Biomedical subjects

D B Sattelle

Publications and source records attributed to D B Sattelle.

At least 37 records · Page 2Linked to original sources

PubMed2000

Symposium overview: mechanism of action of nicotine on neuronal acetylcholine receptors, from molecule to behavior.

Nicotine has long been known to interact with nicotinic acetylcholine (ACh) receptors since Langley used it extensively to chart sympathetic ganglia a century ago. It has also been used as an effective insecticide. However, it was not until the 1990s that the significance of nicotine was increasingly recognized from the toxicological, pharmacological, and environmental points of view. This is partly because studies of neuronal nicotinic ACh receptors are rapidly emerging from orphan status, fueled by several lines of research. Since Alzheimer's disease is known to be associated with down-regulation of cholinergic activity in the brain, a variety of nicotine derivatives are being tested and developed for treatment of the disease. Public awareness of the adverse effects of nicotine has reached the highest level recently. Since insect resistance to insecticides is one of the most serious issues in the pest-control arena, it is an urgent requirement to develop new insecticides that act on target sites not shared by the existing insecticides. The neuronal nicotinic ACh receptor is one of them, and new nicotinoids are being developed. Thus, the time is ripe to discuss the mechanism of action of nicotine from a variety of angles, including the molecular, physiological, and behavioral points of view. This Symposium covered a wide area of nicotine studies: genetic, genomic, and functional aspects of nicotinic ACh receptors were studied, as related to anthelmintics and insecticides; interactions between ethanol and nicotine out the ACh receptor were analyzed, in an attempt to explain the well-known heavy drinker-heavy smoker correlation; the mechanisms that underlie the desensitization of ACh receptors were studied as related to nicotine action; selective pharmacological profiles of nicotine, and descriptions of some derivatives were described; and chronic nicotine infusion effects on memory were examined using animal models.

Animals↗

PubMed1999

Inositol 1,4,5-trisphosphate receptors are strongly expressed in the nervous system, pharynx, intestine, gonad and excretory cell of Caenorhabditis elegans and are encoded by a single gene (itr-1).

Inositol 1,4,5-trisphosphate (InsP3) activates receptors (InsP3Rs) that mediate intracellular Ca(2+ )release, thereby modulating intracellular calcium signals and regulating important aspects of cellular physiology and gene expression. To further our understanding of InsP3Rs we have characterised InsP3Rs and the InsP3R gene, itr-1, from the model organism Caenorhabditis elegans. cDNAs encoding InsP3Rs were cloned enabling us to: (a) identify three putative transcription start sites that result in alternative mRNA 5' ends: (b) detect alternative splicing at three sites and: (c) determine the full genomic organisation of the itr-1 gene. The InsP3R protein (ITR-1) is approximately 42 % identical with known InsP3Rs and possesses conserved structural features. When the putative InsP3 binding domain was expressed in Escherichia coli, specific binding of InsP3 was detected. Using antibodies against ITR-1 we detected a protein of 220 kDa in C. elegans membranes. These antibodies and itr-1::GFP (green fluorescent protein) reporter constructs were used to determine the expression pattern of itr-1 in C. elegans. Strong expression was observed in the intestine, pharynx, nerve ring, excretory cell and gonad. These results demonstrate the high degree of structural and functional conservation of InsP3Rs from nematodes to mammals and the utility of C. elegans as a system for studies on InsP3R mediated signalling.

Amino Acid Sequence↗

PubMed1999

Cross-resistance with dieldrin of a novel tricyclic dinitrile GABA receptor antagonist.

A novel tricyclic dinitrile, KN244, blocked the wild-type (dieldrin-sensitive) homo-oligomeric gamma-aminobutyric acid (GABA)-gated chloride channel of Drosophila melanogaster expressed in Xenopus oocytes. Sensitivity to the block by KN244 of the response to 30 microM GABA (IC50=41.6 nM, wild-type RDLac) was reduced abut 100 fold (IC50=4.5 microM) in the dieldrin-resistant (RDLacA302S) form of RDL.

Animals↗

PubMed1999

Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+ stores in Drosophila melanogaster S2 cell lines.

The role of acidic intracellular calcium stores in calcium homeostasis was investigated in the Drosophila Schneider cell line 2 (S2) by means of free cytosolic calcium ([Ca2+]i) and intracellular pH (pHi) imaging together with measurements of total calcium concentrations within intracellular compartments. Both a weak base (NH4Cl, 15 mM) and a Na+/H+ ionophore (monensin, 10 microM) evoked cytosolic alkalinization followed by Ca2+ release from acidic intracellular Ca2+ stores. Pretreatment of S2 cells with either thapsigargin (1 microM), an inhibitor of endoplasmic reticulum Ca(2+)-ATPases, or with the Ca2+ ionophore ionomycin (10 microM) was without effect on the amplitude of Ca2+ release evoked by alkalinization. Application of the cholinergic agonist carbamylcholine (100 microM) to transfected S2-DM1 cells expressing a Drosophila muscarinic acetylcholine receptor (DM1) emptied the InsP3-sensitive Ca2+ store but failed to affect the amplitude of alkalinization-evoked Ca2+ release. Glycyl-L-phenylalanine-beta-naphthylamide (200 microM), a weak hydrophobic base known to permeabilize lysosomes by osmotic swelling, triggered Ca2+ release from internal stores, while application of brefeldin A (10 microM), an antibiotic which disperses the Golgi complex, resulted in a smaller increase in [Ca2+]i. These results suggest that the alkali-evoked calcium release is largely attributable to lysosomes, a conclusion that was confirmed by direct measurements of total calcium content of S2 organelles. Lysosomes and endoplasmic reticulum were the only organelles found to have concentrations of total calcium significantly higher than the cytosol. However, NH4Cl (15 mM) reduced the level of total calcium only in lysosomes. Depletion of acidic Ca2+ stores did not elicit depletion-operated Ca2+ entry. They were refilled upon re-exposure of cells to normal saline ([Ca2+]o = 2 mM), but not by thapsigargin-induced [Ca2+]i elevation in Ca(2+)-free saline.

Animals↗

PubMed1999

The Caenorhabditis elegans orthologue of the human gene responsible for spinal muscular atrophy is a maternal product critical for germline maturation and embryonic viability.

Spinal muscular atrophy (SMA) is a common disorder characterized by loss of lower motor neurones of the spinal cord. The disease is caused by mutations in the survival motor neurone ( SMN ) gene. SMN is ubiquitously expressed and evolutionarily conserved, and its role in RNA processing has been well established. However, these properties do not explain the observed specificity of motor neurone death. To gain further insight into the function of SMN, we have isolated and characterized the Caenorhabditis elegans orthologue of the SMN gene ( CeSMN ). Here we show that CeSMN is transmitted maternally as a predominantly nuclear factor, which remains present in all the blastomeres throughout embryonic development and onwards into adulthood. In adult nematodes, a CeSMN-green fluorescent protein fusion protein is expressed in a number of cell types including the germline. Both disruption of the endogenous CeSMN function and overexpression of the gene result in a severe decrease in the number of progeny and in locomotive defects. In addition, its transient knockdown leads to sterility caused by a defect in germ cell maturation. The expression pattern and functional properties so far observed for CeSMN, together with its unusual behaviour in the germline, indicate that SMN may be involved in specific gene expression events at these very early developmental stages. We have also identified a deletion in the CeSMN promoter region in egl-32. This mutant may become a useful genetic tool with which to explore regulation of CeSMN and hence provide possible clues for novel therapeutic strategies for SMA.

Amino Acid Sequence↗

PubMed1999

Cultured insect mushroom body neurons express functional receptors for acetylcholine, GABA, glutamate, octopamine, and dopamine.

Fluorescence calcium imaging with fura-2 and whole cell, patch-clamp electrophysiology was applied to cultured Kenyon cells (interneurons) isolated from the mushroom bodies of adult crickets (Acheta domesticus) to demonstrate the presence of functional neurotransmitter receptors. In all cells investigated, 5 microM acetylcholine (ACh, n = 52) evoked an increase in intracellular free calcium ([Ca2+]i). Similar effects were observed in response to 10 microM nicotine. The ACh response was insensitive to atropine (50 microM) but was reduced by mecamylamine (50 microM) and alpha-bungarotoxin (alpha-bgt, 10 microM). ACh-induced inward ion currents (n = 28, EACh approximately 0 mV) were also blocked by 1 microM mecamylamine and by 1 microM alpha-bgt. Nicotine-induced inward currents desensitized more rapidly than ACh responses. Thus functional alpha-bgt-sensitive nicotinic ACh receptors are abundant on all Kenyon cells tested, and their activation leads to an increase in [Ca2+]i. gamma-Aminobutyric acid (GABA, 100 microM) triggered a sustained decrease in [Ca2+]i. Similar responses were seen with a GABAA agonist, muscimol (100 microM), and a GABAB agonist, 3-APPA (1 mM), suggesting that more than one type of GABA receptor can affect [Ca2+]i. This action of GABA was not observed when the extracellular KCl concentration was lowered. All cells tested (n = 26) with patch-clamp electrophysiology showed picrotoxinin (PTX)-sensitive, GABA-induced (30-100 microM) currents with a chloride-sensitive reversal potential. Thus, an ionotropic PTX-sensitive GABA receptor was found on all Kenyon cells tested. Most (61%) of the 54 cells studied responded to -glutamate (100 microM) application either with a biphasic increase in [Ca2+]i or with a single, delayed, sustained [Ca2+]i increase. Nearly all cells tested (95%, n = 19) responded to (100 microM) -glutamate with rapidly desensitizing, inward currents that reversed at approximately -30 mV. Dopamine (100 microM) elicited either a rapid or a delayed increase in [Ca2+]i in 63% of the 26 cells tested. The time course of these responses varied greatly among cells. Dopamine failed to elicit currents in patch-clamped cells (n = 4). A brief decrease in [Ca2+]i was induced by octopamine (100 microM) in approximately 54% of the cells tested (n = 35). However, when extracellular CaCl2 was lowered, octopamine triggered a substantial increase in [Ca2+]i in 35% of the cells tested (n = 26). No octopamine-elicited currents were detected in patched-clamped cells (n = 10).

Animals↗

PubMed1999

Mapping membrane potential transients in crayfish (Procambarus clarkii) optic lobe neuropils with voltage-sensitive dyes.

Voltage-sensitive dyes NK 2761 and RH 155 were employed (in conjunction with a 12 x 12 photodiode array) to study membrane potential transients in optic lobe neuropils in the eye stalk of the crayfish Procambarus clarkii. By this means we investigated a pathway linking deutocerebral projection neurons, via hemiellipsoid body local interneurons, to an unidentified target (most likely neurons processing visual information) in the medulla terminalis. Rapid (10- to 20-ms duration), transient changes in absorption with the characteristics of action potentials were recorded from the optic nerve and the region occupied by deutocerebral projection neurons after stimulation of the olfactory globular tract in the optic nerve and were blocked by 1 microM tetrodotoxin. Action potentials appeared to propagate to the glomerular layer of the hemiellipsoid body where synaptic responses were recorded from a restricted region of the hemiellipsoid body occupied by dendrites of hemiellipsoid body neurons. Action potentials were also recorded from processes of hemiellipsoid body neurons located in the medulla terminalis. Synaptic responses in the hemiellipsoid body and medulla terminalis were eliminated by addition to the saline of 500 microM Cd2+ or 20 mM Co2+, whereas the action potential attributed to branches of deutocerebral projection neurons in the hemiellipsoid body remained unaffected. Action potentials of hemiellipsoid body neurons in the medulla terminalis evoked postsynaptic potentials (50- to 200-ms duration) with an unidentified target in the medulla terminalis. Transient absorption signals were not detected in either the internal or external medulla nor were they recorded from other parts of the optic lobes in response to electrical stimulation of axons of the deutocerebral projection neurons. Functional maps of optical activity, together with electrophysiological and pharmacological findings, suggest that gamma-aminobutyric acid affects synaptic transmission in glomeruli of the hemiellipsoid body. Synapses of the olfactory pathway located in the medulla terminalis may act as a "filter," modifying visual information processing during olfactory stimulation.

Animals↗

PubMed1998

BIDN, a bicyclic dinitrile convulsant, selectively blocks GABA-gated Cl- channels.

BIDN (3,3-bis(trifluoromethyl)bicyclo[2,2,1]heptane-2,2-dicarbonitrile) at 10(-5) M blocked GABA-induced inhibitory postsynaptic potentials (IPSPs) recorded from an identified, giant interneuron (G12) of the cockroach (Periplaneta americana). The same concentration of this bicyclic dinitrile also blocked Cl- -mediated responses of G12 to GABA applied by pressure microinjection into the terminal abdominal ganglion neuropile containing dendrites of G12. BIDN (10(-5) M) was without effect on a response of G12 to GABA known to be mediated by a GABAB type receptor. In studies of the cell body of an identified motor neurone, the fast coxal depressor (Df) in the cockroach metathoracic ganglion, BIDN (10(-5) M) blocked reversibly an extrasynaptic GABA-gated Cl- channel, but not an extrasynaptic L-glutamate-gated Cl- channel. Glycine-gated Cl- channels observed when rat brain messenger RNA was expressed in Xenopus laevis oocytes were unaffected by BIDN at concentrations up to 10(-4) M, whereas this same concentration of BIDN completely blocked GABA-gated Cl- responses recorded from the same preparations. Unlike picrotoxin, which antagonises a variety of ligand-gated Cl- channels, to date BIDN has been found to block only Cl- channels gated by GABA, both in insect and vertebrate preparations.

Animals↗

PubMed1998

Adult insect mushroom body neurons in primary culture: cell morphology and characterization of potassium channels.

The dissociation and maintenance in culture of cells derived from the mushroom bodies of adult crickets (Acheta domesticus) are described. This primary culture was developed in order to investigate maturation and differentiation of mushroom-body cells including Kenyon cells, the major intrinsic interneurons of mushroom bodies, which have been shown to be involved in learning and memory in insects. Three distinct cell types were observed, all identified as neural cells on the basis of their size, morphology and immunocytochemical staining with horseradish peroxidase. These cells appear to correspond to the three cell types observed in vivo: Kenyon cells, ganglion mother cells and neuroblasts. Some cells showed neurite growth, usually with long unipolar processes, occasionally with either bipolar or, more rarely, multipolar processes. Neuronal cell bodies readily formed seals with patch pipettes, allowing stable, whole-cell, patch-clamp electrophysiological recordings. Depolarization of the cell under voltage-clamp resulted in at least two types of outwardly directed potassium currents: a delayed rectifier-type of current that was sensitive to tetraethylammonium, and a cadmium-sensitive current with rapid inactivation. Neither type of current was affected by quinidine, a blocker of potassium currents recorded from pupal honeybee Kenyon cells. Other ionic currents, which have yet to be characterized, were also observed.

Animals↗

PubMed1998

Thapsigargin and receptor-mediated activation of Drosophila TRPL channels stably expressed in a Drosophila S2 cell line.

The Drosophila melanogaster genes, transient receptor potential (trp) and transient receptor potential-like (trpl) encode putative plasma membrane cation channels TRP and TRPL, respectively. We have stably co-expressed Drosophila TRPL with a Drosophila muscarinic acetylcholine receptor (DM1) in a Drosophila cell line (S2 cells). Basal Ca2+ levels measured using Fura-2/AM in unstimulated S2-DM1-TRPL cells were low and indistinguishable from untransfected cells, indicating that the TRPL channels were not constitutively active in this expression system. Activation of DM1 receptor in S2-DM1-TRPL cells by 100 microM carbamylcholine induced Ca2+ release from an intracellular Ca2+ pool followed by a Gd(3+)-insensitive Ca2+ influx. Pretreatment of S2-DM1-TRPL cells with 10 microM atropine abolished Gd(3+)-insensitive Ca2+ influx triggered by carbamylcholine, but the response was not blocked by prior incubation with pertussis toxin. TRPL channels could also be reliably activated by bath application of 1 microM thapsigargin for 10 min or 100 nM thapsigargin for 60 min in Ca(2+)-free solution. In some cells, TRPL channels activated by thapsigargin could further be activated by carbamylcholine. The findings suggest that, when stably expressed in the S2 cell line, TRPL may be regulated by two distinct mechanisms: (i) store depletion; and (ii) stimulation of DM1 receptor via pertussis-toxin insensitive G-protein (or the subsequent activation of PLC), but without further requirement for Ca2+ release.

Animals↗

PubMed1998

Effects of the alpha subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors.

1. Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (alpha4beta2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal alpha subunit (SAD) with the chicken beta2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (-)-nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two-electrode, voltage-clamp electrophysiology. 2. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the alpha4beta2 receptor; (+)-epibatidine, (-)-nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken beta2 receptor, as was (-)-nicotine, whereas (+)-epibatidine and acetylcholine were full agonists. 3. The EC50 of imidacloprid was decreased by replacing the chicken alpha4 subunit with the Drosophila SAD alpha subunit. This alpha subunit substitution also resulted in an increase in the EC50 for (+)-epibatidine, (-)-nicotine and acetylcholine. Thus, the Drosophila (SAD) alpha subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs. 4. Imidacloprid acted as a weak antagonist of ACh-mediated responses mediated by SADbeta2 hybrid receptors and as a weak potentiator of ACh responses mediated by alpha4beta2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the alpha subunit.

Animals↗

PubMed1998

Functional characterization of a mutated chicken alpha7 nicotinic acetylcholine receptor subunit with a leucine residue inserted in transmembrane domain 2.

1. Site-directed mutagenesis was used to create an altered form of the chicken alpha7 nicotinic acetylcholine (ACh) receptor subunit (alpha7x61) in which a leucine residue was inserted between residues Leu9' and Ser10' in transmembrane domain 2. The properties of alpha7x61 receptors are distinct from those of the wild-type receptor. 2. Oocytes expressing wild-type alpha7 receptors responded to 10 microM nicotine with rapid inward currents that desensitized with a time-constant of 710+/-409 ms (mean+/-s.e.mean, n=5). However in alpha7x61 receptors 10 microM nicotine resulted in slower onset inward currents that desensitized with a time-constant of 5684+/-3403 ms (mean+/-s.e.mean, n = 4). No significant difference in the apparent affinity of nicotine or acetylcholine between mutant and wild-type receptors was observed. Dihydro-beta-erythroidine (DHbetaE) acted as an antagonist on both receptors. 3. Molecular modelling of the alpha7x61 receptor channel pore formed by a bundle of M2 alpha-helices suggested that three of the channel lining residues would be altered by the leucine insertion i.e.; Ser10 would be replaced by the leucine insertion, Val13' and Phe14' would be replaced, by Thr and Val, respectively. 4 When present in the LEV-1 nicotinic ACh receptor subunit from Caenorhabditis elegans the same alteration conferred resistance to levamisole anthelmintic drug. Levamisole blocked responses to nicotine of wild-type and alpha7x61 receptors. However, block was more dependent on membrane potential for the alpha7x61 receptors. 5. We conclude that the leucine insertion in transmembrane domain 2 has the unusual effect of slowing desensitization without altering apparent agonist affinity.

Acetylcholine↗

PubMed1998

An extensive and diverse gene family of nicotinic acetylcholine receptor alpha subunits in Caenorhabditis elegans.

Using reverse transcription-polymerase chain reactions the transcription of eight novel candidate nicotinic acetylcholine receptor (nAChR) alpha subunit genes has been demonstrated in the nematode Caenorhabditis elegans. Together with five other alpha subunit genes described elsewhere by ourselves (unc-38) and other workers (deg-3, acr-4, Ce21 and acr-6), this is now the largest known family of nAChR alpha subunit genes in a single species. By homology we have identified four groups of alpha subunits: DEG-3-like; ACR-16[Ce21]-like; UNC-38-like and ACR-8-like. Five C. elegans nAChR alpha subunits contain a modification in loop C of the ACh binding site in which the normally conserved Tyr-x-Cys-Cys, is replaced by a distinct motif (Tyr-x-x-Cys-Cys). Variation is also found in the channel lining M2 regions, including the replacement in four subunits of the highly conserved leucine at the 9' position by valine and most notably, the replacement in all ACR-8-like subunits of the highly conserved glutamic acid at the -1' position by histidine. Restrained molecular dynamics simulations have been used to generate homo-pentameric M2 helix bundle models for alpha subunits and possible functional implications examined. The calculated electrostatic potential energy profile for the M2 region of ACR-8 differs strikingly from that of ACR-16[Ce21] largely due to the presence of histidine at the -1' position, suggesting a possible perturbation of nAChR channel action permeability in the presence of this subunit type.

Amino Acid Sequence↗

PubMed1998

Antagonist profile and molecular dynamic simulation of a Drosophila melanogaster muscarinic acetylcholine receptor.

A stably-transfected, Drosophila cell line (S2-DMl-1) expressing the Drosophila DMl muscarinic acetylcholine receptor (mAChR) exhibits high-affinity, saturable, specific binding of the radiolabelled muscarinic antagonist [3H]-N-methyl scopolamine ([3H]-NMS) with an equilibrium dissociation constant (Kd) of 0.67 +/- 0.02 and a Bmax of 1.53 +/- 0.3 pmol/mg protein. Displacement of [3H]-NMS by mAChR antagonists results in the pharmacological profile: 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) > hexahydrosiladifenidol > p-fluorohexahydrosiladifenidol > nitrocaramiphen > pirenzepine > methoctramine > AFDX-116. This antagonist profile most closely resembles that of the vertebrate M3 mAChR subtype. In this study, however, we have demonstrated that the antagonist profile of DM1 is distinct from those of vertebrate mAChR subtypes. Molecular dynamic simulations of the Drosophila muscarinic receptor are presented in the free, carbamylcholine-bound and NMS-bound forms. Theoretical, quantitative structure-activity relationship models have been developed; a good correlation is observed between the interaction energies of the minimized ligand-receptor complexes and the pharmacological affinities of the antagonists tested.

Animals↗

PubMed1997

Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, and unc-38 encode functional nicotinic acetylcholine receptor subunits.

We show that three of the eleven genes of the nematode Caenorhabditis elegans that mediate resistance to the nematocide levamisole and to other cholinergic agonists encode nicotinic acetylcholine receptor (nAChR) subunits. unc-38 encodes an alpha subunit while lev-1 and unc-29 encode non-alpha subunits. The nematode nAChR subunits show conservation of many mammalian nAChR sequence features, implying an ancient evolutionary origin of nAChR proteins. Expression in Xenopus oocytes of combinations of these subunits that include the unc-38 alpha subunit results in levamisole-induced currents that are suppressed by the nAChR antagonists mecamylamine, neosurugatoxin, and d-tubocurarine but not alpha-bungarotoxin. The mutant phenotypes reveal that unc-38 and unc-29 subunits are necessary for nAChR function, whereas the lev-1 subunit is not. An UNC-29-GFP fusion shows that UNC-29 is expressed in body and head muscles. Two dominant mutations of lev-1 result in a single amino acid substitution or addition in or near transmembrane domain 2, a region important to ion channel conductance and desensitization. The identification of viable nAChR mutants in C. elegans provides an advantageous system in which receptor expression and synaptic targeting can be manipulated and studied in vivo.

Amino Acid Sequence↗

PubMed1997

Prothoracicotropic hormone-producing neurosecretory cells in the silkworm, Bombyx mori, express a muscarinic acetylcholine receptor.

Using an anti-muscarinic acetylcholine receptor (mAChR) antibody and an anti-prothoracicotropic hormone (PTTH) antibody, double immunofluorescence staining was performed on brain sections of the silkworm, Bombyx mori. Four pairs of dorsolateral neurosecretory cells, along with some intercerebral neurosecretory cells, were immunoreactive to anti-mAChR antibody. Among these immunoreactive cells, two pairs of dorsolateral neurosecretory cells were identified to be PTTH-producing neurosecretory cells. Nerve fibers in the median and paramedian protocerebral areas, and nerve terminals in the corpus allatum also showed immunoreactivity to the anti-mAChR antibody. Some of these nerve terminals expressing mAChRs were overlapped by immunostaining with the anti-PTTH antibody. These results indicated that PTTH-producing neurosecretory cells of Bombyx mori expressed an mAChR, and that muscarinic, cholinergic transmission might directly regulate PTTH release from neurosecretory cells.

Animals↗

PubMed1997

Molecular biology of insect neuronal GABA receptors.

Ionotropic gamma-aminobutyric acid (GABA) receptors are distributed throughout the nervous systems of many insect species. As with their vertebrate counterparts, GABAA receptors and GABAC receptors, the binding of GABA to ionotropic insect receptors elicits a rapid, transient opening of anion-selective ion channels which is generally inhibitory. Although insect and vertebrate GABA receptors share a number of structural and functional similarities, their pharmacology differs in several aspects. Recent studies of cloned Drosophila melanogaster GABA receptors have clarified the contribution of particular subunits to these differences. Insect ionotropic GABA receptors are also the target of numerous insecticides and an insecticide-resistant form of a Drosophila GABA-receptor subunit has enhanced our understanding of the structure-function relationship of one aspect of pharmacology common to both insect and vertebrate GABA receptors, namely antagonism by the plant-derived toxin picrotoxinin.

Animals↗

PubMed1997

Effects of [3H]-BIDN, a novel bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates.

1. The radiolabelled bicyclic dinitrile, [3H]-3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([3H]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of gamma-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC50, 35 +/- 3 nM) and dieldrin (IC50, 93 +/- 7 nM), displaced [3H]-BIDN from rootworm membranes. When tested at 100 microM, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]oct ane(EBOB), 4-t-butyl-2,6,7-trioxa-1-phosphabicy-clo[2.2.2]octane-1-thio ne (TBPS), 1-phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [3H]-BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands. 2. Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and alpha and beta endosulphan displace bound [3H]-BIDN from rootworm membranes by a competitive mechanism. 3. Rat brain membranes were also shown to possess a population of saturable, specific [3H]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [3H]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm. 4. Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABA(B) receptors were unaffected. 5. Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl- channels to which a number of insecticidally-active molecules bind.

Animals↗