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Institute
Salivary gland cells of the blowfly Calliphora vicina have a vacuolar-type H+-ATPase (V-ATPase) that lies in their apical membrane and energizes the secretion of a KCl-rich primary saliva upon stimulation with serotonin (5-hydroxytryptamine). Whether and to what extent V-ATPase contributes to intracellular pH (pH(i)) regulation in unstimulated gland cells is unknown. We used the fluorescent dye BCECF to study intracellular pH(i) regulation microfluorometrically and show that: (1) under resting conditions, the application of Na+-free physiological saline induces an intracellular alkalinization attributable to the inhibition of the activity of a Na+-dependent glutamate transporter; (2) the maintenance of resting pHi is Na+, Cl-, concanamycin A and DIDS sensitive; (3) recovery from an intracellular acid load is Na+ sensitive and requires V-ATPase activity; (4) the Na+/H+ antiporter is not involved in pHi recovery after a NH4Cl prepulse; and (5) at least one Na+-dependent transporter and the V-ATPase maintain recovery from an intracellular acid load. Thus, under resting conditions, the V-ATPase and at least one Na+-dependent transporter maintain normal pH(i) values of pH.7.5. We have also detected the presence of a Na+-dependent glutamate transporter, which seems to act as an acid loader. Despite this not being a common pH(i)-regulating transporter, its activity affects steady-state pH(i) in C. vicina salivary gland cells.
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.
Secretion in blowfly (Calliphora vicina) salivary glands is stimulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT), which activates both inositol 1,4,5-trisphosphate (InsP(3))/Ca2+ and cyclic adenosine 3',5'-monophosphate (cAMP) signalling pathways in the secretory cells. In order to characterize the signal-inducing 5-HT receptors, we cloned two cDNAs (Cv5-ht2 alpha, Cv5-ht7) that share high similarity with mammalian 5-HT2 and 5-HT7 receptor genes, respectively. RT-PCR demonstrated that both receptors are expressed in the salivary glands and brain. Stimulation of Cv5-ht2 alpha-transfected mammalian cells with 5-HT elevates cytosolic [Ca2+] in a dose-dependent manner (EC50 = 24 nM). In Cv5-ht7-transfected cells, 5-HT produces a dose-dependent increase in [cAMP](i) (EC50 = 4 nM). We studied the pharmacological profile for both receptors. Substances that appear to act as specific ligands of either Cv5-HT2 alpha or Cv5-HT7 in the heterologous expression system were also tested in intact blowfly salivary gland preparations. We observed that 5-methoxytryptamine (100 nM) activates only the Cv(5)-HT2 alpha receptor, 5-carboxamidotryptamine (300 nM) activates only the Cv5-HT7 receptor, and clozapine (1 mu M) antagonizes the effects of 5-HT via Cv5-HT7 in blowfly salivary glands, providing means for the selective activation of each of the two 5-HT receptor subtypes. This study represents the first comprehensive molecular and pharmacological characterization of two 5-HT receptors in the blowfly and permits the analysis of the physiological role of these receptors, even when co-expressed in cells, and of the modes of interaction between the Ca2+- and cAMP-signalling cascades. Citation: Roser C, Jordan N, Balfanz S, Baumann A, Walz B, et al. (2012) Molecular and Pharmacological Characterization of Serotonin 5-HT2a and 5-HT7 Receptors in the Salivary Glands of the Blowfly Calliphora vicina.
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 in the cockroach Periplaneta americana secrete protein-containing saliva when stimulated by serotonin (5-HT) and protein-free saliva upon dopamine stimulation. In order to obtain information concerning the signalling pathways involved in 5-HT-induced protein secretion, we have determined the protein content of saliva secreted after experimental manipulations that potentially elevate intracellular Ca2+ and cyclic nucleotide concentrations in isolated glands. We have found that 5-HT stimulates the rate of protein secretion in a dose-dependent manner (threshold: 3 x 10(-8) M; EC50 1.5 x 10(-6) M). The maximal rate of 5-HT-induced protein secretion was 2.2 +/- 0.2 mu g/min. Increasing intracellular Ca2+ or cAMP by bath application of ionomycin (5 mu M), db cAMP (10 mM), forskolin (100 mu M) or IBMX (100 mu M), respectively, stimulated protein secretion at significantly lower rates, whereas db cGMP (1 mM) did not activate protein secretion. The high rates and the kinetics of 5-HT-induced protein secretion could only be mimicked by either applying forskolin together with IBMX (with or without ionomycin) or by applying IBMX together with ionomycin. Our measurements suggest that 5-HT-induced protein secretion is mediated by an elevation of [cAMP](i) and that Ca2+ may function as a co-agonist and augment the rate of protein secretion. (c) 2005 Elsevier Ltd. All rights reserved
The acinar salivary glands of the cockroach, Periplaneta americana, are innervated by dopaminergic and serotonergic nerve fibers. Serotonin stimulates the secretion of protein-rich saliva, whereas dopamine causes the production of protein-free saliva. This suggests that dopamine acts selectively on ion-transporting peripheral cells within the acini and the duct cells, and that serotonin acts on the protein-producing central cells of the acini. We have investigated the pharmacology of the dopamine-induced secretory activity of the salivary gland of Periplaneta americana by testing several dopamine receptor agonists and antagonists. The effects of dopamine can be mimicked by the non-selective dopamine receptor agonist 6,7-ADTN and, less effectively, by the vertebrate D1 receptor-selective agonist chloro-APB. The vertebrate D1 receptor-selective agonist SKF 38393 and vertebrate D2 receptor-selective agonist R(-)- TNPA were ineffective. R(+)-Lisuride induces a secretory response with a slower onset and a lower maximal response compared with dopamine-induced secretion. However, lisuride-stimulated glands continue secreting saliva, even after lisuride-washout. Dopamine-induced secretions can be blocked by the vertebrate dopamine receptor antagonists cis(Z)- flupenthixol, chlorpromazine, and S(+)-butaclamol. Our pharmacological data do not unequivocally indicate whether the dopamine receptors on the Periplaneta salivary glands belong to the D1 or D2 subfamily of dopamine receptors, but we can confirm that the pharmacology of invertebrate dopamine receptors is remarkably different from that of their vertebrate counterparts. (C) 2004 Elsevier Ltd. All rights reserved