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First published online April 18, 2006
Journal of Experimental Biology 209, 1716-1724 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02187
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Fluorescence measurements of serotonin-induced V-ATPase-dependent pH changes at the luminal surface in salivary glands of the blowfly Calliphora vicina

Julia Rein1, Bernhard Zimmermann1,2, Carsten Hille1, Ingo Lang1,*, Bernd Walz1 and Otto Baumann1,{dagger}

1 Institut für Biochemie und Biologie, Universität Potsdam, D-14415 Potsdam, Germany
2 Carl Zeiss Jena GmbH, Advanced Imaging Microscopy, D-07745 Jena, Germany


Figure 1
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  		Fig. 1. (A,B) Confocal fluorescence images of a live salivary gland incubated with 30 µmol l–1 HAF in PS. The optical section plane through the gland tubule is indicated (upper right of each image). The broken lines in B indicate the luminal surface of the salivary gland, as deduced from background fluorescence of the cells. HAF staining is present on the basal infoldings (broad arrows) and on the lateral domain of the plasma membrane (arrowheads). Note that staining ends abruptly halfway along the lateral domain, indicating that HAF does not diffuse across septate junctions. (C) The structural organization of the secretory portion of the salivary gland. Asterisks, lumen of the gland; arrowhead, septate junction; long arrow, apical infoldings (=canaliculi); broad arrow, basal labyrinth. Bar, 20 µm.

 

Figure 2
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  		Fig. 2. HAF in the apical membrane domain. (A) Timed series of confocal images through a live salivary gland after pressure-injection of 30 µmol l–1 HAF in PS into its lumen. The first image was recorded ~3 min after dye injection. Subsequent images were taken at an interval of 3.2 min. Note that fluorescence intensity gradually rises within the first four images, indicating the insertion of more dye molecules into the membrane. (B,C) Confocal images of a live salivary gland ~20 min after injection of HAF into its lumen (asterisks). The optical section plane is indicated (upper right of each image). HAF staining is present on the luminal surface of the secretory cells and extends into the canaliculi, which are deep infoldings of the apical membrane (arrows). Double arrows indicate tracheoles that reside on the outer surface of the gland and that exhibit autofluorescence. (D,E) Confocal images through a chemically fixed, Oregon Green–Phalloidin-stained salivary gland (shown for comparative purposes). Phalloidin-labelled actin filaments line the canaliculi (long arrows). Asterisks indicate the lumen of the gland, and broad arrows indicate F-actin at the basal surface. Bars, 50 µm (A); 20 µm (B–E).

 

Figure 3
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  		Fig. 3. pH-dependent changes in HAF fluorescence. A salivary gland was incubated for 5 min with 30 µmol l–1 HAF in PS in order to label the basolateral surface of the epithelial cells and was then superfused with PS of different pH as indicated. (A) HAF fluorescence upon excitation at 470 nm and 410 nm. (B) Ratio of the fluorescence signals obtained at the excitation wavelengths of 470 nm and 410 nm (F470/F410). Acidification is accompanied by a decrease in the ratio and alkalization by an increase in F470/F410.

 

Figure 4
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  		Fig. 4. Spatiotemporal analysis of pH changes at the luminal surface evoked by 30 nmol l–1 5-HT. (A) Series of pseudocolour images of the F470/F410 ratio in a salivary gland with HAF in the apical membrane. The squares in the first image indicate the areas selected to generate the graph. +5-HT, beginning of the 5-HT stimulus; –5-HT, washout of 5-HT. Note that the F470/F410 ratio laterally along the salivary gland tube (asterisks) is not linked to pH because it results from cellular autofluorescence rather than HAF fluorescence. Only F470/F410 in the mid-region of the salivary gland results from HAF and thus relates to luminal surface pH. Image intervals, 50 s; scale bar, 50 µm. (B) Time course of 5-HT-induced changes in luminal surface pH in four different regions along a salivary gland (squares in A).

 

Figure 5
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  		Fig. 5. Dose–response curve for 5-HT-induced changes in luminal surface pH. (A–C) Each salivary gland with HAF at its apical membrane domain was exposed first to 30 nmol l–1 5-HT (reference stimulus), then to a test stimulus of variable 5-HT concentration. An area of about 20 µmx20 µm, representing the size of one cell, was selected in the field of view to generate the graph. Note that the luminal compartment acidified reversibly in response to a 5-HT stimulus. (D) The decrease in F470/F410 ratio evoked by the test stimulus was normalized to the F470/F410 change induced by the reference stimulus. Values represent means ± s.d. of 5 experiments for each concentration.

 

Figure 6
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  		Fig. 6. Effect of concanamycin A on 5-HT-induced pH changes for salivary glands with HAF in their apical membrane domain. (A) Incubation in 1 µmol l–1 concanamycin A/0.02% DMSO (ConA) inhibited the 5-HT-induced changes in F470/F410 ratio almost completely. (B) Control gland, exposed to the same experimental conditions but treated with DMSO only. (C) Quantitative analysis of the effect of concanamycin A (ConA) or 0.02% DMSO (Control) on the basal F470/F410 ratio. Values (means ± s.d., N=6) represent the difference in F470/F410 ratio before and after exposure to concanamycin A or DMSO, but before the second 5-HT stimulus. (D) Quantitative analysis of the effect of concanamycin A on the 5-HT-induced acidification. Response to 5-HT in the presence of concanamycin A/0.02% DMSO (ConA) or 0.02% DMSO only (Control) was normalized to the response induced by the initial 5-HT stimulus. Values represent means ± s.d. (N=6).

 

Figure 7
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  		Fig. 7. 5-HT-induced pH changes in the lumen of the salivary gland as measured with double-barrelled pH-sensitive microelectrodes. (A) Effects of three different 5-HT concentrations (1, 3 and 10 nmol l–1) on transepithelial potential (upper trace) and luminal pH (lower trace). The slightly positive transepithelial potential in the unstimulated gland became at least transiently negative upon 5-HT stimulation and displayed a large positive overshoot after 5-HT withdrawal as described previously (Berridge and Prince, 1971Go). In our hands the time courses of the transepithelial potential changes recorded by the reference barrel of the pH-sensitive microelectrodes differed slightly from those recorded by the classical paraffin oil-gap method (e.g. Berridge and Prince, 1971Go) for unknown reasons. However, similar differences were noted earlier in luminal recordings with double-barrelled K+-sensitive microelectrodes by the same group (see e.g. fig. 5 in Gupta et al., 1978Go). (B) Dose–response relationship of the 5-HT-induced pH changes obtained from recordings similar to those shown in A.

 




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