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Institute
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
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.
gamma-aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the central nervous system (CNS). Its effects are mediated by either ionotropic GABA(A) receptors or metabotropic GABA(B) receptors. GABA(B) receptors regulate, via Gi/o, G-proteins, ion channels, and adenylyl cyclases. In humans, GABA(B) receptor subtypes are involved in the etiology of neurologic and psychiatric disorders. In arthropods, however, these members of the G-protein-coupled receptor family are only inadequately characterized. Interestingly, physiological data have revealed important functions of GABA(B) receptors in the American cockroach, Periplaneta americana. We have cloned cDNAs coding for putative GABA(B) receptor subtypes 1 and 2 of P. americana (PeaGB1 and PeaGB2). When both receptor proteins are co-expressed in mammalian cells, activation of the receptor heteromer with GABA leads to a dose-dependent decrease in cAMP production. The pharmacological profile differs from that of mammalian and Drosophila GABA(B) receptors. Western blot analyses with polyclonal antibodies have revealed the expression of PeaGB1 and PeaGB2 in the CNS of the American cockroach. In addition to the widespread distribution in the brain, PeaGB1 is expressed in salivary glands and male accessory glands. Notably, PeaGB1-like immunoreactivity has been detected in the GABAergic salivary neuron 2, suggesting that GABA(B) receptors act as autoreceptors in this neuron.
Molecular identification and functional characterization of an adenylyl cyclase from the honeybee
(2006)
Cyclic AMP (cAMP) serves as an important messenger in virtually all organisms. In the honeybee (Apis mellifera), cAMP-dependent signal transduction has been implicated in behavioural processes as well as in learning and memory. Key components of cAMP-signalling cascades are adenylyl cyclases. However, the molecular identities and biochemical properties of adenylyl cyclases are completely unknown in the honeybee. We have cloned a cDNA (Amac3) from honeybee brain that encodes a membrane-bound adenylyl cyclase. The Amac3 gene is an orthologue of the Drosophila ac39E gene. The corresponding proteins share an overall amino acid similarity of approximately 62%. Phylogenetically, AmAC3 belongs to group 1 adenylyl cyclases. Heterologously expressed AmAC3 displays basal enzymatic activity and efficient coupling to endogenous G protein signalling pathways. Stimulation of beta-adrenergic receptors induces AmAC3 activity with an EC50 of about 3.1 mu m. Enzymatic activity is also increased by forskolin (EC50 approximately 15 mu m), a specific agonist of membrane-bound adenylyl cyclases. Similar to certain biogenic amine receptor genes of the honeybee, Amac3 transcripts are expressed in many somata of the brain, especially in mushroom body neurones. These results suggest that the enzyme serves in biogenic amine signal transduction cascades and in higher brain functions that contribute to learning and memory of the bee
Molecular characterization of the ebony gene from the American cockroach, Periplaneta americana
(2005)
Biogenic amines are an important class of primary messengers in the central (CNS) and peripheral nervous systems and in peripheral organs. These substances regulate and modulate many physiological and behavioral processes. Various inactivation mechanisms for these substances exist to terminate biogenic amine-mediated signal transduction. In vertebrates, the enzymes monoamine oxidase and/or catechol-O-methyl-transferase are involved in these processes. In insects, however, in which both enzymes are low in abundance or absent, biogenic amines are inactivated mainly by N- acetylation or O-sulphation. In Droso-philo, beta-alanyl conjugation mediated by the Ebony protein has recently been shown to be a novel and alternative pathway for biogenic amine inactivation. Here, we report the cloning of ebony cDNA (Peaebony) from a brain-specific cDNA library of the cockroach Periplaneta americana. The open reading frame encodes a protein of 860 amino acid residues (PeaEbony). The PeaEbony polypeptide shares homology to Ebony sequences from Anopheles gambiae, Apis mellifera, and Drosophila melonogaster. In addition, PeaEbony exhibits sequence similarity to a family of microbial non-ribosomal peptide synthetases. The mRNA encoding PeaEbony is highly expressed in the cockroach brain and to a lesser extent in the salivary glands. PeaEbony is, therefore, probably involved in the inactivation of various biogenic amines through beta-alanyl conjugation in the cockroach CNS. Since the salivary glands in Periplaneta are innervated by dopaminergic and serotonergic neurons, PeaEbony probably also biochemically modifies dopamine and serotonin in these acinar glands. Arch. Insect Biochem. (c) 2005 Wiley-Liss, Inc
Biogenic amines are important messenger substances in the central nervous system and in peripheral organs of vertebrates and of invertebrates. The honeybee, Apis mellifera, is excellently suited to uncover the functions of biogenic amines in behaviour, because it has an extensive behavioural repertoire, with a number of biogenic amine receptors characterised in this insect. In the honeybee, the biogenic amines dopamine, octopamine, serotonin and tyramine modulate neuronal functions in various ways. Dopamine and serotonin are present in high concentrations in the bee brain, whereas octopamine and tyramine are less abundant. Octopamine is a key molecule for the control of honeybee behaviour. It generally has an arousing effect and leads to higher sensitivity for sensory inputs, better learning performance and increased foraging behaviour. Tyramine has been suggested to act antagonistically to octopamine, but only few experimental data are available for this amine. Dopamine and serotonin often have antagonistic or inhibitory effects as compared to octopamine. Biogenic amines bind to membrane receptors that primarily belong to the large gene-family of GTP-binding (G) protein coupled receptors. Receptor activation leads to transient changes in concentrations of intracellular second messengers such as cAMP, IP3 and/or Ca2+. Although several biogenic amine receptors from the honeybee have been cloned and characterised more recently, many genes still remain to be identified. The availability of the completely sequenced genome of Apis mellifera will contribute substantially to closing this gap. In this review, we will discuss the present knowledge on how biogenic amines and their receptor-mediated cellular responses modulate different behaviours of honeybees including learning processes and division of labour.
Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera . The gene was isolated from a brain-specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis . Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher-order brain functions.
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.
Inverse agonist and neutral antagonist actions of synthetic compounds at an insect 5-HT1 receptor
(2010)
Background and purpose: 5-Hydroxytryptamine (5-HT) has been shown to control and modulate many physiological and behavioural functions in insects. In this study, we report the cloning and pharmacological properties of a 5-HT1 receptor of an insect model for neurobiology, physiology and pharmacology. Experimental approach: A cDNA encoding for the Periplaneta americana 5-HT1 receptor was amplified from brain cDNA. The receptor was stably expressed in HEK 293 cells, and the functional and pharmacological properties were determined in cAMP assays. Receptor distribution was investigated by RT-PCR and by immunocytochemistry using an affinity-purified polyclonal antiserum. Key results: The P. americana 5-HT1 receptor (Pea5-HT1) shares pronounced sequence and functional similarity with mammalian 5-HT1 receptors. Activation with 5-HT reduced adenylyl cyclase activity in a dose-dependent manner. Pea5-HT1 was expressed as a constitutively active receptor with methiothepin acting as a neutral antagonist, and WAY 100635 as an inverse agonist. Receptor mRNA was present in various tissues including brain, salivary glands and midgut. Receptor-specific antibodies showed that the native protein was expressed in a glycosylated form in membrane samples of brain and salivary glands. Conclusions and implications: This study marks the first pharmacological identification of an inverse agonist and a neutral antagonist at an insect 5-HT1 receptor. The results presented here should facilitate further analyses of 5-HT1 receptors in mediating central and peripheral effects of 5-HT in insects.
The biogenic amine serotonin (5-HT) plays a key role in the regulation and modulation of many physiological and behavioural processes in both vertebrates and invertebrates. These functions are mediated through the binding of serotonin to its receptors, of which 13 subtypes have been characterized in vertebrates. We have isolated a cDNA from the honeybee Apis mellifera (Am5-ht7) sharing high similarity to members of the 5-HT7 receptor family. Expression of the Am5-HT7 receptor in HEK293 cells results in an increase in basal cAMP levels, suggesting that Am5-HT7 is expressed as a constitutively active receptor. Serotonin application to Am5-ht7-transfected cells elevates cyclic adenosine 3',5'-monophosphate (cAMP) levels in a dose-dependent manner (EC50 = 1.1-1.8 nM). The Am5-HT7 receptor is also activated by 5-carboxamidotryptamine, whereas methiothepin acts as an inverse agonist. Receptor expression has been investigated by RT-PCR, in situ hybridization, and western blotting experiments. Receptor mRNA is expressed in the perikarya of various brain neuropils, including intrinsic mushroom body neurons, and in peripheral organs. This study marks the first comprehensive characterization of a serotonin receptor in the honeybee and should facilitate further analysis of the role(s) of the receptor in mediating the various central and peripheral effects of 5-HT.