Bernard Hue

The modulatory effects of the anxiolytic etifoxine on GABAA receptors are mediated by the β subunit

The anxiolytic compound etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride) potentiates GABA(A) receptor function in cultured neurons (Neuropharmacology 39 (2000) 1523). However, the molecular mechanisms underlying these effects are not known. In this study, we have determined the influence of GABA(A) receptor subunit composition on the effects of etifoxine, using recombinant murine GABA(A) receptors expressed in Xenopus oocytes. Basal chloride currents mediated by homomeric beta receptors were reduced by micromolar concentrations of etifoxine, showing that beta subunits possess a binding site for this modulator. In oocytes expressing alpha(1)beta(x) GABA(A) receptors (x=1, 2 or 3), etifoxine evoked a chloride current in the absence of GABA and enhanced GABA (EC10)-activated currents, in a dose-dependent manner. Potentiating effects were also observed with alpha(2)beta(x), beta(x)gamma(2s) or alpha(1)beta(x)gamma(2s) combinations. The extent of potentiation was clearly beta-subunit-dependent, being more pronounced at receptors containing a beta(2) or a beta(3) subunit than at receptors incorporating a beta(1) subunit. The mutation of Asn 289 in the channel domain of beta(2) to a serine (the homologous residue in beta(1)) did not significantly depress the effects of etifoxine at alpha(1)beta(2) receptors. This specific pattern of inhibition/potentiation was compared with that of other known modulators of GABA(A) receptor function like benzodiazepines, neurosteroids, barbiturates or loreclezole.

Poneratoxin, a novel peptide neurotoxin from the venom of the ant, Paraponera clavata.

1. At concentrations varying from 10(-8) to 10(-6) M synthetic poneratoxin (PoTX) is a strong, but very slowly acting agonist for smooth muscles and its blocks synaptic transmission in the insect CNS in a concentration-dependent manner and depolarizes giant interneurons. 2. However, in isolated dorsal unpaired median cells 10(-6) M PoTX causes only a reversible hyperpolarization of about 5 mV. 3. At concentrations from 10(-8) to 10(-6) M PoTX affects the electrical activity of isolated cockroach axons, as well as isolated frog and rat skeletal muscle fibres. 4. PoTX prolongs action potentials and induces slow automatic activity, due to a slow Na(+)-current activation at very negative values of potential and due to slow deactivation.

M2-like presynaptic receptors modulate acetylcholine release in the cockroach (Periplaneta americana) central nervous system

Abstract The effects of muscarinic agonists on the acetylcholine release in the cercal-afferent giant-interneurone synapses of the cockroach ( Periplaneta americana ) were investigated using the single-fibre oil-gap method. Pressure-ejection of arecoline, carbachol and oxotremorine within the sixth abdominal ganglion reduced the cEPSP amplitude in a concentration-dependent manner without any effect at the postsynaptic site in the range of concentrations used. A maximal inhibitory effect of 56, 54 and 68% was estimated for arecoline, carbachol and oxotremorine, respectively. The rank order of potencies for the agonists tested was: arecoline > carbachol > oxotremorine. McN-A-343 and bethanechol had an inhibitory effect on acetylcholine release but higher doses also acted at the postsynaptic level. The depressive effect of arecoline was only counteracted by AF-DX 116 and methoctramine. In presence of picrotoxin the inhibitory effect of arecoline and oxotremorine was reduced by about 15%. It is concluded that acetylcholine release in the cockroach central nervous system is modulated by functional M 2 -like presynaptic receptors. The influence of presynaptic electrical activity on this modulatory effect is discussed.

Block of synaptic transmission in insect CNS by toxins from the venom of the WASP Megascolia Flavifrons (FAB.)

1. The effects of the venom and its fractions of Megascolia flavifrons have been studied on synaptic transmission and axonal excitability of the giant interneuron of the cockroach. 2. The venom does not affect axonal excitability, but blocks synaptic transmission, and induces postsynaptic depolarization with a delay. 3. Five different active fractions have been recognized. 4. Three fractions of them contain substances already identified as histamine, Thr6 bradykinin and Thr6 bradykinin-Lys-Ala (megascoliakinin). 5. Three fractions contain activities, which have not yet been chemically identified. 6. All of them, and also bradykinin block synaptic transmission; histamine was not active.

Effects of a centipede venom fraction on insect nervous system, a native Xenopus oocyte receptor and on an expressed Drosophila muscarinic receptor.

Centipede venoms are complex protein mixtures; very few is known about their pharmacological actions. Application of a Scolopendra sp. venom fraction (SC1) on the cockroach giant axon induced an increase in the leak current correlated with a decrease in the membrane resistance, suggesting the presence in SC1 of components opening non-specific pores in the axonal membrane. On a cockroach central cholinergic synapse, microinjection of SC1 induced a small transient depolarization of the postsynaptic membrane, followed by a slow stable depolarization and a drastic decrease in the evoked subthreshold excitatory postsynaptic potential amplitude. A pretreatment of the ganglion with atropine or scopolamine reduced the amplitude of the SC1-induced depolarizing wave, suggesting a possible cholinergic muscarinic target. On control Xenopus oocytes, SC1 induced an inward oscillatory Ca2(+)-dependent Cl- current mediated through the activation of native lysophosphatidic acid receptors (LPAr). Indeed, pretreatment of oocytes with 1 microM N-palmitoyl-tyrosine phosphoric acid, a selective competitive antagonist of LPAr, decreased responses to SC1 by 70%. Application of SC1 to oocytes expressing a cloned Drosophila muscarinic receptor (Dml) induced a biphasic response comprising: (1) a large fast Cl- current that was abolished by pretreatment with atropine and scopolamine and (2) a slow and small oscillating Cl- current corresponding to the response observed in control oocytes. These observations confirm the presence of muscarinic agonists in SCI and reveal their direct action on an insect muscarinic receptor subtype homologous to mammalian M1-M3 receptors.

Threonine6-bradykinin in the venom of the wasp Colpa interrupta (F.) presynaptically blocks nicotinic synaptic transmission in the insect CNS

1. The venom of the solitary scoliid wasp Colpa interrupta (F.) shows a kinin-activity, when tested on a cascade of mammalian smooth muscle preparations, and, in addition, a contraction of the rat colon. 2. The venom also irreversibly blocks the nicotinic synaptic transmission from the cercal nerve to a giant interneuron in the sixth abdominal ganglion of the cockroach, Periplaneta americana. 3. The same activities have been found within one HPLC fraction. 4. However, rechromatography of this fraction resulted in four subfractions being active on smooth muscles. 5. One fraction caused contraction of the colon, three other fractions contained kinin-activity. 6. Only the most active kinin fraction blocked synaptic transmission in the insect CNS. 7. This fraction contained threonine-bradykinin. 8. Synthetic Thr-bradykinin causes irreversible presynaptic activation-induced block of transmission in the insect CNS.

Philanthotoxins, a new class of neuroactive polyamines, block nicotinic transmission in the insect CNS

1. The effect of 12 philanthotoxins has been studied on synaptic transmission, resting membrane potential and conductance and axonal excitability of a giant interneuron in the sixth abdominal ganglion of the cockroach Periplaneta americana. 2. All toxins cause a slowly reversible depression of the EPSP amplitude to a steady-state value. 3. Compared with the natural toxin PTX-4.3.3, three analogues are equiactive, five analogues are less active (ranging from 1–20% activity) and three analogues are more active (varying from 160–2800% activity). 4. The most active toxin is trifluoromethyl-PTX-4.3.3. 5. Small hyperpolarization was caused by part of the toxins. 6. Membrane conductance and axonal excitability were not affected.

The venom of Ampulex Compressa—effects on behaviour and synaptic transmission of cockroaches

1. The solitary wasp Ampulex compressa stings a cockroach, Periplaneta americana, twice. 2. The first sting into the ventral thorax results in a transient paralysis. During this paralysis the wasp stings the suboesophageal ganglion, which gradually results in a permanent deactivation. 3. The venom gland is a paired and highly branched organ, with a common ductus venatus. The large lumen is lined with a folded cuticula. No venom reservoir is present. 4. Extract of the venom gland induces a slow contraction of the guinea pig ileum. 5. The agonist present in the venom cannot be identified with a known agonist. 6. Venom gland extract blocks synaptic transmission from the cercal nerve to giant neurons in the sixth abdominal ganglion of the cockroach. 7. The block develops gradually, like the gradual appearance of the effects of the sting into the suboesophageal ganglion on the behaviour of the cockroach.

Pharmacological characterization and chemical fractionation of the venom of the ponerine ant, Paraponera clavata (F.)

1. The neurotoxic action of the venom of the ponerine ant, Paraponera clavata, was studied using a cascade of mammalian smooth muscle preparations and a preparation for investigating transmission from fibres of the cercal nerve to a giant interneuron in the sixth abdominal ganglion of the cockroach. 2. The venom contains three toxic fractions that block synaptic transmission in the insect central nervous system. 3. Two of these fractions have agonistic action on mammalian smooth muscle preparations. 4. One of the later fractions was characterized pharmacologically as containing a kinin. 5. The other, and most active neurotoxic fraction, was rechromatographed, resulting in the purification of a peptide of 25 amino acids residues, called poneratoxin, PoTX: Phe-Leu-Pro-Leu-Leu-Ile-Leu-Gly-Ser-Leu-Leu-Met-Thr-Pro-Pro-Val-Ile-Gln- Ala-Ile-His-Asp-Ala-Gln-Arg-HN2.

Irreversible presynaptic activation-induced block of transmission in the insect CNS by hemicholinium-3 and threonine-6-bradykinin

1. 1. Threonine-6-bradykinin and hemicholinium-3 irreversibly block synaptic transmission from the cercal nerve XI of the cockroach, Periplaneta americana, to a giant interneuron in the sixth abdominal ganglion. 2. 2. This block is frequency dependent. 3. 3. Carbachol potentials are not affected by the kinin. It is concluded that the kinin, like hemicholinium-3, blocks transmission at a presynaptic site, probably by depletion of the readily releasable store of transmitter substance.