, perfusion (for the time shown by the horizontal bars) of high concentration of noradrenaline (Nor; 10 µM, A) or vasopressin (Vp; 10 nM, B), induced a rapid and strong increase in [Ca2+]i in the noninjected cell only. Although this [Ca2+]i increase was maintained for more than 3 minutes in the responding cell, no diffusion of Ca2+ was observed in the connected cell injected with heparin. These results are representative of those obtained using 10 doublets in four independent experiments.
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Fig. 3. Simultaneous determination of gap-junction permeability and [Ca2+]i increase in hepatocyte doublets. Hepatocytes loaded with calcein and Ca2+-orange were imaged by confocal microscopy (as described in Materials and Methods) and appear brightly fluorescent (C). Panel A shows the time course of vasopressin (Vp)-induced [Ca2+]i increase in a doublet of hepatocytes observed at 580 nm using Ca2+-orange excited at 546 nm. Simultaneous determination at 520 nm of calcein fluorescence excited at 488 nm is shown in B (note that traces have been shifted for clarity). In contrast to Ca2+-orange, the small molecular weight of calcein allows it to diffuse freely across gap junctions so that gap-junction permeability can be evaluated by fluorescence recovery after photobleaching (FRAP). Basal fluorescence level of the two dyes was measured for 30 seconds. Then perfusion of Vp (10 nM) induced a rapid and maintained [Ca2+]i increase in both cells of the doublet (A). After about 1 minute the cell indicated as cell 2 was bleached by focused 100% intensity laser excitation at 488 nm for 15 seconds (arrows and open box). Subsequently, the cell’s fluorescence recovered by diffusion of unbleached dye from the adjacent cell through the gap junctions (B, traces 1 and 2; see C). Note that fluorescence of Ca2+-orange was slightly affected by photobleaching, as indicated by the small decrease in fluorescence intensity observed at 580 nm (panel A, trace 2) and the fact that the photobleached cell appeared greener after recovery (C, c). Panel C shows a merged image of a doublet of hepatocytes loaded with calcein and Ca2+-orange before (a), immediately (b) and about 2 minutes after photobleach (c). Cell 2 was photobleached as described above.
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Fig. 5. Coordination of Ca2+ oscillations increase with the increase of agonist concentration in connected hepatocytes. Hepatocytes were loaded or injected with fura2. The figures show successive measures of [Ca2+]i in the same hepatocyte triplet. Cells were sequentially stimulated with increasing concentrations of noradrenaline (0.02, 0.05 and 0.1 µM) for the time shown by the horizontal bars. Addition of the lowest dose of noradrenaline to the bath was followed by oscillations in the three cells that were not coordinated (left). After washing, addition of a higher concentration of noradrenaline (0.05 µM) induced coordinated Ca2+ oscillations (middle part). Finally, very well coordinated oscillations were observed in the presence of 0.1 µM noradrenaline (right). The same results were obtained in hepatocyte doublets and are representative of those obtained using 12 triplets and 21 doublets in five independent experiments. Recording of the traces was interrupted during the washing process (5 minutes).
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Fig. 6. Increase in InsP3 receptor sensitivity induces coordination of Ca2+ oscillations in connected hepatocytes. Hepatocyte multiplets were loaded with fura2. Addition of a low concentration of noradrenaline (0.02 µM, A; 0.05 µM, B) for the time shown by the open box induced uncoordinated Ca2+ oscillations. However, when the same cells were incubated in the presence of 8Bromo-cAMP (10 µM, for the time shown by the black box; A), or when noradrenaline was perfused in the presence of thimerosal (10 µM, for the time shown by the black box; B), the same dose of noradrenaline elicited coordinated Ca2+ oscillations. The same results were obtained in hepatocyte doublets and are representative of those obtained using 7 triplets and 12 doublets in four independent experiments. For technical reasons, recording of the traces was interrupted during the washing process (3 minutes) and frame pairs were captured every 10 seconds during a part of thimerosal perfusion.
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Fig. 7. InsP3 5-phosphatase prevents coordination of Ca2+ oscillations induced by InsP3-dependent agonists in connected hepatocytes. Hepatocyte triplets loaded or injected with fura2 were challenged with noradrenaline (0.1 µM and 1 µM) for the time shown by horizontal bars. Tracings, representing [Ca2+]i in the three connected cells, have been shifted arbitrarily along the y-axis for clarity. For technical convenience, tracings were interrupted but left, middle and right parts of the figure show successive measurement of [Ca2+]i in the same hepatocyte triplet. In the left part, noradrenaline addition to the bath was followed by coordinated [Ca2+]i oscillations. The intermediate cell of the triplet was then injected with InsP3 5-phosphatase, as described in Materials and Methods. After injection (middle), application of noradrenaline did not elevate [Ca2+]i in the injected cell. By contrast, noradrenaline still elicited Ca2+ oscillations, but these oscillations were not coordinated at all. Finally, at the end of the experiment, the same triplet was challenged with a higher concentration of noradrenaline (1 µM), which elicited a Ca2+ response in the three connected cells (right). Note the oscillatory pattern for the InsP3 5-phosphatase injected cell. These results are representative of those obtained using 9 triplets in three independent experiments.
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© The Company of Biologists Ltd 2001
). As shown here, recovery was large (>60%) and rapid (
100 seconds) in the control experiment. By contrast, octanol reduced almost totally the fluorescence recovery after photobleach.