Secretion in blowfly salivary glands is induced by the neurohormone serotonin and powered by a vacuolar-type H+- ATPase (V-ATPase) located in the apical membrane of the secretory cells. We have established a microfluorometric method for analysing pH changes at the luminal surface of the secretory epithelial cells by using the fluorescent dye 5-N- hexadecanoyl-aminofluorescein (HAF). After injection of HAF into the lumen of the tubular salivary gland, the fatty acyl chain of the dye molecule partitions into the outer leaflet of the plasma membrane and its pH-sensitive fluorescent moiety is exposed at the cell surface. Confocal imaging has confirmed that HAF distributes over the entire apical membrane of the secretory cells and remains restricted to this membrane domain. Ratiometric analysis of HAF fluorescence demonstrates that serotonin leads to a reversible dose-dependent acidification at the luminal surface. Inhibition by concanamycin A confirms that the serotonin-induced acidification at the luminal surface is due to H+ transport across the apical membrane via V-ATPase. Measurements with pH-sensitive microelectrodes corroborate a serotonin-induced luminal acidification and demonstrate that luminal pH decreases by about 0.4 pH units at saturating serotonin concentrations. We conclude that ratiometric measurements of HAF fluorescence provide an elegant method for monitoring V-ATPase-dependent H+ transport in the blowfly salivary gland in vivo and for analysing the spatiotemporal pattern of pH changes at the luminal surface
Stimulation with the neurotransmitter dopamine causes an amplitude-modulated increase in the intracellular Ca2+ concentration ([Ca2+](i)) in epithelial cells of the ducts of cockroach salivary glands. This is completely attributable to a Ca2+ influx from the extracellular space. Additionally, dopamine induces a massive [Na+](i) elevation via the Na+- K+-2Cl(-) cotransporter (NKCC). We have reasoned that Ca2+-entry is mediated by the Na+-Ca2+ exchanger (NCE) operating in the Ca2+-entry mode. To test this hypothesis, [Ca2+](i) and [Na+](i) were measured by using the fluorescent dyes Fura- 2, Fluo-3, and SBFI. Inhibition of Na+-entry from the extracellular space by removal of extracellular Na+ or inhibition of the NKCC by 10 mu M bumetanide did not influence resting [Ca2+]i but completely abolished the dopamine-induced [Ca2+](i) elevation. Simultaneous recordings of [Ca2+](i) and [Na+](i) revealed that the dopamine-induced [Na+](i) elevation preceded the [Ca2+](i) elevation. During dopamine stimulation, the generation of an outward Na+ concentration gradient by removal of extracellular Na+ boosted the [Ca2+](i) elevation. Furthermore, prolonging the dopamine-induced [Na+](i) rise by blocking the Na+/K+-ATPase reduced the recovery from [Ca2+](i) elevation. These results indicate that dopamine induces a massive NKCC-mediated elevation in [Na+](i), which reverses the NCE activity into the reverse mode causing a graded [Ca2+](i) elevation in the duct cells.
Reversible assembly of the V0V1 holoenzyme from V-0 and V-1 subcomplexes is a widely used mechanism for regulation of vacuolar-type H+-ATPases (V-ATPases) in animal cells. in the blowfly (Calliphora vicina) salivary gland, V- ATPase is located in the apical membrane of the secretory cells and energizes the secretion of a KCl-rich saliva in response to the hormone serotonin. We have examined whether the CAMP pathway, known to be activated by serotonin, controls V-ATPase assembly and activity. Fluorescence measurements of pH changes at the luminal surface of isolated glands demonstrate that CAMP, Sp-adenosine-3',5'-cyclic monophosphorothioate, or forskolin, similar to serotonin, cause V-ATPase-dependent luminal acidification. In addition, V-ATPase-dependent ATP hydrolysis increases upon treatment with these agents. Immunofluorescence microscopy and pelleting assays have demonstrated further that V, components become translocated from the cytoplasm to the apical membrane and V-ATPase holoenzymes are assembled at the apical membrane during conditions that increase intracellular cAMP. Because these actions occur without a change in cytosolic Ca2+, our findings suggest that the cAMP pathway mediates the reversible assembly and activation of V-ATPase molecules at the apical membrane upon hormonal stimulus
The acinar salivary glands of cockroaches receive a dual innervation from the subesophageal ganglion and the stomatogastric nervous system. Acinar cells are surrounded by a plexus of dopaminergic and serotonergic varicose fibers. In addition, seroton-ergic terminals lie deep in the extracellulor spaces between acinar cells. Excitation-secretion coupling in cockroach salivary glands is stimulated by both dopamine and serotonin. These monoamines cause increases in the intracellular concentrations of cAMP and Ca2+. Stimulation of the glands by serotonin results in the production of a protein-rich saliva, whereas stimulation by dopamine results in saliva that is protein-free. Thus, two elementary secretary processes, namely electrolyte/water secretion and protein secretion, are triggered by different aminergic transmitters. Because of its simplicity and experimental accessibility, cockroach salivary glands have been used extensively as a model system to study the cellular actions of biogenic amines and to examine the pharmacological properties of biogenic amine receptors. In this review, we summarize current knowledge concerning the aminergic control of cockroach salivary glands and discuss our efforts to characterize Periplaneta biogenic amine receptors molecularly
The salivary glands of the blowfly were injected with luminescent oxygen-sensitive microbeads. The changes in oxygen content within individual gland tubules during hormone-induced secretory activity were quantified. The measurements are based on an upgraded phase-modulation technique, where the phase shift of the sensor phosphorescence is determined independently from concentration and background signals. We show that the combination of a lock-in amplifier with a fluorescence microscope results in a convenient setup to measure oxygen concentrations within living animal tissues at the cellular level.