Spatiotemporal calcium signaling in a Drosophila melanogaster cell line stably expressing a Drosophila muscarinic acetylcholine receptor.

A muscarinic acetylcholine receptor (mAChR), DM1, expressed in the nervous system of Drosophila melanogaster, has been stably expressed in a Drosophila S2 cell line (S2-DM1) and used to investigate spatiotemporal calcium changes following agonist activation. Carbamylcholine (CCh) and oxotremorine are potent agonists, whereas application of the vertebrate M1 mAChR agonist, McN-A-343, results in a weak response. Activation of S2-DM1 receptors using CCh resulted in an increase in intracellular calcium ([Ca(2+)](i)) that was biphasic. Two distinct calcium sources were found to contribute to calcium signaling: (1) internal stores that are sensitive to both thapsigargin and 2-aminoethoxydiphenyl borate and (2) capacitative calcium entry. Spatiotemporal imaging of individual S2-DM1 cells showed that the CCh-induced [Ca(2+)](i) transient resulted from a homogeneous calcium increase throughout the cell, indicative of calcium release from internal stores. In contrast, ionomycin induced the formation of a "calcium ring" at the cell periphery, consistent with external calcium influx.

Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors.

Imidacloprid is increasingly used worldwide as an insecticide. It is an agonist at nicotinic acetylcholine receptors (nAChRs) and shows selective toxicity for insects over vertebrates. Recent studies using binding assays, molecular biology and electrophysiology suggest that both alpha- and non-alpha-subunits of nAChRs contribute to interactions of these receptors with imidacloprid. Electrostatic interactions of the nitroimine group and bridgehead nitrogen in imidacloprid with particular nAChR amino acid residues are likely to have key roles in determining the selective toxicity of imidacloprid. Chemical calculation of atomic charges of the insecticide molecule and a site-directed mutagenesis study support this hypothesis.

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.

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.

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.

Polycyclic dinitriles: a novel class of potent GABAergic insecticides provides a new radioligand, [3H]BIDN.

The polycyclic dinitriles are a potent class of insecticides which are non-competitive GABA (gamma-aminobutyric acid) antagonists acting at the convulsant site. Comparison with other classes of GABA convulsant site ligands using molecular modelling has shown significant structural similarities. We have developed a pharmacophore model which unifies this class and some previous classes of GABA convulsants. Key pharmacophore elements are a polarizable functionality separated by a fixed distance from two H-bond accepting elements. This model is based on information from X-ray crystal structures and Sybyl using the Tripos force field. Using this pharmacophore model, numerous structural modifications were explored to enhance understanding of structure-activity relationships at the GABA receptor convulsant site of insects and mammals. A radiolabelled bicyclic dinitrile, [3H]BIDN [3H]3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile+ ++), was prepared from this area of chemistry and was used as a probe for the interaction of polycyclic dinitriles at the target site.

Immunocytochemical mapping of a C-terminus anti-peptide antibody to the GABA receptor subunit, RDL in the nervous system in Drosophila melanogaster.

An antibody raised against a peptide based on the C-terminal derived amino acid sequence from a cloned Drosophila melanogaster (fruit fly) gene, Rdl (resistant to dieldrin), was used to investigate localization of a GABA receptor subunit in adult male D. melanogaster. Many regions in the brain and thoracic ganglia were stained with this antibody. For example, staining was detected in the medulla, lobula and lobular plate optic neurpiles. Also stained were the antennal lobe glomeruli, the ellipsoid body of the central complex and the mushroom bodies. These results suggest possible roles for an RDL-like GABA receptor subunit in the processing of olfactory, visual and mechanosensory information in the nervous system of D. melanogaster.

Blocking actions of BIDN, a bicyclic dinitrile convulsant compound, on wild-type and dieldrin-resistant GABA receptor homo-oligomers of Drosophila melanogaster expressed in Xenopus oocytes.

The receptor antagonist actions are described for a novel bicyclic dinitrile compound (BIDN, 3,3-bis-(trifluoromethyl)-bicyclo [2.2.1] heptane-2,2-dicarbonitrile) on a Drosophila melanogaster homo-oligomeric GABA receptor expressed in Xenopus oocytes. BIDN blocked the wild-type form of the receptor in a neither purely competitive, nor purely non-competitive manner, being dependent on the GABA concentration yet insurmountable, and block was independent of the membrane potential. BIDN was found to be less effective against a mutant (A(302) --> S) form of the receptor resistant to dieldrin and picrotoxinin. This cross resistance of dieldrin-resistant receptors to BIDN is of interest in the light of recent findings that BIDN binding to insect membranes is displaced competitively by dieldrin, but not by picrotoxinin.

[35S]t-butylbicyclophosphorothionate binding sites in susceptible and cyclodiene-resistant houseflies.

4-aminobutyric acid (GABA)-gated chloride ion channels are important molecular targets for a number of polychlorocycloalkane compounds including cyclodiene insecticides. Previous radioligand binding studies have indicated that cyclodiene insecticides are potent inhibitors of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to housefly thorax and abdomen membranes. In the present study, a laboratory-reared, cyclodiene-resistant (CYW) housefly strain (Musca domestica) showed resistance to a number of cyclodiene insecticides. Specific, saturable [35S]TBPS binding was detected in thorax and abdomen membranes prepared from housefly strains susceptible (CSMA) and resistant (CYW) to cyclodienes. Scatchard analysis of [35S]TBPS binding data from CSMA and CYW membranes revealed no significant differences between the two strains in either the affinity (Kd) or the density (Bmax) of specific, saturable binding sites. There were no differences in the comparative effectiveness of a range of polychlorocycloalkanes, including cyclodiene insecticides, as inhibitors of specific [35S]TBPS binding to CSMA and CYW thorax and abdomen membranes. Therefore, if an alteration in target site is a mechanism for resistance to cyclodienes in the CYW strain, it is not readily measurable using [35S]TBPS.

Pharmacology of insect GABA receptors.

A GABA-operated Cl- channel that is bicuculline-insensitive is abundant in the nervous tissue of cockroach, in housefly head preparations and thorax/abdomen preparations, and in similar preparations from several insect species. Bicuculline-insensitive GABA-operated Cl- channels, which are rare in vertebrates, possess sites of action of benzodiazepines, steroids and insecticides that are pharmacologically-distinct from corresponding sites on vertebrate GABAA receptors. The pharmacological profile of the benzodiazepine-binding site linked to an insect CNS GABA-operated Cl- channel resembles more closely that of vertebrate peripheral benzodiazepine-binding sites. Six pregnane steroids and certain polychlorocycloalkane insecticides, which are active at t-butylbicyclophosphorothionate (TBPS)-binding sites, also differ in their effectiveness on vertebrate and insect GABA receptors. Radioligand binding and physiological studies indicate that in insects there may be subtypes of the GABA receptor. Molecular biology offers experimental approaches to understanding the basis of this diversity.

Pharmacological and biochemical properties of insect GABA receptors.

The first evidence for the existence of GABA receptors in any tissue was provided by studies on an invertebrate preparation but, until recently, characterization of GABA receptors from such lower organisms has advanced slowly. The identification of GABA receptors as putative target sites for a variety of insecticidal agents has contributed to the resurgence of interest in amino acid receptors of insects and other invertebrates. In this review, James Rauh and colleagues describe the properties of GABA receptors of insects and detail some striking pharmacological differences between the well-characterized GABA receptors of vertebrates and those of insects and other invertebrate organisms. A detailed understanding of invertebrate receptor pharmacology will be increasingly important for defining the mode of action of numerous modern pesticides.

Blocking actions of heptachlor at an insect central nervous system GABA receptor.

The actions of the polychlorocycloalkane insecticide heptachlor, and its epoxide metabolite, were examined on GABA receptors in insects and vertebrates. Electrophysiological experiments on the cell body of the cockroach (Periplaneta americana) fast coxal depressor motor neuron (Df), and GABA-activated 36Cl- uptake experiments on microsacs prepared from cockroach ventral nerve cords showed that both heptachlor and heptachlor epoxide blocked functional GABA receptors. The block appeared to be non-competitive and was voltage-independent over the membrane potential range -75 mV to -110 mV. There was no significant difference between the potencies of heptachlor and heptachlor epoxide in the functional assays for insect GABA receptors. Both compounds inhibited [35S]-t-butylbicyclophosphorothionate [( 35S]TBPS) binding in insects and vertebrates. The findings provide further evidence for block of an insect GABA receptor/Cl- channel by the cyclodiene class of polychlorocycloalkanes, and reveal differences in the insecticide-[35S]TBPS binding site interactions of insects and vertebrates.

Glycoprotein properties of muscarinic acetylcholine receptors from bovine cerebral cortex.

Muscarinic acetylcholine receptors from bovine cerebral cortex were solubilized in digitonin for the subsequent determination of several biochemical properties. The digitonin-solubilized receptors were representative of the entire membrane-bound population of muscarinic receptors with respect to carbohydrate content, isoelectric point, and molecular weight. The glycoprotein nature of the solubilized receptors was demonstrated by their quantitative binding to wheat germ agglutinin-agarose. The presence of a bound antagonist did not decrease the extent of receptor binding to this lectin. Treatment of receptors with neuraminidase to remove N-acetylneuraminic acid residues reduced binding to wheat germ agglutinin-agarose by 40%; further treatment with endoglycosidases D and H, to remove all N-linked carbohydrate, decreased binding by a total of 67%. Removal of N-acetylneuraminic acid residues had no effect on agonist binding properties of the membrane-bound receptors. The carbohydrate-specific enzymes were further used to assess the contribution of carbohydrate to the isoelectric point and molecular weight of the receptor. Muscarinic receptors solubilized in either digitonin or Triton X-100 focused as one major species with a pI of 4.3. Neuraminidase treatment resulted in an increase of 0.17 units in the pI of the receptor. Muscarinic receptors labeled with the covalent muscarinic antagonist propylbenzilylcholine mustard migrated as a single major polypeptide with a molecular weight of 73,000 on sodium dodecyl sulfate-urea-polyacrylamide gels. The exclusion of urea from these gels severely retarded receptor mobility, indicating a strong tendency for aggregation of receptors in SDS. Removal of N-linked carbohydrate by endoglycosidase treatment reduced the molecular weight of the antagonist binding polypeptide by no more than 5%. These results demonstrate the glycoprotein nature of muscarinic receptors from mammalian cerebral cortex and provide evidence for their heterogeneity with respect to carbohydrate content.

Two molecular weight forms of muscarinic acetylcholine receptors in the avian central nervous system: switch in predominant form during differentiation of synapses.

Muscarinic acetylcholine receptors from the avian central nervous system were examined for developmental changes that correlated with the differentiation of cholinergic synapses. In contrast to previous studies that showed a single molecular weight form of muscarinic receptors in the mature central nervous system, the current study of receptors from embryonic and newly hatched chick retina showed the presence of two electrophoretic forms having apparent molecular weights of 86,200 +/- 400 and 72,200 +/- 300. Two receptor forms also were observed in embryonic cerebrum, optic tectum, and cerebellum. Each form was present, although decreased in molecular weight by 6000, after treatment with deglycosylating enzymes, consistent with molecular differences occurring in the protein portions, rather than the carbohydrate portions, of the molecules. The relative proportions of the high and low molecular weight receptors in retina showed a striking inversion during development. Before synaptogenesis, receptors were mainly of Mr 86,000, whereas after synaptogenesis, receptors were mainly of Mr 72,000. Development of a predominantly low molecular weight receptor population also occurred in aggregate, but not monolayer, cell culture, suggesting a possible role for cell-cell interactions in triggering the change. Pulse-chase labeling of receptors on cultured cells indicated that both forms were present on the cell surface; the labeled Mr 86,000 population had a half-life of 5 hr, whereas the labeled Mr 72,000 population had a half-life of 19 hr. The change in size of muscarinic receptors during development may reflect the action of regulatory mechanisms critical to the proper assembly and function of synapses in the central nervous system.

A Ca2+-activated ATPase specifically released by Ca2+ shock from Paramecium tetraurelia.

Deciliation of Paramecium tetraurelia by a Ca2+ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca2+-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca2+, Sr2+, or Ba2+, but not by Mg2+ or by monovalent cations. The crude enzyme has a specific activity of 2-3 mumol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50 degrees C, and which has a sedimentation coefficient of 8-10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed.

Calmodulin is a major component of extruded trichocysts from Paramecium tetraurelia.

Extruded trichocysts are composed of a family of proteins with molecular weights between 15,000 and 20,000. We have used heat treatment and affinity chromatography on fluphenazine-Sepharose to purify calmodulinlike proteins from whole cells and from extruded trichocysts. The purified protein from trichocysts is indistinguishable from that of whole cells; it is heat-stable, activates brain phosphodiesterase in a Ca++-dependent fashion, changes mobility on SDS polyacrylamide gels in the presence of Ca++, contains 1 mol of trimethyllysine/17 kdaltons, and has the amino acid composition characteristic of calmodulins. Calmodulin is a major component of purified, extruded trichocysts, of which it represents between 1 and 10% by mass. The other proteins of the trichocyst also resemble calmodulin in several properties. Possible roles for calmodulin in the Ca++-activated extrusion of trichocysts is discussed.