Amtyr1: characterization of a gene from honeybee (Apis mellifera) brain encoding a functional tyramine receptor

Biogenic amine receptors are involved in the regulation and modulation of various physiological and behavioral processes in both vertebrates and invertebrates. We have cloned a member of this gene family from the CNS of the honeybee, Apis mellifera. The deduced amino acid sequence is homologous to tyramine receptors cloned from Locusta migratoria and Drosophila melanogaster as well as to an octopamine receptor cloned from Heliothis virescens. Functional properties of the honeybee receptor were studied in stably transfected human embryonic kidney 293 cells. Tyramine reduced forskolin-induced cyclic AMP production in a dose-dependent manner with an EC50 of approximately 130 nM. A similar effect of tyramine was observed in membrane homogenates of honeybee brains. Octopamine also reduced cyclic AMP production in the transfected cell line but was both less potent (EC50 of approximately 3 microM) and less efficacious than tyramine. Receptor-encoding mRNA has a wide-spread distribution in the brain and subesophageal ganglion of the honeybee, suggesting that this tyramine receptor is involved in sensory signal processing as well as in higher-order brain functions.     

Isolation, cloning, and tissue expression of a putative octopamine/tyramine receptor from locust visceral muscle tissues

Octopamine has been shown to play major roles in invertebrate nervous systems as a neurotransmitter, neuromodulator, and neurohormone. Tyramine is the biochemical precursor of octopamine and its neuromodulatory role is now being investigated and clarified in invertebrates, particularly in insects. Both octopamine and tyramine mediate their actions via G protein-coupled receptors (GPCRs) and are believed to play important functions in the regulation of physiological processes in locust oviduct. Here we report the isolation, cloning, and tissue expression of a putative octopamine/tyramine receptor from the locust, Locusta migratoria. Degenerate oligonucleotides in PCR reactions were first used to obtain partial cDNA sequences and then these partial sequences were used in screens to obtain a full-length cDNA. The cloned cDNA is about 3.1 kb long and encodes a protein of 484 amino acid residues with typical characteristics of GPCRs including seven transmembrane domains and many signature residues. The amino acid sequence of the cloned cDNA displays sequence similarities with known GPCRs, particularly octopamine/tyramine receptors. Screening of the locust genomic DNA library resulted in isolation of a genomic DNA with the same size as the cDNA, indicating that the gene is intron-less. RT-PCR and Northern blot analyses revealed the expression of the receptor mRNA in brain, ventral nerve cord, oviduct, and midgut tissues. Southern blot analyses using EcoRI and HindIII restriction endonucleases recognized at least two distinct gene bands.     

Octopamine receptors in the honeybee (Apis mellifera) brain and their disruption by RNA-mediated interference

Octopamine plays important neuromodulatory roles in the honeybee brain. Accordingly, mRNA from a recently identified honeybee octopamine receptor (AmOA1) is distributed throughout the brain. We have evaluated the occurrence of AmOA1 in the antennal lobe (AL) as well as rest of the brain (RB) by western blotting using an antiserum raised against a peptide selected from AmOA1 sequence. In addition to an expected band (78 kDa in the AL), one additional band (72 kDa) was identified from the AL and four bands (48, 60, 72 and 78 kDa) were observed in the RB. These bands were also recognized with antiserum against a different peptide segment from an octopamine receptor ortholog from the fruitfly (OAMB). Significant sequence identity with the peptide segment used to generate the antiserum was only found with OAMB and its splice variants in fruitfly; it was less conserved in other biogenic amine receptors from honeybee and other insects. Furthermore, western blot analysis performed on brains with dsRNA-treated antennal lobes showed a decrease in the intensity of all four bands. This suggests that AmOA1 antiserum specifically recognizes one or more types of AmOA1 receptors in the honeybee brain. We extend our earlier study of RNAi to quantify the rate of spread of dsRNA from a localized injection to other neuropils.     

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.     

Glutathione and Signal Transduction in the Mammalian CNS

The tripeptide glutathione (GSH) has been thoroughly investigated in relation to its role as antioxidant and free radical scavenger. In recent years, novel actions of GSH in the nervous system have also been described, suggesting that GSH may serve additionally both as a neuromodulator and as a neurotransmitter. In the present article, we describe our studies to explore further a potential role of GSH as neuromodulator/neurotransmitter. These studies have used a combination of methods, including radioligand binding, synaptic release and uptake assays, and electrophysiological recording. We report here the characteristics of GSH binding sites, the interrelationship of GSH with the NMDA receptor, and the effects of GSH on neural activity. Our results demonstrate that GSH binds via its gamma-glutamyl moiety to ionotropic glutamate receptors. At micromolar concentrations GSH displaces excitatory agonists, acting to halt their physiological actions on target neurons. At millimolar concentrations, GSH, acting through its free cysteinyl thiol group, modulates the redox site of NMDA receptors. As such modulation has been shown to increase NMDA receptor channel currents, this action may play a significant role in normal and abnormal synaptic activity. In addition, GSH in the nanomolar to micromolar range binds to at least two populations of binding sites that appear to be distinct from all known excitatory amino acid receptor subtypes. GSH bound to these sites is not displaceable by glutamatergic agonists or antagonists. These binding sites, which we believe to be distinct receptor populations, appear to recognize the cysteinyl moiety of the GSH molecule. Like NMDA receptors, the GSH binding sites possess a coagonist site(s) for allosteric modulation. Furthermore, they appear to be linked to sodium ionophores, an interpretation supported by field potential recordings in rat cerebral cortex that reveal a dose-dependent depolarization to applied GSH that is blocked by the absence of sodium but not by lowering calcium or by NMDA or (S)-2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate antagonists. The present data support a reevaluation of the role of GSH in the nervous system in which GSH may be involved both directly and indirectly in synaptic transmission. A full accounting of the actions of GSH may lead to more comprehensive understanding of synaptic function in normal and disease states.     

Control of postsynaptic Ca2+ influx in developing neocortex by excitatory and inhibitory neurotransmitters.

We assessed the pathways by which excitatory and inhibitory neurotransmitters elicit postsynaptic changes in [Ca2+]i in brain slices of developing rat and cat neocortex, using fura 2. Glutamate, NMDA, and quisqualate transiently elevated [Ca2%]i in all neurons. While the quisqualate response relied exclusively on voltage-gated Ca2+ channels, almost all of the NMDA-induced Ca2+ influx was via the NMDA ionophore itself, rather than through voltage-gated Ca2+ channels. Glutamate itself altered [Ca2+]i almost exclusively via the NMDA receptor. Furthermore, synaptically induced Ca2+ entry relied almost completely on NMDA receptor activation, even with low-frequency stimulation. The inhibitory neurotransmitter GABA also increased [Ca2+]i, probably via voltage-sensitive Ca2+ channels, whereas the neuromodulator acetylcholine caused Ca2+ release from intracellular stores via a muscarinic receptor. Low concentrations of these agonists produced nonperiodic [Ca2+]i oscillations, which were temporally correlated in neighbouring cells. Optical recording with Ca2(+)-sensitive indicators may thus permit the visualization of functional networks in developing cortical circuits.     

Calcium response after stimulation by substance P of U373 MG cells: inhibition of store-operated calcium entry by protein kinase C

In this paper we investigate the Ca2+ response after Substance P (SP) stimulation of U373 MG cells. SP is a tachykinin and physiologically acts as a neurotransmitter and neuromodulator in the nervous system, but pathologically triggers malignant glial cells, such as U373 MG, to release cytokines and increase proliferation rate.In this paper we show that SP increases the proliferation rate of U373 MG cells and the intracellular Ca2+ concentration by mobilizing Ca2+ only from thapsigargin-sensitive stores. In fact, Ca2+ entry through store-operated calcium entry (SOCE) channels, which was observed after thapsigargin treatment, was not detected after stimulation by SP. The inhibition of SOCE after SP stimulation must be mediated by protein kinase C (PKC), because it was not observed in the presence of calphostin C (an inhibitor of PKC). Moreover, stimulation by SP-induced membrane potential hyperpolarization. Our results are consistent with the following sequence of events: (i) SP interacts with NK(1) receptors; (ii) fast homologous receptor desensitization occurs; (iii) reuptake by endoplasmic reticulum Ca(2+)-ATPase quantitatively overwhelms the extrusion by plasma membrane Ca2+-ATPase. These results have two important consequences. In U373 MG cells the SOCE does not contribute to the Ca2+ response after SP, and is not necessarily involved in promoting cell proliferation.     

Modulation of glutamate receptor functions by glutathione

In addition to its well-known antioxidant effects, glutathione apparently has an additional double role in the central nervous system as a neurotransmitter and neuromodulator. A number of recent neurochemical, neuropharmacological and electrophysiological studies have yielded evidence on both functions. As an excitatory neurotransmitter, glutathione depolarizes neurons by acting as ionotropic receptors of its own which are different from any other excitatory amino acid receptors. As a neuromodulator, it displaces ionotropic glutamate receptor ligands from their binding sites and regulates calcium influx through N-methyl-D-aspartate receptor-governed ionophores. In brain slices glutathione has been shown to regulate the release of other transmitters, e.g., gamma-aminobutyrate and dopamine, mediated by N-methyl-D-aspartate receptors. In the present article, we review recent findings on the neuromodulatory actions of glutathione and discuss possible physiological and pathophysiological consequences.     

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.     

Molecular,pharmacological, and signaling properties of octopamine receptors from honeybee (Apis mellifera) brain

G protein‐coupled receptors are important regulators of cellular signaling processes. Within the large family of rhodopsin‐like receptors, those binding to biogenic amines form a discrete subgroup. Activation of biogenic amine receptors leads to transient changes of intracellular Ca²⁺‐([Ca²⁺]_i) or 3′,5′‐cyclic adenosine monophosphate ([cAMP]_i) concentrations. Both second messengers modulate cellular signaling processes and thereby contribute to long‐lasting behavioral effects in an organism. In vivo pharmacology has helped to reveal the functional effects of different biogenic amines in honeybees. The phenolamine octopamine is an important modulator of behavior. Binding of octopamine to its receptors causes elevation of [Ca²⁺]_i or [cAMP]_i. To date, only one honeybee octopamine receptor that induces Ca²⁺ signals has been molecularly and pharmacologically characterized. Here, we examined the pharmacological properties of four additional honeybee octopamine receptors. When heterologously expressed, all receptors induced cAMP production after binding to octopamine with EC_50s in the nanomolar range. Receptor activity was most efficiently blocked by mianserin, a substance with antidepressant activity in vertebrates. The rank order of inhibitory potency for potential receptor antagonists was very similar on all four honeybee receptors with mianserin >> cyproheptadine > metoclopramide > chlorpromazine > phentolamine. The subroot of octopamine receptors activating adenylyl cyclases is the largest that has so far been characterized in arthropods, and it should now be possible to unravel the contribution of individual receptors to the physiology and behavior of honeybees.

     

Octopamine--a single modulator with double action on the heart of two insect species (Apis mellifera macedonica and Bactrocera oleae): Acceleration vs. inhibition

The effects of octopamine, the main cardioacceleratory transmitter in insects, were investigated, in the isolated hearts of the honeybee, Apis mellifera macedonica, and the olive fruit fly, Bactrocera oleae. Octopamine induced a biphasic effect on the frequency and force of cardiac contractions acting as an agonist, with a strong acceleratory effect, at concentrations higher than 10⁻¹² M for the honeybee and higher than 50 × 10⁻⁹ M for the olive fruit fly. The heart of the honeybee is far more sensitive than the heart of olive fruit fly. This unusual sensitivity is extended to the blockers of octopaminergic receptors, where phentolamine at 10⁻⁵ M stopped the spontaneous contractions of the honeybee heart completely and permanently, while the same blocker at the same concentration caused only 50% inhibition in the heart of the olive fruit fly. Phentolamine and mianserin at low concentrations of 10⁻⁷ M also blocked the heart octopaminergic receptors, but for a short period of time, of less than 15.0 min, while a partial recovery in heart contraction started in spite of the presence of the antagonist. The unusual response of the honeybee heart in the presence of phentolamine and/or mianserin suggests excitatory effects of octopamine via two different receptor subtypes. At lower concentrations, 10⁻¹⁴ M, the agonist octopamine was converted to an antagonist, inducing a hyperpolarization in the membrane potential of the honeybee cardiac pacemaker cells and inhibiting the firing rate of the heart. The inhibitory effects of octopamine on certain parameters of the rhythmic bursts of the heart of the honeybee, were similar to those of mianserin and phentolamine, typical blockers of octopaminergic receptors. The heart of the olive fruit fly was 10⁵ times less sensitive to octopamine, since a persistent inhibition of heart contractions occurred at 10⁻⁹ M. In conclusion, the acceleration of the insect heart is achieved by increasing the levels of octopamine, while there is a passive but also an active decrease in heart activity due to the minimization of octopamine.     

Octopamine-Mediated Neuromodulation of Insect Senses

Octopamine functions as a neuromodulator, neurotransmitter, and neurohormone in insect nervous systems. Octopamine has a prominent role in influencing multiple physiological events: (a) as a neuromodulator, it regulates desensitization of sensory inputs, arousal, initiation, and maintenance of various rhythmic behaviors and complex behaviors such as learning and memory; (b) as a neurotransmitter, it regulates endocrine gland activity; and (c) as a neurohormone, it induces mobilization of lipids and carbohydrates. Octopamine exerts its effects by binding to specific proteins that belong to the superfamily of G protein-coupled receptors and share the structural motif of seven transmembrane domains. The activation of octopamine receptors is coupled with different second messenger pathways depending on species, tissue source, receptor type and cell line used for the expression of cloned receptor. The second messengers include adenosine 3′,5′-cyclic monophosphate (cAMP), calcium, diacylglycerol (DAG), and inositol 1,4,5-trisphosphate (IP₃). The cAMP activates protein kinase A, calcium and DAG activate protein kinase C, and IP₃ mobilizes calcium from intracellular stores. Octopamine-mediated generation of these second messengers is associated with changes in cellular response affecting insect behaviors. The main objective of this review is to discuss significance of octopamine-mediated neuromodulation in insect sensory systems.     

Postaggregative differentiation induction by cyclic AMP in Dictyostelium: intracellular transduction pathway and requirement for additional stimuli

Cyclic AMP induces postaggregative differentiation in aggregation competent cells of Dictyostelium by interacting with cell surface cAMP receptors. We investigated the transduction pathway of this response and additional requirements for the induction of postaggregative differentiation. Optimal induction of postaggregative gene expression requires that vegetative cells are first exposed to 2-4 hr of nanomolar cAMP pulses, and subsequently for 4-6 hr to steady-state cAMP concentrations in the micromolar range. Cyclic AMP pulses, which are endogenously produced before and during aggregation, induce full responsiveness to cAMP as a morphogen. The transduction pathway from the cell surface cAMP receptor to postaggregative gene expression may involve Ca2+ ions as intracellular messengers. A cAMP-induced increase in intracellular cAMP or cGMP levels is not involved in the transduction pathway.     

Calcium signaling and synaptic modulation: regulation of endocannabinoid-mediated synaptic modulation by calcium

Postsynaptic Ca2+ signal influences synaptic transmission through multiple mechanisms. Some of them involve retrograde messengers that are released from postsynaptic neurons in a Ca2+-dependent manner and modulate transmitter release through activation of presynaptic receptors. Recent studies have revealed essential roles of endocannabinoids in retrograde modulation of synaptic transmission. Endocannabinoid release is induced by either postsynaptic Ca2+ elevation alone or activation of postsynaptic Gq/11-coupled receptors with or without Ca2+ elevation. The former pathway is independent of phospholipase Cbeta (PLCbeta) and requires a large Ca2+ elevation to a micromolar range. The latter pathway requires PLCbeta and is facilitated by a moderate Ca2+ elevation to a submicromolar range. This facilitation is caused by Ca2+-dependency of receptor-driven PLCbeta activation. The released endocannabinoids then activate presynaptic cannabinoid receptor type 1 (CB1), and suppress transmitter release from presynaptic terminals. Both CB1 receptors and Gq/11-coupled receptors are widely distributed in the brain. Thus, the endocannabinoid-mediated retrograde modulation may be an important and widespread mechanism in the brain, by which postsynaptic events including Gq/11-coupled receptor activation and Ca2+ elevation can retrogradely influence presynaptic function.     

Structure of a novel InsP3 receptor.

Inositol 1,4,5-trisphosphate (InsP3) constitutes a major intracellular second messenger that transduces many growth factor and neurotransmitter signals. InsP3 causes the release of Ca2+ from intracellular stores by binding to specific receptors that are coupled to Ca2+ channels. One such receptor from cerebellum has previously been extensively characterized. We have now determined the full structure of a second, novel InsP3 receptor which we refer to as type 2 InsP3 receptor as opposed to the cerebellar type 1 InsP3 receptor. The type 2 InsP3 receptor has the same general structural design as the cerebellar type 1 InsP3 receptor with which it shares 69% sequence identity. Expression of the amino-terminal 1078 amino acids of the type 2 receptor demonstrates high affinity binding of InsP3 to the type 2 receptor with a similar specificity but higher affinity than observed for the type 1 receptor. These results demonstrate the presence of several types of InsP3 receptor in brain and raise the possibility that intracellular Ca2+ signaling may involve multiple pathways with different regulatory properties dependent on different InsP3 receptors.     

Octopaminergic modulation of the membrane currents in the central feeding system of the pond snail Lymnaea stagnalis

Octopamine is released by the intrinsic OC interneurons in the paired buccal ganglia and serves both as a neurotransmitter and a neuromodulator in the central feeding network of the pond snail Lymnaea stagnalis. The identified B1 buccal motoneuron receives excitatory inputs from the OC interneurons and is more excitable in the presence of 10 microM octopamine in the bath. This modulatory effect of octopamine on the B1 motoneuron was studied using the two electrode voltage clamp method. In normal physiological saline depolarising voltage steps from the holding potential of -80 mV evoke a transient inward current, presumably carried by Na(+) ions. The peak values of this inward current are increased in the presence of 10 microM octopamine in the bath. In contrast, both the transient (IA) and delayed (IK) outward currents are unaffected by octopamine application. Replacing the normal saline with a Na(+)-free bathing solution containing K(+) channel blockers (50 mM TEACl, 4 mM 4AP) revealed the presence of an additional inward current of the B1 neurons, carried by Ca(2+). Octopamine (10 microM) in the bath decreased the amplitudes of this current. These results suggest that the membrane mechanisms which underlie the modulatory effect of octopamine on the B1 motoneuron include selective changes of the Na(+)- and Ca(2+)-channels.     

Isolation of a cDNA for an octopamine-like, G-protein coupled receptor from the cattle tick, Boophilus microplus.

Octopamine is a biogenic amine neurotransmitter of invertebrates that binds to a G-protein coupled receptor that has seven transmembrane domains. Formamidine pesticides like amitraz are highly specific agonists of the octopamine receptor. Amitraz is used extensively to control the cattle tick, Boophilus microplus, and many other ticks but now there are strains of ticks that are resistant to amitraz. We have isolated a cDNA from the cattle tick, B. microplus, that belongs to the biogenic amine family of receptors. The predicted amino acid sequence from this cDNA is most similar to octopamine receptors from insects. The nucleotide sequence of this gene from amitraz-resistant and amitraz-susceptible cattle ticks was identical. Thus, a point mutation/s did not confer resistance to amitraz in the strains we studied. Alternative explanations for resistance to amitraz in B. microplus are discussed.