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。     

章鱼胺及其衍生物的抗氧化性研究

章鱼胺是一种天然的β3-肾上腺素能受体激动剂,关于其生物活性,文献报道大多为肥胖症的治疗和II型糖尿病防治方面的研究。既往研究指出,章鱼胺的前体化合物酪胺具有较高的抗氧化活性,且与其浓度呈正相关。本文以章鱼胺为原料合成十种衍生物。为比较章鱼胺、酪胺及章鱼胺衍生物的抗氧化活性,本文测定了这些物质对DPPH自由基、超氧阴离子自由基和羟基自由基的清除率。研究结果表明,章鱼胺及其两种衍生物OA07和OA08清除DPPH自由基、超氧阴离子自由基和羟基自由基的能力均高于酪胺;另一章鱼胺衍生物OA05具有较高的超氧阴离子自由基清除能力,值得进一步关注和研究。     

昆虫5-羟色胺及其受体的研究进展

5-羟色胺（5-hydroxytryptamine, 5-HT）是昆虫体内一种重要的生物胺。5-HT在昆虫神经组织和非神经组织中均可合成,它可被5-HT转运体重吸收进入突触前结构中。5-HT通过结合特异性的G蛋白偶联受体在昆虫体内发挥不同的神经调控作用,调节昆虫主要的行为活动,比如取食、生物钟、聚集、学习和记忆等。昆虫体内5-HT受体有5种,分别为5-HT1A,5-HT1B, 5-HT2A,5-HT2B 和5-HT7。其中5-HT1A和5-HT1B偶联胞内cAMP的降低, 5-HT2A和5-HT2B偶联胞内Ca²⁺的释放, 5 HT7偶联胞内cAMP的升高。近年来,昆虫体内5-HT及其受体的研究有了很大的进展,昆虫体内越来越多的5-HT受体被克隆,并进行了功能和药理学性质分析。不同昆虫5 HT受体药理学性质存在差异,将为以5-HT受体为靶标,设计新型特异性杀虫剂提供理论基础。     

昆虫体内的酪胺:合成、降解与生理功能

作为神经活性物质,昆虫体内的酪胺(tyramine, TA)主要在酪胺能神经元中合成,但也可在马氏管主细胞中合成。TA在结合其受体发挥生理功能后,可被突触前膜的转运体(transporter)转运回突触前膜重复利用。N-酰基化可能是昆虫体内TA降解的主要途径。目前,昆虫体内发现的TA受体均属于G蛋白偶联受体,通过与Gi或Gq结合导致cAMP或(和) Ca~(2+)水平的变化,实现信号转导。此外,果蝇神经系统内星型胶质细胞、瞬时感受器电位通道Waterwitch (Wtrw)以及多巴胺能神经元也参与TA的信号转导。TA参与昆虫求偶与交配后行为的调节,与章鱼胺(octopamine, OA)、FMIRFamide神经肽协同调节精子和卵的贮存和排放;还参与调节马氏管排泄,与多巴胺(dopamine, DA)协同调节蜜蜂工蜂的生殖分化,与OA以相互拮抗的方式调节昆虫的运动。飞蝗群居型和散居型个体的分化也受TA和OA的协同调节。TA还可以调节采集蜂资源利用与开发的平衡。现综述该领域相关研究进展并展望未来研究方向。     

昆虫多巴胺及其受体的研究进展

多巴胺（dopamine, DA）是脊椎动物和无脊椎动物体内一种重要的生物胺, 其参与调控了昆虫的多种生理反应和行为过程, 如学习与记忆、 认知、 性取向、 抉择、 运动以及型变等。多巴胺主要通过结合特异性的G蛋白偶联受体, 即多巴胺受体（dopamine receptors, DARs）来发挥生理作用。本文综述了多巴胺在昆虫中的调控、 分布及所参与的生理功能, 如多巴胺调控昆虫的交配、 发育、 嗅觉以及运动行为等, 特别对DARs的信号转导、 生理功能以及药理学等方面进行了详细评述。昆虫的DARs大致可分为两大类： D1-like DARs和D2-like DARs。D1-like DARs包含有2种亚型, 分别为DOP1和DOP2。DOP1仅能偶联胞内cAMP的上升, 而DOP2不仅可以起胞内cAMP的上升, 还可偶联胞内Ca²⁺的释放。 D2-like DARs仅包含有1种亚型DOP3, 其被激活后引起胞内cAMP的降低。DA通过激活不同的DARs可偶联不同的第二信使系统, 所产生的下游细胞反应则与昆虫的各种行为相关, 而对昆虫DARs的药理学研究将有助于我们开发特异性的杀虫剂用于害虫防治。     

棉铃虫章鱼胺受体2基因的分子鉴定

【目的】为了探讨章鱼胺受体2基因(Octopamine receptor 2,OA2B2)在棉铃虫Helicoverpa armigera(Hübner)体内表达谱及激素对其调控作用,为今后研究Ha OA2B2受体基因在转录调控中的作用,开发新型杀虫剂提供一定的理论依据。【方法】本实验采用QRT-PCR技术、激素处理、RNAi介导的蜕皮激素相关核受体沉默等方法对Ha OA2B2受体基因在棉铃虫不同发育阶段及组织中的表达谱与激素调控进行探究。【结果】生物信息学分析表明,Ha OA2B2受体基因编码框长度1 191 bp,编码396个氨基酸。Ha OA2B2受体基因氨基酸序列与家蚕Bombyx mori一致性高达89%。Ha OA2B2受体基因在棉铃虫各时期均有表达,尤其在5龄第3天转录水平达到最高;Ha OA2B2受体基因在棉铃虫幼虫头部表达量最高。蜕皮激素可促进Ha OA2B2受体基因的表达。【结论】分析了该基因的序列特征和表达谱,蜕皮激素对其表达量的影响,为今后研究Ha OA2B2受体基因在转录调控中的作用以及开发新型杀虫剂提供一定的理论依据。     

昆虫嗅觉受体的研究进展

昆虫的嗅觉对昆虫的栖息地选择、觅食、群集、趋避、繁殖以及信息传递等行为具有重要的影响。对昆虫嗅觉机理的深入研究和嗅觉信号传导途径的完整阐述,是探索农业害虫的专一性防治的基础。嗅觉受体(olfactory receptors,Ors)是G蛋白偶联受体(G protein-coupled receptor)的一种,是嗅觉系统的关键成分。近年来嗅觉受体的研究日益受到关注。本文对昆虫嗅觉的基本过程、基因结构和表达调控特征、蛋白分子结构、生理功能、分布部位和相关配体的研究等进行了综述。     

中华蜜蜂酪胺受体基因克隆及表达分析

酪胺(tyramine)属于生物多聚胺类,是昆虫中枢神经系统内重要的神经递质、神经调质和神经激素,参与调控昆虫的多种行为和生理过程,如酪胺受体基因参与调控动物的学习与记忆。本研究首次克隆获得中华蜜蜂(Apis cerana cerana)酪胺受体基因Actyr1和Actyr2的全长cDNA序列,利用qRT-PCR方法鉴定了Actyr1和Actyr2在中华蜜蜂不同组织器官中的表达谱,采用地高辛原位杂交技术对Actyr1和Actyr2在大脑中的表达进行了定位。中华蜜蜂Actyr1、Actyr2的cDNA全长序列分别为1241 bp(GenBank登录号：KC814693)和1270 bp(GenBank登录号：KC814694),分别编码297、399个氨基酸残基。qRT-PCR分析结果表明,Actyr1和Actyr2在不同组织中的表达量为头部最高,其次是腹部表皮,触角和胸部肌肉的表达量最低,并且头部的表达量显著高于其他组织的表达量;原位杂交结果显示,Acytr1和Actyr2在中华蜜蜂大脑蘑菇体的凯尼恩细胞、触角叶周围的细胞处均有较强阳性着色。这些研究表明,Acytr1和Actyr2基因可能参与了蜜蜂的学习记忆,并且在相同的细胞中互相作用,共同调控蜜蜂的生物学功能。     

甜味机制的研究进展

甜味的感受细胞是味觉细胞,味觉细胞是个双极细胞,味觉受体是一类G蛋白偶联受体,根据甜味物质性质的不同,通过两种途径－－cAMP途径与IP3和DAG途径进行甜味转导。PKA,PKC,味素和转导素在甜味转导中发挥了不同的功能。     

G蛋白偶联受体二聚化研究进展

G蛋白偶联受体是细胞膜受体最大的家族,参与调节多种生理过程,在信号识别及转导中具有重要作用,传统观点认为G蛋白偶联受体作为单体起作用,近年来,越来越多的证据表明,G蛋白偶联受体不仅能以二聚体形式存在,而且在细胞信号转导中起重要作用,尤其是对阿片受体异源二聚体的研究,推动了这一领域的研究。本文综述了G蛋白偶联受体二聚化研究进展,以及同源和异源二聚体的结构与功能。     

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.     

Tyramine and octopamine: antagonistic modulators of behavior and metabolism

The phenolamines tyramine and octopamine are decarboxylation products of the amino acid tyrosine. Although tyramine is the biological precursor of octopamine, both compounds are independent neurotransmitters, acting through various G-protein coupled receptors. Especially, octopamine modulates a plethora of behaviors, peripheral and sense organs. Both compounds are believed to be homologues of their vertebrate counterparts adrenaline and noradrenaline. They modulate behaviors and organs in a coordinated way, which allows the insects to respond to external stimuli with a fine tuned adequate response. As these two phenolamines are the only biogenic amines whose physiological significance is restricted to invertebrates, the attention of pharmacologists was focused on the corresponding receptors, which are still believed to represent promising targets for new insecticides. Recent progress made on all levels of octopamine/tyramine research enabled us to better understand the molecular events underlying the control of complex behaviors.     

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.     

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.     

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.     

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.     

Neuromodulator octopamine attenuates extrajunctional glutamate sensitivity in insect muscle

Octopamine was found to decrease extrajunctional, but not junctional glutamate responses, in mealworm neuromuscular preparations. This action of octopamine was mimicked by forskolin, 8-(4-chlorophenylthio)-adenosine 3′:5′-cyclic monophosphate (CPT-cyclic AMP), and 8-bromoguanosine 3′:5′-cyclic monophosphate (8-bromo-cyclic GMP), but not by 1,2-oleoylacetylglycerol (OAG), a protein kinase C activator. We suggest that the octopamine-induced reduction in the glutamate sensitivity of extrajunctional membranes may enable the muscle to more closely follow its neuronal input by preventing a depolarization (and hence a conductance increase) due to the discharge of unsequestered transmitter molecules at nonsynaptic sites.