Betulin binds to gamma-aminobutyric acid receptors and exerts anticonvulsant action in mice

The lupane type pentacyclic triterpenes: lupeol, betulin, and betulinic acid are widely distributed natural compounds. Recently, pharmaceutical compositions from plant extracts (family Marcgraviaceae) containing betulinic acid, have been patented as anxiolytic remedies. To extend our knowledge of the CNS effects of the triterpenes, we suggest here that the chemically related lupeol, betulin and betulinic acid may interact with the brain neurotransmitter γ-aminobutyric acid (GABA) receptors in vitro and in vivo. Using radioligand receptor-binding assay, we showed that only betulin bound to the GABA_A-receptor sites in mice brain in vitro and antagonised the GABA_A-receptor antagonist bicuculline-induced seizures in mice after intracisternal and intraperitoneal administration. Neither betulinic acid nor lupeol bound to GABA_A receptor nor did they inhibit bicuculline-induced seizures in vivo. These findings demonstrate for the first time the CNS effects of betulin in vivo, and they also show distinct GABA_A-receptor-related properties of lupane type triterpenes.These findings may open new avenues in understanding the central effects of betulin, and they also indicate possibilities for novel drug design on the basis of betulin structure.     

Betulin binds to γ-aminobutyric acid receptors and exerts anticonvulsant action in mice

The lupane type pentacyclic triterpenes: lupeol, betulin, and betulinic acid are widely distributed natural compounds. Recently, pharmaceutical compositions from plant extracts (family Marcgraviaceae) containing betulinic acid, have been patented as anxiolytic remedies. To extend our knowledge of the CNS effects of the triterpenes, we suggest here that the chemically related lupeol, betulin and betulinic acid may interact with the brain neurotransmitter γ-aminobutyric acid (GABA) receptors in vitro and in vivo. Using radioligand receptor-binding assay, we showed that only betulin bound to the GABA_A-receptor sites in mice brain in vitro and antagonised the GABA_A-receptor antagonist bicuculline-induced seizures in mice after intracisternal and intraperitoneal administration. Neither betulinic acid nor lupeol bound to GABA_A receptor nor did they inhibit bicuculline-induced seizures in vivo. These findings demonstrate for the first time the CNS effects of betulin in vivo, and they also show distinct GABA_A-receptor-related properties of lupane type triterpenes.These findings may open new avenues in understanding the central effects of betulin, and they also indicate possibilities for novel drug design on the basis of betulin structure.     

Distinct functional and pharmacological properties of tonic and quantal inhibitory postsynaptic currents mediated by gamma-aminobutyric acid(A) receptors in hippocampal neurons

gamma-Aminobutyric acid (GABA), the principal inhibitory neurotransmitter, activates a persistent low amplitude tonic current in several brain regions in addition to conventional synaptic currents. Here we demonstrate that GABA(A) receptors mediating the tonic current in hippocampal neurons exhibit functional and pharmacological properties different from those of quantal synaptic currents. Patch-clamp techniques were used to characterize miniature inhibitory postsynaptic currents (mIPSCs) and the tonic GABAergic current recorded in CA1 pyramidal neurons in rat hippocampal slices and in dissociated neurons grown in culture. The competitive GABA(A) receptor antagonists, bicuculline and picrotoxin, blocked both the mIPSCs and the tonic current. In contrast, mIPSCs but not the tonic current were inhibited by gabazine (SR-95531). Coapplication experiments and computer simulations revealed that gabazine bound to the receptors responsible for the tonic current but did not prevent channel activation. However, gabazine competitively inhibited bicuculline blockade. The unitary conductance of the GABA(A) receptors underlying the tonic current (approximately 6 pS) was less than the main conductance of channels activated during quantal synaptic transmission (approximately 15--30 pS). Furthermore, compounds that potentiate GABA(A) receptor function including the benzodiazepine, midazolam, and anesthetic, propofol, prolonged the duration of mIPSCs and increased tonic current amplitude in cultured neurons to different extents. Clinically-relevant concentrations of midazolam and propofol caused a greater increase in tonic current compared with mIPSCs, as measured by total charge transfer. In summary, the receptors underlying the tonic current are functionally and pharmacologically distinct from quantally activated synaptic receptors and these receptors represent a novel target for neurodepressive drugs.     

Agonist and potentiation actions of n-octanol on gamma-aminobutyric acid type A receptors.

The n-octanol effects on the gamma-aminobutyric acid type A (GABAA) receptor were studied in human embryonic kidney 293 cells transfected with alpha1, beta2, and gamma2S subunit cDNAs. GABA-evoked currents had an EC50 of 13.3 +/- 1.7 microM and a Hill coefficient (nH) of 1.4 +/- 0.1. n-Octanol was also capable of evoking a small current with an EC50 of 1000 microM and an nH of 2. In addition, n-octanol modulated GABA-induced currents in a concentration-dependent manner. Coapplications of n-octanol increased peak currents evoked by 3 microM GABA with an EC50 of 190 microM and an nH of 1.8. The extent of potentiation decreased with increasing GABA concentrations and no potentiation was observed when n-octanol was coapplied with 1000 microM GABA. One-minute preapplication of 1000 microM n-octanol slightly potentiated 3 microM GABA-induced current, whereas it suppressed 300 microM GABA-induced current to 16% of the control, suggesting that 84% of the receptors underwent desensitization. Two models were used to explain n-octanol agonistic and potentiating actions on the alpha1beta2gamma2S GABAA receptor: n-octanol binds to multiple sites to exert multiple actions, or n-octanol acts as a partial agonist to manifest these actions. The partial agonist model is unique because it is a simpler model to explain n-octanol actions on the GABAA receptor.     

Maturation of glutamatergic and GABAergic synapse composition in hippocampal neurons

It is commonly accepted that glutamatergic and GABAergic presynaptic terminals form perfectly matched appositions opposite their appropriate receptors and associated binding proteins. However, recent reports indicate that certain synaptic proteins that are commonly used to identify excitatory or inhibitory synapses can be mismatched, particularly during development. In order to construct a more comprehensive scheme of synapse composition during development, we co-immunolabeled for several principle excitatory and inhibitory proteins over the course of synaptogenesis in cultured hippocampal neurons. We find that although the majority of synaptic appositions are composed of matched clusters of pre- and postsynaptic proteins appropriate for a particular neurotransmitter, many are initially mismatched, even in dendrites receiving both glutamatergic and GABAergic innervation. Over time, the fidelity of GABAergic synapse composition increases such that, despite the persistence of some mismatched components at glutamatergic sites, the incidence of mismatch diminishes at both inhibitory and excitatory synapses. Activation of either GABA-A or NMDA receptors promotes fidelity at GABAergic sites, but NMDA receptor activation promotes mismatching among glutamatergic synapses. Thus, apposition of pre- and postsynaptic elements can occur independent of neurotransmitter specificity and synaptic activity modifies these associations. Our findings support the idea that synapse maturation occurs in several distinct stages, and that these stages are regulated by a combination of activity-dependent and -independent factors.     

Pharmacokinetics and in vitro effects of a 4-aminobutyric acid derivative with anticonvulsant action

N-trimethyl-acetyl-4-aminobutyric acid, called PG2, is a new anticonvulsant acting as a 4-aminobutyric acid (GABA) pro-drug. The stimulatory effect of PG2 on synaptosomal 14C-GABA release and its inhibitory effect of synaptosomal 14C-GABA uptake were studied together with its pharmacokinetics after intracardiac and oral administration to rat. PG2 is a weak inhibitor of the GABAse system in vitro with an apparent Ki value of 0.83 mmol/l.     

Differential trafficking of adenosine receptors in hippocampal neurons monitored using GFP- and super-ecliptic pHluorin-tagged receptors

Adenosine receptors (ARs) modulate many cellular and systems-level processes in the mammalian CNS. However, little is known about the trafficking of ARs in neurons, despite their importance in controlling seizure activity and in neuroprotection in cerebral ischaemia. To address this we examined the agonist-dependent internalisation of C-terminal GFP-tagged A(1)Rs, A(2A)Rs and A(3)Rs in primary hippocampal neurons. Furthermore, we developed a novel super-ecliptic pHluorin (SEP)-tagged A(1)R which, via the N-terminal SEP tag, reports the cell-surface expression and trafficking of A(1)Rs in real-time. We demonstrate the differential trafficking of ARs in neurons: A(3)Rs internalise more rapidly than A1Rs, with little evidence of appreciable A(2A)R trafficking over the time-course of the experiments. Furthermore, the novel SEP-A(1)R construct revealed the time-course of internalisation and recovery of cell-surface expression to occur within minutes of agonist exposure and removal, respectively. These observations highlight the labile nature of A(1)R and A(3)Rs when expressed at the neuronal plasma membrane. Given the high levels of adenosine in the brain during ischaemia and seizures, internalisation of the inhibitory A(1)R may result in hyperexcitability, increased brain damage and the development of chronic epileptic states.     

Distinct actions of etomidate and propofol at β3-containing γ-aminobutyric acid type A receptors

Etomidate and propofol have clearly distinguishable effects on the central nervous system. However, studies in knock-in mice provided evidence that these agents produce anesthesia via largely overlapping molecular targets, namely GABA_A receptors containing β3 subunits. Here the authors address the question as to whether etomidate and propofol are targeting different subpopulations of β3 subunit containing GABA_A receptors.The effects of etomidate and propofol (0.5 μM and 1.0 μM) on spontaneous activity of neocortical neurons were investigated in organotypic slice cultures from wild-type and β3(N265M) knock-in mice. Firing patterns were characterized by mean burst length and number of action potentials per burst. Additionally, etomidate and propofol actions on GABA_A receptor-mediated currents were investigated by whole-cell voltage clamp recordings.On the network level, the duration of spontaneously occurring bursts of action potentials was decreased by etomidate but increased by propofol in the wild-type. The effects of etomidate were abolished in β3(N265M) mutant slices while those of propofol were qualitatively inverted. On the receptor level, GABA_A receptor-mediated inhibition of cortical neurons was modulated by etomidate and propofol in different ways. Again, drug-specific actions of etomidate and propofol were largely attenuated by the β3(N265M) mutation.Etomidate and propofol alter the firing patterns and GABA_A receptor-mediated inhibition of neocortical neurons in different ways. This suggests that etomidate and propofol act via non-uniform molecular targets. Because the major effects induced by these anesthetics were attenuated by the β3(N265M) mutation, different subpopulations of β3-containing GABA_A receptors are likely to be involved.     

Development of GABAB subunits and functional GABAB receptors in rat cultured hippocampal neurons

Metabotropic gamma-aminobutyric acid receptors (GABA(B)Rs) play a critical role in inhibitory synaptic transmission in the hippocampus but the ontogeny of their subunit synthesis and synaptic localisation has not been determined. Here we report the distributions and developmental profiles of GABA(B1) and GABA(B2) subunits in cultured rat embryonic hippocampal neurons. Limited levels of GABA(B1) and GABA(B2) immunoreactivity were present at 3 days in vitro (DIV). At 7 DIV, when baclofen-evoked inwardly rectifying K(+) channel-mediated responses first appear in the cells, there was a more widespread expression within the soma and proximal dendrites. Levels of the K(+) channel GIRK 1 were relatively constant at all time points suggesting channel availability does not limit the appearance of functional GABA(B)Rs. At 14 DIV the staining displayed a punctate dendritic distribution and near maximal GABA(B)R-mediated electrophysiological responses were obtained. About half of the puncta for each GABA(B)R subunit in dendrites co-localised with the synaptic marker SV2a suggesting that these subunits are at or very near to synapses. Interestingly, at all ages strong GABA(B)R immunoreactivity was also present in the nuclei of neurons. These results provide an important developmental baseline for future studies aimed at investigating, for example, the trafficking and functional regulation of these receptors.     

Gamma-aminobutyric acid and benzodiazepine receptors in cultured cerebellar granule cells: effects of taurine and its lipophilic derivatives

The effects of taurine and some lipophilic derivatives of taurine on binding to GABA and benzodiazepine receptors were studied in intact cerebellar granule cells. The phenylsuccinylimido derivatives of taurine appeared to increase the binding of muscimol in micromolar concentrations, while taurine decreased it slightly. Only minor changes were seen in the basal binding of flunitrazepam, whereas stimulation of the binding by GABA was strongly reduced by piperidino, benzamido and phenyl-succinylimidotaurine with taurine itself again showing only a weak effect. Diphenylhydantoin, which bears structural resemblance to the phenylsuccinylimido group, had a strong effect on the stimulated binding of flunitrazepam and it also slightly reduced the basal level of binding. Thus, it seems possible that the effects of the phenylsuccinylimido derivatives of taurine on the binding of flunitrazepam were due to this chemical structure and not to the taurine-like core of the molecules. The phthalimido derivative of taurine, taltrimide, which has been tested in clinical trials with epileptic patients, did not show any activity in the binding studies.     

Effects of bromowillardiine and willardiine on non-N-methyl-D-aspartate receptors in postnatal rat hippocampal neurons.

The physiology and pharmacology of willardiine and bromowillardiine, structural analogues of quisqualate, were studied in cultured postnatal rat hippocampal neurons using whole-cell voltage-clamp techniques. These agonists appear to act at a shared non-N-methyl-D-aspartate (non-NMDA) receptor-channel complex and gate nonselective cationic currents. Willardiine currents desensitize rapidly and to a much greater degree than bromowillardiine currents. In addition, the brominated compound produces steady state currents that are 5 times larger than those produced by willardiine at saturation. Bromowillardiine is also a more efficacious excitotoxin, producing about 3-fold greater acute neuronal damage than willardiine at saturating concentrations. These results suggest that agonist structure affects the ability of non-NMDA agonists to induce desensitization and add support to the hypothesis that receptor desensitization serves to limit acute excitotoxicity in cultured neurons.     

gamma-aminobutyric acid B receptors are negatively coupled to adenylate cyclase in brain, and in the cerebellum these receptors may be associated with granule cells

Baclofen and gamma-aminobutyric acid (GABA) are shown to inhibit basal adenylate cyclase activity in brain of rat. The response is mediated through the GABAB receptor, and the rank order of potency for agonists is (-)-baclofen (EC50 = 4 microM) greater than GABA (EC50 = 17 microM) greater than muscimol greater than (+)-baclofen. GABAA agonists are not effective inhibitors of cyclase activity. The response is bicuculline-insensitive, and diazepam does not modify the GABA or (-)-baclofen inhibition of adenylate cyclase. Studies with neurologically mutant mice correlated a loss in GABAB receptor-mediated inhibition of cyclase with a loss in cerebellar granule cells. Thus, the GABAB receptor is negatively coupled to adenylate cyclase in various brain areas, and, in the cerebellum, data suggest a granule cell localization of this activity.     

盐酸埃他卡林对大鼠海马神经元谷氨酸受体功能及突触活动的影响

目的 :研究盐酸埃他卡林 (Ipt)对脑神经元谷氨酸受体功能及突触活动的影响。方法 :采用原代培养的大鼠海马神经元 ,应用膜片钳全细胞记录技术 ,记录Ipt对培养的海马神经元谷氨酸或天冬氨酸(NMDA)诱发电流及神经元突触后电流的影响。结果 :Ipt(1～ 1 0 0 μmol·L- 1)可浓度依赖性地对抗培养的海马神经元谷氨酸或NMDA诱发电流 ,并为ATP敏感性钾通道拮抗剂格列本脲 30 μmol·L- 1所对抗。Ipt抑制培养的海马神经元之间突触联系形成的自发兴奋性突触后电流 ,降低其发放频率 ,抑制其电流幅度 ;但对微小兴奋性突触后电流无显著性影响。结论 :Ipt可阻断脑神经元谷氨酸受体功能 ,抑制脑神经元谷氨酸的兴奋性突触传递 ,其作用与ATP敏感性钾通道相关     

Comparison of the actions of phencyclidine and sigma ligands on CA1 hippocampal pyramidal neurons in the rat

To compare the actions of prototypic drugs which are selective for phencyclidine and sigma receptors, the electrophysiological effects of phencyclidine (PCP),3-[3-hydroxyphenyl]-N-(1-propyl)piperidine [+)3-PPP), and 1,3-di(2-tolyl)guanidine (DTG) on CA1 hippocampal pyramidal neurons were examined. A wide range of concentrations of drug was tested to differentiate specific, receptor-mediated effects from nonselective, anesthetic-like actions. At relatively large concentrations (0.1-1 mM), each compound reversibly increased the threshold of action potentials driven by Schaffer collaterals, the duration of action potentials and membrane resistance. The low potencies and rank order of potency suggested that phencyclidine, (+)3-PPP, and DTG were not acting through either high affinity sigma or phencyclidine receptors. These compounds did have receptor-mediated effects at smaller concentrations. Since none of the compounds affected evoked excitatory or inhibitory postsynaptic potentials (EPSP or IPSP) or driven action potentials at subanesthetic concentrations (less than 100 microM), no evidence was found to support the hypothesis that the actions of phencyclidine result from enhanced release of transmitter, caused by the inhibition of a presynaptic potassium conductance. As observed in other neurons, phencyclidine blocked excitations in CA1 pyramidal cells mediated by N-methyl-D-aspartic acid (NMDA) at behaviorally relevant concentrations (1-10 microM). However, (+)3-PPP (1 microM-1 mM) enhanced the pyramidal cell response to NMDA. Alone, DTG did not effect the NMDA-induced response but did inhibit the enhancement induced by (+)3-PPP. The agonist and antagonist actions of the sigma-selective ligands, (+)3-PPP and DTG, suggests that they modify NMDA-induced responses by acting at the sigma receptor.     

司帕沙星、联苯乙酸对大鼠海马神经元GABA-A和离子型谷氨酸受体的作用(英文)

目的 :观察司帕沙星单用和与联苯乙酸合用时 ,对γ 氨基丁酸 (GABA) ,NMDA ,AMPA和海人藻酸诱导电流的作用。方法 :应用全细胞式膜片钳技术在急性打散的新生大鼠海马锥体神经元上记录GABA ,NMDA ,AMPA和海人藻酸的诱导电流。结果 :司帕沙星 1mmol·L- 1对GABA诱导的电流有迅速可逆的抑制作用 ,抑制率为 (4 7.6±s2 .9) % ,而联苯乙酸 10 μmol·L- 1无抑制作用。但联苯乙酸 (10 μmol·L- 1)与司帕沙星 1mmol·L- 1合用时 ,稍稍增强抑制作用 ,司帕沙星单用或合用联苯乙酸 (10 μmol·L- 1)对GABA诱导电流的IC50 分别为 (2 38± 2 1) μmol·L- 1和 (89± 5 ) μmol·L- 1。司帕沙星、联苯乙酸或司帕沙星合用联苯乙酸对NM DA ,AMPA和海人藻酸诱导的电流无明显作用。结论 :提示司帕沙星 ,或与联苯乙酸合用时所产生的中枢神经毒性可能与抑制GABA诱导的电流有关 ,而与NMDA ,AMPA和海人藻酸诱导的电流无关。     

Drosophila GABA(B) receptors are involved in behavioral effects of gamma-hydroxybutyric acid (GHB)

Gamma-hydroxybutyric acid (GHB) can be synthesized in the brain but is also a known drug of abuse. Although putative GHB receptors have been cloned, it has been proposed that, similar to the behavior-impairing effects of ethanol, the in vivo effects of pharmacological GHB may involve metabotropic gamma-aminobutyric acid (GABA) GABA(B) receptors. We developed a fruitfly (Drosophila melanogater) model to investigate the role of these receptors in the behavioral effects of exogenous GHB. Injecting GHB into male flies produced a dose-dependent motor impairment (measured with a computer-assisted automated system), which was greater in ethanol-sensitive cheapdate mutants than in wild-type flies. These effects of pharmacological concentrations of GHB require the presence and activation of GABA(B) receptors. The evidence for this was obtained by pharmacological antagonism of GABA(B) receptors with CGP54626 and by RNA interference (RNAi)-induced knockdown of the GABA(B(1)) receptor subtype. Both procedures inhibited the behavioral effects of GHB. GHB pretreatment diminished the behavioral response to subsequent GHB injections; i.e., it triggered GHB tolerance, but did not produce ethanol tolerance. On the other hand, ethanol pretreatment produced both ethanol and GHB tolerance. It appears that in spite of many similarities between ethanol and GHB, the primary sites of their action may differ and that recently cloned putative GHB receptors may participate in actions of GHB that are not mediated by GABA(B) receptors. These receptors do not have a Drosophila orthologue. Whether Drosophila express a different GHB receptor should be explored.     

NMDA and AMPA/kainate receptors are involved in the anticonvulsant activity of riluzole in DBA/2 mice

The anticonvulsant activity of riluzole against sound-induced seizures was studied in the DBA/2 mouse model. Riluzole (0.1-4 mg kg(-1), intraperitoneal (i.p.)) produced dose-dependent effects with ED(50) values for the suppression of tonic, clonic and wild running phases of 0.72, 1.38 and 2.71 mg kg(-1), respectively. Riluzole also protected DBA/2 mice from seizures induced by an intracerebroventricular (i.c.v.) injection of N-methyl-D-aspartate (NMDA) with ED(50) values of 3.03 and 5.0 mg kg(-1) for tonus and clonus, respectively. Pretreatment with glycine, an agonist to the glycine/NMDA receptors, shifted the dose-response effect of riluzole to the right (ED(50)=6.53 against tonus and 9.34 mg kg(-1) vs. clonus). Similarly, D-serine, an agonist at the glycine site, shifted the ED(50) of riluzole against the tonic component of audiogenic seizures from 0.72 to 1.97, and that against clonus from 1.38 to 2.77 mg kg(-1). Riluzole was also potent to prevent seizures induced by administration of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), an AMPA/kainate receptor agonist (ED(50)=1.80 and 3.35 mg kg(-1), against tonus and clonus, respectively). Pretreatment with aniracetam, a positive allosteric modulator of AMPA/kainate receptors, shifted the dose-response curve of riluzole to the right (ED(50)=1.78 against tonus and 2.58 mg kg(-1) vs. clonus). The data indicate that riluzole is an effective anticonvulsant drug in the genetic model of seizure-prone DBA/2 mice. Our findings suggest that the anticonvulsant properties of riluzole depend upon its interaction with neurotransmission mediated by both the glycine/NMDA and the AMPA/kainate receptor complex.     

Piperazine derivatives including the putative anxiolytic drugs,buspirone and ipsapirone,are agonists at 5-HT1A receptors negatively coupled with adenylate cyclase in hippocampal neurons

Summary Two putative anxiolytic drugs [ipsapirone (TVXQ 7821) and buspirone], structurally unrelated to benzodiazepines, have negligible ataxic and sedative side effects. These drugs are piperazine analogs which interact at 5-HT₁ binding sites. It is demonstrated here that these drugs and two other piperazine derivatives, trifluoromethylphenylpiperazine (TFMPP) and m-chlorophenylpiperazine (mCPP), are agonists at 5-HT_1A receptors, a subclass of the 5-HT₁ receptor, mediating inhibition of forskolin (100 M) stimulated adenylate cyclase in particulate fractions of guinea pig hippocampus as well as inhibition of the formation of cyclic AMP promoted by vasoactive intestinal polypeptide (0.1 M) plus forskolin (1 M) in mouse hippocampal neurons in primary culture. This study demonstrates that these piperazine based drugs act in both brain homogenate preparations and in intact neurons in a similar manner. The biochemical models described here may aid in the development of even more active drugs in this class.This work was supported by grants from the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Direction des Recherches Etudes et Techniques (DRET, N°85/188) of the French Defense Ministry and by the Direction and Ministère de la Recherche Scientifique Send offprint requests to J. Bockaert at the above address     

The actions of lovastatin on platelet function and platelet eicosanoid receptors in type II hypercholesterolaemia

Summary We have studied the effects of 12 weeks of lovastatin (20 mg per day) on platelet function and thromboxane formation in 18 patients with type II hypercholesterolaemia in a double-blind, placebo-controlled, prospective study.Lovastatin significantly reduced total serum and LDL-cholesterol by 20% and 25% respectively. Washed platelets of lovastatin-treated patients had significantly reduced collagen-induced aggregation and thromboxane formation ex vivo. There was no change in ADP-induced platelet aggregation, but a significant increase in prostacyclin (iloprost)-stimulated platelet cyclic AMP concentrations in lovastatin-treated patients. This was associated with a significant increase in the number of prostacyclin receptors in platelet membranes prepared from lovastatin-treated patients. There was also an increase in platelet thromboxane receptors. There were no such changes in the placebo group.These data confirm our original observation of normalization of platelet function in hypercholesterolaemia by HMGCoA reductase inhibitors and suggest changes in platelet membrane composition at the megakaryocyte level as a possible site of action.     

Blockade of nicotinic currents in hippocampal neurons defines methyllycaconitine as a potent and specific receptor antagonist.

Methyllycaconitine, a toxin isolated from the seeds of Delphinium brownii, inhibited acetylcholine- and anatoxin-induced whole-cell currents in cultured fetal rat hippocampal neurons, at picomolar concentrations. This antagonism was specific, concentration dependent, reversible, and voltage independent. Furthermore, methyllycaconitine inhibited 125I-alpha-bungarotoxin binding to adult rat hippocampal membranes, protected against the alpha-bungarotoxin-induced pseudoirreversible blockade of nicotinic currents, and shifted the concentration-response curve of acetylcholine to the right in fetal rat hippocampal neurons, suggesting a possible competitive mode of action for this toxin. Remarkably low concentrations of methyllycaconitine (1-1000 fM) decreased the frequency of anatoxin-induced single-channel openings, with no detectable decrease in the mean channel open time. These actions of methyllycaconitine commend this neurotoxin for the characterization of the alpha-bungarotoxin-sensitive subclass of neuronal nicotinic receptors, which has hitherto eluded functional demonstration.