David J. Beadle

Responses to GABA by isolated insect neuronal somata: pharmacology and modulation by a benzodiazepine and a barbiturate

Mechanically dissociated neuronal somata from the thoracic ganglia of Locusta migratoria and Schistocerca gregaria were viable in vitro for hours and were current- and voltage-clamped to record the responses evoked by brief pressure applications of gamma-aminobutyric acid (GABA) in the presence of various modulators. The application of GABA and muscimol, but not baclofen, produced a hyperpolarization and concurrent increase in the membrane conductance. The current underlying this response reversed at -65 mV, was evoked in all cells tested and showed outward rectification. In 6 of 74 Locusta neurones but not in the neurones of Schistocerca, GABA and muscimol evoked a biphasic response. The initial, fast phase was indistinguishable from the GABA-evoked current seen in all neurones. The remaining predominant, slow and long-duration component of the response was an inward current over the membrane potential range 0 to -80 mV, increasing with hyperpolarization. The GABAA antagonists bicuculline and pitrazepin were without effect on the fast GABA response while picrotoxin was a potent blocker of both the fast and the slow GABA responses. Flunitrazepam enhanced the amplitude of the fast response by up to 70% without increasing its duration. Sodium pentobarbital enhanced both the amplitude and the duration of the fast GABA response. We conclude that the locust thoracic neuronal GABA receptor/channel complex resembles the vertebrate GABAA receptor in having associated modulatory receptor sites for benzodiazepines and barbiturates, but differs from it in terms of the pharmacology of the GABA receptor itself.

Actions of insecticides on the insect GABA receptor complex.

The actions of insecticides on the insect gamma-aminobutyric acid (GABA) receptor were investigated using [35S]t-butylbicyclophosphorothionate [( 35S]TBPS) binding and voltage-clamp techniques. Specific binding of [35S]TBPS to a membrane homogenate derived from the brain of Locusta migratoria locusts is characterised by a Kd value of 79.3 +/- 2.9 nM and a Bmax value of 1770 +/- 40 fmol/mg protein. [35S]TBPS binding is inhibited by mM concentrations of barbiturates and benzodiazepines. In contrast dieldrin, ivermectin, lindane, picrotoxin and TBPS are inhibitors of [35S]TBPS binding at the nanomolar range. Bicuculline, baclofen and pyrethroid insecticides have no effect on [35S]TBPS binding. These results are similar to those obtained in electrophysiological studies of the current elicited by GABA in both Locusta and Periplaneta americana central neurones. Noise analysis of the effects of lindane, TBPS, dieldrin and picrotoxin on the cockroach GABA responses reveals that these compounds decrease the variance of the GABA-induced current but have no effect on its mean open time. All these compounds, with the exception of dieldrin, significantly decrease the conductance of GABA-evoked single current.

Synthesis of functional GABAA receptors in stable insect cell lines

We have synthesised the β1‐subunit of the bovine GABAA receptor in stable, continuous insect (Spodoptera frugiperda) cell lines. A cDNA was integrated randomly into the insect cell genome under control of a baculovirus immediate early (IE‐1) gene promoter. Transformed cells were obtained by co‐transfection of the insect cells with pIEK 1 .GRβ1, encoding the β1 subunit cDNA, and pIEK1 .neo, encoding the neomycin resistance gene. G‐418‐resistant clones were selected and expanded into continuous cell lines synthesising functional, GABA‐gated, homo‐oligomeric chloride channels. These cell lines had significant advantages over the transient baculovirus expression system for the characterisation of receptors using electrophysiological recording techniques.

Mutational analysis of the mouse 5-HT7 receptor: importance of the third intracellular loop for receptor-G-protein interaction

The mouse serotonin (5‐HT) receptor subtype, 5‐HT7, belongs to the family of seven transmembrane G‐protein‐coupled receptors. To identify the structural basis for the coupling of 5‐HT7 receptor to GαS we constructed a number of receptor mutants in which amino acid residues were either substituted or deleted from the second and third intracellular loops. Wild‐type and mutant 5‐HT7 receptors were expressed in insect cells using the baculovirus vectors. Two mutant receptor species, 5‐HT7(E325G) and 5‐HT7(K327S), demonstrated markedly impaired abilities to stimulate adenylyl cyclase. The results suggest the importance of the C‐terminal region of the third intracellular loop in receptor–G‐protein interaction and that specific charged residues, E325 and K327, may play a critical role in this interaction.

Functional characterisation of the Drosophila 5-HT dro1 and 5-HT dro2B serotonin receptors in insect cells: activation of a Gαs -like protein by 5-HT dro1 but lack of coupling to inhibitory G-proteins by 5-HT dro2B

Insect cells are routinely used for the production of receptor proteins. Expression of the Drosophila 5‐HT dro1 serotonin receptor resulted in positive coupling of the receptor to adenylyl cyclase via the Gαs G‐protein subtype. The Drosophila 5‐HT dro2B receptor stimulated the metabolism of inositol phospholipid via a pertussis toxin‐insensitive G‐protein, but exhibited no detectable inhibition of adenylyl cyclase. Immunoblot analysis of the endogenous G‐proteins revealed that Sf9 cells lack the G‐protein subtypes Gαi1–3 and Gα0, but express the subtypes Gαs and Gαq .

Voltage-dependent calcium channel subtypes controlling somatic substance P release in the peripheral nervous system

1. Isolated rat dorsal root ganglia (DRG) neurones support vesicular, non synaptic release of substance P in a depolarisation and Ca2+ dependent manner. 2. In vivo this process may mediate cross-communication between DRG cells in some neuropathological conditions and is therefore a putative area for drug intervention. 3. The authors investigated the voltage-dependent Ca2+ channel (VDCC) subtypes involved in somatic release of substance P. Fresh (< 1 day) cultures of DRG neurones were incubated with high K+ depolarising saline in the presence and absence of subtype selective VDCC blockers. Substance P released into the external media was collected and quantified using a radioimmunoassay. 4. The results show that L-type and N-type, but not P-type, VDCCs play an important role in high K+ evoked substance P release from rat DRG neurones.

The pharmacological profile of GABA receptors on cultured insect neurones

Neuronal cultures of the cockroach, Periplaneta americana, were used to study the pharmacological profile of GABA receptors using the whole-cell-voltage clamp technique. The results indicated that insect GABA receptors are linked to a chloride channel that can be activated by both GABA(A) and GABA(C) receptor agonists. The receptors are blocked by GABA(A) chloride channel blockers and some insecticides but not by competitive GABA(A) receptor antagonists. The GABA(C) receptor competitive antagonists were either full or partial agonists of the cockroach GABA receptors. The receptors were modulated by the enantiomers of lindane. In conclusion, insect GABA receptors appear to have a distinct pharmacological profile that does not conform to either vertebrate GABA(A) or GABA(C) receptors.

Assembly of functional GABAA receptors in insect cells using baculovirus expression vectors.

We have constructed recombinant baculoviruses containing cDNAs encoding either the alpha 1- or the beta 1-subunit of the bovine GABAA receptor. In Spodoptera frugiperda (IPLB-Sf-21) cells infected with recombinant virus expressing either the alpha 1- or beta 1-subunit, or in cells co-infected with both viruses, functional GABAA receptors were detected by whole-cell electrophysiological recordings. The threshold for the responses mediated by the homo-oligomeric channels (alpha- or beta-) was 2-3 x 10(-6) M GABA, and for the co-infected cells was 8 x 10(-8) M GABA, suggesting that hetero-oligomeric channels formed in these cells. All GABA-induced currents were found to be inhibited by bicuculline and picrotoxin, potentiated by pentobarbital but were insensitive to benzodiazepines.

Choline uptake by cultured neurones from the central nervous system of embryonic cockroaches

Abstract Cultured neurones from the cockroach, Periplaneta americana , have been used to investigate the uptake of [ 3 H]choline. The neurones take up choline from the extracellular medium by both high and low affinity transport systems. Analysis of the high affinity system showed it to be sodium-dependent, temperature-sensitive and largely inhibited by μ M hemicholinium-3, but relatively insensitive to metabolic inhibitors. Elevated potassium concentrations markedly decreased it, suggesting that high affinity uptake is dependent on membrane potential. This transport component is associated with a considerable degree of acetylcholine synthesis: the possibility that these results indicate the presence of presynaptic cholinergic neurones in this culture system is discussed.

Electrophysiological activity of the C-peptide of the Locusta insulin-related peptide Effect on the membrane conductance of Locusta neurones in vitro

The C‐peptide of Locusta insulin‐related peptide, which is a 50 residue peptide originally isolated from the corpora cardiaca of the insect Locusta migratoria and to which we refer as 5‐kDa peptide, has been synthesised chemically by the solid‐phase metho, using a BOC strategy. Since this peptide contains in its sequence a potential monobasic cleavage site, we also synthesised its 1–38 residue‐related fragment, named 4‐kDa peptide, although we have no hints of its natural occurrence in the corpora cardiaca. Electrophysiological studies have shown that both the 5‐kDa and 4‐kDa peptides depolarise the membrane and increase the membrane conductance of neurones freshly isolated from the thoracic ganglia of Locusta. Under voltage‐clamp conditions, the current underlying these effects was inwardly directed and could be resolved into 2 components. One component, I(5‐kDa)1, activated at potentials more hyperpolarised than −50 mV, peaked at about −75 mV and was blocked by the potassium channel blockers cesium and rubidium. The second component, I(5‐kDa)2 was activated at potentials more depolarised than −50 mV, increased with depolarisation and was not blocked by cesium and rubidium. The effects of the 5‐kDa and 4‐kDa peptides on the membrane potential and membrane conductance of Locusta neurones suggest that these peptides may have a physiological role in the central nervous system of insects.