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Distribution and serotonin-induced activation of vacuolar-type H⁺-ATPase in the salivary glands of the blowfly Calliphora vicina

Bernhard Zimmermann^1,2, Petra Dames¹, Bernd Walz¹ and Otto Baumann^1,*

¹ Institut für Biochemie und Biologie, Zoophysiologie, Universität Potsdam, Lennéstr. 7a, D-14471 Potsdam, Germany
² Carl Zeiss Jena GmbH, Advanced Imaging Microscopy, Carl-Zeiss-Promenade 10, D-07745 Jena, Germany

View larger version (214K): [in a new window]   Fig. 1. Morphology of secretory cells in a high-pressure-frozen freeze-substituted salivary gland. (A) Part of a cross-section through a non-stimulated salivary gland. The gland is composed of a single layer of secretory cells and enclosed by a basal lamina (bl). The apical surface (a) of the secretory cells is covered by spatulate microvilli and invaginates deeply into the cell. Numerous mitochondria (m) reside close to the apical (a) and the basolateral (b) domain of the plasma membrane. lu, lumen of the salivary gland; nu, nucleus. (B) High-magnification view of spatulate microvilli on the secretory cell apical domain. The cytoplasmic aspect of the plasma membrane is studded with particles (arrowheads). In most areas of the plasma membrane, these particles are too densely packed to be revealed individually (arrows). (C) The basolateral domain of the plasma membrane lacks such particles. Scale bars, 2.5 µm (A), 200 nm (B,C).

View larger version (23K): [in a new window]   Fig. 2. Detection of Na+/K+-ATPase and V-ATPase within salivary glands. Western blots of salivary gland proteins were probed with an antibody against Na+/K+-ATPase -subunit and antibodies against various subunits of V-ATPase. All antibodies intensely label a band of the appropriate molecular mass.

View larger version (13K): [in a new window]   Fig. 3. Bafilomycin A1-sensitive and ouabain-sensitive ATPase activity in homogenized salivary glands. (A) Dose–response curve for the effect of bafilomycin A1 on the total ATPase activity within the homogenate. Values represent the means ± S.D. of 4–11 experiments for each concentration. Half-maximal inhibition (IC50) is reached at approximately 3 nmol l–1 bafilomycin A1. (B) Effect of 5-hydroxytryptamine (5-HT) on total ATPase activity, bafilomycin-sensitive ATPase activity and ouabain-sensitive ATPase activity. Values represent the mean ± S.D. of 5–16 experiments; asterisks indicate a significance level of P<0.001. In 5-HT-stimulated salivary glands, bafilomycin-sensitive ATPase activity is enhanced by a factor of approximately 2, whereas ouabain-sensitive ATPase activity does not change significantly.

View larger version (116K): [in a new window]   Fig. 4. Immunofluorescence localization of Na+/K+-ATPase and V-ATPase. (A,E,I) Labelling with OregonGreen–phalloidin (green) reveals the organization of the apical domain of the secretory cells. (A) An optical cross-section through an entire salivary gland and (E,I) two horizontal planes through a gland as indicated by broken lines in (A). The apical surface of the secretory cells is involuted deeply into the epithelium to form a branching system of canaliculi (arrows) that embrace the DAPI-stained nuclei (blue in E,I). (B,F,J) Double-labelling of a longitudinal section through a salivary gland with antibody 5 against Na+/K+-ATPase -subunit (red) and phalloidin (green); (C,G,K) a longitudinal section co-stained with an antibody against V-ATPase subunit E (red) and phalloidin (green); (D,H,L) a cross-section co-labelled with an antibody against V-ATPase subunit d (red) and phalloidin (green); (J,K,L) composite images of phalloidin and antibody labelling. The basolateral domain but not the apical domain of the plasma membrane is intensely labelled for Na+/K+-ATPase. Antibodies against the various V-ATPase subunits stain the luminal side of the cell and the canaliculi (arrows). Moreover, some vesicular structures within the secretory cells are labelled by V-ATPase antibodies (yellow arrowheads in C,D,K,L). Immunofluorescence associated with the basal lamina (white arrowheads) surrounding the epithelial tubule is due to nonspecific binding of secondary antibodies. Scale bars, 20 µm.

View larger version (21K): [in a new window]   Fig. 5. Assembly state of V-ATPase in control and 5-hydroxytryptamine (5-HT)-stimulated salivary glands. The glands were homogenized and separated by high-speed centrifugation into a pellet fraction (P) and a supernatant (S). The relative distribution of various proteins between the two fractions was then probed by western blot analysis. Upon stimulation with 5-HT, the amount of the V1 component proteins (subunits A and E) increases within the pellet and diminishes in the supernatant. No significant change in response to 5-HT stimulation is observed in the distribution of integral membrane proteins (Na+/K+-ATPase -subunit; V0 component proteins, subunits a and d), of -tubulin or of the total amount of protein between both fractions. Values represent the mean ± S.D. of six independent experiments. Asterisks indicate a significant difference (P<0.001).