Pharmacological characterization of a β‐adrenergic‐like octopamine receptor in Plutella xylostella

The β‐adrenergic‐like octopamine receptor (OA2B2) belongs to the class of G‐protein coupled receptors. It regulates important physiological functions in insects, thus is potentially a good target for insecticides. In this study, the putative open reading frame sequence of the Pxoa2b2 gene in Plutella xylostella was cloned. Orthologous sequence alignment, phylogenetic tree analysis, and protein sequence analysis all showed that the cloned receptor belongs to the OA2B2 protein family. PxOA2B2 was transiently expressed in HEK‐293 cells. It was found that PxOA2B2 could be activated by both octopamine and tyramine, resulting in increased intracellular cyclic AMP (cAMP) levels, whereas dopamine and serotonin were not effective in eliciting cAMP production. Further studies with series of PxOA2B2 agonists and antagonists showed that all four tested agonists (e.g., naphazoline, clonidine, 2‐phenylethylamine, and amitraz) could activate the PxOA2B2 receptor, and two of tested antagonists (e.g., phentolamine and mianserin) had significant antagonistic effects. However, antagonist of yohimbine had no effects. Quantitative real‐time polymerase chain reaction analysis showed that Pxoa2b2 gene was expressed in all developmental stages of P. xylostella and that the highest expression occurred in male adults. Further analysis with fourth‐instar P. xylostella larvae showed that the Pxoa2b2 gene was mainly expressed in Malpighian tubule, epidermal, and head tissues. This study provides both a pharmacological characterization and the gene expression patterns of the OA2B2 in P. xylostella, facilitating further research for insecticides using PxOA2B2 as a target.     

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.     

Cloning, localization, and permanent expression of a Drosophila octopamine receptor

A cDNA for a member of the G protein-coupled receptor family was isolated from Drosophila using a probe derived from a human beta 2-adrenergic receptor cDNA. This Drosophila receptor gene is localized at 99A10-B1 on the right arm of chromosome 3 and is preferentially expressed in Drosophila heads. The insect octopamine receptor has been permanently expressed in mammalian cells, where it mediates the attenuation of adenylate cyclase activity and exhibits a pharmacological profile consistent with an octopamine type 1 receptor. Sequence and pharmacological comparisons indicate that the octopamine receptor is unique but closely related to mammalian adrenergic receptors, perhaps as an evolutionary precursor.     

Agonist-induced desensitization of an octopamine receptor

The octopamine-sensitive adenylate cyclase associated with haemocytes of the American cockroach, Periplaneta americana, has been used as a model system with which to study desensitization of the octopamine receptor. Preincubation of the haemocytes with octopamine results in a large decrease in subsequent maximal stimulation of cyclic AMP production by octopamine with little change in affinity of the receptor for the agonist. This effect of preincubation is dependent upon the concentration of octopamine in the preincubation media and on the duration of exposure. The attenuation appears to be a receptor-mediated event rather than an artifact of the preincubation. Octopamine receptor agonists (octopamine, synephrine, N-demethylchlordimeform) induce desensitization while biogenic amines with poor octopamine receptor affinity (dopamine, serotonin, norepinephrine) are without affect. In contrast, the octopamine receptor antagonist, phentolamine, appears to enhance subsequent stimulation by octopamine. The attenuation of octopamine stimulation of adenylate cyclase is conserved in broken-cell preparations with no alteration of responses to NaF or forskolin. Incubation of the cells with dibutyryl cyclic AMP or forskolin does not induce desensitization. The data indicate that the OA receptors coupled to AC in cockroach haemocytes undergo an homologous desensitization in response to exposure to agonists.     

Cloning, expression and functional analysis of an octopamine receptor from Periplaneta americana

Octopamine regulates multiple physiological functions in invertebrates. The biological effects of octopamine and the pharmacology of octopamine receptors have been extensively studied in the American cockroach, Periplaneta americana. This paper reports the cloning of the first octopamine receptor from Periplaneta americana. A cDNA encoding a putative 7 transmembrane receptor was isolated from the head of Periplaneta americana. The encoded protein contains 628 amino acids and has sequence similarity to other biogenic amine receptors. This protein was expressed in COS-7 cells for radioligand binding studies using the antagonist 3H-yohimbine. Competitive binding comparing biogenic amines that could potentially function as endogenous ligands demonstrated this receptor had the highest affinity for octopamine (Ki = 13.3 microM) followed by tyramine, dopamine, serotonin and histamine. Octopamine increased both cAMP levels (EC50 = 1.62 microM) and intracellular concentrations of calcium through the receptor expressed in HEK-293 cells. Tyramine increased levels of both of these second messengers but only at significantly higher concentrations than octopamine. The cAMP increase by octopamine was independent of the increase in calcium. Competitive binding with antagonists revealed this receptor is similar to Lym oa1 from Lymnaea stagnalis. The data indicate that this cDNA is the first octopamine receptor cloned from Periplaneta americana and therefore has been named Pa oa1.     

Honey bee dopamine and octopamine receptors linked to intracellular calcium signaling have a close phylogenetic and pharmacological relationship

Background

Three dopamine receptor genes have been identified that are highly conserved among arthropod species. One of these genes, referred to in honey bees as Amdop2, shows a close phylogenetic relationship to the a-adrenergic-like octopamine receptor family. In this study we examined in parallel the functional and pharmacological properties of AmDOP2 and the honey bee octopamine receptor, AmOA1. For comparison, pharmacological properties of the honey bee dopamine receptors AmDOP1 and AmDOP3, and the tyramine receptor AmTYR1, were also examined.

Methodology/Principal Findings

Using HEK293 cells heterologously expressing honey bee biogenic amine receptors, we found that activation of AmDOP2 receptors, like AmOA1 receptors, initiates a rapid increase in intracellular calcium levels. We found no evidence of calcium signaling via AmDOP1, AmDOP3 or AmTYR1 receptors. AmDOP2- and AmOA1-mediated increases in intracellular calcium were inhibited by 10 µM edelfosine indicating a requirement for phospholipase C-β activity in this signaling pathway. Edelfosine treatment had no effect on AmDOP2- or AmOA1-mediated increases in intracellular cAMP. The synthetic compounds mianserin and epinastine, like cis-(Z)-flupentixol and spiperone, were found to have significant antagonist activity on AmDOP2 receptors. All 4 compounds were effective antagonists also on AmOA1 receptors. Analysis of putative ligand binding sites offers a possible explanation for why epinastine acts as an antagonist at AmDOP2 receptors, but fails to block responses mediated via AmDOP1.

Conclusions/Significance

Our results indicate that AmDOP2, like AmOA1, is coupled not only to cAMP, but also to calcium-signalling and moreover, that the two signalling pathways are independent upstream of phospholipase C-β activity. The striking similarity between the pharmacological properties of these 2 receptors suggests an underlying conservation of structural properties related to receptor function. Taken together, these results strongly support phylogenetic analyses indicating that the AmDOP2 and AmOA1 receptor genes are immediate paralogs.     

Identification and characterization of a novel family of Drosophila beta-adrenergic-like octopamine G-protein coupled receptors

Insect octopamine receptors carry out many functional roles traditionally associated with vertebrate adrenergic receptors. These include control of carbohydrate metabolism, modulation of muscular tension, modulation of sensory inputs and modulation of memory and learning. The activation of octopamine receptors mediating many of these actions leads to increases in the levels of cyclic AMP. However, to date none of the insect octopamine receptors that have been cloned have been convincingly shown to be capable of directly mediating selective and significant increases in cyclic AMP levels. Here we report on the identification and characterization of a novel, neuronally expressed family of three Drosophila G-protein coupled receptors that are selectively coupled to increases in intracellular cyclic AMP levels by octopamine. This group of receptors, DmOct beta1R (CG6919), DmOct beta2R (CG6989) and DmOct beta3R (CG7078) shows homology to vertebrate beta-adrenergic receptors. When expressed in Chinese hamster ovary cells all three receptors show a strong preference for octopamine over tyramine for the accumulation of cyclic AMP but show unique pharmacological profiles when tested with a range of synthetic agonists and antagonists. Thus, the pharmacological profile of individual insect tissue responses to octopamine might vary with the combination and the degree of expression of the individual octopamine receptors present.     

The cloning of one putative octopamine receptor and two putative serotonin receptors from the tobacco hawkmoth, Manduca sexta

Serotonin and octopamine (OA) are biogenic amines that are active throughout the nervous systems of insects, affecting sensory processing, information coding and behavior. As an initial step towards understanding the modulatory roles of these amines in olfactory processing we cloned two putative serotonin receptors (Ms5HT1A and Ms5HT1B) and one putative OA (MsOAR) receptor from the moth Manduca sexta. Ms5HT1A and Ms5HT1B were both similar to 5HT1-type receptors but differed from each other in their N-terminus and 3rd cytoplasmic loop. Ms5HT1A was nearly identical to a serotonin receptor from Heliothis virescens and Ms5HT1B was almost identical to a serotonin receptor from Bombyx mori. The sequences for homologs of Ms5HT1A from B. mori and Ms5HT1B from H. virescens were also obtained, suggesting that the Lepidoptera likely have at least two serotonin receptors. The MsOAR shares significant sequence homology with pharmacologically characterized OA receptors, but less similarity to putative OA/tyramine receptors from the moths B. mori and H. virescens. Using the MsOAR sequence, fragments encoding putative OA receptors were obtained from B. mori and H. virescens, suggesting that MsOAR is the first OA receptor cloned from a lepidopteran.     

Pharmacological profile of octopamine and 5HT receptors on the lateral oviducts of the cockroach,Periplaneta americana

The effects of the amines 5HT and octopamine on the myogenic activity of Periplaneta americana (L.) oviducts and the pharmacological profile of octopamine and 5HT receptors on the lateral oviducts have been determined. Application of 5HT to the oviducts resulted in a dose-dependent increase in basal tonus and amplitude of contractions. Antagonist studies revealed that the 5HT receptor on the cockroach oviduct most resembles the mammalian 5HT₂ receptor. Application of octopamine resulted in a decrease in basal tonus and had a biphasic effect on the amplitude of contractions, being stimulatory at low doses and inhibitory at higher ones. The inhibitory effects of octopamine appear to be mediated via cAMP and are blocked by antagonists which indicate that the octopamine receptor is of the octopamine-2 type. © 1995 Wiley-Liss, Inc.     

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。     

A possible new class of octopamine receptors coupled to adenylate cyclase in the brain of the dipterous Ceratitis capitata. Pharmacological characterization and regulation of 3H-octopamine binding

Octopamine exerts its effects in insects through interaction with at least two classes of receptors, designated octopamine-1 and octopamine-2. Octopamine-2 receptors are positively coupled to adenylate cyclase, while octopamine-1 receptors are not coupled to this enzyme system. Ceratitis capitata brain appears to have octopamine receptors as unique aminergic receptors coupled to adenylate cyclase. These receptors show some pharmacological analogies with respect to octopamine-2 receptors, however they should constitute a new class of octopamine receptors. C. capitata brain octopamine receptors have also been characterized by [3H]octopamine-binding studies, exhibiting similar regulatory mechanisms to other receptors coupled to adenylate cyclase activation.     

Photoaffinity Labeling of a Neuronal Octopamine Receptor

Abstract: The invertebrate aminergic neurotransmitter and neuromodulator octopamine (OA) acts at both neuronal and nonneuronal receptors that appear to have distinct pharmacological characteristics. The current work uses a potent and specific OA photoaffinity ligand, tritiated 2(2,6-diethyl-4-azidophenylimino)imidazolidine ([³H]NC-5Z), to identify and characterize a putative neuronal OA receptor protein in membranes from nerve tissue of the desert locust, Schistocerca gregaria . Under nonphotolyzing conditions, [³H]NC-5Z demonstrated high-affinity binding ( K _D = 2.5 ± 0.3 n M ; B _max = 702 fmol/mg of protein) to a single class of noninteracting sites. The absolute and rank order potency of binding of both agonists and antagonists was highly correlated ( r = 0.99) with their known ability to displace [³H]OA binding to locust neuronal membranes and was consistent with the labeling of a class 3 OA receptor. Under photolyzing conditions, [³H]NC-5Z demonstrated irreversible binding that was resistant to trichloroacetic acid and methanol, displaceable by OA and other octopaminergic agonists and antagonists, soluble in sodium dodecyl sulfate, and only sparingly soluble in nonionic detergents. Membrane-bound [³H]NC-5Z, solubilized with Nonidet P-40, bound specifically only to immobilized concanavalin A or lentil lectin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of photolyzed proteins under reducing conditions revealed a single peak of radioactivity with a molecular mass of 53 ± 5 kDa. Taken together, these biochemical and pharmacological results support the identity of this protein peak as that of the neuronal OA3 receptor.     

Pharmacological characterisation and functional roles for egg-laying of a β-adrenergic-like octopamine receptor in the brown planthopper Nilaparvata lugens

Octopamine, the invertebrate counterpart of adrenaline and noradrenaline, controls and modulates many physiological and behavioral processes in protostomes. It mediates its effects by binding to specific receptors belonging to the superfamily of G-protein coupled receptors. We report the cloning of a cDNA from the brown planthopper (Nloa2b2) sharing high similarity with members of the OA2B2 receptor class. Activation of NlOA2B2 by octopamine increased the production of cAMP in a dose-dependent manner (EC₅₀ = 114 nM). Tyramine also activated the receptor but with much less potency than octopamine. Using a series of known agonists and antagonists of octopamine receptors and cAMP measurements, we observed a rather unique pharmacological profile of NlOA2B2. The potency ranking of the tested agonists was naphazoline > clonidine. The activated effect of octopamine is abolished by co-incubation with epinastine, mianserin, phentolamine, methiothepin, butaclamol or methysergide. Nloa2b2 was expressed in different developmental stages and in various tissues including female reproductive regions known to be involved in egg-laying behavior. Using in vivo pharmacology and RNAi methodology, we demonstrated that interference of NlOA2B2 signaling pathway had a strong impact on the egg-laying behavior of female brown planthopper. The data presented here mark the first comprehensive study—from gene to behavior—of a OA2B2 receptor in the rice brown planthopper.     

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.     

Functional and pharmacological characterization of a β-adrenergic-like octopamine receptor from the silkworm Bombyx mori

A cDNA encoding a seven-transmembrane receptor was cloned from the nervous tissues of silkworm (Bombyx mori) larvae. Sequence analysis indicated that the gene is an ortholog of CG6989, which encodes a Drosophila β-adrenergic-like octopamine (OA) receptor (DmOctβ2R). As very little information is available regarding this class of receptors, we generated a cell line that stably expressed the gene in HEK-293 cells and we then performed functional and pharmacological studies of this receptor. [³H]OA-binding assays using membrane preparations of this cell line showed that the receptor possesses a higher affinity for OA than for tyramine (TA) or dopamine (DA). The cell line elicited a bell-shaped, OA concentration-dependent increase in intracellular cAMP levels, with a maximum at 100 nM. (R)-OA was more potent than (S)-OA. TA and DA had weak or marginal effects on cAMP production. The OA receptor agonist demethylchlordimeform elicited a similar biphasic response, although the maximum response was attained at a concentration as low as 1 nM. The rank order of potency of other agonists was as follows: naphazoline > tolazoline, clonidine. Among the antagonists tested, only chlorpromazine significantly attenuated the OA-induced increase in cAMP levels. No increase in intracellular Ca²⁺ levels was observed with OA at concentrations up to 100 μM. These findings indicate that the cloned receptor is a β-adrenergic-like OA receptor with unique functional and pharmacological 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.     

Molecular and functional characterization of an octopamine receptor from honeybee (Apis mellifera) brain

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.     

Identification of critical structural determinants responsible for octopamine binding to the alpha-adrenergic-like Bombyx mori octopamine receptor

Octopamine (OA) is a biogenic amine with a widespread distribution in the insect nervous system. OA modulates and/or regulates various behavioral patterns of insects as a neurotransmitter, neuromodulator, and neurohormone. OA receptors (OARs) belong to one of the families of G protein-coupled receptors (GPCRs). The binding of OA to OARs is coupled to the activation of the specific G proteins, which induces the release of intracellular second messengers such as cAMP and/or calcium. We previously reported the isolation of an OAR (BmOAR1) from Bombyx mori. In the study presented here, five mutated BmOAR1s were constructed with a point mutation in the putative binding crevice and expressed in HEK-293 cells. The S202A mutant receptor was found to retain the cAMP response to OA as does the wild-type receptor, but such function was impaired in the other four mutants (D103A, S198A, Y412F, and S198A/S202A). Furthermore, competition binding assays using [3H]OA and calcium mobilization assays gave results that were approximately consistent with those of the cAMP assays. Taken together, the results indicate that D103 and S198 are involved in the binding and activation of BmOAR1 with OA through electrostatic or hydrogen bond interactions, but S202 does not appear to participate in this process. Y412 seems to be involved in one of the active forms of BmOAR1. These findings should prove helpful in designing new pest control chemicals.