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。     

Positive and negative modulation of Bombyx mori adenylate cyclase by 5-phenyloxazoles: identification of octopamine and tyramine receptor agonists

Nineteen 5-phenyloxazoles (5POs) were examined for their ability to modulate adenylate cyclase by measuring cAMP produced in head membrane homogenates of fifth instar larvae of the silkworm Bombyx mori. Among the compounds tested, 5-(4-methoxyphenyl)oxazole (9) and the 2,6-dichlorophenyl congener showed the highest activation of adenylate cyclase; both compounds produced approximately half the level of cAMP produced by the action of octopamine (OCT). The OCT receptor antagonists chlorpromazine, mianserin, and metoclopramide attenuated 9-stimulated cAMP production. In contrast, 5-(4-hydroxyphenyl)oxazole (8) and the 4-cyanophenyl congener attenuated both OCT-stimulated and basal cAMP production. The tyramine (TYR) receptor antagonist yohimbine inhibited the negative effect of 8. These findings indicate that the 5PO class of compounds includes both positive and negative modulators of adenylate cyclase in the heads of B. mori larvae, and that 9 and 8 are OCT and TYR receptor agonists, respectively. These compounds might prove useful for a pharmacological dissection of biogenic amine receptors.     

Single amino acid of an octopamine receptor as a molecular switch for distinct G protein couplings

Octopamine (OA) is thought to be the invertebrate counterpart of noradrenaline and regulates various behavioral patterns of invertebrates by activating OA receptors. As a typical G protein-coupled receptor, BmOAR1, a Bombyx mori α-adrenergic-like OA receptor, is coupled to both G_s and G_q proteins to induce the release of the intracellular second messengers cAMP and Ca²⁺. In this study, we examined the pharmacological and functional properties of the cloned OA receptor, using OA enantiomers. The wild-type OA receptor exhibited significant stereoselectivity for OA enantiomers in cAMP production and binding affinity, but not in calcium signaling response. On the contrary, the Y412F mutant abolished the discrimination between OA enantiomers in the binding affinity and did not evoke any cAMP signaling response. This mutant exhibited levels of potency and efficacy similar to those of the wild-type receptor in the calcium assays. Taken together, these results suggest that Tyr412 might act as a molecular switch to regulate distinct G protein couplings, and a sequential activation model is proposed for such specific-residue-dependent, selective activation in receptors that are coupled to multiple G proteins.     

Substituent-dependent, positive and negative modulation of Bombyx mori adenylate cyclase by synthetic octopamine/tyramine analogues

Octopamine (OCT)/tyramine (TYR) analogues, mainly including p- and beta-substituted phenylethylamines, were prepared as probes for the ligand-binding site(s) of adenylate cyclase-coupled OCT or TYR receptors, and were examined for their effects on cAMP production in the head membranes of Bombyx mori larvae. Small structural changes in OCT and TYR proved to lead to three types of OCT/TYR analogues: (1) compounds that increase the cAMP level by themselves and also depress OCT-stimulated cAMP production, (2) compounds that do not stimulate cAMP production by themselves but inhibit OCT-stimulated cAMP production, and (3) compounds that are not active in either the stimulation of cAMP production or the inhibition of OCT-stimulated cAMP production. Tyramine, which belongs to the second group, also inhibited the basal level of cAMP production at high concentrations. The data indicate that two biogenic amine systems that positively and negatively regulate the level of the second messenger cAMP are present in the head part of B. mori larvae. This finding points to the necessity of separately evaluating the positive and negative regulatory effects in order to quantitatively understand the structure-activity relationships of OCT receptor ligands. Arch.     

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.     

Trace amines differentially regulate adult locomotor activity, cocaine sensitivity, and female fertility in Drosophila melanogaster

The trace biogenic amines tyramine and octopamine are found in the nervous systems of animals ranging in complexity from nematodes to mammals. In insects such as Drosophila melanogaster, the trace amine octopamine is a well-established neuromodulator that mediates a diverse range of physiological processes, but an independent role for tyramine is less clear. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC). We previously reported the identification of two Tdc genes in Drosophila: the peripherally-expressed Tdc1 and the neurally-expressed Tdc2. To further clarify the neural functions of the trace amines in Drosophila, we examined normal and cocaine-induced locomotor activity in flies that lack both neural tyramine and octopamine because of mutation in Tdc2 (Tdc2(RO54)). Tdc2(RO54) flies have dramatically reduced basal locomotor activity levels and are hypersensitive to an initial dose of cocaine. Tdc2-targeted expression of the constitutively active inward rectifying potassium channel Kir2.1 replicates these phenotypes, and Tdc2-driven expression of Tdc1 rescues the phenotypes. However, flies that contain no measurable neural octopamine and an excess of tyramine due to a null mutation in the tyramine beta-hydroxylase gene (TbetaH(nM18)) exhibit normal locomotor activity and cocaine responses in spite of showing female sterility due to loss of octopamine. The ability of elevated levels of neural tyramine in TbetaH(nM18) flies to supplant the role of octopamine in adult locomotor and cocaine-induced behaviors, but not in functions related to female fertility, indicates mechanistic differences in the roles of trace amines in these processes.     

Insulin‐like receptor substrate gene chico regulates octopamine metabolism in Drosophila melanogaster

Octopamine, one of the main insect biogenic amines, plays an important role in the control of fitness in Drosophila melanogaster Meigen. The present study examines the effects of a null mutation of the gene of the insulin‐like receptor substrate (chico), in the heterozygous state, on octopamine metabolism, heat stress resistance and fecundity of D. melanogaster. A rise in the activity of one of the key enzymes of octopamine synthesis, tyrosine decarboxylase, as well as that of an enzyme of its degradation, octopamine‐dependent N‐acetyl transferase, is observed in chico^1/+ females. It is also found that the resistance to heat stress is decreased and fecundity is reduced dramatically in chico^1/+ flies. Such changes in these parameters in D. melanogaster females result from a rise in octopamine titre, which suggests that chico affects the octopamine level by regulating the activity of tyrosine decarboxylase.     

Genetic Control of Biogenic-Amine Systems in Drosophila Under Normal and Stress Conditions

The contents of octopamine and its precursors (tyrosine and tyramine) were studied in adults of two lines of Drosophila virilis with contrasting stress responses. It was demonstrated that in individuals responding to stress by a hormonal stress reaction (line 101), the contents of octopamine and tyrosine are lower than in nonresponding flies (line 147). It was found that there is no difference between the lines in the level of tyramine under normal conditions. The dopamine response to stressor was also studied. Genetic analysis of these differences revealed that they are controlled by a single gene and that the gene is not sex-linked. The gene controlling the response was found to be linked to chromosome 6 of D. virilis.     

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.     

昆虫体内章鱼胺的分布、功能及其研究进展

章鱼胺是无脊椎动物神经系统中普遍存在的多种微量生物胺之一。章鱼胺的分布及含量变化对于昆虫的生长、取食、代谢等多种生理、生物效应具有重要的作用。文章对昆虫体内章鱼胺的功能性、昆虫体内章鱼胺的分布及其主要功能、章鱼胺痕量测定方法、生存环境的改变及化学药剂的作用对章鱼胺含量的影响以及外界刺激对章鱼胺含量影响的生理及生物化学机理研究进行了综述。     

表皮生长因子受体酪氨酸激酶结构域与其抑制剂染料木素的模拟对接

染料木素是表皮生长因子受体酪氨酸激酶结构域(EGFR-TK)高度特异的非竞争性抑制剂.本研究采用AUTODOCK3.05分子对接软件包对EGFR-TK与染料木素进行了模拟对接研究,探究了二者的相互作用机制,为染料木素的抗肿瘤机制提供理论依据.对接结果表明,染料木素结合在EGFR-TK的活性腔中,与EGFR-TK发生了强烈的相互作用,结合自由能△G为-31.2 kJ/mol;染料木素通过干扰TK催化活性结构中Lys721/Glu738离子对的形成而抑制了EGFR-TK的活性,属于非竞争性结合和抑制作用;在结合中,疏水力和氢键发挥了重要作用.     

Accelerated molecular dynamics simulations of the octopamine receptor using GPUs: discovery of an alternate agonist‐binding position

Octopamine receptors (OARs) perform key biological functions in invertebrates, making this class of G‐protein coupled receptors (GPCRs) worth considering for insecticide development. However, no crystal structures and very little research exists for OARs. Furthermore, GPCRs are large proteins, are suspended in a lipid bilayer, and are activated on the millisecond timescale, all of which make conventional molecular dynamics (MD) simulations infeasible, even if run on large supercomputers. However, accelerated Molecular Dynamics (aMD) simulations can reduce this timescale to even hundreds of nanoseconds, while running the simulations on graphics processing units (GPUs) would enable even small clusters of GPUs to have processing power equivalent to hundreds of CPUs. Our results show that aMD simulations run on GPUs can successfully obtain the active and inactive state conformations of a GPCR on this reduced timescale. Furthermore, we discovered a potential alternate active‐state agonist‐binding position in the octopamine receptor which has yet to be observed and may be a novel GPCR agonist‐binding position. These results demonstrate that a complex biological system with an activation process on the millisecond timescale can be successfully simulated on the nanosecond timescale using a simple computing system consisting of a small number of GPUs. Proteins 2016; 84:1480–1489. © 2016 Wiley Periodicals, Inc.     

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.     

Dopamine counteracts octopamine signalling in a neural circuit mediating food response in C. elegans

Animals assess food availability in their environment by sensory perception and respond to the absence of food by changing hormone and neurotransmitter signals. However, it is largely unknown how the absence of food is perceived at the level of functional neurocircuitry. In Caenorhabditis elegans, octopamine is released from the RIC neurons in the absence of food and activates the cyclic AMP response element binding protein in the cholinergic SIA neurons. In contrast, dopamine is released from dopaminergic neurons only in the presence of food. Here, we show that dopamine suppresses octopamine signalling through two D2‐like dopamine receptors and the G protein Gi/o. The D2‐like receptors work in both the octopaminergic neurons and the octopamine‐responding SIA neurons, suggesting that dopamine suppresses octopamine release as well as octopamine‐mediated downstream signalling. Our results show that C. elegans detects the absence of food by using a small neural circuit composed of three neuron types in which octopaminergic signalling is activated by the cessation of dopamine signalling.     

Tyramine receptor (SER-2) isoforms are involved in the regulation of pharyngeal pumping and foraging behavior in Caenorhabditis elegans

Octopamine regulates essential processes in nematodes; however, little is known about the physiological role of its precursor, tyramine. In the present study, we have characterized alternatively spliced Caenorhabditis elegans tyramine receptor isoforms (SER-2 and SER-2A) that differ by 23 amino acids within the mid-region of the third intracellular loop. Membranes prepared from cells expressing either SER-2 or SER-2A bind [3H]lysergic acid diethylamide (LSD) in the low nanomolar range and exhibit highest affinity for tyramine. Similarly, both isoforms exhibit nearly identical Ki values for a number of antagonists. In contrast, SER-2A exhibits a significantly lower affinity than SER-2 for other physiologically relevant biogenic amines, including octopamine. Pertussis toxin treatment reduces affinity for both tyramine and octopamine, especially for octopamine in membranes from cells expressing SER-2, suggesting that the conformation of the mid-region of the third intracellular loop is dictated by G-protein interactions and is responsible for the differential tyramine/octopamine affinities of the two isoforms. Tyramine reduces forskolin-stimulated cAMP levels in HEK293 cells expressing either isoform with nearly identical IC50 values. Tyramine, but not octopamine, also elevates Ca2+ levels in cells expressing SER-2 and to a lesser extent SER-2A. Most importantly, ser-2 null mutants (pk1357) fail to suppress head movements while reversing in response to nose-touch, suggesting a role for SER-2 in the regulation of foraging behavior, and fail to respond to tyramine in assays measuring serotonin-dependent pharyngeal pumping. These are the first reported functions for SER-2. These results suggest that C. elegans contains tyramine receptors, that individual SER-2 isoforms may differ significantly in their sensitivity to other physiologically relevant biogenic amines, such as octopamine (OA), and that tyraminergic signaling may be important in the regulation of key processes in nematodes.     

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.     

Tyramine and octopamine have opposite effects on the locomotion of Drosophila larvae

Biogenic amines are believed to play important roles in producing behaviors. Although some biogenic amines have been extensively studied in both vertebrates and invertebrates, little is known about the effects of trace amines like tyramine and octopamine. We investigated how trace amines affect behaviors using quantitative morphometric methods on Drosophila Tbetah(nM18) and iav(N) mutants that have altered levels of tyramine and octopamine. Locomotion of wild-type and mutant third instar larvae was analyzed using Dynamic Image Analysis System (DIAS) software. We found that Tbetah(nM18) mutants, with elevated tyramine levels and reduced octopamine levels, had a severe locomotion phenotype. Mutant larvae spent much more time in pausing episodes than wild-type larvae and displayed a reduction in speed and linear translocation. The locomotion phenotype was partially rescued by feeding Tbetah(nM18) larvae octopamine, an effect that could be nullified with simultaneous feeding of tyramine. Feeding Tbetah(nM18) larvae yohimbine, an agent that inhibits the activity of Drosophila tyramine receptors, also improved some locomotion parameters. Feeding both octopamine and yohimbine further improved rescue efficiency. Simultaneously reducing the octopamine and tyramine levels as in iav(N) larvae, in contrast, led to a less severe behavioral phenotype than that of Tbetah(nM18) mutants. Feeding iav(N) larvae either tyramine or octopamine exerted only a minor improvement in locomotion. These results suggest that tyramine and octopamine have opposite effects on Drosophila larval locomotion regulation and that a balance between the two is important in producing normal behavior.     

Pharmacological characterization of a Bombyx mori α‐adrenergic‐like octopamine receptor stably expressed in a mammalian cell line

Series of agonists and antagonists were examined for their actions on a Bombyx moriα‐adrenergic‐like octopamine receptor (OAR) stably expressed in HEK‐293 cells. The rank order of potency of the agonists was clonidine>naphazoline>tolazoline in Ca²⁺ mobilization assays, and that of the antagonists was chlorpromazine>yohimbine. These findings suggest that the B. mori OAR is more closely related to the class‐1 OAR in the intact tissue than to the other classes. N′‐(4‐Chloro‐o‐tolyl)‐N‐methylformamidine (DMCDM) and 2‐(2,6‐diethylphenylimino)imidazolidine (NC‐5) elevated the intracellular calcium concentration ([Ca²⁺]_i) with EC₅₀s of 92.8 µM and 15.2 nM, respectively. DMCDM and NC‐5 led to increases in intracellular cAMP concentration ([cAMP]_i) with EC₅₀s of 234 nM and 125 nM, respectively. The difference in DMCDM potencies between the cAMP and Ca²⁺ assays might be due to “functional selectivity.” The Ca²⁺ and cAMP assay results for DMCDM suggest that the elevation of [cAMP]_i, but not that of [Ca²⁺]_i, might account for the insecticidal effect of formamidine insecticides. © 2009 Wiley Periodicals, Inc.