Studies On The Pharmacological Properties And Physiological Roles Of Biogenic Amine Receptors In The Rice Stem Borer Chilo Suppressalis

The biogenic amines, octopamine, tyramine and dopamine regulate many essential processes in insects, such as egg-laying, circadian rhythm, olfactory, decision-making, phase transition, fight and flight, learning and memory. They are products of the amino acid tyrosine and tyramine is the biological precursor of octopamine. All of them are independent neurotransmitters, exert their effects by binding to specific receptor proteins that belong to the superfamily of G-protein-coupled receptors. Because these receptors have been recognized as very suitable and specific insecticide and acaricide targets, the studies of octopamine, tyramine and dopamine in insects, especially the studies about their receptors, have been paid more attentions in recent years. In this study, we chose the rice striped stem borer, Chilo suppressalis Walker (Lepidoptera: Crambidae), one of the main herbivorous pests that cause serious damage to rice production in Asia, as object. Using modern molecular biology methods, we investigated the pharmacological properties and physiological roles of octopamine receptors, tyramine receptors and dopamine receptors in the rice striped stem borer.1. Characterization of an a-adrenergic-like octopamine receptor from the rice stem borer(Chilo suppressalis) and its potential role involved in insect cellular immunityOctopamine, the insect equivalent of norepinephrine, links the nervous system and immune system in insects. This study examines the underlying molecular mechanisms (i.e. second messenger systems) mediating octopamine effects on insect immune cells. At low concentrations (<1μM), octopamine stimulated hemocyte spreading and phagocytosis in the larval Lepidopteran (caterpillar) Chilo suppressalis, whereas at high concentrations (>10μM), octopamine inhibited hemocyte spreading and phagocytosis. Similarly, octopamine concentration had differential effects on two intracellular signaling pathways, Ca2+and cAMP. Low concentrations of octopamine increased intracellular Ca2+, but only high concentrations of octopamine (>1μM) led to an increase in both Ca2+and cAMP. We identified an a-adrenergic-like octopamine receptor in this species (CsOA1) and confirmed that it is expressed in hemocytes. After heterologous expression in HEK-293cells, the CsOAl receptor produced the same octopamine concentration-dependent responses on intracellular Ca2+and cAMP as had been observed in hemocytes. These findings support earlier work showing that octopamine has both stimulatory and suppressive effects on immune responses, depending on the octopamine concentration. Our evidence suggests that these biphasic effects are mediated by an octopamine receptor signaling through intracellular Ca2+and cAMP second messenger pathways. Stress hormones/neuromodulators have complex effects on immune function in animals across phyla. This complexity may be mediated, in part, by conserved connections between adrenergic-like G-coupled-protein receptors and second messenger systems.2. Characterization of a P-adrenergic-like octopamine receptor from the rice stem borer (Chilo suppressalis)Octopamine, the invertebrate counterpart of adrenaline and noradrenaline, plays a key role in regulation of many physiological and behavioral processes in insects. It modulates these functions through binding to specific octopamine receptors, which are typical rhodopsin-like G-protein-coupled receptors. A cDNA encoding a seven-transmembrane receptor was cloned from the nerve cord of the rice stem borer, Chilo suppressalis, viz. CsOA2B2, which shares high sequence similarity to CG6989, a Drosophila β-adrenergic-like octopamine receptor (DmOctβ2R). We generated an HEK-293cell line that stably expresses CsOA2B2in order to examine the functional and pharmacological properties of this receptor. Activation of CsOA2B2by octopamine increased the production of cAMP in a dose-dependent manner (EC50=2.33×10-9M), with a maximum response at100nM. Tyramine also activated the receptor but with much less potency than octopamine. Dopamine and serotonin had marginal effects on cAMP production. Using a series of known agonists and antagonists for octopamine receptors, we observed a rather unique pharmacological profile for CsOA2B2through measurements of cAMP. The rank order of potency of the agonists was naphazoline> clonidine. The activated effect of octopamine is abolished by co-incubation with phentolamine, mianserin or chlorpromazine. Using in vivo pharmacology, CsOA2B2antagonists mianserin and phentolamine impaired the motor ability of individual rice stem borers. The results of the present study are important for a better functional understanding of this receptor as well as for practical applications in the development of environmentally sustainable pesticides.3. Two splicing variants of a novel family of octopamine receptors with different signaling propertiesThe octopamine and tyramine, as the invertebrate counterparts of the vertebrate adrenergic transmitters, control, regulate, and modulate many physiological and behavioral processes. Both compounds mediate their effects by binding to specific receptors belonging to the superfamily of G-protein-coupled receptors. Activation of different receptor types is coupled with different second messenger pathways. Here we described the functional characterization of one octopamine receptor gene from Chilo suppressails. As a result of alternative splicing, this octopamine receptor gene (CsOA3) encodes two molecularly distinct isoforms, CsOA3S and CsOA3L. CsOA3L differs from CsOA3S by the presence of an additional30amino acids within the third intracellular loop. This region is implicated in the receptor interaction with G-proteins. When heterologously expressed, both receptors cause increases of intracellular Ca2+concentration in response to applying nanomolar concentrations of octopamine. The short form, CsOA3S, was activated by both octopamine and tyramine, resulting in decreased intracellular cAMP levels ([cAMP]i) in a dose-dependent manner (EC50～39nM, octopamine;～94nM, tyamine). Tyramine activated the receptor but much lesser potency than octopamine, whereas dopamine and serotonin are not-effective. While CsOA3L did not show this properties. Tissues distribution pattern of CsOA3genes were assessed by qRT-PCR. Overlapping but not identical expression patterns were observed for the individual transcrpts. The CsOA3is, to our knowledge, a new family of insect octopamine receptors.4. Molecular cloning and pharmacological characterisation of a tyramine receptor from the rice stem borer, Chilo suppressalis (Walker)A full-length cDNA (designated CsTyR1) from the rice stem borer, Chilo suppressalis (Walker), has been obtained through homology cloning in combination with rapid amplification of cDNA ends/polymerase chain reaction (RACE-PCR). The mRNA of CsTyR1is present in various tissues, including hemocytes, fat body, midgut, Malpighian tubules, nerve cord and epidermis, and it is found predominantly in the larval nerve cord with16-80-fold enrichment compared with other tissues. The authors generated a HEK-293cell line stably expressing CsTyRl in order to examine functional and pharmacological properties of this receptor. Both TA and OA at0.01-100μM can reduce forskolin-stimulated intracellular cAMP levels in a dose-dependent manner (TA, EC50=369nm; OA, EC50=978nm). In agonist assays, activation of CsTyR1by clonidine and amitraz but not by naphazoline and chlordimeform can also significantly inhibit forskolin-stimulated cAMP production. The inhibitory effect of TA at10μM is eliminated by coincubation with yohimbine, phentolamine or chlorpromazine (each10μM).5. Cloning, expression and functional analysis of a tyramine receptor type2from striped stem borer, Chilo suppressalisBiogenic amines such as octopamine, tyramine, dopamine and serotonin play various important physiological roles in insects by activating distinct G-protein-coupled receptors (GPCRs) that share a putative seven transmembrane domain structure. Here, we report the first cloning and pharmacological characterization of a tyramine receptor type2in the striped stem borer, Chilo suppressalis, viz., CsTyR2, which shares high sequence similarity to members of the invertebrate tyramine receptor family. The CsTyR2receptor was stably expressed in HEK-293cells, and its ligand response has been examined. Receptor activation with TA induced a dose-dependent increase in intracellular Ca2+concentration ([Ca2+]i) in cells, with an EC50value of20nM, whereas octopamine, dopamine and serotonin increased ([Ca2+]i) only at concentrations above100μM. The mRNA is present in various tissues including hemocytes, fatbody, midgut, Malpighian tubules, nerve cord and epidermis in the larva stage. The CsTyR2transcript was detected predominantly in the Malpighian tubules and nerve cord. This expression plasticity indicates that CsTyR2might be involved in excretory system and nervous system in C. suppressalis. This study marks the first comprehensive molecular, pharmacological, and functional characterization of a tyramine receptor in the rice stem borer.6. Molecular and pharmacological characterization of dopamine receptors in the rice stem borer, Chilo suppressalisWe identified two full (CsDOP1and CsDOP3) and one partial (CsDOP2) sequences of putative dopamine receptors in the rice stem borer Chilo suppressalis. The genes CsDOP1and CsDOP2were predicted as orthologs of previously characterized D1-like dopamine receptors in the silkworm Bombyx mori and fruitfly Dmsophila melanogaster. CsDOP3shows high sequence homology with other D2-like dopamine receptors. Heterologous expression of CsDOP1and CsDOP3in HEK-293cells demonstrated dose-dependent responses to dopamine (EC50:CsDOP1=2.0±0.19×10-8M; CsDOP3=6.7±0.58×10-7M). Experiments with agonists and antagonists revealed that the rank order of potency for the active synthetic agonists and antagonists tested was different for each of the receptors (CsDOP1:agonists,6,7-ADTN> dopamine> bromocriptine> pramipexole; antagonists, SCH23390>butaclamol>chlorpromazine>phentolamine> yohimbine>propranolol>ketanserin>flupenthixol> mianserin; CsDOP3:agonists, bromocriptine>DA; antagonists, epinastine=mianserin> SCH23390=chlorpromazine).In this study, we have cloned three octopamine receptors, two tyramine receptors and three dopamine receptors from the rice stem borer, Chilo suppressalis. And we have investigated the pharmacological properties and physiological roles related to innate immunity and motor ability in insects of these receptors.