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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