Effects of ginsenosides,active components of ginseng,on nicotinic acetylcholine receptors expressed in Xenopus oocytes

We investigated the effects of ginsenosides, the active ingredient of ginseng, on neuronal or muscle-type nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal alpha3beta4, alpha7 or human muscle alphabetadeltavarepsilon subunits. Treatment with acetylcholine elicited an inward peak current (I(ACh)) in oocytes expressing nicotinic acetylcholine receptor subtypes. Cotreatment with ginsenoside Rg2 and acetylcholine inhibited I(ACh) in oocytes expressing with alpha3beta4 or alphabetadeltavarepsilon but not in oocytes expressing alpha7 nicotinic acetylcholine receptors. The inhibition of I(ACh) by ginsenoside Rg2 was reversible and dose-dependent. The half-inhibitory concentrations (IC50) of ginsenoside Rg2 were 60.2+/-14.1 and 15.7+/-3.5 microM in oocytes expressing alpha3beta4 and alphabetadeltavarepsilon nicotinic acetylcholine receptors, respectively. The inhibition of I(ACh) by ginsenoside Rg2 was voltage-independent and noncompetitive. Other ginsenosides besides ginsenoside Rg2 also inhibited I(ACh) in oocytes expressing alpha3beta4 or alphabetadeltavarepsilon nicotinic acetylcholine receptors. The order of potency for the inhibition of I(ACh) was ginsenoside Rg2>Rf>Re>Rg1>Rc>Rb2>Rb1 in oocytes expressing alpha3beta4 nicotinic acetylcholine receptors and was ginsenoside Rg2>Rf>Rg1>Re>Rb1>Rc>Rb2 in oocytes expressing alphabetadeltavarepsilon nicotinic acetylcholine receptors. These results indicate that ginsenosides might regulate nicotinic acetylcholine receptors in a differential manner and this regulation might be one of the pharmacological actions of Panax ginseng.     

Quercetin Inhibits ��3��4 Nicotinic Acetylcholine Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes

Quercetin mainly exists in the skin of colored fruits and vegetables as one of flavonoids. Recent studies show that quercetin, like other flavonoids, has diverse pharmacological actions. However, relatively little is known about quercetin effects in the regulations of ligand-gated ion channels. In the previous reports, we have shown that quercetin regulates subsets of homomeric ligand-gated ion channels such as glycine, 5-HT_3A and α7 nicotinic acetylcholine receptors. In the present study, we examined quercetin effects on heteromeric neuronal α3β4 nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal α3 and β4 subunits. Treatment with acetylcholine elicited an inward peak current (I_ACh) in oocytes expressing α3β4 nicotinic acetylcholine receptor. Co-treatment with quercetin and acetylcholine inhibited I_ACh in oocytes expressing α3β4 nicotinic acetylcholine receptors. The inhibition of I_ACh by quercetin was reversible and concentration-dependent. The half-inhibitory concentration (IC₅₀) of quercetin was 14.9±0.8 µM in oocytes expressing α3β4 nicotinic acetylcholine receptor. The inhibition of I_ACh by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate α3β4 nicotinic acetylcholine receptor and this regulation might be one of the pharmacological actions of quercetin in nervous systems.     

Differential effect of bovine serum albumin on ginsenoside metabolite-induced inhibition of α3β4 nicotinic acetylcholine receptor expressed in<Emphasis Type="Italic">Xenopus</Emphasis> oocytes

Ginsenosides, major active ingredients of Panax ginseng, that exhibit various pharmacological and physiological actions are transformed into compound K (CK) or M4 by intestinal microorganisms. CK is a metabolite derived from protopanaxadiol (PD) ginsenosides, whereas M4 is a metabolite derived from protopanaxatriol (PT) ginsenosides. Recent reports shows that ginsenosides might play a role as pro-drugs for these metabolites. In present study, we investigated the effect of bovine serum albumin (BSA), which is one of major binding proteins on various neurotransmitters, hormones, and other pharmacological agents, on ginsenoside Rg2-, CK-, or M4-induced regulation of alpha3beta4 nicotinic acetylcholine (ACh) receptor channel activity expressed in Xenopus oocytes. In the absence of BSA, treatment of ACh elicited inward peak current (I(ACh)) in oocytes expressing alpha3beta4 nicotinic ACh receptor. Co-treatment of ginsenoside Rg2, CK, or M4 with ACh inhibited I(ACh) in oocytes expressing (alpha3beta4 nicotinic ACh receptor with reversible and dose-dependent manner. In the presence of 1% BSA, treatment of ACh still elicited I(ACh) in oocytes expressing alpha3beta4 nicotinic ACh receptor and co-treatment of ginsenoside Rg2 or M4 but not CK with ACh inhibited I(ACh) in oocytes expressing alpha3beta4 nicotinic ACh receptor with reversible and dose-dependent manner. These results show that BSA interferes the action of CK rather than M4 on the inhibitory effect of I(ACh) in oocytes expressing alpha3beta4 nicotinic ACh receptor and further suggest that BSA exhibits a differential interaction on ginsenoside metabolites.     

Effects of quercetin on α9α10 nicotinic acetylcholine receptor-mediated ion currents

Quercetin, one of the flavonoids, is a low molecular weight substance found in fruits and vegetables. Quercetin, like other flavonoids, has a wide range of neuropharmacological actions and antioxidant effects. The α9α10 nicotinic acetylcholine receptor is one of the numerous nicotinic acetylcholine receptors that exist as a heteropentameric form between efferent olivocochlear fibers and hair cells of the cochlea. In this study, we report the effects of quercetin on rat α9α10 nicotinic acetylcholine receptor-mediated ion currents using the two-electrode voltage-clamp technique. Treatment with acetylcholine evoked inward currents (I_ACh) in oocytes heterologously expressing the α9α10 nicotinic acetylcholine receptor. Quercetin blocked I_ACh in concentration-dependent and reversible manners, and the blocking effect on I_ACh was stronger with pre-application than co-application of quercetin. The half maximal inhibitory concentration (IC₅₀) of quercetin was 45.4 ± 10.1 μM. Quercetin-mediated I_ACh inhibition was not affected by acetylcholine concentration and was independent of membrane-holding potential. Although the inhibitory effect of quercetin was significantly attenuated in the absence of extracellular Ca²⁺, the action of quercetin was independent of extracellular Ca²⁺ concentration, indicating that the presence of extracellular Ca²⁺ might be needed for quercetin-related effects and might play an important role in quercetin-mediated regulation of the α9α10 nicotinic acetylcholine receptor. These results indicate that quercetin-mediated regulation of the α9α10 nicotinic acetylcholine receptor could provide a molecular basis for quercetin actions at the cellular level.     

Selective effects of a 4-oxystilbene derivative on wild and mutant neuronal chick alpha7 nicotinic receptor

1. We assessed the pharmacological activity of triethyl-(beta-4-stilbenoxy-ethyl) ammonium (MG624), a drug that is active on neuronal nicotinic receptors (nicotinic AChR). Experiments on the major nicotinic AChR subtypes present in chick brain, showed that it inhibits the binding of [125I]-alphaBungarotoxin (alphaBgtx) to the alpha7 subtype, and that of [3H]-epibatidine (Epi) to the alpha4beta2 subtype, with Ki values of respectively 106 nM and 84 microM. 2. MG624 also inhibited ACh elicited currents (I(ACh)) in the oocyte-expressed alpha7 and alpha4beta2 chick subtypes with half-inhibitory concentrations (IC50) of respectively 109 nM and 3.2 microM. 3. When tested on muscle-type AChR, it inhibited [125I]-alphaBgtx binding with a Ki of 32 microM and ACh elicited currents (I(ACh)) in the oocyte-expressed alpha1beta1gammadelta chick subtype with an IC50 of 2.9 microM. 4. The interaction of MG624 with the alpha7 subtype was investigated using an alpha7 homomeric mutant receptor with a threonine-for-leucine 247 substitution (L247T alpha7). MG624 did not induce any current in oocytes expressing the wild type alpha7 receptor, but did induce large currents in the oocyte-expressed L247T alpha7 receptor. The MG624 elicited current (I(MG62)) has an EC50 of 0.2 nM and a Hill coefficient nH of 1.9, and is blocked by the nicotinic receptor antagonist methyllycaconitine (MLA). 5. These binding and electrophysiological studies show that MG624 is a potent antagonist of neuronal chick alpha7 nicotinic AChR, and becomes a competitive agonist following the mutation of the highly conserved leucine residue 247 located in the M2 channel domain.     

Magnesium sulfate enhances non-depolarizing muscle relaxant vecuronium action at adult muscle-type nicotinic acetylcholine receptor in vitro

Aim:

To investigate the effect of magnesium sulfate and its interaction with the non-depolarizing muscle relaxant vecuronium at adult muscle-type acetylcholine receptors in vitro.

Methods:

Adult muscle-type acetylcholine receptors were expressed in HEK293 cells. Drug-containing solution was applied via a gravity-driven perfusion system. The inward currents were activated by brief application of acetylcholine (ACh), and recorded using whole-cell voltage-clamp technique.

Results:

Magnesium sulfate (1–100 mmol/L) inhibited the inward currents induced ACh (10 μmol/L) in a concentration-dependent manner (IC₅₀=29.2 mmol/L). The inhibition of magnesium sulfate was non-competitive. In contrast, vecuronium produced a potent inhibition on the adult muscle-type acetylcholine receptor (IC₅₀=8.7 nmol/L) by competitive antagonism. Magnesium sulfate at the concentrations of 1, 3, and 6 mmol/L markedly enhanced the inhibition of vecuronium (10 nmol/L) on adult muscle-type acetylcholine receptors.

Conclusion:

Clinical enhancement of vecuronium-induced muscle relaxation by magnesium sulfate can be attributed partly to synergism between magnesium sulfate and non-depolarizing muscle relaxants at adult muscle-type acetylcholine receptors.     

Effects of dextrorotatory morphinans on alpha3beta4 nicotinic acetylcholine receptors expressed in Xenopus oocytes

We previously demonstrated that dextromethorphan (DM; 3-methoxy-17-methylmorphinan) analogs have neuroprotective effects, and a recent report showed that DM reduces the adverse effects of morphine and blocks alpha3beta4 nicotinic acetylcholine receptors, a major target of anti-addictive agents. Here, we investigated the effects of DM, three of its analogs (DF, 3-methyl-17-methylmorphinan; AM, 3-allyloxy-17-methoxymorphian; and CM, 3-cyclopropyl-17-methoxymorphinan) and one of its metabolites (HM; 3-methoxymorphinan), on neuronal alpha3beta4 nicotinic acetylcholine receptor channel activity expressed in Xenopus laevis oocytes, using the two-microelectrode voltage clamp technique. We found that intraoocyte injection of neuronal alpha3 and beta4 nicotinic acetylcholine receptor subunit cRNAs elicited an inward current (IACh) in the presence of acetylcholine. Co-treatment with DM, DF, AM, CM or HM inhibited IACh in a dose-dependent, voltage-independent and reversible manner. The IC50 values for DM, DF, AM, CM and HM were 19.5+/-5.2, 15.8+/-4.5, 16.3+/-1.7, 10.1+/-2.8, and 13.5+/-4.0 microM, respectively. The order of potency for the inhibition of IACh was CM>HM>DF=AM>DM in oocytes expressing alpha3beta4 nicotinic acetylcholine receptors. The inhibitions of (IACh) by DM, DF and HM, AM and CM were non-competitive. These results indicate that AM, CM and HM could be novel non-competitive agents regulating alpha3beta4 nicotinic acetylcholine receptor channel activity.     

Block by 5-hydroxytryptamine and apomorphine of recombinant human neuronal nicotinic receptors.

The effects of 5-hydroxytryptamine and apomorphine on human neuronal nicotinic acetylcholine receptor/channels were examined by expressing these channels in Xenopus oocytes. Functional channels were expressed by combining one type of alpha subunits (alpha3 or alpha4) and one type of beta subunits (beta2 or beta4). 5-Hydroxytryptamine (100 microM to 1 mM) and apomorphine (10 to 100 microM) inhibited an inward current activated by acetylcholine in the oocytes expressing the channels. The sensitivity to 5-hydroxytryptamine or apomorphine depended on subunit combinations. When concentration-response relationship was obtained for the acetylcholine-activated current, the maximal response was reduced by these compounds. The inhibition by these compounds exhibited voltage-dependence: the inhibition was augmented at negative potentials. The results suggest that 5-hydroxytryptamine and apomorphine noncompetitively inhibits human recombinant nicotinic acetylcholine receptor/channels, presumably by acting on channel pores.     

Differential regulations of quercetin and its glycosides on ligand-gated ion channels

Quercetin, one of the flavonoids, is a compound of low molecular weight found in various plants and shows a wide range of diverse neuropharmacological actions. In fruits and vegetables, quercetin exists as monomer- (quercetin-3-O-rhamnoside) (Rham1), dimer- (Rutin), or trimer-glycosides [quercetin-3-(2G-rhamnosylrutinoside)] (Rham2) at carbon-3. In the previous studies, we demonstrated that quercetin inhibits both glycine and 5-hydroxytryptamine type 3, (5-HT3A) receptor channel activities expressed in Xenopus oocytes. However, the effects of quercetin glycosides on glycine and 5-HT3A receptor channel activities are not well known. In the present study, we investigated the effects of quercetin glycosides on the human glycine alpha1 receptor and mouse 5-HT3A receptor channel activities expressed in Xenopus oocytes using a two-electrode voltage clamp technique. In oocytes expressing glycine or 5-HT3A receptors, quercetin- or its glycosides-induced inhibitions on glycine- (IGly) and 5-HT-induced current (I5-HT) were dose-dependent and reversible. Applications of quercetin and its glycosides inhibited IGly in order of quercetin>Rutin> or =Rham1>Rham2. Applications of quercetin and its glycosides inhibited I5-HT in order of Rham2> or =quercetin>Rutin=Rham1. The inhibitions of IGly by quercetin glycosides were non-competitive and voltage-sensitive, whereas the inhibitions of I5-HT by quercetin glycosides were competitive and voltage-insensitive manners. These results also indicate that quercetin glycosides might regulate the human glycine alpha1 and mouse 5-HT3A receptors with differential manners.     

Identification of ginsenoside interaction sites in 5-HT3A receptors

We previously demonstrated that 20(S)-ginsenoside Rg(3) (Rg(3)), one of the active components of Panax ginseng, non-competitively inhibits 5-HT(3A) receptor channel activity on extracellular side of the cell. Here, we sought to elucidate the molecular mechanisms underlying Rg(3)-induced 5-HT(3A) receptor regulation. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg(3) on 5-HT-mediated ion currents (I(5-HT)) in Xenopus oocytes expressing wild-type or 5-HT(3A) receptors harboring mutations in the gating pore region of transmembrane domain 2 (TM2). In oocytes expressing wild-type 5-HT(3A) receptors, Rg(3) dose-dependently inhibited peak I(5-HT) with an IC(50) of 27.6+/-4.3microM. Mutations V291A, F292A, and I295A in TM2 greatly attenuated or abolished the Rg(3)-induced inhibition of peak I(5-HT). Mutation V291A but not F292A and I295A induced constitutively active ion currents with decrease of current decay rate. Rg(3) accelerated the rate of current decay with dose-dependent manner in the presence of 5-HT. Rg(3) and TMB-8, an open channel blocker, dose-dependently inhibited constitutively active ion currents. The IC(50) values of constitutively active ion currents in V291A mutant receptor were 72.4+/-23.1 and 6.5+/-0.7microM for Rg(3) and TMB-8, respectively. Diltiazem did not prevent Rg(3)-induced inhibition of constitutively active ion currents in occlusion experiments. These results indicate that Rg(3) inhibits 5-HT(3A) receptor channel activity through interactions with residues V291, F292, and I295 in the channel gating region of TM2 and further demonstrate that Rg(3) regulates 5-HT(3A) receptor channel activity in the open state at different site(s) from those of TMB-8 and diltiazem.     

Comparison of the actions of carbamate anticholinesterases on the nicotinic acetylcholine receptor

Neostigmine (Neo), pyridostigmine (Pyr), and physostigmine (Phy) at low concentrations inhibited acetylcholine (ACh) esterase, thereby indirectly potentiating ACh enhancement of [3H]perhydrohistrionicotoxin (H12-HTX) binding to the channel sites of the nicotinic ACh receptor of Torpedo membranes. However, at higher concentrations, they inhibited ACh action due to their direct binding to the ACh receptor. They displaced binding of [3H]ACh and 125I-alpha-bungarotoxin (alpha-BGT) to the receptor sites with the following order of decreasing potency: Neo greater than Phy greater than Pyr. Furthermore, Neo and Pyr potentiated [3H] H12-HTX binding to the receptor's channel sites. Preincubation of ACh receptors with any of the three carbamates reduced the rate of binding of 125I-alpha-BGT and increased the potency of carbamylcholine in inhibiting 125I-alpha-BGT binding, suggesting that the three carbamates act as partial agonists and potentiate receptor desensitization. Although none of the three carbamates inhibited [3H]H12-HTX binding to the receptor's closed channel conformation, only Phy was a potent inhibitor of [3H]H12-HTX binding to the carbamylcholine-activated conformation. The potency of Phy was not due to the absence of positive charge since Phy methiodide acted similarly. The data suggest that the major action of the three carbamates at nicotinic cholinergic synapses is inhibition of ACh-esterase. Their interactions with the nicotinic ACh receptor are with its "receptor" as well as allosteric "channel" sites, but they differ in their effects. Neo and Pyr act mainly as partial agonists, while Phy is mostly an inhibitor of the channel in the activated receptor conformation.     

Effects of ginsenosides on GABAA receptor channels expressed in xenopus oocytes

Ginsenosides, major active ingredients of Panax ginseng, are known to regulate excitatory ligand-gated ion channel activity such as nicotinic acetylcholine and NMDA receptor channel activity. However, it is not known whether ginsenosides affect inhibitory ligand-gated ion channel activity. We investigated the effect of ginsenosides on human recombinant GABA(A) receptor (alpha1beta1gamma2S) channel activity expressed in Xenopus oocytes using a two-electrode voltage-clamp technique. Among the eight individual ginsenosides examined, namely, Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg2, we found that Rc most potently enhanced the GABA-induced inward peak current (I(GABA)). Ginsenoside Rc alone induced an inward membrane current in certain batches of oocytes expressing the GABA(A) receptor. The effect of ginsenoside Rc on I(GABA) was both dose-dependent and reversible. The half-stimulatory concentration (EC50) of ginsenoside Rc was 53.2 +/- 12.3 microM. Both bicuculline, a GABA(A) receptor antagonist, and picrotoxin, a GABA(A) channel blocker, blocked the stimulatory effect of ginsenoside Rc on I(GABA). Niflumic acid (NFA) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), both Cl- channel blockers, attenuated the effect of ginsenoside Rc on I(GABA). This study suggests that ginsenosides regulated GABA(A) receptor expressed in Xenopus oocytes and implies that this regulation might be one of the pharmacological actions of Panax ginseng.     

Interactions of chlordecone (kepone) and mirex with the nicotinic acetylcholine receptor—ion channel complex

Interactions of chlordecone (kepone) and mirex with nicotinic acetylcholine (ACh) receptor complexes from Torpedo californica electric organs were studied using biochemical probes for ACh and ion-channel binding sites. Neither compound inhibited the binding of [¹²⁵I]-bungarotoxin (BGT) to the receptor; chlordecone, however, enhanced carbamylcholine affinity for the receptor 5-fold. Chlordecone, but not mirex, also inhibited the binding of [³H]perhydrohistrionicotoxin and [³H]phencyclidine to sites associated with the receptor-gated ion channel. Ion-channel inhibition by chlordecone was enhanced in the presence of carbamylcholine. These results indicate that chlordecone, but not mirex, interacts with the ion-translocation mechanism associated with nicotinic ACh receptors, where it may sterically block ion flux as well as stabilize a desensitized conformation of the receptor complex.     

Effect of galantamine on the human alpha7 neuronal nicotinic acetylcholine receptor, the Torpedo nicotinic acetylcholine receptor and spontaneous cholinergic synaptic activity

1. Various types of anticholinesterasic agents have been used to improve the daily activities of Alzheimer's disease patients. It was recently demonstrated that Galantamine, described as a molecule with anticholinesterasic properties, is also an allosteric enhancer of human alpha4beta2 neuronal nicotinic receptor activity. We explored its effect on the human alpha7 neuronal nicotinic acetylcholine receptor (nAChR) expressed in Xenopus oocytes. 2. Galantamine, at a concentration of 0.1 microM, increased the amplitude of acetylcholine (ACh)-induced ion currents in the human alpha7 nAChR expressed in Xenopus oocytes, but caused inhibition at higher concentrations. The maximum effect of galantamine, an increase of 22% in the amplitude of ACh-induced currents, was observed at a concentration of 250 microM Ach. 3. The same enhancing effect was obtained in oocytes transplanted with Torpedo nicotinic acetylcholine receptor (AChR) isolated from the electric organ, but in this case the optimal concentration of galantamine was 1 microM. In this case, the maximum effect of galantamine, an increase of 35% in the amplitude of ACh-induced currents, occurred at a concentration of 50 microM ACh. 4. Galantamine affects not only the activity of post-synaptic receptors but also the activity of nerve terminals. At a concentration of 1 microM, quantal spontaneous events, recorded in a cholinergic synapse, increased their amplitude, an effect which was independent of the anticholinesterasic activity associated with this compound. The anticholinesterasic effect was recorded in preparations treated with a galantamine concentration of 10 microM. 5. In conclusion, our results show that galantamine enhances human alpha7 neuronal nicotinic ACh receptor activity. It also enhances muscular AChRs and the size of spontaneous cholinergic synaptic events. However, only a very narrow range of galantamine concentrations can be used for enhancing effects.     

5-hydroxytryptamine interaction with the nicotinic acetylcholine receptor

The present study examines the interaction of the neurotransmitter 5-hydroxytryptamine (5-HT) with muscle-type nicotinic acetylcholine receptors. 5-HT inhibits the initial rate of [125I]alpha-bungarotoxin binding to Torpedo acetylcholine receptor membranes (IC(50)=8.5+/-0.32 mM) and [3H]5-HT can be photoincorporated into acetylcholine receptor subunits, with labeling of the alpha-subunit inhibitable by both agonists and competitive antagonists. Within the agonist-binding domain, [3H]5-HT photoincorporates into alphaTyr(190), alphaCys(192) and alphaCys(193). Functional studies using the human clonal cell line TE671/RD, show that 5-HT is a weak inhibitor (IC(50)=1.55+/-0.25 mM) of acetylcholine receptor activity. In this regard, agonist-response profiles in the absence and presence of 5-HT indicate a noncompetitive mode of inhibition. In addition, 5-HT displaces high affinity [3H]thienylcyclohexylpiperidine binding to the desensitized Torpedo acetylcholine receptor channel (IC(50)=1.61+/-0.07 mM). Collectively, these results indicate that 5-HT interacts weakly with the agonist recognition site and inhibits receptor function noncompetitively by binding to the acetylcholine receptor channel.     

The effects of subunit composition on the inhibition of nicotinic receptors by the amphipathic blocker 2,2,6,6-tetramethylpiperidin-4-yl heptanoate

The therapeutic targeting of nicotinic receptors in the brain will benefit from the identification of drugs that may be selective for their ability to activate or inhibit a limited range of nicotine acetylcholine receptor subtypes. In the present study, we describe the effects of 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), a novel compound that is a potent inhibitor of neuronal nicotinic receptors. Evaluation of nicotinic acetylcholine receptor (nAChR) subunits expressed in Xenopus laevis oocytes indicated that TMPH can produce a potent and long-lasting inhibition of neuronal nAChR formed by the pairwise combination of the most abundant neuronal alpha (i.e., alpha3 and alpha4) and beta subunits (beta2 and beta4), with relatively little effect, because of rapid reversibility of inhibition, on muscle-type (alpha1beta1gammadelta) or alpha7 receptors. However, the inhibition of neuronal beta subunit-containing receptors was also decreased if any of the nonessential subunits alpha5, alpha6, or beta3 were coexpressed. This decrease in inhibition is shown to be associated with a single amino acid present in the second transmembrane domain of these subunits. Our data indicate great potential utility for TMPH to help relate the diverse central nervous system effects to specific nAChR subtypes.     

Block by phytoestrogens of recombinant human neuronal nicotinic receptors

The effects of phytoestrogens on neuronal nicotinic acetylcholine receptor/channels were examined by expressing recombinant channels in Xenopus oocytes. When functional channels were expressed with human alpha4 and beta2 subunits, daidzein (10 and 100 microM) partially inhibited the ionic current activated by acetylcholine. The current inhibition was also observed when functional channels were expressed with human alpha3 and beta4 subunits or rat homologues. Genistin (100 microM) also inhibited the acetylcholine-activated current. Tamoxifen (100 microM), an antiestrogen did not antagonize the inhibition by daidzein. The results suggest that phytoestrogens, like estrogens and xenoestrogens, block human neuronal acetylcholine receptors through non-genomic mechanisms.     

The effects of 3,4-methylenedioxymethamphetamine (MDMA) on nicotinic receptors: Intracellular calcium increase, calpain/caspase 3 activation, and functional upregulation

Previous work by our group demonstrated that homomeric α7 nicotinic acetylcholine receptors (nAChR) play a role in the neurotoxicity induced by 3,4-methylenedioxymethamphetamine (MDMA), as well as the binding affinity of this drug to these receptors. Here we studied the effect of MDMA on the activation of nAChR subtypes, the consequent calcium mobilization, and calpain/caspase 3 activation because prolonged Ca²⁺ increase could contribute to cytotoxicity. As techniques, we used fluorimetry in Fluo-4-loaded PC12 cells and electrophysiology in Xenopus oocytes. MDMA produced a rapid and sustained increase in calcium without reaching the maximum effect induced by ACh. It also concentration-dependently inhibited the response induced by ACh, nicotine, and the specific α7 agonist PNU 282987 with IC₅₀ values in the low micromolar range. Similarly, MDMA induced inward currents in Xenopus oocytes transfected with human α7 but not with α4β2 nAChR and inhibited ACh-induced currents in both receptors in a concentration-dependent manner. The calcium response was inhibited by methyllycaconitine (MLA) and α-bungarotoxin but not by dihydro-β-erythroidine. These results therefore indicate that MDMA acts as a partial agonist on α7 nAChRs and as an antagonist on the heteromeric subtypes. Subsequently, calcium-induced Ca²⁺ release from the endoplasmic reticulum and entry through voltage-operated calcium channels are also implicated as proved using specific antagonists. In addition, treatment with MDMA for 24 h significantly increased basal Ca²⁺ levels and induced an increase in α-spectrin breakdown products, which indicates that calpain and caspase 3 were activated. These effects were inhibited by pretreatment with MLA. Moreover, pretreatment with MDMA induced functional upregulation of calcium responses to specific agonists of both heteromeric and α7 nAChR. Sustained calcium entry and calpain activation could favor the activation of Ca²⁺-dependent enzymes such as protein kinase C and nitric oxide synthase, which are involved in the generation of ROS and the blockade of the dopamine transporter. This, together with caspase 3 activation, must play a role in MDMA-induced cytotoxicity.     

Modulation by estrogens and xenoestrogens of recombinant human neuronal nicotinic receptors

The effects of estrogens and xenoestrogens on human neuronal nicotinic acetylcholine receptor/channels were examined by expressing recombinant channels in Xenopus oocytes. When functional channels were expressed with 3 and β4 subunits, estrogens (17β-estradiol, 17-estradiol, 17-ethynylestradiol and diethylstilbestrol) and xenoestrogens (bisphenol A, p-nonylphenol and p-octylphenol) inhibited an ionic current activated by acetylcholine at concentrations up to 100 μM. When the subunit combination was changed to 4β2, diethystilbestrol and the xenoestrogens inhibited the acetylcholine-activated current, but 17β-estradiol or 17-estradiol did not. For 17-ethynylestradiol, the current through the 4β2 receptor/channel was inhibited at 1 μM, but it was markedly enhanced at 10 and 100 μM. Tamoxifen (10 μM), an antiestrogen, itself inhibited the acetylcholine-activated current but did not antagonize the current modulations induced by the estrogens and the xenoestrogens. These and additional results suggest that human neuronal nicotinic acetylcholine receptors are the targets of non-genomic actions of estrogens and xenoestrogens.     

The novel Na(+)/Ca(2+) exchange inhibitor KB-R7943 also blocks native and expressed neuronal nicotinic receptors

We studied the effects of the novel Na(+)/Ca(2+) exchange inhibitor KB-R7943, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate, on the native nicotinic receptors present at the bovine adrenal chromaffin cells, as well as on rat brain alpha(3)beta(4) and alpha(7) nicotinic acetylcholine receptors (AChRs) expressed in XENOPUS: oocytes. As expected, KB-R7943 blocked the Na(+)-gradient dependent (45)Ca(2+) uptake into chromaffin cells (IC(50) of 5.5 microM); but in addition, the compound also inhibited the (45)Ca(2+) entry and the increase of cytosolic Ca(2+) concentration, [Ca(2+)](c), stimulated by 5 s pulses of ACh (IC(50) of 6.5 and 1.7 microM, respectively). In oocytes expressing alpha(3)beta(4) and alpha(7) nicotinic AChRs, voltage-clamped at -60 mV, inward currents elicited by 1 s pulses of 100 microM ACh (I(ACh)) were blocked by KB-R7943 with an IC(50) of 0.4 microM and a Hill coefficient of 0.9. Blockade of alpha(3)beta(4) currents by KB-R7943 was noncompetitive; moreover, the blocker (0.3 microM) became more active as the ACh concentration increased (34 versus 66% blockade at 30 microM and 1 mM ACh, respectively). Inhibition of alpha(3)beta(4) currents by 0.3 microM KB-R7943 was more pronounced at hyperpolarized potentials. If given within the ACh pulse (10 microM), the inhibition amounted to 33, 64 and 80% in oocytes voltage-clamped at -40, -60 and -100 mV, respectively. The onset of blockade was faster and the recovery slower at -100 mV; the reverse was true at -40 mV. In conclusion, KB-R7943 is a potent blocker of nicotinic AChRs; moreover, it displays many features of an open-channel blocker at the rat brain alpha(3)beta(4) AChR. These results should be considered when KB-R7943 is to be used to study Ca(2+) homeostasis in cells expressing nicotinic AChRs and the Na(+)/Ca(2+) exchanger.