Pyrethroid insecticides evoke neurotransmitter release from rabbit striatal slices

The effects of the synthetic pyrethroid insecticide fenvalerate ([R,S]-alpha-cyano-3-phenoxybenzyl[R,S]-2-(4-chlorophenyl)-3- methylbutyrate) on neurotransmitter release in rabbit brain slices were investigated. Fenvalerate evoked a calcium-dependent release of [3H]dopamine and [3H]acetylcholine from rabbit striatal slices that was concentration-dependent and specific for the toxic stereoisomer of the insecticide. The release of [3H]dopamine and [3H]acetylcholine by fenvalerate was modulated by D2 dopamine receptor activation and antagonized completely by the sodium channel blocker, tetrodotoxin. These findings are consistent with an action of fenvalerate on the voltage-dependent sodium channels of the presynaptic membrane resulting in membrane depolarization, and the release of dopamine and acetylcholine by a calcium-dependent exocytotic process. In contrast to results obtained in striatal slices, fenvalerate did not elicit the release of [3H]norepinephrine or [3H]acetylcholine from rabbit hippocampal slices indicative of regional differences in sensitivity to type II pyrethroid actions.     

3H]tetraphenylphosphonium accumulation in cerebral cortical synaptosomes as a measure of nicotine-induced changes in membrane potential.

The effect of nicotine on synaptosomal membrane potential in P2 preparations of rat cerebral cortex was investigated using a membrane permeant, lipophilic cation, [3H]tetraphenylphosphonium ([3H]TPP+). [3H]TPP+ accumulated in synaptosomes in a time-dependent manner and its accumulation was decreased when the extracellular potassium concentration was increased and in the presence of the sodium channel toxin, veratridine. Nicotine (1-1000 microM) decreased the accumulation of [3H]TPP+ in both P2 synaptosomal preparations and in synaptosomes purified using Percoll gradients. This effect of nicotine was mimicked by other nicotinic agonists (1,1-dimethyl-4-phenylpiperazinium iodide, cytisine, suberyldicholine and acetylcholine) and was partially blocked by 10 microM mecamylamine and 30 microM hexamethonium. Atropine (1 microM) and the removal of calcium from the incubation mixture both enhanced the effect of nicotine while the addition of physostigmine (10 microM) reduced the nicotine-induced decrease in [3H]TPP+ accumulation, evidence that acetylcholine released from the synaptosomes by nicotine may produce hyperpolarization of synaptosomes via stimulation of presynaptic muscarinic receptors. It is concluded that the effect of nicotine on [3H]TPP+ accumulation is mediated by nicotine stimulation of a ganglionic-type nicotinic cholinergic receptor and that this method of determining synaptosomal membrane potential will provide a functional measure of presynaptic nicotinic receptor activation.     

Repression of activity-dependent c-fos and brain-derived neurotrophic factor mRNA expression by pyrethroid insecticides accompanying a decrease in Ca(2+) influx into neurons

Permethrin, a type I pyrethroid insecticide, is known to affect sodium channels of neurons and prolong sodium currents. On the other hand, the expression of brain-derived neurotrophic factor (BDNF) and c-fos genes is activated through Ca(2+) influx into neurons, in an activity-dependent manner. In this study, therefore, we investigated whether permethrin influenced the Ca(2+) signal-induced expression of these genes. In primary culture of mouse cerebellar granule cells (CGCs), stimulation with veratridine, a potent agonist for sodium channels, which causes membrane depolarization in neurons, induced c-fos and BDNF mRNA expression accompanying the Ca(2+) influx into neurons. Pretreatment with permethrin at doses nontoxic to CGCs repressed the induction of these genes dose dependently, with trans-permethrin more potent than cis-permethrin. Consistent with this, the increase in Ca(2+) influx caused by veratridine was repressed by permethrin. The membrane depolarization induced by elevating the potassium (K(+)) concentration in medium (high K(+)) caused the activation of c-fos and BDNF genes, which was also repressed by permethrin. Immunoblotting analysis of c-Fos and a gel-mobility assay of AP-1 DNA-binding activity supported the decrease in c-Fos synthesis in permethrin-treated CGCs. The type II pyrethroid cypermethrin also affected the expression of these genes but less effectively than permethrin. Thus, pyrethroids inhibit the activity-dependent gene expression in neurons.     

Kinetics of modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels by tetramethrin and deltamethrin.

Pyrethroid insecticides may be classified into two groups: type I pyrethroids lack a cyano group in the alpha-position, whereas type II pyrethroids have a cyano group. Both types prolong the sodium channel current thereby causing hyperexcitability, yet details of modulation of current kinetics remain largely to be seen. The mechanism of pyrethroid modulation of sodium currents was studied by the whole-cell patch-clamp technique with rat dorsal root ganglion neurons. Both deltamethrin (type II) and tetramethrin (type I) acted on both tetrodotoxin-sensitive and tetrodotoxin-resistant channels in a qualitatively similar manner and some quantitative differences were derived from different kinetics. During repetitive stimulation in the presence of deltamethrin, leak current increased due to accumulation of prolonged tail currents, explaining the apparent use-dependent modification. For tetramethrin-modified channels, such accumulation was much less because of faster kinetics. Slowing of the kinetics of sodium channel activation by deltamethrin was revealed even after the fast inactivation had been removed by papain. The kinetics of deltamethrin-modified sodium channels was fitted better by the equation that contained two activation components than that with one component. Deltamethrin caused a large shift of the conductance-voltage curve in the direction of hyperpolarization. Cell-attached patch-clamp experiments revealed that deltamethrin had much smaller mobility in the cell membrane than tetramethrin. It was concluded that the apparent use dependence of deltamethrin modification of sodium channels was due primarily to the accumulation of prolonged tail currents during repetitive stimulation and that the sodium channel activation mechanism is the major target of pyrethroids.     

Modulation of levels of free calcium within synaptosomes by organochlorine insecticides

Effects of the organochlorine insecticides chlordecone, mirex, 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2,2-trichloroethane and 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane on free intrasynaptosomal Ca2+ [( Ca++]i), synaptosomal 45Ca uptake and synaptosomal plasma and mitochondrial membrane potentials in vitro were studied. Chlordecone (10-50 microM) increased [Ca++]i from the resting level of 370 nM in a dose- and time-dependent manner to above 1.5 microM. This took place in the presence of 1 mM extrasynaptosomal Ca++ but not in nominally Ca++-free medium. Verapamil, a voltage sensitive Ca++ channel blocker, inhibited the initial increase of [Ca++]i caused by chlordecone, by 40%. Chlordecone also elevated [Ca++]i in synaptosomes in which mitochondrial Ca++ uptake had been abolished by valinomycin. Chlordecone depolarized partially the synaptosomal plasma membrane and, to a lesser extent, the potential of mitochondria within synaptosomes. However, chlordecone appeared to inhibit synaptosomal K+-stimulated and unstimulated 45Ca++ uptake by 20 to 30%. Inasmuch as chlordecone also stimulated release of 45Ca++ and the fluorescent dye fura-2 from preloaded synaptosomes, the apparent inhibition of uptake might be due to lysis of some synaptosomes by chlordecone. The effect of chlordecone on [Ca++]i decreased when the total amount of tissue in incubations was increased. [Ca++]i was only elevated marginally by mirex at the same concentration range. The results suggest that chlordecone increases free intrasynaptosomal Ca++ mainly by increasing influx of extrasynaptosomal Ca++. The principal mechanism appears to be a nonspecific leakage of Ca++ through the plasma membrane but some Ca++ may pass through voltage-sensitive Ca++ channels due to chlordecone-induced membrane depolarization.     

Induction of cytochrome P450 CYP6B7 and cytochrome b5 mRNAs from Helicoverpa armigera (Hubner) by pyrethroid insecticides in organ culture

An organ culture system derived from Helicoverpa armigera has been used to study the expression of cytochrome P450 and cytochrome b5 mRNAs. Northern analysis showed that levels of the mRNAs for cytochrome P450s, CYP6B2, CYP6B6 and CYP6B7, and cytochrome b5 in control tissue were commensurate with those in the tissue of whole larvae. Substantial induction of cytochrome P450, CYP6B7 and cytochrome b5 mRNAs by alpha-pinene, and the pyrethroids, fenvalerate, cypermethrin and permethrin were observed in fat body culture. Neither mRNA was induced, either in midgut or integument organ cultures. In contrast, the relatively water-soluble compound phenobarbital, could induce CYP6B7 mRNA but not cytochrome b5 mRNA in fat body cultures. As for pyrethroids, phenobarbital had no effect on the other tissues in culture. These results confirm a previous conclusion that pyrethroids could induce CYP6B7 mRNA, which was based upon a very slight induction observed in living insects. Because many cytochrome P450 substrates can act as their inducers, these results support a previous conclusion that CYP6B7 could be the enzyme that is involved in pyrethroid resistance in H. armigera.     

Modulation of the Ca++-evoked release of [3H]dopamine from striatal synaptosomes by dopamine (D2) agonists and antagonists

Release of [3H]dopamine ([3H]DA) from striatal synaptosomes is evoked most commonly by elevating potassium levels in the presence of calcium. However, it has been difficult to show that DA agonists or antagonists can modify K+-evoked release of [3H]DA. DA. In this study [3H]DA release evoked by exposure of synaptosomes (isolated and superfused previously with 0.0 mM Ca++ and 0.1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) to 1.25 mM Ca++ can be modulated by the DA (D2) agonists apomorphine, pergolide and quinpirole and antagonists l-sulpiride and domperidone. The release was evoked under low potassium (6 mM or less) concentrations and the potassium concentration in the superfusion medium was not elevated before or during Ca++ exposure. Analysis of the superfusates obtained during Ca++ exposure revealed that approximately 80% of the tritium released was [3H]DA. The ability of DA (D2) agonists to inhibit the Ca++-evoked release from synaptosomes superfused with 9 mM K+ was greatly reduced. Therefore, prolonged depolarization may block DA (D2) regulation of [3H]DA release from synaptosomes. The Ca++-evoked release of [3H]DA was reduced greatly when 1 microM tetrodotoxin was present indicating sodium channels play a role in triggering the processes involved in Ca++-evoked [3H]DA release.     

Alpha-2 adrenergic inhibition of Ca(++)-evoked [3H]norepinephrine release from synaptosomes is blocked by depolarization

The Ca(++)-evoked release of [3H]norepinephrine was used in these studies to investigate presynaptic regulation of norepinephrine release. In hippocampal synaptosomes, previously unexposed to Ca++ during isolation and superfusion, 1.25 mM Ca++ evoked a modest (4 to 7% of total stores) release of [3H]norepinephrine with 4.5 mM [K+] present. The alpha-2 adrenergic agonist clonidine inhibited 60% of the Ca(++)-evoked [3H]norepinephrine release. The alpha-2 adrenergic antagonists idazoxan and yohimbine reversed clonidine inhibition of release whereas the alpha-1 antagonist prazosin did not. Increasing the [K+] before Ca++ exposure increased [3H]norepinephrine release, and at 20 [K+] the release increased to over 20% of total stores. However, at [K+] above 9 mM, inhibition of Ca(++)-evoked release by clonidine decreased, and by 20 mM [K+] clonidine no longer inhibited release. Release was unaffected by 5 microM idazoxan or the opiate antagonist naloxone at 15 or 20 mM [K+]. The K+ channel blockers tetraethylammonium (5 mM) and 4-aminopyridine (0.1 mM) increased Ca(++)-evoked release almost 4-fold above control (4.5 mM [K+] present). Neither clonidine nor idazoxan affected Ca(++)-evoked release with the K+ channel blockers present. Therefore, even though K+ channel blockers and 20 mM [K+] increase neurotransmitter release, it is not autoreceptor activation by released endogenous norepinephrine that is responsible for blocking alpha-2 inhibition, but the depolarization produced by these treatments. The 20 mM [K+] blockade of alpha-2 inhibition was decreased by lowering the [Ca++] in the superfusion buffer. Therefore, synaptosomal accumulation of Ca++ may partially explain the loss of alpha-2 inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism and subcellular distribution of N-amino-N,N-dimethylaminoethanol (N-aminodeanol) in rat striatal synaptosomes

The metabolism and subcellular distribution of a novel choline analog, N-amino-N,N-dimethylaminoethanol (N-aminodeanol) in rat striatal synaptosomes was studied using combined gas chromatography mass spectrometry for simultaneous estimation of N-aminodeanol, choline, tracer choline and their acetate esters. The enzymes choline acetyltransferase, acetylcholinesterase and choline kinase were assayed in kinetic studies using N-aminodeanol or acetyl-N-aminodeanol as substrates. The results demonstrate that [2H4]N-aminodeanol is transported and acetylated in synaptosomes at rates approximately 30% of those measured for [2H4]choline. Of the [2H4]N-aminodeanol that was transported by the high affinity choline uptake system, the proportion acetylated was similar to that measured for [2H4]choline. [2H4]Acetyl-N-aminodeanol replaced endogenous acetylcholine stores and was released. The combined release of endogenous and false transmitters from synaptosomes in the presence of [2H4]N-aminodeanol was reduced compared to controls in the presence of [2H4]choline, although combined tissue stores did not change significantly. After coincubation with [2H4]N-aminodeanol and [2H4]choline, the molar ratios of true and false transmitter in the tissue appeared to reflect the kinetic parameters for high affinity transport of the precursors. Subcellular fractionation experiments indicated that [2H4]acetyl-N-aminodeanol was incorporated into vesicles more slowly than [2H4]acetylcholine. These results indicate that the reduced rate of turnover in the presence of false precursor is not due to its rate of acetylation or to the rate of release of previously formed false transmitter, but rather to the slower membrane transport of N-aminodeanol by the high affinity uptake system. The replacement of endogenous acetylcholine in synaptosomes by acetyl-N-aminodeanol, which has 4% the potency of acetylcholine at muscarinic receptors, suggests that N-aminodeanol may be useful in studying the in vivo effects of a false cholinergic transmitter.     

Multiple cellular mechanisms mediate the effect of lobeline on the release of norepinephrine

The complex effect of lobeline on [(3)H]norepinephrine ([(3)H]NE) release was investigated in this study. Lobeline-induced release of [(3)H]NE from the vas deferens was strictly concentration-dependent. In contrast, electrical stimulation-evoked release was characterized by diverse effects of lobeline depending on the concentration used: at lower concentration (10 microM), it increased the release and at high concentration (100 and 300 microM), the evoked release of [(3)H]NE was abolished. The effect of lobeline on the basal release was [Ca(2+)]-independent, insensitive to mecamylamine, a nicotinic acetylcholine receptor antagonist, and to desipramine, a noradrenaline uptake inhibitor. However, lobeline-induced release was temperature-dependent: at low temperature (12 degrees C), at which the membrane carrier proteins are inhibited, lobeline failed to increase the basal release. Lobeline dose dependently inhibited the uptake of [(3)H]NE into rat hippocampal synaptic vesicles and purified synaptosomes with IC(50) values of 1.19 +/- 0.11 and 6.53 +/- 1.37 microM, respectively. Lobeline also inhibited Ca(2+) influx induced by KCl depolarization in sympathetic neurons measured with the Fura-2 technique. In addition, phenylephrine, an alpha(1)-adrenoceptor agonist, contracted the smooth muscle of the vas deferens and enhanced stimulation-evoked contraction. Both effects were inhibited by lobeline. Our results can be best explained as a reversal of the monoamine uptake by lobeline that is facilitated by the increased intracellular NE level after lobeline blocks vesicular uptake. At high concentrations, lobeline acts as a nonselective Ca(2+) channel antagonist blocking pre- and postjunctional Ca(2+) channels serving as a counterbalance for the multiple transmitter releasing actions.     

Stereoselective secretion of atenolol from PC12 cells

Stereoselective storage and release of the cardioselective beta adrenergic receptor antagonist atenolol was studied using cultured PC12 cells as a neural model. [3H]Atenolol efflux from preloaded PC12 cells was increased 4-fold in response to membrane depolarization by elevated extracellular potassium (50 mM). [3H]Norepinephrine release was enhanced 4.5-fold under the same conditions. Potassium-induced release of both [3H] atenolol and [3H]norepinephrine was inhibited completely in the absence of extracellular calcium. [3H]Atenolol release from PC12 cells was also reduced by the calcium channel antagonist nifedipine (IC50 = 1.6 +/- 0.5 nM). In addition, the calcium channel agonist BAY K8644 (1 microM) significantly enhanced potassium-induced [3H]atenolol efflux. After loading overnight, accumulation and storage of the (-)-enantiomer of atenolol by PC12 cells was found to be approximately 3-fold greater than that of the (+)-enantiomer. The (-)-enantiomer of atenolol was also preferentially released by 50 mM potassium with a (-)/(+)-enantiomer ratio of 3.6 to 1. The results support the existence within neurosecretory cells of storage and calcium-dependent release mechanisms which result in stereoselective secretion of the (-)-or active enantiomer of atenolol in response to membrane depolarization.     

Cholecystokinin, but not gastrin, induces gamma-aminobutyric acid release from myenteric neurons of the guinea pig ileum

Effects of cholecystokinin (CCK) and gastrin on the release of acetylcholine (ACh) and gamma-aminobutyric acid (GABA) were examined in the longitudinal muscle with myenteric plexus (LM-MP) preparations of the guinea pig small intestine. CCK and gastrin induced the Ca++-dependent and tetrodotoxin-sensitive release of [3H]ACh from the LM-MP preparations preloaded with [3H]choline. Proglumide, but not scopolamine, hexamethonium and [D-Pro2,D-Trp7,9]substance P inhibited the release of [3H]ACh induced by CCK and gastrin. The desensitization to CCK and gastrin was observed with a 30-min exposure of the preparation to CCK and gastrin, respectively, and the cross-desensitization to peptides was not observed, thereby indicating that these peptides induce the release of ACh mainly via respective receptors. Bicuculline which inhibited completely the release of [3H]ACh induced by GABA inhibited the release of [3H]ACh induced by CCK but not by gastrin by 42.3 +/- 4.22%. CCK, but not gastrin, produced the Ca++-dependent and tetrodotoxin-sensitive release of endogenous GABA and [3H]GABA from LM-MP preparations preloaded with [3H]GABA. The release of [3H]GABA induced by CCK was antagonized by proglumide, but not by scopolamine, hexamethonium and [D-Pro2,D-Trp7,9]substance P. These results provide evidence that the GABAergic neuron is stimulated by CCK, but not by gastrin and stimulates the cholinergic neuron.     

Actions of pyrethroid insecticides on sodium currents, action potentials, and contractile rhythm in isolated mammalian ventricular myocytes and perfused hearts

Pyrethroid insecticides are known to modify neuronal sodium channels, inducing persistent, steady-state sodium current at depolarized membrane potentials. Cardiac myocytes are also rich in sodium channels but comparatively little is known about the effect of pyrethroids on the heart, or on the cardiac sodium channel isoform. In the present study therefore, we determined the actions of type I and type II pyrethroids against rat and guinea pig ventricular myocytes under current and voltage clamp, and on isolated perfused rat hearts. In myocytes, tefluthrin (type I) and fenpropathrin and alpha-cypermethrin (type II) prolonged action potentials and evoked afterdepolarizations. The time course of sodium current (I(Na)) was also prolonged by these compounds. Pyrethroids delayed I(Na) inactivation, when measured under selective conditions as current sensitive to 30 microM tetrodotoxin, by increasing the proportion of slowly inactivating current at the expense of fast inactivating current. Further experiments, focusing on fenpropathrin, revealed that its effects on I(Na) inactivation time course were dose-dependent, and the Na(+) "window-current" was increased in its presence. In unstimulated, isolated hearts perfused with the same pyrethroids, the variability in contraction amplitude increased due to variations in the intervals between heartbeats. These potentially arrhythmogenic changes are consistent with the effects observed at the cellular level. The type I pyrethroid tetramethrin had little effect in any of the preparations. These findings suggest that some pyrethroids possess considerable mammalian cardiac arrhythmogenic potential, the manifestation of which in vivo may depend on the route of exposure.     

Pyrethroid Insecticides: Poisoning Syndromes,Synergies, and Therapy

Background: Pyrethroid insecticides are widely used, but there have been relatively few reports of systemic poisoning. These reports have, however, shown that pharmacotherapy is difficult and that the duration of poisoning can be unexpectedly long. Pyrethroids are ion channel toxins prolonging neuronal excitation, but are not directly cytotoxic. Two basic poisoning syndromes are seen. Type I pyrethroids produce reflex hyperexcitability and fine tremor. Type II pyrethroids produce salivation, hyperexcitability, choreoathetosis, and seizures. Both produce potent sympathetic activation. Local effects are also seen: skin contamination producing paresthesia and ingestion producing gastrointestinal irritation. The slow absorption of pyrethroids across the skin usually prevents systemic poisoning, although a significant reservoir of pyrethroid may remain bound to the epidermis. Carboxyesterase inhibitors can enhance pyrethroid toxicity in high-dose experimental studies. Hence, the unauthorized pyrethroid/organophosphate mixtures marketed in some developing countries may precipitate human poisoning. Pyrethroid paresthesia can be treated by decontamination of the skin, but systemic poisoning is difficult to control with anticonvulsants. Pentobarbitone, however, is surprisingly effective as therapy against systemic type II pyrethroid poisoning in rats, probably due to its dual action as a chloride channel agonist and a membrane stabilizer.     

Inhibitory effects of amphetamine on potassium-stimulated release of [3H]dopamine from striatal slices and synaptosomes

Amphetamine, 10(-7) M or greater, evoked the release of [3H]dopamine ([3H]DA) and inhibited subsequent K+-evoked [3H]DA release from striatal synaptosomes superfused at a flow rate (1 ml/min) that prevented reuptake. Amphetamine inhibited the K+-evoked release of [3H]DA to a lesser extent in striatal slices or in synaptosomes superfused at a flow rate (0.35 ml/min) that allowed reuptake. The observed decrease in amphetamine inhibition of K+-evoked release was primarily due to amphetamine blocking [3H]DA reuptake. Interneuronal interactions may account for some of the inhibitory effects of amphetamine on K+-evoked release in the slice. Inhibition of K+-evoked release from either slices or synaptosomes was still evident when 10(-6) M amphetamine was removed from the superfusion buffer and the spontaneous release had returned to control levels. The presence of Ca++ during amphetamine exposure was required for subsequent inhibition of K+-evoked release in synaptosomes. Amphetamine in the presence of Ca++ did not affect the subsequent release of [3H]DA evoked by the Ca++ ionophore, A23187. Therefore, amphetamine inhibition of the K+-evoked release of [3H]DA cannot be explained by prior depletion of Ca++-releasable pools. Nifedipine, 1 microM, failed to block either the Ca++-dependent release of [3H]DA or the inhibition of K+-evoked release by amphetamine. However, 1 mM cobalt inhibited the Ca++-dependent release of [3H]DA by amphetamine and antagonized the inhibition of K+-evoked release after amphetamine exposure. This suggests that amphetamine may open voltage-dependent Ca++ channels sensitive to cobalt but not nifedipine. Amphetamine may desensitize these voltage-dependent Ca++ channels and inhibit their activation by K+ depolarization.     

A reassessment of the neurotoxicity of pyrethroid insecticides

The pyrethroids are a widely used class of insecticides to which there is significant human exposure. They are however generally regarded as safe to man, and there have been few reports of human fatalities. Their acute toxicity is dominated by pharmacological actions upon the central nervous system (CNS), predominantly mediated by prolongation of the kinetics of voltage-gated sodium channels, although other mechanisms operate. This review summarizes our present understanding of such actions and the pharmacological options to antagonize them. One significant problem is the very clear heterogeneity of pyrethroid sensitivity that is seen across sodium channel subtypes; however, the distribution and function of these across the central nervous system are poorly characterized. The review also provides an overview of recent studies that suggest additional effects of pyrethroids: developmental neurotoxicity, the production of neuronal death, and action mediated via pyrethroid metabolites. The evidence for these is at present equivocal, but all 3 carry important implications for human health.     

Benzodiazepines and barbiturate potentiate the pre- and postsynaptic gamma-aminobutyric acid (GABA)A receptor-mediated response in the enteric nervous system of guinea pig small intestine

To determine whether or not presynaptic gamma-aminobutyric acid (GABA) receptors regulate the release of GABA, we examined properties of the presynaptic GABA receptor and compared the findings within the case of the postsynaptic GABA receptor, using the longitudinal muscle with myenteric plexus (L-M) preparation of guinea pig small intestine. Muscimol, but not baclofen, reduced the Ca++-dependent release of [3H]GABA evoked by high K+ in the presence of tetrodotoxin from L-M preparation of the small intestine preloaded with [3H]GABA. Bicuculline, picrotoxin and furosemide antagonized the effect of muscimol. Diazepam, clonazepam and pentobarbital potentiated the muscimol-induced inhibition of high K+-evoked release of [3H]GABA. The potentiating effect of clonazepam was antagonized by Ro 15-1788. Muscimol induced a Ca++-dependent and tetrodotoxin-sensitive release of [3H]acetylcholine from L-M preparation preloaded with [3H]choline. The effect of muscimol was antagonized by bicuculline, picrotoxin and furosemide. Diazepam, clonazepam and pentobarbital potentiated the muscimol-evoked release of [3H]ACh. The potentiation of muscimol effect by clonazepam was inhibited by Ro 15-1788. These results indicate that both the GABA autoreceptor and postsynaptic receptor may possess the same property which is related to benzodiazepine and barbiturate binding sites in the enteric nervous system of the guinea pig small intestine. The benzodiazepine binding site seems to be of central type.     

A survey of pyrethroid‐resistant populations of Meligethes aeneus F. in Poland indicates the incidence of numerous substitutions in the pyrethroid target site of voltage‐sensitive sodium channels in individual beetles

The pollen beetle (Meligethes aeneus F.) is the most devastating pest of oilseed rape (Brassica napus) and is controlled by pyrethroid insecticides. However, resistance to pyrethroids in Europe is becoming widespread and predominant. Pyrethroids target the voltage‐sensitive sodium channel (VSSC), and mutations in VSSC may be responsible for pyrethroid insensitivity. Here, we analysed individual beetles that were resistant to esfenvalerate, a pyrethroid, from 14 populations that were collected from oilseed rape fields in Poland. We screened the VSSC domains that were presumed to directly interact with pyrethroids. We identified 18 heterozygous nucleic acid substitutions, amongst which six caused an amino acid change: N912S, G926S, I936V, R957G, F1538L and E1553G. Our analysis of the three‐dimensional structure of these domains in VSSC revealed that some of these changes may slightly influence the protein structure and hence the docking efficiency of esfenvalerate. Therefore, these mutations may impact the susceptibility of the sodium channel to the action of this insecticide.     

Induced release of acetylcholine from guinea pig ileum longitudinal muscle-myenteric plexus by anatoxin-a.

Anatoxin-a (ANTX), a nicotinic agonist, has been shown to induce contraction of guinea pig ileum, which was abrogated by the muscarinic antagonist atropine and the nicotinic antagonists tubocurarine and hexamethonium. We showed here that the ganglionic nicotinic antagonist mecamylamine was a better inhibitor of the contraction of ileum induced by ANTX. The sodium channel blocker tetrodotoxin also abolished ANTX-induced contraction. In contrast, alpha-bungarotoxin, the muscle type nicotinic receptor blocker, had no effect on ANTX-induced contraction of guinea pig ileum. Longitudinal muscle-myenteric plexus prepared from guinea pig ileum, labeled with [3H]choline and then incubated with ANTX was shown for the first time to release [3H]acetylcholine (ACh) in a dose-dependent manner. Pretreatment of longitudinal muscle-myenteric plexus with tubocurarine, hexamethonium or mecamylamine blocked ANTX-induced release of [3H]ACh. In contrast, atropine was without effect. Mecamylamine was the most potent antagonist. As observed in ileum contraction, tetrodotoxin completely and potently blocked the release of [3H]ACh induced by ANTX. Neither alpha-bungarotoxin nor the neuromuscular junction blockers conotoxin G1 or M1 could inhibit the [3H]ACh release. Taken together, these results suggested that ANTX activated nicotinic receptors on ganglionic interneurons to trigger a release of ACh, which next stimulated muscarinic receptors and induced ileum contraction.