Peter D. Evans

Insect octopamine receptors: a new classification scheme based on studies of cloned Drosophila G-protein coupled receptors

SummaryInsect 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.

Inhibition of Caenorhabditis elegans social feeding by FMRFamide-related peptide activation of NPR-1

Social and solitary feeding in natural Caenorhabditis elegans isolates are associated with two alleles of the orphan G-protein-coupled receptor (GPCR) NPR-1: social feeders contain NPR-1 215F, whereas solitary feeders contain NPR-1 215V. Here we identify FMRFamide-related neuropeptides (FaRPs) encoded by the flp-18 and flp-21 genes as NPR-1 ligands and show that these peptides can differentially activate the NPR-1 215F and NPR-1 215V receptors. Multicopy overexpression of flp-21 transformed wild social animals into solitary feeders. Conversely, a flp-21 deletion partially phenocopied the npr-1(null) phenotype, which is consistent with NPR-1 activation by FLP-21 in vivo but also implicates other ligands for NPR-1. Phylogenetic studies indicate that the dominant npr-1 215V allele likely arose from an ancestral npr-1 215F gene in C. elegans. Our data suggest a model in which solitary feeding evolved in an ancestral social strain of C. elegans by a gain-of-function mutation that modified the response of NPR-1 to FLP-18 and FLP-21 ligands.

Stress-induced changes in the octopamine levels of insect haemolymph

Abstract The relationship between stress and haemolymph octopamine levels has been examined in the locust Schistocerca americana gregaria and the cockroach Periplaneta americana . The resting level of octopamine ( ± SE , n = 24 ) in haemolymph sampled in the first quarter of the light cycle from the metathoracic leg of locusts was 7.5 ± 0.7 pg/μl and 4.4 ± 0.8 pg/μl from the cockroach thorax. In P. americana , 74% of the octopamine in the haemolymph was present in the plasma. Significant variation in resting levels was found when haemolymph was taken from different sampling sites in locusts and when haemolymph was removed at various times during a 24 hr period from cockroaches, with the highest concentration coinciding with the peak of locomotor activity. This pattern was reversed in cockroach nervous tissue with octopamine accumulating in the photophase and being reduced in the scotophase. An increase in haemolymph octopamine was a common response to a variety of stressful stimuli in insects, with rises of up to 10-fold in locusts and 3-fold in cockroaches. The results are discussed in relation to insect activity levels and the proposed neurohormonal role of octopamine in mediating the fight or flight response in insects.

Rapid, Nongenomic Responses to Ecdysteroids and Catecholamines Mediated by a Novel Drosophila G-Protein-Coupled Receptor

Nongenomic response pathways mediate many of the rapid actions of steroid hormones, but the mechanisms underlying such responses remain controversial. In some cases, cell-surface expression of classical nuclear steroid receptors has been suggested to mediate these effects, but, in a few instances, specific G-protein-coupled receptors (GPCRs) have been reported to be responsible. Here, we describe the activation of a novel, neuronally expressed Drosophila GPCR by the insect ecdysteroids ecdysone (E) and 20-hydroxyecdysone (20E). This is the first report of an identified insect GPCR interacting with steroids. The Drosophila melanogaster dopamine/ecdysteroid receptor (DmDopEcR) shows sequence homology with vertebrate β-adrenergic receptors and is activated by dopamine (DA) to increase cAMP levels and to activate the phosphoinositide 3-kinase pathway. Conversely, E and 20E show high affinity for the receptor in binding studies and can inhibit the effects of DA, as well as coupling the receptor to a rapid activation of the mitogen-activated protein kinase pathway. The receptor may thus represent the Drosophila homolog of the vertebrate “γ-adrenergic receptors,” which are responsible for the modulation of various activities in brain, blood vessels, and pancreas. Thus, DmDopEcR can function as a cell-surface GPCR that may be responsible for some of the rapid, nongenomic actions of ecdysteroids, during both development and signaling in the mature adult nervous system.

Isolation, primary structure and bioactivity of schistoflrf-amide, a FMRF-amide-like neuropeptide from the locust, Schistocerca gregaria

We have isolated a neuropeptide, related to the Phe-Met-Arg-Phe-NH2 family, from the thoracic nervous system of the locust, Schistocerca gregaria, using a purification system based on the radioimmunoassay of high pressure liquid chromatography fractions. The primary sequence of this locust peptide is Pro-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH2. The bioactivities of the native and synthetic neuropeptide are identical in both the locust heart and hindleg extensor-tibiae muscle bioassays.

Cloning and Functional Characterization of a Novel Dopamine Receptor from Drosophila melanogaster

A cDNA clone is described that encodes a novel G-protein-coupled dopamine receptor (DopR99B) expressed in Drosophila heads. The DopR99B receptor maps to 99B3–5, close to the position of the octopamine/tyramine receptor gene at 99A10–B1, suggesting that the two may be related through a gene duplication. Agonist stimulation of DopR99B receptors expressed in Xenopus oocytes increased intracellular Ca2+ levels monitored as changes in an endogenous inward Ca2+-dependent chloride current. In addition to initiating this intracellular Ca2+ signal, stimulation of DopR99B increased cAMP levels. The rank order of potency of agonists in stimulating the chloride current is: dopamine > norepinephrine > epinephrine > tyramine. Octopamine and 5-hydroxytryptamine are not active (<100 μm). This pharmacological profile plus the second-messenger coupling pattern suggest that the DopR99B receptor is a D1-like dopamine receptor. However, the hydrophobic core region of the DopR99B receptor shows almost equal amino acid sequence identity (40–48%) with vertebrate serotonergic, α1- and β-adrenergic, and D1-like and D2-like dopaminergic receptors. Thus, thisDrosophila receptor defines a novel structural class of dopamine receptors. Because DopR99B is the second dopamine receptor cloned from Drosophila, this work establishes dopamine receptor diversity in a system amenable to genetic dissection.

OCTOPAMINE DISTRIBUTION IN THE INSECT NERVOUS SYSTEM

Abstract— Octopamine distribution has been surveyed in the nervous systems of two insect species, the locust, Schistocerca americana gregaria, and the cockroach Periplaneta americana. It is essentially similar for both species, being highly localised in the ganglia of the ventral nerve cord. Large amounts of octopamine are also found in the optic lobes especially, in the locust where it is concentrated in the medulla of the optic lobe. Octopamine can also be shown to be associated with insect neurohae‐mal structures such as the corpora cardiaca and the neurohaemal organs of the medial nervous system. The significance of the distribution is discussed.

Identification and characterization of a novel family of Drosophilaβ-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, DmOctβ1R (CG6919), DmOctβ2R (CG6989) and DmOctβ3R (CG7078) shows homology to vertebrate β‐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.

Biogenic amines in the nervous system of the cockroach, Periplaneta americana: Association of octopamine with mushroom bodies and dorsal unpaired median (DUM) neurones

Abstract The distribution of dopamine and noradrenaline in the CNS of the cockroach Periplaneta americana is examined using a radioenzymatic assay. Histochemical techniques for amine localisation (neutral red staining and histochemical fluorescence) have also been applied to the cockroach CNS. The results suggest that both the dorsal unpaired median (DUM) neurones in the thoracic and abdominal ganglia and the globuli cells of the mushroom bodies of the cockroach cerebral ganglia are octopaminergic. These results are confirmed by radioenzymatic assays for octopamine on microdissected somata.

OCTOPAMINE: A HIGH‐AFFINITY UPTAKE MECHANISM IN THE NERVOUS SYSTEM OF THE COCKROACH

Abstract— In the CNS of the cockroach, Periplaneta americana, the uptake of the biogenic amine, octopamine, can be divided into three components. High and low affinity Na+‐sensitive components (Kms 0.5 μ and 19.8 UM respectively) are present, together with a Na+‐insensitive component which shows no saturation kinetics between 0.07 and 100 μ The structure‐specificity dependence of the components and their drug sensitivity have been examined. The significance of the high‐affinity uptake component is discussed in terms of amine inactivation, and its parallels with noradrenaline uptake in the vertebrate nervous system are considered.