Pharmacological characterization of a tyramine receptor from the southern cattle tick,Rhipicephalus (Boophilus) microplus

The southern cattle tick (Rhipicephalus (Boophilus) microplus) is a hematophagous external parasite that vectors the causative agents of bovine babesiosis or cattle tick fever, Babesia bovis and B. bigemina, and anaplasmosis, Anaplasma marginale. The southern cattle tick is a threat to the livestock industry in many locations throughout the world. Control methods include the use of chemical acaricides including amitraz, a formamidine insecticide, which is proposed to activate octopamine receptors. Previous studies have identified a putative octopamine receptor from the southern cattle tick in Australia and the Americas. Furthermore, this putative octopamine receptor could play a role in acaricide resistance to amitraz. Recently, sequence data indicated that this putative octopamine receptor is probably a type-1 tyramine receptor (TAR1). In this study, the putative TAR1 was heterologously expressed in Chinese hamster ovary (CHO-K1) cells, and the expressed receptor resulted in a 39-fold higher potency for tyramine compared to octopamine. Furthermore, the expressed receptor was strongly antagonized by yohimbine and cyproheptadine, and mildly antagonized by mianserin and phentolamine. Tolazoline and naphazoline had agonistic or modulatory activity against the expressed receptor, as did the amitraz metabolite, BTS-27271; however, this was only observed in the presence of tyramine. The southern cattle tick's tyramine receptor may serve as a target for the development of anti-parasitic compounds, in addition to being a likely target of formamidine insecticides.     

A comparison of the signalling properties of two tyramine receptors from Drosophila

In invertebrates, the phenolamines, tyramine and octopamine, mediate many functional roles usually associated with the catecholamines, noradrenaline and adrenaline, in vertebrates. The α‐ and β‐adrenergic classes of insect octopamine receptor are better activated by octopamine than tyramine. Similarly, the Tyramine 1 subgroup of receptors (or Octopamine/Tyramine receptors) are better activated by tyramine than octopamine. However, recently, a new Tyramine 2 subgroup of receptors was identified, which appears to be activated highly preferentially by tyramine. We examined immunocytochemically the ability of CG7431, the founding member of this subgroup from Drosophila melanogaster, to be internalized in transfected Chinese hamster ovary (CHO) cells by different agonists. It was only internalized after activation by tyramine. Conversely, the structurally related receptor, CG16766, was internalized by a number of biogenic amines, including octopamine, dopamine, noradrenaline, adrenaline, which also were able to elevate cyclic AMP levels. Studies with synthetic agonists and antagonists confirm that CG16766 has a different pharmacological profile to that of CG7431. Species orthologues of CG16766 were only found in Drosophila species, whereas orthologues of CG7431 could be identified in the genomes of a number of insect species. We propose that CG16766 represents a new group of tyramine receptors, which we have designated the Tyramine 3 receptors.     

Bicuculline-insensitive GABA-gated Cl<Superscript>−</Superscript> channels in the larval nervous system of the moth<Emphasis Type="Italic"> Manduca sexta</Emphasis>

GABA-gated Cl^– channels were studied in the nervous system of the larval tobacco hawk moth, Manduca sexta, using electrophysiology, ³⁶Cl^– uptake into membrane microsacs and immunocytochemistry. A GABA-induced increase in Cl^– conductance was recorded from a visually identifiable neurone (fg1) in the desheathed frontal ganglion. The response was insensitive to the vertebrate GABA_A receptor antagonist, bicuculline, but was blocked by picrotoxinin. Bicuculline-insensitive, picrotoxinin-sensitive, GABA-stimulated ³⁶Cl^– uptake was also detected in membrane microsacs prepared from the isolated larval M. sexta nervous system. Such receptors appear to be the major type of GABA receptor in larval nervous system membrane microsac preparations. An antibody raised against a 17 amino acid peptide, based on the predicted C-terminus of the Drosophila GABA receptor subunit (RDL), stained not only cell bodies, including that of fg1, but also the neuropile in the frontal ganglion, indicating the existence of RDL-like GABA receptor subunits in neurones of this ganglion. Thus, bicuculline-insensitive GABA-gated Cl^– channels are present in the larval nervous system of M. sexta.     

Insect octopamine receptors: a new classification scheme based on studies of cloned Drosophila G-protein coupled receptors

Summary Insect octopamine receptors are G-protein coupled receptors. They can be coupled to second messenger pathways to mediate either increases or decreases in intracellular cyclic AMP levels or the generation of intracellular calcium signals. Insect octopamine receptors were originally classified on the basis of second messenger changes induced in a variety of intact tissue preparations. Such a classification system is problematic if more than one receptor subtype is present in the same tissue preparation. Recent progress on the cloning and characterization in heterologous cell systems of octopamine receptors from Drosophila and other insects is reviewed. A new classification system for insect octopamine receptors into “α-adrenergic-like octopamine receptors (OctαRs)”, “β-adrenergic-like octopamine receptors (OctβRs)” and “octopamine/tyramine (or tyraminergic) receptors” is proposed based on their similarities in structure and in signalling properties with vertebrate adrenergic receptors. In future studies on the molecular basis of octopamine signalling in individual tissues it will be essential to identify the relative expression levels of the different classes of octopamine receptor present. In addition, it will be essential to identify if co-expression of such receptors in the same cells results in the formation of oligomeric receptors with specific emergent pharmacological and signalling properties.     

Octopamine receptor subtypes and their modes of action

Octopamine receptor subclasses were first proposed to explain differences in the pharmacological profiles of a range of physiological responses to octopamine obtained in the extensor-tibiae neuromuscular preparation of the locust. Thus, OCTOPAMINE₁ receptors which inhibit an endogenous myogenic rhythm, increase intracellular calcium levels. Also OCTOPAMINE₂ receptors which modulate neuromuscular transmission in this preparation, increase the level of adenylate cyclase activity. The current status of this classification is reviewed by examining the pharmacology of responses to octopamine in a range of preparations. It is concluded that the distinction between OCTOPAMINE₁ and OCTOPAMINE₂ receptor types is still valid, but that OCTOPAMINE₂ receptors exhibit some tissue specific variations. Studies on a clonedDrosophila octopamine/tyramine (phenolamine) receptor are discussed and illustrate many of the difficulties presently encountered in making a definitive classification of octopamine receptors. These include the possibilities that single receptors may activate multiple second messenger systems and that different agonists may differentially couple the same receptor to different second messenger systems.     

Tyramine injections reduce locust viability

In the locust nervous system, tyramine is the direct precursor for octopamine synthesis and, as an octopamine analogue, it can activate octopamine receptors. Furthermore, the identification of specific tyramine receptors in Locusta migratoria and Drosophila melanogaster suggests that it is an important transmitter or modulator candidate. In this paper, we report that repeated tyramine injections reduced the viability of last instar larvae of Locusta and Schistocerca. In addition, a retardation of the last ecdysis was observed as a sublethal effect of the repeated tyramine treatment. Moreover, egg deposition by adult females was also retarded and/or drastically reduced. These effects show similarity to sublethal effects described for certain "insecticidal" octopamine receptor agonists, such as formamidines and phenyliminoimidazolidines. Since certain formamidine compounds were also shown to be agonists for the cloned tyramine receptors, it cannot be excluded that some lethal or sublethal consequences of tyramine administration are the result of an interaction with specific tyramine receptors.     

Distribution of two GABA receptor-like subunits in theDrosophila CNS

Previously we have described the distribution of theRdl GABA receptor subunit in theDrosophila CNS. Knowing thatRdl can coassemble with LCCH3 (aDrosophila GABA receptor-like subunit showing sequence similarity to vertebrate subunit GABA_A receptors) in baculovirus infected insect cells, we compared the localization of these two receptor subunits in order to identify any potential overlap in their spatial or temporal distribution. The two subunits show very different patterns of localization. Early in development LCCH3 is found in the majority of developing neuroblasts and later is localized to the cell bodies of the embryonic nerve cord and brain, and the neuronal cell bodies surrounding the adult brain. In contrast,Rdl receptor subunits appear confined to the neuropil in all developmental stages. These results have two important implications. Firstly, they suggest that although these two subunits can coassemble in heterologous expression systems, they may not be found in the same tissues in the nervous system. Secondly, production of LCCH3 before neuronal differentiation leads us to speculate on the role of that LCCH3 containing receptors in the developing nervous system.     

A new family of insect tyramine receptors

The Drosophila Genome Project database contains a gene, CG7431, annotated to be an "unclassifiable biogenic amine receptor." We have cloned this gene and expressed it in Chinese hamster ovary cells. After testing various ligands for G protein-coupled receptors, we found that the receptor was specifically activated by tyramine (EC(50), 5x10(-7)M) and that it showed no cross-reactivity with beta-phenylethylamine, octopamine, dopa, dopamine, adrenaline, noradrenaline, tryptamine, serotonin, histamine, and a library of 20 Drosophila neuropeptides (all tested in concentrations up to 10(-5) or 10(-4)M). The receptor was also expressed in Xenopus oocytes, where it was, again, specifically activated by tyramine with an EC(50) of 3x10(-7)M. Northern blots showed that the receptor is already expressed in 8-hour-old embryos and that it continues to be expressed in all subsequent developmental stages. Adult flies express the receptor both in the head and body (thorax/abdomen) parts. In addition to the Drosophila tyramine receptor gene, CG7431, we found another closely related Drosophila gene, CG16766, that probably also codes for a tyramine receptor. Furthermore, we annotated similar tyramine-like receptor genes in the genomic databases from the malaria mosquito Anopheles gambiae and the honeybee Apis mellifera. These four tyramine or tyramine-like receptors constitute a new receptor family that is phylogenetically distinct from the previously identified insect octopamine/tyramine receptors. The Drosophila tyramine receptor is, to our knowledge, the first cloned insect G protein-coupled receptor that appears to be fully specific for tyramine.     

Structural characterization and agonist binding to human α4β2 nicotinic receptors

The Cys-loop receptor super-family of neurotransmitter-gated ion channels mediates fast synaptic transmission throughout the human nervous system. These receptors exhibit widely varying pharmacologies, yet their structural characterization has relied heavily on their homology with the naturally abundant muscle-type Torpedo nicotinic acetylcholine receptor. Here we examine for the first time the structure of a human α4β2 neuronal nicotinic acetylcholine receptor. We show that human α4β2 nicotinic receptors adopt a secondary/tertiary fold similar to that of the Torpedo nicotinic receptor with a large proportion of both α-helix and β-sheet, but exhibit a substantially increased thermal stability. Both receptors bind agonist, but with different patterns of agonist recognition – particularly in the nature of the interactions between aromatic residues and the agonist quaternary amine functional group. By comparing α4β2 and Torpedo receptors, we begin to delineate their structural similarities and differences.     

Properties of putative nicotinic and muscarinic cholinergic receptors in the central nervous system of Locusta migratoria

Nervous tissue preparations from Locusta migratoria specifically bind potent nicotinic (α-bungarotoxin) and muscarinic (quinuclidinyl benzilate) ligands. Binding properties and pharmacological data indicate that the central nervous system of the locust contains at least two distinct classes of receptors. Subcellular fractionation experiments revealed that the receptor activity is enriched in the synaptosomal fraction. In the head as well as in the thoracic ganglia the nicotinic acetylcholine receptors were found to be much more abundant than the muscarinic binding sites; whereas in mouse brain the muscarinic receptor type predominates.     

Biogenic amine biosynthetic and transduction genes in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

Biogenic amines (BAs), such as octopamine, tyramine, dopamine, serotonin, and acetylcholine regulate various behaviors and physiological functions in insects. Here, we identified seven genes encoding BA biosynthetic enzymes and 16 genes encoding BA G protein-coupled receptors in the genome of the endoparasitoid wasp, Pteromalus puparum. We compared the genes with their orthologs in its host Pieris rapae and the related ectoparasitic wasp Nasonia vitripennis. All the genes show high (>90%) identity to orthologs in N. vitripennis. P. puparum and N. vitripennis have the smallest number of BA receptor genes among the insect species we investigated. We then analyzed the expression profiles of the genes, finding those acting in BA biosynthesis were highly expressed in adults and larvae and those encoding BA receptors are highly expressed in adults than immatures. Octα1R and 5-HT₇ genes were highly expressed in salivary glands, and a high messenger RNA level of 5-HT_1A was found in venom apparatuses. We infer that BA signaling is a fundamental component of the organismal organization, homeostasis and operation in parasitoids, some of the smallest insects.     

Characterization of insect neuronal octopamine receptors (OA3 receptors)

Octopamine receptors in the nervous tissue of insects were investigated using a ligand-receptor assay with [³H]NC-5Z or [³H]octopamine as the radioligands. Both ligands recognized a homogenous class of binding sites with the properties of an octopamine receptor. This receptor has been characterized pharmacologically. Both high-affinity agonists (e.g. NC 7, K₁=0.3 nM) and antagonists (e.g. maroxepine, K₁=1.02 nM) were investigated. The neuronal octopamine receptor belongs to a receptor class that can easily be distinguished from peripheral octopamine receptors. Initial investigations of the localization of octopamine receptors within the insect nervous tissue show the greatest receptor density in the optic lobes.     

The role of octopamine in locusts and other arthropods

The biogenic amine octopamine and its biological precursor tyramine are thought to be the invertebrate functional homologues of the vertebrate adrenergic transmitters. Octopamine functions as a neuromodulator, neurotransmitter and neurohormone in insect nervous systems and prompts the whole organism to “dynamic action”. A growing number of studies suggest a prominent role for octopamine in modulating multiple physiological and behavioural processes in invertebrates, as for example the phase transition in Schistocerca gregaria. Both octopamine and tyramine exert their effects by binding to specific receptor proteins that belong to the superfamily of G protein-coupled receptors. Since these receptors do not appear to be present in vertebrates, they may present very suitable and specific insecticide and acaricide targets.     

昆虫体内章鱼胺和酪胺的研究进展

章鱼胺(octopamine, OA)和酪胺(tyramine, TA)在昆虫体内扮演着各种重要的生理角色。它们协调控制着昆虫的各种器官和行为, 如调节外周淋巴器官功能和影响昆虫的学习与记忆、昼夜节律等, 使得昆虫能够以合理的方式来应对外界刺激, 并被认为在功能上对应于脊椎动物体内的肾上腺素和去甲肾上腺素。虽然都是酪氨酸脱羧基产物, 且酪胺是章鱼胺的生物合成前体, 但它们都通过不同的G蛋白偶联受体在昆虫体内发挥不同的神经调控作用。近年来, 对昆虫体内章鱼胺和酪胺, 尤其是它们与对应受体作用的研究, 日益受到关注。本文对昆虫体内章鱼胺和酪胺的生物合成, 在神经和非神经组织中的分布, 被突触前结构的再摄取以及它们在昆虫体内的不同生理功能等方面的研究进展进行了综述, 特别对章鱼胺和酪胺受体基因的克隆、信号转导途径以及药理作用特性等相关研究的最新进展进行了详细评述。     

Characterization of a Prawn OA/TA Receptor in Xenopus Oocytes Suggests Functional Selectivity between Octopamine and Tyramine

Here we report the characterization of an octopamine/tyramine (OA/TA or TyrR1) receptor (OA/TA_Mac) cloned from the freshwater prawn, Macrobrachium rosenbergii, an animal used in the study of agonistic social behavior. The invertebrate OA/TA receptors are seven trans-membrane domain G-protein coupled receptors that are related to vertebrate adrenergic receptors. Behavioral studies in arthropods indicate that octopaminergic signaling systems modulate fight or flight behaviors with octopamine and/or tyramine functioning in a similar way to the adrenalins in vertebrate systems. Despite the importance of octopamine signaling in behavioral studies of decapod crustaceans there are no functional data available for any of their octopamine or tyramine receptors. We expressed OA/TA_Mac in Xenopus oocytes where agonist-evoked trans-membrane currents were used as readouts of receptor activity. The currents were most effectively evoked by tyramine but were also evoked by octopamine and dopamine. They were effectively blocked by yohimbine. The electrophysiological approach we used enabled the continuous observation of complex dynamics over time. Using voltage steps, we were able to simultaneously resolve two types of endogenous currents that are affected over different time scales. At higher concentrations we observe that octopamine and tyramine can produce different and opposing effects on both of these currents, presumably through the activity of the single expressed receptor type. The pharmacological profile and apparent functional-selectivity are consistent with properties first observed in the OA/TA receptor from the insect Drosophila melanogaster. As the first functional data reported for any crustacean OA/TA receptor, these results suggest that functional-selectivity between tyramine and octopamine is a feature of this receptor type that may be conserved among arthropods.     

Cloning and expression pattern of a putative octopamine/tyramine receptor in antennae of the noctuid moth <Emphasis Type="Italic">Mamestra brassicae</Emphasis>

In insects, biogenic amines have been shown to play an important role in olfactory plasticity. In a first attempt to decipher the underlying molecular mechanisms, we report the molecular cloning and precise expression pattern of a newly identified octopamine/tyramine-receptor-encoding gene in the antennae of the noctuid moth Mamestra brassicae (MbraOAR/TAR). A full-length cDNA has been obtained through homology cloning in combination with rapid amplification of cDNA ends/polymerase chain reaction; the deduced protein exhibits high identities with previously identified octopamine/tyramine receptors in other moths. In situ hybridization within the antennae has revealed that MbraOAR/TAR is expressed at the bases of both pheromone-sensitive and non-sensitive olfactory sensilla and in cells with a neurone-like shape. In accordance with previous physiological studies that have revealed a role of biogenic amines in the electrical activity of the receptor neurones, our results suggest that biogenic amines (either octopamine or tyramine) target olfactory receptor neurones to modulate olfactory coding as early as the antennal level.     

20-OH-Ecdysone-induced morphogenetic movements in a Chironomus cell line are accompanied by expression of an embryonic muscarinic system

The permanent epithelial insect cell line used was derived from Chironomus tentans embryos. Cells are maintained in suspension culture, where they grow as single-layered vesicles. On treatment with the moulting hormone 20-OH-ecdysone cell division ceases. Patches of cuboidal epithelium appear in the vesicles which finally become multilayered and form bud-like protrusions at the outside. In the present study, we localized cholinesterase activity in the cell protrusions by histochemistry and demonstrated coexpression of cholinergic muscarinic receptors by immunofluorescence. Muscarinic receptors were visualized with the monoclonal antibody M35. Six hours after treatment with 20-OH-ecdysone, muscarinic receptors appeared in a few individual cells of the epithelial vesicles before morphological changes became visible. After 24 h, immunofluorescence was concentrated in multilayered patches which now also showed cholinesterase activity. After 3 days, muscarinic receptors and cholinesterase activity were localized in the epithelium protrusions. The results are discussed in the context of an embryonic cholinergic muscarinic system the expression of which has been described in vertebrate and non-vertebrate embryos and is correlated with phases of morphogenesis.     

GABAA receptor subunit polypeptides increase in parallel but exhibit distinct distributions in the developing rat cerebellum

The GABA_A receptor, a multisubunit ligand-gated ion channel, plays a central role in cell–cell communication in the developing and adult nervous system. Although the developmental expression of mRNAs encoding many subunit isoforms has been extensively characterized throughout the central nervous system, little is known concerning the relationship between subunit mRNA and polypeptide expression. To address this issue, we examined the developmental expression of the α1, β2/3, and γ2 subunit polypeptides, subunits that are thought to coassemble in many brain regions. Western blot analysis using subunit-specific antibodies revealed that the levels of these polypeptides in both the cerebral cortex and cerebellum increased severalfold during the second postnatal week. Whereas polypeptide expression in the cerebellum paralleled that of the corresponding subunit mRNAs, increase in β2/3 and γ2 polypeptide expression in the cerebral cortex occurred in the absence of detectable changes in the mRNA levels. To determine whether the increases in subunit polypeptide expression in the cerebellum were accompanied by changes in distribution, immunohistochemistry was performed. These studies demonstrated that the subunits exhibited different but partially overlapping distributions that remained constant throughout postnatal development. Our findings suggest that although GABA_A receptor subunit polypeptide expression may be regulated primarily at the level of the mRNA, additional regulatory mechanisms may play role. Furthermore, the observation that subunit distribution remains constant in the cell bodies of cerebellar Purkinje neurons, which express the α1, β2, β3, and γ2 subunit mRNAs exclusively, suggests that GABA_A receptor subunit composition in this cell population does not change during postnatal maturation. 1994 John Wiley & Sons, Inc.     

Isolation and functional characterization of the chick M5 muscarinic acetylcholine receptor gene

The chick is a widely used system for study of the actions of muscarinic acetylcholine receptors in the cardiovascular, visual, and nervous systems. We report the isolation and functional analysis of the gene encoding the chick M5 muscarinic receptor. RT-PCR analysis indicates that the M5 receptor is expressed at low levels in embryonic chick brain and heart. When expressed in stably transfected Chinese hamster ovary cells, the M5 receptor exhibits high-affinity binding to muscarinic antagonists and mediates robust activation of phospholipase C activity.