Effects of triflumezopyrim on the reproduction of brown planthopper,Nilaparvata lugens

The brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae), is a notorious pest on rice in Asia and has evolved serious resistance to traditional chemical insecticides in recent years. Triflumezopyrim, a newly commercialized mesoionic insecticide, exhibited an excellent control effect on sucking insects, including N. lugens. However, the effects of triflumezopyrim on the reproduction of N. lugens have not yet been fully elucidated. In this study, we investigated the impacts of different concentrations of triflumezopyrim (LC₁₀, LC₅₀ and LC₉₀) on the reproduction and the reproduction related genes of N. lugens exposure to the insecticide via systemic and contact route. The results indicated that the preoviposition periods of N. lugens females in the LC₅₀ and LC₉₀ triflumezopyrim-treated groups (♀ t × ♂ t) were prolonged in contact toxicity treatment, compared with control (acetone treatment). The fecundities for three pair combinations (♀ c ×♂ t, ♀ t ×♂ c and ♀ t ×♂ t) treated with LC₅₀ and LC₉₀ concentrations of triflumezopyrim showed a significant decrease compared to water/acetone-treated control via contact and systemic route. No significant effects of LC₁₀ triflumezopyrim on preoviposition period and fecundity were found in contact and systemic toxicity treatments. However, the triflumezopyrim did not influence the egg hatchability. By microscope observation, the LC₅₀ and LC₉₀ triflumezopyrim impended the ovarian development in females. qRT-PCR analysis showed that the vitellogenin (NlVg) relative expression level of female adults exposed to LC₅₀ and LC₉₀ triflumezopyrim decreased by 30.3 – 39.5% in two toxicity treatments. The titers of another reproduction related hormones juvenile hormone III (JH III) in LC₅₀ and LC₉₀ triflumezopyrim-treatments were 27.4% and 25.8% lower than control in contact toxicity treatment and 27.08% and 37.18% lower than control in systemic toxicity treatment. In summary, these results suggested that the median lethal and high lethal concentrations of triflumezopyrim inhibited the reproduction and reduced the expression of NlVg and JH III in N. lugens. This study provided a reference for the efficient and scientific utilization of triflumezopyrim in the field.     

Desensitization of nicotinic acetylcholine receptors in central nervous system neurons of the stick insect (Carausius morosus) by imidacloprid and sulfoximine insecticides

Imidacloprid, sulfoxaflor and two experimental sulfoximine insecticides caused generally depressive symptoms in stick insects, characterized by stillness and weakness, while also variably inducing postural changes such as persistent ovipositor opening, leg flexion or extension and abdomen bending that could indicate excitation of certain neural circuits. We examined the same compounds on nicotinic acetylcholine receptors in stick insect neurons, which have previously been shown to desensitize in the presence of ACh. Brief U-tube application of 10⁻⁴ M solutions of insecticides for 1 s evoked currents that were much smaller than ACh-evoked currents, and depressed subsequent ACh-evoked currents for several minutes, indicating that the compounds are low-efficacy partial agonists that potently desensitize the receptors. Much lower concentrations of insecticides applied in the bath for longer periods did not activate currents, but inhibited ACh-evoked currents via desensitization of the receptors. Previously described fast- and slowly-desensitizing nACh currents, I_ACh1 and I_ACh2 respectively, were each found to consist of two components with differing sensitivities to the insecticides. Imidacloprid applied in the bath desensitized high-sensitivity components, I_ACh1H and I_ACh2H with IC₅₀s of 0.18 and 0.13 pM, respectively. It desensitized the low-sensitivity slowly desensitizing component, I_ACh2L, with an IC₅₀ of 2.6 nM, while a component of the fast-desensitizing current, I_ACh1L, was least sensitive, with an IC₅₀ of 81 nM I_ACh1L appeared to be insensitive to the three sulfoximines tested, whereas all three sulfoximines potently desensitized I_ACh1H and both slowly desensitizing components, with IC₅₀s between 2 and 7 nM. We conclude that selective desensitization of certain nAChR subtypes can account for the insecticidal actions of imidacloprid and sulfoximines in stick insects.     

Native subunit composition of two insect nicotinic receptor subtypes with differing affinities for the insecticide imidacloprid

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively to control a variety of insect pest species. The brown planthopper (Nilaparvata lugens), an insect pest of rice crops throughout Asia, is an important target species for control with neonicotinoid insecticides such as imidacloprid. Studies with nAChRs purified from N. lugens have identified two [³H]imidacloprid binding sites with different affinities (K_d = 3.5 ± 0.6 pM and 1.5 ± 0.2 nM). Co-immunoprecipitation studies with native preparations of N. lugens nAChRs, using subunit-selective antisera, have demonstrated the co-assembly of Nlα1, Nlα2 and Nlβ1 subunits into one receptor complex and of Nlα3, Nlα8 and Nlβ1 into another. Immunodepletion of Nlα1 or Nlα2 subunits resulted in the selective loss of the lower affinity imidacloprid binding site, whereas immunodepletion of Nlα3 or Nlα8 caused the selective loss of the high-affinity site. Immunodepletion of Nlβ1 resulted in a complete absence of specific imidacloprid binding. In contrast, immunodepletion with antibodies selective for other N. lugens nAChR subunits (Nlα4, Nlα6, Nlα7 and Nlβ2) had no significant effect on imidacloprid binding. Taken together, these data suggest that nAChRs containing Nlα1, Nlα2 and Nlβ1 constitute the lower affinity binding site, whereas nAChRs containing Nlα3, Nlα8 and Nlβ1 constitute the higher affinity binding site for imidacloprid in N. lugens.     

Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats

This study aimed to investigate the effect of magnolol (5,5′-diallyl-2,2′-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca²⁺ currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3–100 μM). In the presence of Bay K8644 (100 nM), magnolol (10–100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and N_ώ-nitro-l-arginine methyl ester (l-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3–100 μM) inhibited the L-type Ca²⁺ currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca²⁺ channel activity.     

Phospholipases A2 isolated from snake venoms block acetylcholine-elicited currents in identified Lymnaea stagnalis neurons

Phospholipases A₂ (PLA₂s) are the most abundant family of snake venom proteins and play a significant role in prey envenomation. Their content in venoms is rather high. PLA₂s not only have enzyme activity but exhibit other types of biological activities including neurotoxicity. We have earlier shown that a protein bitanarin from the venom of the puff adder Bitis arietans is capable to block the responses of Lymnaea stagnalis neurons to acetylcholine and represents an active PLA₂ at the same time. Further investigation of PLA₂s isolated from the venoms of snakes of two families revealed their capability to interact with nicotinic acetylcholine receptors (nAChRs): PLA₂ from Vipera ursinii (Viperidae family), Naja kaouthia, and Bungarus fasciatus (Elapidae family) suppressed acetylcholine-induced current in identified neurons of L. staganlis. The effect was evident at PLA₂ concentration in the range of tens micromoles. The data obtained suggest the presence in a PLA₂ molecule of a site interacting with nAChR and a possible involvement of nAChR block in toxic action of PLA₂s.     

Functional characterisation of a nicotinic acetylcholine receptor α subunit from the brown dog tick, Rhipicephalus sanguineus

Ticks and tick-borne diseases have a major impact on human and animal health worldwide. Current control strategies rely heavily on the use of chemical acaricides, most of which target the CNS and with increasing resistance, new drugs are urgently needed. Nicotinic acetylcholine receptors (nAChRs) are targets of highly successful insecticides. We isolated a full-length nAChR α subunit from a normalised cDNA library from the synganglion (brain) of the brown dog tick, Rhipicephalus sanguineus. Phylogenetic analysis has shown this R. sanguineus nAChR to be most similar to the insect α1 nAChR group and has been named Rsanα1. Rsanα1 is distributed in multiple tick tissues and is present across all life-stages. When expressed in Xenopus laevis oocytes Rsanα1 failed to function as a homomer, with and without the addition of either Caenorhabditis elegans resistance-to-cholinesterase (RIC)-3 or X. laevis RIC-3. When co-expressed with chicken β2 nAChR, Rsanα1 evoked concentration-dependent, inward currents in response to acetylcholine (ACh) and showed sensitivity to nicotine (100 μM) and choline (100 μM). Rsanα1/β2 was insensitive to both imidacloprid (100 μM) and spinosad (100 μM). The unreliable expression of Rsanα1 in vitro suggests that additional subunits or chaperone proteins may be required for more robust expression. This study enhances our understanding of nAChRs in arachnids and may provide a basis for further studies on the interaction of compounds with the tick nAChR as part of a discovery process for novel acaricides.     

The actions of the catch relaxing peptide,carp, on identified Helix central neurons

1. The action of the catch relaxing peptide CARP (H-Ala-Met-Pro-Met-Leu-Arg-Leu-amide, AMPMLRL-amide) has been studied on Helix central neurons.2. CARP, at thresholds of 1 nM or less, excited some cells while inhibiting others. CARP modulated the action of acetylcholine, e.g. at 10–100 nM CARP, the acetylcholine response was greatly reduced.3. The inhibitory action of CARP is mediated through an increase in chloride permeability while CARP excitation was dependent on sodium and calcium.4. The use of blocking agents suggested that CARP and acetylcholine acted on separate receptors.     

Neonicotinoid Binding,Toxicity and Expression of Nicotinic Acetylcholine Receptor Subunits in the Aphid Acyrthosiphon pisum

Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [³H]-IMI (Kd of 0.16±0.04 nM and 41.7±5.9 nM) and for the nicotinic antagonist [¹²⁵I]-α-bungarotoxin (Kd of 0.008±0.002 nM and 1.135±0.213 nM). Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [¹²⁵I]-α-bungarotoxin binding sites while CLT affinity was similar for both [¹²⁵I]-α-bungarotoxin and [³H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC₅₀ = 0.038 µg/ml) and TMX (LC₅₀ = 0.034 µg/ml) were more toxic than CLT (LC₅₀ = 0.118 µg/ml). The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumβ1 expressions levels, whereas Apisumβ2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies.     

The Activity of GAT107, an Allosteric Activator and Positive Modulator of α7 Nicotinic Acetylcholine Receptors (nAChR), Is Regulated by Aromatic Amino Acids That Span the Subunit Interface

GAT107, the (+)-enantiomer of racemic 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, is a strong positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptor (nAChR) activation by orthosteric agonists with intrinsic allosteric agonist activities. The direct activation produced by GAT107 in electrophysiological studies is observed only as long as GAT107 is freely diffusible in solution, although the potentiating activity primed by GAT107 can persist for over 30 min after drug washout. Direct activation is sensitive to α7 nAChR antagonist methyllycaconitine, although the primed potentiation is not. The data are consistent with GAT107 activity arising from two different sites. We show that the coupling between PAMs and the binding of orthosteric ligands requires tryptophan 55 (Trp-55), which is located at the subunit interface on the complementary surface of the orthosteric binding site. Mutations of Trp-55 increase the direct activation produced by GAT107 and reduce or prevent the synergy between allosteric and orthosteric binding sites, so that these mutants can also be directly activated by other PAMs such as PNU-120596 and TQS, which do not activate wild-type α7 in the absence of orthosteric agonists. We identify Tyr-93 as an essential element for orthosteric activation, because Y93C mutants are insensitive to orthosteric agonists but respond to GAT107. Our data show that both orthosteric and allosteric activation of α7 nAChR require cooperative activity at the interface between the subunits in the extracellular domain. These cooperative effects rely on key aromatic residues, and although mutations of Trp-55 reduce the restraints placed on the requirement for orthosteric agonists, Tyr-93 can conduct both orthosteric activation and desensitization among the subunits.     

Effects of mibefradil on synaptic transmission in the hippocampus and on voltage-dependent currents in isolated hippocampal and thalamic neurons of the rat

The effects of a novel anti-hypertensive drug, mibefradil, on voltage-dependent currents in isolated thalamic and hippocampal neurons, as well as on synaptic transmission in the hippocampus have been studied. Mibefradil exerted a potent inhibitory action on low-threshold calcium currents in thalamic neurons (IC₅₀=160 nM). In higher concentrations (1–20 μM), this drug blocked not only low-threshold calcium current but also voltage-dependent sodium and delayed potassium currents in pyramidal hippocampal neurons. The amplitude of population action potentials in hippocampal slices decreased by 55% in the presence of 20μM mibefradil. All of the effects of mibefradil were almost completely reversible. In our experiments, the sensitivity of low-threshold calcium channels in thalamic neurons to mibefradil was higher than that observed on other objects. The ability of mibefradil to block not only calcium currents but also other types of voltage-dependent ion conductances in hippocampal neurons may be considered an essential factor that determines the specificity of the pharmacological profile of this drug.     

Two Subtypes of Nicotinic Acetylcholine Receptors in <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> Neurons Control Chloride Conductance

Giant neurons of the mollusc Lymnaea stagnalis contain heterogeneous population of nicotinic acetylcholine receptors (nAChRs) according to their relative sensitivity to antagonists. All these receptors are involved in the total response to acetylcholine (ACh). To evaluate activity of different pharmacological agents correctly it is necessary to know ionic selectivity of nAChRs which participate in transmembrane ionic current. In this work we studied the influence of ionic composition of the external and intracellular solutions on the current amplitude and current–voltage relation under the action of ACh or other nAChR agonists on the identified neurons of the left and right parietal ganglia of Lymnaea. After non-permeable cation N-methyl-D-glucamine was completely substituted for external Na⁺ ions there were no changes in the current characteristics. After a 10-fold decrease in Cl–concentration in the external solution there was a considerable shift of the current–voltage curve to the right, outward currents at the holding potential (V_h) up to 30 mV were not observed. On the contrary, a 10-fold decrease of Cl^– concentration in the intracellular solution led to a shift of the current–voltage curve to hyperpolarizing direction, the reversal potential shift was in the average –42 mV. When ACh and nicotinic agonists with higher selectivity towards vertebrate α7 neuronal nAChR type and one of the two subtypes of Lymnaea nAChRs were compared, no differences in changes of ionic current characteristics were found. Neurons with distinct relative fraction of one or another nAChR subtype reacted to Cl^– concentration change in the same way. Our results support earlier data on Cl^– mechanism of Lymnaea neuron responses to ACh and evidence identical ionic selectivity of the two nAChR subtypes in identified neurons tested.     

Whole-cell patch-clamp recording of nicotinic acetylcholine receptors in adult Brugia malayi muscle

Lymphatic filariasis is a debilitating disease caused by clade III parasites like Brugia malayi and Wuchereria bancrofti. Current recommended treatment regimen for this disease relies on albendazole, ivermectin and diethylcarbamazine, none of which targets the nicotinic acetylcholine receptors in these parasitic nematodes. Our aim therefore has been to develop adult B. malayi for electrophysiological recordings to aid in characterizing the ion channels in this parasite as anthelmintic target sites. In that regard, we recently demonstrated the amenability of adult B. malayi to patch-clamp recordings and presented results on the single-channel properties of nAChR in this nematode. We have built on this by recording whole-cell nAChR currents from adult B. malayi muscle. Acetylcholine, levamisole, pyrantel, bephenium and tribendimidine activated the receptors on B. malayi muscle, producing robust currents ranging from > 200 pA to ~ 1.5 nA. Levamisole completely inhibited motility of the adult B. malayi within 10 min and after 60 min, motility had recovered back to control values.     

Differential resistance of insect sodium channels with kdr mutations to deltamethrin, permethrin and DDT

Knockdown resistance (kdr) in insects, caused by inherited nucleotide polymorphisms in the voltage-gated sodium channel (VGSC) gene, is a major threat to the efficacy of pyrethroid insecticides. Classic kdr, resulting from an L1014F substitution in the VGSC is now present in numerous pest species. Two other substitutions at the L1014 locus have also been reported, L1014S and L1014H. Here we have used expression of L1014 modified Drosophila para VGSCs in Xenopus oocytes with two-electrode voltage clamp to characterise all three mutations. The mutations L1014F and L1014H caused significant depolarizing shifts in the half activation voltage (V_50,act) from −17.3 mV (wild-type) to −13.1 and −13.5 mV respectively, whereas L1014S caused no shift in V_50,act but its currents decayed significantly faster than wild-type channels. Treatment of the wild-type channel with deltamethrin (≥1 nM), permethrin (≥30 nM) or DDT (≥1 ??M) resulted in hyperpolarizing shifts in V_50,act. Deltamethrin, permethrin and DDT also produced “tail currents” with EC₅₀s of 0.043, 0.40 and 65 ??M and maximum modifications of 837, 325 and 7% respectively. L1014F provided a high level of resistance against all insecticides for both measured parameters. L1014H most effectively combated deltamethrin induced tail currents while L1014S strongly resisted the large DDT induced shifts in V_50,act. We conclude that L1014H and L1014S may have arisen through heavy exposure to specific pyrethroids and DDT respectively.     

Differential interactions of 5-(4-piperidyl)-3-isoxazolol analogues with insect γ-aminobutyric acid receptors leading to functional selectivity

γ-Aminobutyric acid (GABA) receptors (GABARs) mediate fast inhibitory synaptic transmission and are also targets for drugs and insecticides. To better understand the molecular interactions of ligands with the orthosteric sites of GABARs, we examined 4-aryl/arylalkyl-5-(4-piperidyl)-3-isoxazolol, 4-aryl-5-(4-piperidyl)-3-isothiazolol, and 5-aryl-4-(4-piperidyl)-1-hydroxypyrazole for their antagonism with regard to three insect GABARs. The 3-isoxazolol was preferable to the 3-isothiazolol and 1-hydroxypyrazole in antagonism to common cutworm and housefly GABARs. Of the tested analogues, 4-(3-biphenylyl)-5-(4-piperidyl)-3-isoxazolol (2a) displayed the greatest antagonism for common cutworm and housefly GABARs, with IC₅₀ values of 3.4 and 10.2 μM, respectively. In contrast to the antagonism of the two GABARs, 2a showed partial agonism for the case of small brown planthopper GABARs, with an EC₅₀ value of 31.3 μM. Homology models and docking simulations revealed that a cation-π interaction between an analogue and an Arg residue in loop C or E of the orthosteric site is a key component of antagonism. This specific phenomenon was lacking in the interactions between 2a and the orthosteric site of small brown planthopper GABARs. These findings provide important insights into designing and developing novel drugs and insecticides.     

Characterization of a T-superfamily conotoxin TxVC from Conus textile that selectively targets neuronal nAChR subtypes

T-superfamily conotoxins have a typical cysteine pattern of “CC–CC”, and are known to mainly target calcium or sodium ion channels. Recently, we screened the targets of a series of T-superfamily conotoxins and found that a new T-superfamily conotoxin TxVC (KPCCSIHDNSCCGL-NH₂) from the venom of Conus textile. It selectively targeted the neuronal nicotinic acetylcholine receptor (nAChR) subtypes α4β2 and α3β2, with IC₅₀ values of 343.4 and 1047.2 nM, respectively, but did not exhibit obvious pharmacological effects on voltage-gated potassium, sodium or calcium channel in DRG cells, the BK channels expressed in HEK293 cells, or the Kv channels in LβT2 cells. The changes in the inhibitory activities of its Ala mutants, the NMR structure, and molecular simulation results based on other conotoxins targeting nAChR α4β2, all demonstrated that the residues Ile⁶ and Leu¹⁴ were the main hydrophobic pharmacophores. To our best knowledge, this is the first T-superfamily conotoxin that inhibits neuronal nAChRs and possesses high binding affinity to α4β2. This finding will expand the knowledge of the targets of T-superfamily conotoxins and the motif information could help the design of new nAChR inhibitors.     

Spermine biphasically affects N-type calcium channel currents in adult dorsal root ganglion neurons of the rat

Spermine (Spe) is a polyamine co-secreted with neurotransmitters. In this work its effects on N-type Ca²⁺ channel (Ca_V2.2) have been studied on adult sensory neurons of the rat by means of whole-cell patch-clamp. Spe exerted biphasic effects when added to the external solution: at 500 μM decreased N-type Ca²⁺ channel currents, reducing the maximum whole-cell conductance, shifting the activation curve to the right on the voltage axes and decreasing its slope; conversely, at lower concentration (500 nM) Spe induced completely opposite effects. In 62% of the neurons the inhibitory effects were accompanied by a slowing down of the activation kinetics relieved by a conditioning pre-pulse to + 50 mV. The biphasic effects and their rapid onset and offset time course may be explained if multiple sites of action with a different affinity for Spe are present directly on the channel. The effects of Spe on HVA Ca²⁺ currents were strongly dependent on [Ca²⁺]_ext, high [Ca²⁺] powerfully reducing Spe effects. This may be explained if we take into account that as Spe has four positive charges at physiological pH; it may compete with divalent cations for some negatively charged regulatory sites. In these experiments, Spe was effective at concentrations possibly reached in physiological conditions.     

Trypanosoma evansi: Pharmacological evidence of a nicotinic acetylcholine receptor

The role of calcium and its relevance have been deeply revised with respect to trypanosomatids, as the mechanism by which calcium enters trypanosomes was, until now, not well understood. There is evidence supporting the presence of a nAChR in another member of the trypanosomatidae family, Trypanosoma cruzi, these receptors being one entry path to calcium ions. The aims of this work were to determine if there was a nicotinic acetylcholine receptor (nAChR) in Trypanosoma evansi, and to subsequently perform a partial pharmacological characterization of this receptor.After being loaded with FURA-2AM, individual cells of T. evansi, were exposed to cholinergic compounds, and the cells displayed a dose-dependent response to carbachol. This observation indicated that a cholinergic receptor may be present in T. evansi. Although a dose-dependent response to muscarine could not be demonstrated, nicotine could promote an incremental dose-dependent response. The relative potency of this specific agonist of nAChR is in agreement with previous reports. The estimated affinity values were a K_d1 value of 29.6 ± 5.72 nM and a K_d2 value of 315.9 ± 26.6 nM, which is similar to the K_d value reported for the α4 nicotinic receptor. The Hill coefficients were determined to be an n₁ of 1.2 ± 0.3 and an n₂ of 4.2 ± 1.3. Finally, our calculations indicated that there are about 1020 receptors in each T. evansi parasite, which is approximately 15-fold lower than the number reported in Torpedo californica electric cells. These results suggest the presence of a nAChR in T. evansi, which is able to bind nicotinic ligands and induce calcium signals.     

Erythrina mulungu Alkaloids Are Potent Inhibitors of Neuronal Nicotinic Receptor Currents in Mammalian Cells

Crude extracts and three isolated alkaloids from Erythrina mulungu plants have shown anxiolytic effects in different animal models. We investigated whether these alkaloids could affect nicotinic acetylcholine receptors and if they are selective for different central nervous system (CNS) subtypes. Screening experiments were performed using a single concentration of the alkaloid co-applied with acetylcholine in whole cell patch-clamp recordings in three different cell models: (i) PC12 cells natively expressing α3* nicotinic acetylcholine receptors; (ii) cultured hippocampal neurons natively expressing α7* nicotinic acetylcholine receptors; and (iii) HEK 293 cells heterologoulsy expressing α4β2 nicotinic acetylcholine receptors. For all three receptors, the percent inhibition of acetylcholine-activated currents by (+)-11á-hydroxyerysotrine was the lowest, whereas (+)-erythravine and (+)-11á-hydroxyerythravine inhibited the currents to a greater extent. For the latter two substances, we obtained concentration-response curves with a pre-application protocol for the α7* and α4β2 nicotinic acetylcholine receptors. The IC₅₀ obtained with (+)-erythravine and (+)-11á-hydroxyerythravine were 6 µM and 5 µM for the α7* receptors, and 13 nM and 4 nM for the α4β2 receptors, respectively. Our data suggest that these Erythrina alkaloids may exert their behavioral effects through inhibition of CNS nicotinic acetylcholine receptors, particularly the α4β2 subtype.     

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for α-bungarotoxin. Using radiolabeled ligand binding techniques, we show that the binding of [³H]-(±)-epibatidine is heterogeneous and is characterized by two classes of binding sites with equilibrium dissociation constants of about 15 nM and 1 μM. These classes of sites exist in approximately equal numbers and all [³H]-(±)-epibatidine binding is competitively displaced by acetylcholine, suberyldicholine and d-tubocurarine. These results provide further evidence for the complexity of agonist binding to the nAChR and underscore the difficulties in determining simple relationships between site occupancy and functional responses.     

Caffeine has a dual influence on NMDA receptor–mediated glutamatergic transmission at the hippocampus

Caffeine, a stimulant largely consumed around the world, is a non-selective adenosine receptor antagonist, and therefore caffeine actions at synapses usually, but not always, mirror those of adenosine. Importantly, different adenosine receptors with opposing regulatory actions co-exist at synapses. Through both inhibitory and excitatory high-affinity receptors (A₁R and A₂R, respectively), adenosine affects NMDA receptor (NMDAR) function at the hippocampus, but surprisingly, there is a lack of knowledge on the effects of caffeine upon this ionotropic glutamatergic receptor deeply involved in both positive (plasticity) and negative (excitotoxicity) synaptic actions. We thus aimed to elucidate the effects of caffeine upon NMDAR-mediated excitatory post-synaptic currents (NMDAR-EPSCs), and its implications upon neuronal Ca²⁺ homeostasis. We found that caffeine (30–200 μM) facilitates NMDAR-EPSCs on pyramidal CA1 neurons from Balbc/ByJ male mice, an action mimicked, as well as occluded, by 1,3-dipropyl-cyclopentylxantine (DPCPX, 50 nM), thus likely mediated by blockade of inhibitory A₁Rs. This action of caffeine cannot be attributed to a pre-synaptic facilitation of transmission because caffeine even increased paired-pulse facilitation of NMDA-EPSCs, indicative of an inhibition of neurotransmitter release. Adenosine A_2ARs are involved in this likely pre-synaptic action since the effect of caffeine was mimicked by the A_2AR antagonist, SCH58261 (50 nM). Furthermore, caffeine increased the frequency of Ca²⁺ transients in neuronal cell culture, an action mimicked by the A₁R antagonist, DPCPX, and prevented by NMDAR blockade with AP5 (50 μM). Altogether, these results show for the first time an influence of caffeine on NMDA receptor activity at the hippocampus, with impact in neuronal Ca²⁺ homeostasis.