Differential sensitivity of two insect GABA-gated chloride channels to dieldrin,fipronil and picrotoxinin

In the central nervous system of both vertebrates and invertebrates inhibitory neurotransmission is mainly achieved through activation of γ-aminobutyric acid (GABA) receptors. Extensive studies have established the structural and pharmacological properties of vertebrate GABA receptors. Although the vast majority of insect GABA-sensitive responses share some properties with vertebrate GABAA receptors, peculiar pharmacological properties of these receptors led us to think that several GABA-gated chloride channels are present in insects. We describe here the pharmacological properties of two GABA receptor subtypes coupled to a chloride channel on dorsal unpaired median (DUM) neurones of the adult male cockroach. Long applications of GABA induce a large biphasic hyperpolarization, consisting of an initial transient hyperpolarization followed by a slow phase of hyperpolarization that is not quickly desensitized. With GABA, the transient hyperpolarization is sensitive to picrotoxinin, fipronil and dieldrin whereas the slow response is insensitive to these insecticides.When GABA is replaced by muscimol and cis-4-aminocrotonic acid (CACA) a biphasic hyperpolarization consisting of an initial transient hyperpolarization followed by a sustained phase is evoked which is blocked by picrotoxinin and fipronil. Exposure to dieldrin decreases only the early phase of the muscimol and CACA-induced biphasic response, suggesting that two GABA-gated chloride channel receptor subtypes are present in DUM neurones. This study describes, for the first time, a dieldrin resistant component different to the dieldrin- and picrotoxinin-resistant receptor found in several insect species.     

GABA receptors on the cell-body membrane of an identified insect motor neuron

The pharmacology of a gamma-aminobutyric acid (GABA) receptor on the cell body of an identified motor neuron of the cockroach (Periplaneta americana) was investigated by current-clamp and voltage-clamp methods. Iontophoretic application of GABA increased membrane conductance to chloride ions, and prolonged application resulted in desensitization. Hill coefficients, determined from dose-response data, indicated that binding of at least two GABA molecules was required to activate the chloride channel. Differences between vertebrate GABAA receptors and insect neuronal GABA receptors were detected. For the GABA receptor of motor neuron Df, the following rank order of potency was observed: isoguvacine greater than muscimol greater than or equal to GABA greater than 3-aminopropanesulphonic acid. The GABAB receptor agonist baclofen was inactive. Of the potent vertebrate GABA receptor antagonists (bicuculline, pitrazepin, RU5135 and picrotoxin), only picrotoxin (10(-7) M) produced a potent, reversible block of the response to GABA of motor neuron Df. Both picrotoxinin and picrotin also blocked GABA-induced currents. Bicuculline hydrochloride (10(-4) M) and bicuculline methiodide (10(-4) M) were both ineffective when applied at resting membrane potential (-65 mV), although at hyperpolarized levels partial block of GABA-induced current was sometimes observed. Pitrazepin (10(-4) M) caused a partial, voltage-independent block of GABA-induced current. The steroid derivative RU5135 was inactive at 10(-5) M. In contrast to the potent competitive blockade of vertebrate GABAA receptors by bicuculline, pitrazepin and RU5135, none of the weak antagonism caused by these drugs on the insect GABA receptor was competitive. Flunitrazepam (10(-6) M) potentiated GABA responses, providing evidence for a benzodiazepine site on an insect GABA-receptor-chloride-channel complex.     

Receptors for L-glutamate and GABA in the nervous system of an insect (Periplaneta americana)

The nervous system of the cockroach Periplaneta americana is well suited to studies of invertebrate amino acid receptors. Using a combination of radioligand binding and electrophysiological techniques, several distinct receptors have now been identified. These include an l-glutamate-gated chloride channel which has no known counterpart in the vertebrate nervous system, and a putative kainate/quisqualate receptor with pharmacological properties different from those of the existing categories of vertebrate excitatory amino acid receptors. GABA receptors have also been characterized in the cockroach nervous system. Bicuculline, benzodiazepines and steroids have revealed important differences between certain insect GABA-gated chloride channels and vertebrate GABA receptors. Identifiable neurones may facilitate the allocation of specific functions to amino acid receptor subtypes. In view of the existence of subtypes of amino acid receptors in insects, it is of interest to examine how this is reflected at the molecular level in terms of receptor subunit composition and amino acid sequence. Preliminary molecular cloning studies on insect GABA receptors are described.     

Effects of modulators of N-methyl-D-aspartate receptor-mediated neurotransmission on diazepam discrimination in rats

N-methyl-D-aspartate (NMDA) antagonists share a number of pharmacological effects with GABA(A) agonists, including anxiolytic and anticonvulsant effects. This study evaluated the effects of site-selective NMDA antagonists in rats trained to discriminate the benzodiazepine diazepam from vehicle. As expected, diazepam produced robust discriminative stimulus effects and dose-dependently substituted for the training dose. Mixed results were obtained with competitive NMDA antagonists: whereas NPC 17742 partially substituted for diazepam, SDZ EAA 494 did not elicit responding on the diazepam-associated lever. Other site-selective NMDA antagonists, including the open channel blocker phencyclidine, the glycine-site antagonists ACEA 1021 and MDL 102,288, the polyamine-site antagonist arcaine, and the glutamate release inhibitor riluzole, failed to substitute for diazepam. Agonists at nonbenzodiazepine sites of the GABA(A) receptor complex were also tested for comparison purposes. The barbiturate pentobarbital and the neurosteroid Co 2-1068 partially substituted for diazepam. In contrast, the anticonvulsant carbamazepine failed to substitute even at a dose that substantially reduced response rates. These results suggest that substitution of NMDA antagonists for GABA(A) agonists is dependent upon the site at which the NMDA antagonist binds. Further, they suggest that similarities between the stimulus properties of GABA(A) agonists and NMDA antagonists are at least as strong as similarities among agonists acting at different sites on GABA(A) receptors.     

Microscopic kinetics and energetics distinguish GABA(A) receptor agonists from antagonists

Although agonists and competitive antagonists presumably occupy overlapping binding sites on ligand-gated channels, these interactions cannot be identical because agonists cause channel opening whereas antagonists do not. One explanation is that only agonist binding performs enough work on the receptor to cause the conformational changes that lead to gating. This idea is supported by agonist binding rates at GABA(A) and nicotinic acetylcholine receptors that are slower than expected for a diffusion-limited process, suggesting that agonist binding involves an energy-requiring event. This hypothesis predicts that competitive antagonist binding should require less activation energy than agonist binding. To test this idea, we developed a novel deconvolution-based method to compare binding and unbinding kinetics of GABA(A) receptor agonists and antagonists in outside-out patches from rat hippocampal neurons. Agonist and antagonist unbinding rates were steeply correlated with affinity. Unlike the agonists, three of the four antagonists tested had binding rates that were fast, independent of affinity, and could be accounted for by diffusion- and dehydration-limited processes. In contrast, agonist binding involved additional energy-requiring steps, consistent with the idea that channel gating is initiated by agonist-triggered movements within the ligand binding site. Antagonist binding does not appear to produce such movements, and may in fact prevent them.     

Characteristics of GABA receptors on the ocellar L-neurons of American cockroach Periplaneta americana

The ocellar L-neurons of cockroach Periplaneta americana were used in the present study as model systems to investigate the pharmacological properties of the GABA receptors. To do so, a glass microelectrode was impaled into the axon of the L-neurons to record the membrane potential intracellularly and to monitor membrane response to GABA treatment and cercal stimulation by air puff. The traditional GABA and their receptor agonists were introduced through perfusion and/or iontophoresis to monitor their effects on the L-neurons. The GABA receptor antagonists were administered by perfusion to examine if the response of the L-neurons to GABA and/or cercal stimulation was changed. The results revealed that administration of GABA, muscimol and imidazole acetic acid, two GABAA agonists, produced depolarization on the L-neurons. However, treatment of 3-APS and guanidine acetic acid, another two GABAA agonists, evoked hyperpolarization on the L-neurons. Among those tested antagonists, only picrotoxin, GABAA antagonist, antagonize the depolarization induced by GABA and/or cercal stimulation. More interestingly, administration of strychnine, glycine receptor antagonist, largely attenuated the depolarization response of the L-neurons to cercal stimulation. This attenuation caused by strychnine was even stronger than that initiated by varied GABA antagonists. In addition, phaclofen, a GABAB receptor antagonist, showed no antagonistic effect. These results strongly suggest that the characteristics of GABA receptors of the ocellar L-neurons may differ from those in vertebrates. It may be more likely to be a novel GABA receptor.     

Glutamate receptors on the somata of dorsal unpaired median neurons in cockroach,Periplaneta americana,thoracic ganglia

Effects of application of glutamate and glutamatergic ligands were studied to characterize the receptors for glutamate present on the soma membrane of the dorsal unpaired median (DUM) neurons in the thoracic ganglia of the cockroach, Periplaneta americana, using the intracellular recording technique. Application of L-glutamate did not block the GABA-response, and application of beta-guanidino-propionic acid, a competitive antagonist for GABA, failed to block the response to L-glutamate. These results indicate that most of L-glutamate action may not be mediated by a GABA-activated channel. To examine glutamate receptor types on the DUM neurons, glutamate receptor agonists were applied. The ionotropic glutamate receptor (iGluR) agonists evoked depolarizations with the following relative rank of order of potency: kainate > AMPA > quisqualate. Metabotropic glutamate receptor (mGluR) agonists also elicited membrane depolarizations or hyperpolarizations associated with an increase in membrane conductance. The mGluR agonists evoked depolarizations or hyperpolarizations with the following relative rank of order: L-CCG-1 > 1S, 3R-ACPD > L-AP4. Depolarization of the same DUM neuron was detected following exposure of kainate and L-CCG-I, suggesting the coexistence of distinct iGluR and mGluR types. A membrane permeable cAMP analog, CPT-cAMP, could not mimic the effect of mGluR agonists. The mGluR selective antagonists, MCCG and MCPG, failed to antagonize the response to mGluR agonists. The involvement of cAMP in the mGluR response was not confirmed in DUM neurons. Although the functional roles of these receptors are unknown, it might be possible then that these extrasynaptic receptors have a modulatory effect on the excitability of the DUM neurons.     

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 GABA_A 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 att-butylbicy-clophosphorothionate (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.Special issue dedicated to Dr. Eugene Roberts     

Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone

The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D_f) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D_f motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.     

Pharmacological characterization of a 5-hydroxytryptamine-sensitive receptor/adenylate cyclase complex in the mandibular closer muscles of the cricket, Gryllus domestica.

The mandibular closer muscles of the cricket, Gryllus domestica, contain a 5-hydroxytryptamine (5-HT)-sensitive receptor that is coupled to adenylate cyclase. A structure-activity study of the 5-HT molecule indicates that the integrity of the ethylamine sidegroup and the presence of a negatively charged moiety at the 5 position (-OH, -OCH3) are essential for activity. A pharmacological profile is presented for this receptor. The receptor differs from any reported mammalian 5-HT receptor in that none of the mammalian agonists tested were effective. However, the mammalian antagonists for 5-HT receptors, spiperone, mianserin, and ketanserin as well as the anti-histaminic cyproheptadine were all effective antagonists in this preparation. Preliminary analysis of antagonism, particularly by spiperone, shows that these antagonists are probably acting non-competitively. On the basis of the pharmacological data, and comparisons with other insect systems, the 5-HT receptor present in the cricket mandibular muscles has been tentatively classified as 5-HT2-like.     

Block of an insect central nervous system GABA receptor by cyclodiene and cyclohexane insecticides

The effects of a cyclodiene (endrin) and a cyclohexane (lindane) insecticide have been tested on gamma-aminobutyric acid (GABA) receptors in the central nervous system of the cockroach (Periplaneta americana), by using electrophysiological methods and an in vitro functional receptor assay. In electrophysiological experiments on an identified motor neuron (Df), endrin blocked the GABA response with a 50% inhibition concentration of 5.0 x 10(-7) M in a non-competitive manner. The actions of endrin were irreversible under the experimental conditions adopted. Increasing the intracellular chloride concentration reduced the effectiveness of endrin, whereas a change in the potassium concentration failed to influence the block by endrin of GABA responses. Lindane exhibited similar actions to endrin on insect GABA receptors, but was approximately an order of magnitude less effective. In a microsac preparation from cockroach nerve cords, endrin, at a concentration of 1.0 x 10(-5) M, completely blocked GABA-stimulated 36Cl- uptake, whereas the same concentration of lindane was less potent, only blocking about 40% of uptake under similar conditions. Neither insecticide had any effect on L-glutamate-activated chloride channels. The results demonstrate that endrin and lindane block functional insect neuronal GABA receptors.     

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.     

A new octopamine receptor class in locust nervous tissue, the octopamine 3 (OA3) receptor.

The insect neuronal 3H-octopamine binding site represents a new type of octopamine receptor. This receptor has pharmacological features that are characteristic for all known octopamine receptors, but it is possible to distinguish this receptor class from all others using either agonists or antagonists. The quantitative determination of the pharmacological relationships to the other octopamine receptor classes could demonstrate greatest homology with both class 2 (OA2A and OA2B) receptors. Therefore, the neuronal octopamine receptor should be named a class 3 receptor (OA3). A new and simple classification scheme for octopamine receptors which enables classification of the new receptor class is established using antagonists.     

Enantioselective actions of 4-amino-3-hydroxybutanoic acid and (3-amino-2-hydroxypropyl)methylphosphinic acid at recombinant GABA(C) receptors

The R- and S-enantiomers of 4-amino-3-hydroxybutanoic acid (GABOB) were full agonists at human recombinant rho1 GABA(C) receptors. Their enantioselectivity (R>S) matched that reported for their agonist actions at GABA(B) receptors, but was the opposite to that reported at GABA(A) receptors (S>R). The corresponding methylphosphinic acid analogues proved to be rho1 GABA(C) receptor antagonists with R(+)-CGP44533 being more potent than S(-)-CGP44532, thus showing the opposite enantioselectivity to the agonists R(-)- and S(+)-GABOB. These studies highlight the different stereochemical requirements for the hydroxy group in these analogues at GABA(A), GABA(B) and GABA(C) receptors.     

Electrophysiological evidence for 4-isobutyl-3-isopropylbicyclophosphorothionate as a selective blocker of insect GABA-gated chloride channels

Invertebrate γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and glutamate-gated chloride channels (GluCls), which function as inhibitory neurotransmitter receptors, are important targets of insecticides and antiparasitic agents. The antagonism of GABACls and GluCls by 4-isobutyl-3-isopropylbicyclophosphorothionate (PS-14) was examined in cultured cockroach and rat neurons using a whole-cell patch-clamp method. The results indicated that PS-14 selectively blocks cockroach GABACls relative to cockroach GluCls and rat GABACls. PS-14 represents a useful probe for the study of insect GABA receptors.     

Functional modifications of acid-sensing ion channels by ligand-gated chloride channels

Together, acid-sensing ion channels (ASICs) and epithelial sodium channels (ENaC) constitute the majority of voltage-independent sodium channels in mammals. ENaC is regulated by a chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Here we show that ASICs were reversibly inhibited by activation of GABA(A) receptors in murine hippocampal neurons. This inhibition of ASICs required opening of the chloride channels but occurred with both outward and inward GABA(A) receptor-mediated currents. Moreover, activation of the GABA(A) receptors modified the pharmacological features and kinetic properties of the ASIC currents, including the time course of activation, desensitization and deactivation. Modification of ASICs by open GABA(A) receptors was also observed in both nucleated patches and outside-out patches excised from hippocampal neurons. Interestingly, ASICs and GABA(A) receptors interacted to regulate synaptic plasticity in CA1 hippocampal slices. The activation of glycine receptors, which are similar to GABA(A) receptors, also modified ASICs in spinal neurons. We conclude that GABA(A) receptors and glycine receptors modify ASICs in neurons through mechanisms that require the opening of chloride channels.     

Interactions between rho and gamma2 subunits of the GABA receptor

The gamma-aminobutyric acid type C (GABA(C)) receptor is a ligand-gated chloride channel with distinct physiological and pharmacological properties. Although the exact subunit composition of native GABA(C) receptors has yet to be firmly established, there is general agreement that GABA rho subunits participate in their formation. Recent studies on white perch suggest that certain GABA rho subunits can co-assemble with the GABA(A) receptor gamma2 subunit to form a heteromeric receptor with electrophysiological properties that correspond more closely to the native GABA(C) receptor on retinal neurons than any of the homomeric rho receptors. In the present study we examined the interactions among various perch GABA rho and gamma2 subunits. When co-expressed in Xenopus oocytes, the gamma2 subunit co-immunoprecipitated with Flag-tagged perch rho1A, rho1B, and rho2B subunits, but not with the Flag-tagged perch rho2A subunit. Immunocytochemical studies indicated that the membrane surface expression of the gamma2 subunit was detected only when it was co-expressed with perch rho1A, rho1B, or rho2B subunit, but not with the perch rho2A subunit or when expressed alone. In addition, co-immunoprecipitation of perch rho1B and gamma2 subunits was also detected in protein samples of the teleost retina. Taken together, these findings suggest that a heteromeric rho(gamma2) receptor could represent one form of GABA(C) receptor on retinal neurons.     

Pharmacologic characterization of muscarinic receptors of insect brains.

Muscarinic receptors in brain membranes from honey bees, houseflies, and the American cockroach were identified by their specific binding of the non-selective muscarinic receptor antagonist [3H]quinuclidinyl benzilate ([3H]QNB) and the displacement of this binding by agonists as well as subtype-selective antagonists, using filtration assays. The binding parameters, obtained from Scatchard analysis, indicated that insect muscarinic receptors, like those of mammalian brains, had high affinities for [3H]QNB (KD = 0.47 nM in honey bees, 0.17 nM in houseflies and 0.13 nM in the cockroach). However, the receptor concentration was low (108, 64.7, and 108 fmol/mg protein for the three species, respectively). The association and dissociation rates of [3H]QNB binding to honey bee brain membranes, sensitivity of [3H]QNB binding to muscarinic agonists, and high affinity for atropine were also features generally similar to muscarinic receptors of mammalian brains. In order to further characterize the three insect brain muscarinic receptors, the displacement of [3H]QNB binding by subtype-selective antagonists was studied. The rank order of potency of pirenzepine (PZ), the M1 selective antagonist, 11-[2-[dimethylamino)-methyl)1-piperidinyl)acetyl)-5,11- dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepin-6 one (AF-DX 116), the M2-selective antagonist, and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) the M3-selective antagonist, was also the same as that of mammalian brains, i.e., 4-DAMP greater than PZ greater than AF-DX 116. The three insect brain receptors had 27-50-fold lower affinity for PZ (Ki 484-900 nM) than did the mammalian brain receptor (Ki 16 nM), but similar to that reported for the muscarinic receptor subtype cloned from Drosophila. Also, the affinity of insect receptors for 4-DAMP (Ki 18.9-56.6 nM) was much lower than that of the M3 receptor, which predominates in rat submaxillary gland (Ki of 0.37 nM on [3H]QNB binding). These drug specificities of muscarinic receptors of brains from three insect species suggest that insect brains may be predominantly of a unique subtype that is close to, though significantly different from, the mammalian M3 subtype.     

Activation of picrotoxin-resistant GABA receptors by GABA and related compounds induces modulation of cockroach dorsal paired median (DPM) neuron firing

Activation of gamma-aminobutyric acid (GABA) receptors in insect dorsal paired median (DPM) neurons induced two types of response which appeared to be mediated by two different GABA receptor subtypes. When activated by bath application of GABA, one receptor subtype, insensitive to picrotoxin (PTX), mediated a drastic reduction in the firing frequency, leading to a blockade of the spontaneous electrical activity. These effects were accompanied by decreases in the amplitude and duration of the plateau action potential (AP) and the spike after-hyperpolarization (AHP). In most cases, a slight depolarization of the resting membrane potential occurred. Bath application of the vertebrate GABA(B) receptor agonists 3-aminopropyl(methyl)phosphinic acid (SKF 97541) and 3-aminopropylphosphinic acid (CGA 147823/CGP 27492) induced similar responses. Another GABA receptor subtype, less sensitive to GABA, mediated a chloride dependent hyperpolarization that was suppressed by bath application of PTX. The approximate locations of these two GABA receptor subtypes were determined by local pressure microapplications of GABA and vertebrate GABAergic agonists. The PTX-sensitive receptors were located predominantly on the surface of the ganglion where the apical pole of the soma is situated, while the PTX-resistant receptors appeared to be located deeper within the ganglion.These results reveal the existence of two GABA receptor subtypes on the DPM neurons and provide evidence for a functional role for PTX-resistant GABA receptors in the regulation of spontaneous firing.     

Binding sites for [3H]GABA, [3H]flunitrazepam and [35S]TBPS in insect CNS

The CNS of the cockroach Periplaneta americana contains saturable, specific binding sites for [³H]GABA, [³H]flunitrazepam and [³⁵S]TBPS. The [³H]GABA binding site exhibits a pharmacological profile distinct from that reported for mammalian GABA_A and GABA_B receptors. The most potent inhibitors of [³H]GABA binding were GABA and muscimol, whereas isoguvacine, thiomuscimol and 3-aminopropane sulphonic acid were less effective. Bicuculline methiodide and baclofen were ineffective. Binding of [³⁵S]TBPS was partially inhibited by 1.0 × 10⁻⁶ M GABA, whilst binding of [³H]flunitrazepam was enhanced by 1.0 × 10⁻⁷ M GABA. The pharmacological profile of the [³H]flunitrazepam binding site showed some similarities with the peripheral benzodiazepine binding sites of vertebrates, with Ro-5-4864 being a far more effective inhibitor of binding than clonazepam. Thus a class of GABA receptors with pharmacological properties distinct from mammalian GABA receptor subtypes is present in insect CNS.