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The new pi-conjugated 1,2,3-triazol-1,4-diyl fluoroionophore 1 generated via Cu(I) catalyzed [3 + 2] cycloaddition shows high fluorescence enhancement factors (FEF) in the presence of Na+ (FEF = 58) and K+ (FEF = 27) in MeCN and high selectivity towards K+ under simulated physiological conditions (160 mM K+ or Na+, respectively) with a FEF of 2.5 for K+.
The paired salivary glands in the cockroach are composed of acini with ion-transporting peripheral P-cells and protein-secreting central C-cells, and a duct system for the modification of the primary saliva. Secretory activity is controlled by serotonergic and dopaminergic neurons, whose axons form a dense plexus on the glands. The spatial relationship of release sites for serotonin and dopamine to the various cell types was determined by anti-synapsin immunofluorescence confocal microscopy and electron microscopy. Every C-cell apparently has only serotonergic synapses on its surface. Serotonergic and dopaminergic fibres on the acini have their release zones at a distance of similar to0.5 mum from the P-cells. Nerves between acinar lobules may serve as neurohaemal organs and contain abundant dopaminergic and few serotonergic release sites. Some dopaminergic and serotonergic release sites reside in the duct epithelium, the former throughout the duct system, the latter only in segments next to acini. These findings are consistent with the view that C-cells respond exclusively to serotonin, P-cells to serotonin and dopamine, and most duct cells only to dopamine. Moreover, the data suggest that C-cells are stimulated by serotonin released close to their surface, whereas P-cells and most duct cells are exposed to serotonin/dopamine liberated at some distance
The blowfly salivary gland - A model system for analyzing the regulation of plasma membrane V-ATPase
(2012)
Vacuolar H+-ATPases (V-ATPases) are heteromultimeric proteins that use the energy of ATP hydrolysis for the electrogenic transport of protons across membranes. They are common to all eukaryotic cells and are located in the plasma membrane or in membranes of acid organelles. In many insect epithelia, V-ATPase molecules reside in large numbers in the apical plasma membrane and create an electrochemical proton gradient that is used for the acidification or alkalinization of the extracellular space, the secretion or reabsorption of ions and fluids, the import of nutrients, and diverse other cellular activities. Here, we summarize our results on the functions and regulation of V-ATPase in the tubular salivary gland of the blowfly Calliphora vicina. In this gland, V-ATPase activity energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). Because of particular morphological and physiological features, the blowfly salivary glands are a superior and exemplary system for the analysis of the intracellular signaling pathways and mechanisms that modulate V-ATPase activity and solute transport in an insect epithelium.
The phenolamines octopamine and tyramine control, regulate, and modulate many physiological and behavioral processes in invertebrates. Vertebrates possess only small amounts of both substances, and thus, octopamine and tyramine, together with other biogenic amines, are referred to as “trace amines.” Biogenic amines evoke cellular responses by activating G-protein-coupled receptors. We have isolated a complementary DNA (cDNA) that encodes a biogenic amine receptor from the American cockroach Periplaneta americana, viz., Peatyr1, which shares high sequence similarity to members of the invertebrate tyramine-receptor family. The PeaTYR1 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with tyramine reduces adenylyl cyclase activity in a dose-dependent manner (EC50 350 nM). The inhibitory effect of tyramine is abolished by co-incubation with either yohimbine or chlorpromazine. Receptor expression has been investigated by reverse transcription polymerase chain reaction and immunocytochemistry. The mRNA is present in various tissues including brain, salivary glands, midgut, Malpighian tubules, and leg muscles. The effect of tyramine on salivary gland acinar cells has been investigated by intracellular recordings, which have revealed excitatory presynaptic actions of tyramine. This study marks the first comprehensive molecular, pharmacological, and functional characterization of a tyramine receptor in the cockroach.
Source, topography and excitatory effects of GABAergic innervation in cockroach salivary glands
(2009)
Cockroach salivary glands are innervated by dopaminergic and serotonergic neurons. Both transmitters elicit saliva secretion. We studied the distribution pattern of neurons containing gamma-aminobutyric acid ( GABA) and their physiological role. Immunofluorescence revealed a GABA-immunoreactive axon that originates within the subesophageal ganglion at the salivary neuron 2 (SN2) and this extends within the salivary duct nerve towards the salivary gland. GABA-positive fibers form a network on most acinar lobules and a dense plexus in the interior of a minor fraction of acinar lobules. Co-staining with anti-synapsin revealed that some putative GABAergic terminals seem to make pre-synaptic contacts with GABA-negative release sites. Many putative GABAergic release sites are at some distance from other synapses and at distance from the acinar tissue. Intracellular recordings from isolated salivary glands have revealed that GABA does not affect the basolateral membrane potential of the acinar cells directly. When applied during salivary duct nerve stimulation, GABA enhances the electrical response of the acinar cells and increases the rates of fluid and protein secretion. The effect on electrical cell responses is mimicked by the GABA(B) receptor agonists baclofen and SKF97541, and blocked by the GABAB receptor antagonists CGP52432 and CGP54626. These findings indicate that GABA has a modulatory role in the control of salivation, acting presynaptically on serotonergic and/or dopaminergic neurotransmission.
The acinar salivary gland of the cockroach, Periplaneta americana, is innervated by dopaminergic and serotonergic nerve fibers. Stimulation of the glands by serotonin (5-hydroxytryptamine, 5-HT) results in the production of a protein-rich saliva, whereas stimulation by dopamine results in saliva that is protein-free. Thus, dopamine acts selectively on ion-transporting peripheral cells within the acini, and 5-HT acts on protein-producing central cells. We have investigated the pharmacology of the 5-HT-induced secretory activity of isolated salivary glands of P. americana by testing several 5-HT receptor agonists and antagonists. The effects of 5-HT can be mimicked by the non-selective 5-HT receptor agonist 5-methoxytryptamine. All tested agonists that display at least some receptor subtype specificity in mammals, i.e., 5-carboxamidotryptamine, (+/-)-8-OH-DPAT, (+/-)-DOI, and AS 19, were ineffective in stimulating salivary secretion. 5-HT-induced secretion can be blocked by the vertebrate 5-HT receptor antagonists methiothepin, cyproheptadine, and mianserin. Our pharmacological data indicate that the pharmacology of arthropod 5-HT receptors is remarkably different from that of their vertebrate counterparts. (C) 2007 Elsevier Ltd. All rights reserved.
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
Ca2+ and cAMP signalling pathways interact in a complex manner at multiple sites. This crosstalk fine-tunes the spatiotemporal patterns of Ca2+ and cAMP signals. In salivary glands of the blowfly Calliphora vicina fluid secretion is stimulated by serotonin (5-hydroxytryptamine, 5-HT) via activation of two different 5-HT receptors coupled to the InsP(3)/Ca2+ (Cv5-HT2 alpha) or the cAMP pathway (Cv5-HT7), respectively. We have shown recently in permeabilized gland cells that cAMP sensitizes InsP(3)-induced Ca2+ release to InsP(3). Here we study the effects of the CAMP signalling pathway on 5-HT-induced oscillations in transepithelial potential (TEP) and in intracellular [Ca2+]. We show: (1) Blocking the activation of the cAMP pathway by cinanserin suppresses the generation of TEP and Ca2+ oscillations, (2) application of 8-CPT-cAMP in the presence of cinanserin restores 5-HT-induced TEP and Ca2+ oscillations, (3) 8-CPT-cAMP sensitizes the InsP(3)/Ca2+ signalling pathway to 5-HT and the Cv5-HT2 alpha, receptor agonist 5-MeOT, (4) 8-CPT-cAMP induces Ca2+ oscillations in cells loaded with subthreshold concentrations of InsP(3), (5) inhibition of protein kinase A by H-89 abolishes 5-HT-induced TEP and Ca2+ spiking and mimics the effect of cinanserin. These results suggest that activation of the cyclic AMP pathway promotes the generation of 5-HT-induced Ca2+ oscillations in blowfly salivary glands.
Three-dimensional organization of endoplasmatic reticulum in the ventral photoreceptors of Limulus
(1994)
Crosstalk between intracellular signalling pathways is a functionally important and widespread phenomenon in cell physiology across phyla. In the salivary gland of the blowfly, serotonin induces fluid secretion via parallel activation of both the InsP(3)/Ca2+ and the cAMP/PKA signalling pathways, which interact on multiple levels. We have determined the molecular identity of a link between both pathways that mediates a Ca2+-dependent rise of intracellular cAMP. Whereas hydrolysis of cAMP via phosphodiesterases is largely independent of Ca2+, cAMP synthesis by adenylyl cyclases (AC) is potentiated in a Ca2+/calmodulin (Ca2+/CaM)-dependent manner. The existence of a Ca2+/CaM-dependent AC is supported by physiological data and a molecular approach. We have cloned Cv rutabaga cDNA, encoding the first blowfly AC, and confirmed its expression in the salivary gland via reverse transcription followed by polymerase chain reaction. The putative gene product of Cv rutabaga is a Ca2+/CaM-dependent type I AC and shows highest homology to Rutabaga from Drosophila. Thus, a Ca2+/CaM-dependent AC serves as a link between the InsP(3)/Ca2+ and the cAMP/PKA signalling pathways in the salivary gland of the blowfly and might be important for the amplification and optimization of the secretory response.
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.