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

Abstract

SUMMARYSecretory activity in blowfly salivary glands is activated by the hormone serotonin. We have investigated the distribution and activity of two cation pumps that are possibly involved with transepithelial ion transport, i.e. Na+/K+-ATPase and vacuolar-type H+-ATPase(V-ATPase). By immunofluorescence labelling of secretory cells,Na+/K+-ATPase was localized on the basolateral plasma membrane and V-ATPase on the highly folded apical membrane. Activities of both ATPases were probed in salivary gland homogenates by applying specific inhibitors for these ion pumps, namely ouabain and bafilomycin A1. In control glands, bafilomycin-A1-sensitive V-ATPase activity and ouabain-sensitive Na+/K+-ATPase activity accounted for 36% and 19%, respectively, of the total ATPase activity. V-ATPase activity increased approximately twofold after stimulation with serotonin, whereas Na+/K+-ATPase activity was not significantly affected. Biochemical assays provided evidence that the serotonin-induced activation of V-ATPase activity was accompanied by a recruitment of peripheral V1subunits from the cytosol to the plasma membrane, indicative of the assembly of V0V1 holoenzymes.These data show that a V-ATPase located in the apical plasma membranes of the secretory cells is a component of the apical `potassium pump' that has been identified previously by physiological approaches. The V-ATPase energizes the apical membrane and provides the primary driving force for fuelling a putative K+/nH+ antiporter and, thus, for fluid secretion. Serotonin-induced assembly of V0V1holoenzymes might constitute a regulatory mechanism for the control of pump activity.

Development of apical membrane organization and V-ATPase regulation in blowfly salivary glands

“…Cross-reactivity of these antibodies with the homologous proteins in C. vicina has been demonstrated previously (Zimmermann et al, 2003;Dames et al, 2006;Voss et al, 2007;Voss et al, 2009;Baumann and Walz, 2012). Monoclonal anti-actin (clone C4) was obtained from Merck (Darmstadt, Germany), rabbit antiserum (cat.…”

“…To determine the structural reorganization of the apical membrane domain during cell differentiation, cryosections through salivary glands at various developmental stages were labelled with fluorophore-tagged phalloidin. Since microplicae have actin filaments in their interior, staining with the F-actin probe phalloidin visualizes the entire apical membrane of fully differentiated secretory cells in the adult salivary gland (Zimmermann, 2000;Zimmermann et al, 2003;Rotte et al, 2008).…”

“…To serve this task, the plasma membrane of the secretory cells is enormously expanded, providing the area for the positioning of an enormous number of ion transporters and channels (Oschman and Berridge, 1970;Baumann and Walz, 2012). Whereas the basolateral domain of the plasma membrane is folded into a basal labyrinth, the apical membrane forms an extensive system of branching canaliculi that are covered by long and closely stacked pleats of membrane, termed microplicae (Oschman and Berridge, 1970;Zimmermann et al, 2003). These secretory cells with their elaborate architecture arise from simple undifferentiated epithelial cells during the pupal and early adult stage (Berridge et al, 1976).…”

The V-type H+ ATPase: molecular structure and function,physiological roles and regulation

“…Apparently, the serotonininduced increase in cyclic AMP (cAMP) activates an electrogenic K + transport mechanism (Berridge et al, 1976) that today is thought to derive from the V-type H + ATPase working in parallel with a K + /H + antiporter (Wieczorek et al, 1999). Immunofluorescent labeling of different V-ATPase subunits, as well as measurements of enzyme activity, have shown that serotonin recruits V 1 subunits from the cytosol, consistent with the assembly of the V 1 V 0 holoenzyme (Zimmermann et al, 2003). In Malpighian tubules of insects, where intracellular second messengers of diuresis and antidiuresis have been identified, it remains unknown whether cAMP, cGMP, Ca 2+ and NO affect the disassembly/reassembly of the V 1 V 0 holoenzyme.…”

Intracellular pH regulation in unstimulatedCalliphorasalivary glands is Na+ dependent and requires V-ATPase activity

“…A vacuolar H + -ATPase (V-ATPase) located in the apical membrane of the gland cells is of fundamental importance for fluid secretion. Active VATPase generates an inward-directed electrochemical H + -gradient that is used by a putative nH + /K + antiporter for K + transport into the glandular lumen (Dames et al, 2006;Rein et al, 2008;Wieczorek et al, 1991;Zimmermann et al, 2003).…”