Agonist-induced up-regulation of alpha4beta2 nicotinic acetylcholine receptors in M10 cells: pharmacological and spatial definition

Chronic nicotine up-regulates the number of high affinity nicotinic acetylcholine receptors (nAChRs) in mammalian brain. Here, we studied up-regulation of the nAChR composed of alpha4 and beta2 subunits in the M10 cell line by using [3H]epibatidine to measure nAChR in cells in situ and in membrane preparations. Cultures were exposed to drugs for 2 days before assay. All agonists up-regulated [3H]epibatidine binding sites with EC50 values typically 10-100-fold higher than their respective Ki values from competition binding assays. Maximum up-regulation ranged from 40% to 250% above control values. Maximally effective concentrations of the less efficacious agonists methylcarbamylcholine or (+/-)-epibatidine together with nicotine resulted in less up-regulation than that produced by nicotine alone, showing that they are partial up-regulatory agonists. The antagonists dihydro-beta-erythroidine, methyllycaconitine, d-tubocurarine, hexamethonium, decamethonium, and mecamylamine either failed to up-regulate [3H]epibatidine binding sites or up-regulated mildly at high concentrations. When tested at non-up-regulating concentrations, only d-tubocurarine significantly inhibited agonist-induced up-regulation; this inhibition seemed to be noncompetitive. Comparison of [3H]epibatidine displacement in intact M10 cells and membrane preparations by membrane-impermeant ligands indicated that 85% of [3H]epibatidine binding sites are intracellular. On chronic treatment with agonist, the proportion of surface receptors did not change significantly, indicating that most up-regulated [3H]epibatidine binding sites are internal. However, up-regulation is mediated at the cell surface because the impermeant ligand tetramethylammonium was as efficacious as nicotine in eliciting up-regulation, and methylcarbamylcholine (i.e., impermeant but with low efficacy) blocked nicotine induced up-regulation. Thus, agonists elicit up-regulation (mainly of intracellular receptors) by interacting with cell surface nAChRs that are not compatible with either an active or high affinity desensitized conformation.     

Chronic ethanol treatment decreases [3H]epibatidine and [3H]nicotine binding and differentially regulates mRNA levels of nicotinic acetylcholine receptor subunits expressed in M10 and SH-SY5Y neuroblastoma cells

For a study of the underlying mechanisms of a possible interaction between ethanol and nicotinic receptors during ethanol dependence, the aim of this work was to investigate the effect of chronic ethanol exposure on nicotinic receptor subtypes in a transfected fibroblast cell line (M10 cells) stably expressing alpha4beta2 nicotinic receptor subtype and an SH-SY5Y neuroblastoma cell line expressing alpha3, alpha5, alpha7, beta2, and beta4 nicotinic acetylcholine receptor (nAChR) subunits. A significant dose-related decrease (-30-80%) in number of [3H]nicotine binding sites was observed in ethanol-treated (25-240 mM) compared with untreated M10 cells. Similarly, 4-day treatment with ethanol in concentrations relevant to chronic alcoholism (100 mM) decreased the number of nicotinic receptor binding sites in the SH-SY5Y cells when measured using [3H]epibatidine. When M10 cells were chronically treated with nicotine, ethanol partly inhibited the up-regulation of nicotinic receptors when present in the cells together with nicotine. Chronic treatment for 4 days with 100 mM ethanol significantly decreased the mRNA level for the alpha3 nAChR subunit (-39%), while the mRNA levels for the alpha7 (+30%) and alpha4 (+22%) subunits were significantly increased. Chronic ethanol treatment did not affect the mRNA levels for the beta2 nAChR subunit. Changes in the levels of nAChR protein and mRNA may have adaptive significance and be involved in the development of dependence, tolerance, and addiction to chronic ethanol and nicotine exposure. They also may be targets for therapeutic strategies in the treatment of ethanol and nicotine dependence.     

Rat alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor stably expressed in a transfected cell line: pharmacology of ligand binding and function

We stably transfected human kidney embryonic 293 cells with the rat neuronal nicotinic acetylcholine receptor (nAChR) alpha3 and beta4 subunit genes. This new cell line, KXalpha3 beta4R2, expresses a high level of the alpha3/beta4 receptor subtype, which binds (+/-)- [3H]epibatidine with a Kd value of 304+/-16 pM and a Bmax value of 8942 +/- 115 fmol/mg protein. Comparison of nicotinic drugs in competing for alpha3/beta4 receptor binding sites in this cell line and the binding sites in rat forebrain (predominantly alpha4/beta2 receptors) revealed marked differences in their Ki values, but similar rank orders of potency for agonists were observed, with the exception of anatoxin-A. The affinity of the competitive antagonist dihydro-beta-erythroidine is >7000 times higher at alpha4/beta2 receptors in rat forebrain than at the alpha3/beta4 receptors in these cells. The alpha3/beta4 nAChRs expressed in this cell line are functional, and in response to nicotinic agonists, 86Rb+ efflux was increased to levels 8-10 times the basal levels. Acetylcholine, (-)-nicotine, cytisine, carbachol, and (+/-)-epibatidine all stimulated 86Rb+ efflux, which was blocked by mecamylamine. The EC50 values for acetylcholine and (-)-nicotine to stimulate 86Rb+ effluxes were 114 +/- 24 and 28 +/- 4 microM, respectively. The rank order of potency of nicotinic antagonists in blocking the function of this alpha3/beta4 receptor was mecamylamine > d-tubocurarine > dihydro-beta-erythroidine > hexamethonium. Mecamylamine, d-tubocurarine, and hexamethonium blocked the function by a noncompetitive mechanism, whereas dihydro-beta-erythroidine blocked the function competitively. The KXalpha3 beta4R2 cell line should prove to be a very useful model for studying this subtype of nAChRs.     

The heterocyclic substituted pyridine derivative (+/-)-2-(-3-pyridinyl)-1-azabicyclo[2.2.2]octane (RJR-2429): a selective ligand at nicotinic acetylcholine receptors

The present report describes in vitro studies demonstrating that the heterocyclic substituted pyridine compound (+/-)-2-(3-pyridinyl)-1-azabicyclo[2.2.2]octane (RJR-2429) is extremely potent in activating human muscle nicotine ACh receptor (nAChR) (EC50 = 59 +/- 17 nM; Emax = 110 +/- 09% vs. nicotine). RJR-2429 is markedly less potent in activating nAChRs in the clonal cell line PC12, with EC50 = 1100 +/- 230 nM and Emax = 85 +/- 20% when compared with nicotine. The activation of a putative alpha 3 beta 4-containing nAChR in PC12 by RJR-2429 reveals a potency intermediate between nicotine and epibatidine (EC50 of 20,000 nM for nicotine and 30 nM for epibatidine). Dose-response curves for agonist-induced ileum contraction indicate that RJR-2429 is equipotent with nicotine, having an EC30 of approximately 2 microM. RJR-2429 binds with high affinity to alpha 4 beta 2 receptor subtype (Ki = 1.0 +/- 0.3 nM), and chronic exposure results in significant up-regulation of the high-affinity [3H]nicotine binding sites. In addition, RJR-2429 does not activate nAChRs present in rat thalamic preparations but is a potent inhibitor of this receptor subtype. It antagonizes nicotine-stimulated ion flux in thalamic synaptosomes with an IC50 of 154 +/- 37 nM. It also is a potent partial agonist at nAChRs mediating dopamine release from rat synaptosomal preparations (EC50 = 2 +/- 1 nM; Emax = 40%; epibatidine and nicotine have EC50 values of 0.4 and 100 nM, respectively). A model for the structure-activity profile of RJR-2429, nicotine and epibatidine was derived by molecular forcefield and quantum mechanics calculations and may provide important clues for the development of ligands selective for nAChR subtypes as probes in the life sciences or as potential therapeutic tools.     

Pharmacological evaluation of [11C]A-84543: an enantioselective ligand for in vivo studies of neuronal nicotinic acetylcholine receptors

[11C]A-84543, 3-[(1-[11C]methyl-2(S)-pyrrolidinyl)methoxy]pyridine, is a specific and enantioselective neuronal nicotinic acetylcholine receptor (nAChR) radiotracer. The in vivo biodistribution of this radiotracer in mice showed high brain uptake and a distribution consistent with the density of nAChRs. Highest uptake was observed in the thalamus (9.6 %ID/g), cortex (9.9 %ID/g), superior colliculus (7.6 %ID/g) and hippocampus (7.6 %ID/g) at 5 min followed by clearance. As a measure of specificity, the thalamus/cerebellar ratio reached a maximum of 2.3 at 30 min post-injection. Radioactivity in the thalamus and superior colliculus was reduced by 33% by pre-administration of unlabeled A-84543. The nAChR agonists (-)nicotine, cytisine, and (+) epibatidine reduced the radioactivity due to [11C]A-84543 in the superior colliculus by 41%, 38%, and 27%, respectively, while lobeline, which also interacts with central nAChRs, produced a 24% inhibition. The noncompetitive nAChR ligand, mecamylamine displayed no inhibitory effect on [11C]A-84543 accumulation in any brain region. Ketanserin (5-HT2/5-HT2C), scopolamine (mAChR antagonist), (+)butaclamol (DA receptor antagonist), and haloperidol (D2/sigma) also displayed no inhibitory effect in any brain region studied. With the pharmacologically less active enantiomer, 3-[(1-[11C]methyl-2(R)-pyrrolidinyl)methoxy] pyridine, high brain uptake was also observed, but with a low thalamus/cerebellar ratio of 1.4 at 30 min post-injection. [11C]A-84543 displays enantioselectivity for nAChRs and may deserve further investigation as a possible PET radiotracer.     

A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors

We have isolated a 16-amino acid peptide from the venom of the marine snail Conus magus which potently blocks nicotinic acetylcholine receptors (nAChRs) composed of alpha3beta2 subunits. This peptide, named alpha-conotoxin MII, was identified by electrophysiologically screening venom fractions against cloned nicotinic receptors expressed in Xenopus oocytes. The peptide's structure, which has been confirmed by mass spectrometry and total chemical synthesis, differs significantly from those of all previously isolated alpha-conotoxins. Disulfide bridging, however, is conserved. The toxin blocks the response to acetylcholine in oocytes expressing alpha3beta2 nAChRs with an IC50 of 0.5 nM and is 2-4 orders of magnitude less potent on other nAChR subunit combinations. We have recently reported the isolation and characterization of alpha-conotoxin ImI, which selectively targets homomeric alpha7 neuronal nAChRs. Yet other alpha-conotoxins selectively block the muscle subtype of nAChR. Thus, it is increasingly apparent that alpha-conotoxins represent a significant resource for ligands with which to probe structure-function relationships of various nAChR subtypes.     

Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1, 1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++ concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.     

Alpha 5 subunit forms functional alpha 3 beta 4 alpha 5 nAChRs in transfected human cells

nAChRs heterologously expressed in human cells after transient transfection with alpha 3 beta 4 alpha 5 or alpha 3 beta 4 subunit cDNAs exhibited similar sensitivities to antagonists and comparable functional channel profiles. However, the sum of two Hill equations was required for best fitting the ACh dose-current response curves after co-expression of alpha 5, alpha 3 and beta 4 subunits. One component was comparable to that obtained in alpha 3 beta 4-transfected cells, while the additional component, putatively attributed to an alpha 3 beta 4 alpha 5 nAChR population, showed a Hill coefficient > 2 and a nine-fold greater half-maximal ACh concentration (EC50). These results suggest that the alpha 5 subunit participates in the assembly of alpha 3 beta 4 alpha 5 nAChRs complexes in human cells, adding a new member to the family of neuronal nicotinic receptors.     

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HEK293 cells were stably transfected with rat neuronal nicotinic alpha4 and beta2 subunits. Binding of tritiated cytisine and nicotine to cell homogenates revealed the presence of a single class of high-affinity sites (dissociation constants 0.1 nM and 0.4 nM, respectively). Activation of nicotinic receptors was studied using whole-cell patch clamp methods, and acetylcholine, nicotine, dimethylphenylpiperazinium, and cytisine all produced a conductance increase. Responses desensitized to prolonged applications, at both positive and negative membrane potentials. The conductance was strongly rectifying, and outward currents were essentially absent. Responses were maximal at about 2 mM external calcium ion concentration and were reduced by about one-half at either nominally 0 or 10 mM external calcium. Di-hydro-beta-erythroidine blocked physiological responses to acetylcholine and nicotine (IC50, 2.5 nM), and reduced cytisine binding in a competitive manner (Ki 20 nM). Physostigmine enhanced the response to low concentrations of acetylcholine or nicotine. The anesthetic steroid (+)-3alpha-hydroxy-5alpha-androstane-17beta-carbonitrile blocked responses to acetylcholine (IC50, 1.3 microM), but had no effect on cytisine binding at a concentration of 30 microM. The binding properties of the receptors are those expected for rat neuronal nicotinic receptors composed of alpha4 and beta2 subunits. The pharmacological properties indicate that the responsiveness of the receptors may be allosterically enhanced or inhibited.     

Epibatidine activates muscle acetylcholine receptors with unique site selectivity

We recently showed that at desensitized muscle nicotinic receptors, epibatidine selects by 300-fold between the two agonist binding sites. To determine whether receptors in the resting, activatible state show similar site selectivity, we studied epibatidine-induced activation of mouse fetal and adult receptors expressed in 293 HEK cells. Kinetic analysis of single-channel currents reveals that (-)-epibatidine binds with 15-fold selectivity to sites of adult receptors and 75-fold selectivity to sites of fetal receptors. For each receptor subtype, site selectivity arises solely from different rates of epibatidine dissociation from the two sites. To determine the structural basis for epibatidine selectivity, we introduced mutations into either the gamma or the delta subunit and measured epibatidine binding and epibatidine-induced single-channel currents. Complexes formed by alpha and mutant gamma(K34S+F172I) subunits bind epibatidine with increased affinity compared to alphagamma complexes, whereas the kinetics of alpha2betadeltagamma(K34S+F172I) receptors reveal no change in affinity of the low-affinity site, but increased affinity of the high-affinity site. Conversely, complexes formed by alpha and mutant delta(S36K+I178F) subunits bind epibatidine with decreased affinity compared to alphadelta complexes, whereas the kinetics of alpha2betagammadelta(S36K+I178F) and alpha2betaepsilondelta(S36K+I178F) receptors show markedly reduced sensitivity to epibatidine. The overall data show that epibatidine activates muscle receptors by binding with high affinity to alphagamma and alphaepsilon sites, but with low affinity to the alphadelta site.     

Differential inhibition by alpha-conotoxin-MII of the nicotinic stimulation of [3H]dopamine release from rat striatal synaptosomes and slices

The presynaptic nicotinic modulation of dopamine release from striatal nerve terminals is well established, but the subtype(s) of neuronal nicotinic acetylcholine receptor (nAChR) underlying this response has not been identified. Recently, alpha-conotoxin-MII has been reported to inhibit potently and selectively the rat alpha3beta2 combination of nAChR subunits. Here we have synthesised the peptide, confirmed its specificity, and examined its effect on the (+/-)-anatoxin-a-evoked release of [3H]dopamine from rat striatal synaptosomes and slices. Alpha-conotoxin-MII (112 nM) completely blocked acetylcholine-evoked currents of alpha3beta2 nAChRs expressed in Xenopus oocytes (IC50 = 8.0 +/- 1.1 nM). Pairwise combinations of other nicotinic subunits were not blocked by 112 nM alpha-conotoxin-MII. On perfused striatal synaptosomes and slices, alpha-conotoxin-MII dose-dependently inhibited [3H]dopamine release evoked by 1 microM (+/-)-anatoxin-a with IC50 values of 24.3 +/- 2.9 and 17.3 +/- 0.1 nM, respectively. The dose-response curve was shifted to the right with increasing agonist concentrations. However, the maximal inhibition of responses achieved by alpha-conotoxin-MII (112 nM) was 44.9 +/- 5.4% for synaptosomes and 25.0 +/- 4.1% for slices, compared with an inhibition by 10 microM mecamylamine of 77.9 +/- 3.7 and 88.0 +/- 2.1%, respectively. These results suggest the presence of presynaptic alpha3beta2-like nAChRs on striatal dopaminergic terminals, but the incomplete block of (+/-)-anatoxin-a-evoked [3H]dopamine release by alpha-conotoxin-MII also supports the participation of nAChRs composed of other subunits. The lower inhibition found in slices is consistent with an additional indirect nicotinic stimulation of dopamine release via an alpha-conotoxin-MII-insensitive nAChR.     

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Functional effects of human alpha 5 nicotinic ACh receptor (AChR) subunits coassembled with alpha 3 and beta 2 or with alpha 3 and beta 4 subunits, were investigated in Xenopus oocytes. The presence of alpha 5 subunits altered some properties of both alpha 3 AChRs and differentially altered other properties of alpha 3 beta 2 AChRs vs. alpha 3 beta 4 AChRs. alpha 5 subunits increased desensitization and Ca++ permeability of all alpha 3 AChRs. The Ca++ permeabilities of both alpha 3 beta 2 alpha 5 and alpha 3 beta 4 alpha 5 AChRs were comparable to that of alpha 7 AChRs. As we have shown previously, alpha 5 subunits increased the ACh sensitivity of alpha 3 beta 2 AChRs 50-fold but had little effect on alpha 3 beta 4 AChRs. alpha 5 caused only subtle changes in the activation potencies of alpha 3 AChRs for nicotine, cytisine and 1,1-dimethyl-4-plenylpiperazinium (DMPP). However, alpha 5 increased the efficacies of nicotine and DMPP on alpha 3 beta 2 AChRs but decreased them on alpha 3 beta 4 AChRs. Immunoisolation of cloned human AChRs expressed in oocytes showed that alpha 5 efficiently coassembled with alpha 3 plus beta 2 and/or beta 4 subunits. As expected, human AChRs immunoisolated from SH-SY5Y neuroblastoma cells showed that AChRs containing alpha 3 and probably alpha 5 subunits were present, but alpha 4 AChRs were not. In brain, by contrast, alpha 4 beta 2 AChRs were shown to predominate over alpha 3 AChRs. Some of the brain alpha 4 beta 2 AChRs were found to contain alpha 5 subunits.     

5-[I-125/123]lodo-3(2(S)-azetidinylmethoxy)pyridine, a radioiodinated analog of A-85380 for in vivo studies of central nicotinic acetylcholine receptors

The in vivo biodistribution profile of the novel nicotinic acetylcholine receptor (nAChR) radioligand 5-[I-125/123]Iodo-3(2(S)-azetidinylmethoxy)pyridine, [I-125/123]-5-IA, in mouse brain was examined. This radiotracer displayed good brain penetration (3.1% of the injected dose (ID) in whole brain at 15 min post-radioligand injection). Radioligand distribution was consistent with the density of high affinity nAChRs with highest uptake observed in the nAChR-rich thalamus (14.9 %ID/g at 60 min), moderate uptake in cortex (8.5 %ID/g at 60 min), and lowest uptake in the cerebellum (2.4 %ID/g at 60 min). Pretreatment with several different nAChR agonists (A-85380, (-)-nicotine, cytisine) significantly inhibited [I-125]-5-IA binding in all brain regions studied (P < 0.01) demonstrating the high specificity of the radioligand for nAChRs. Blocking doses of the muscarinic antagonist scopolamine and the non-competitive nAChR channel blocker mecamylamine had no significant effect on radioactive uptake supporting the in vitro selectivity of [I-125]-5-IA for the nAChR component of the cholinergic system. [I-125]-5-IA binding sites were shown to be saturable with unlabeled 5-IA. With a relatively low acute toxicity (LD50 > 3 mg/kg via intravenous injection in mice) and high in vivo specificity and selectivity, 5-IA labeled with the imaging radionuclide I-123 may prove useful for single photon emission computed tomography (SPECT) studies of nAChRs in human subjects.     

Functional characterization of the novel neuronal nicotinic acetylcholine receptor ligand GTS-21 in vitro and in vivo

(2.4)-Dimethoxybenzylidene anabaseine dihydrochloride (GTS-21), a compound that interacts with rat neuronal nicotinic acetylcholine receptors (nAChRs), was evaluated using human recombinant nAChRs in vitro and various pharmacokinetic and behavioral models in rodents, dogs and monkeys. GTS-21 bound to human alpha 4 beta 2 nAChR (K1-20 nM) 100-fold more potently than to human alpha 7 nAChR, and was 18- and 2-fold less potent than (-)-nicotine at human alpha 4 beta 2 and alpha 7 nAChR, respectively. Functionally. GTS-21 stimulated [5H]dopamine release from rat striatal slices with an EC50 of 10 +/- 2 microM (250-fold less potent and 70% as efficacious as (-)-nicotine), an effect blocked by the nAChR antagonist dihydro-beta-erythroidine. However, GTS-21 did not stimulate human alpha 4 beta 2 nor human ganglionic nAChRs significantly. In vivo, GTS-21 had no adverse effect on dog blood pressure (< or = 2.5 micromol/kg i.v. bolus infusion), in marked contrast with (-)-nicotine, GTS-21 (-62 micromol/kg.s.e.) also did not cross-discriminate significantly with (-)-nicotine in rats and did not reduce temperature or locomotion in mice. Neither was it active in the elevated plus maze anxiety model (0.19-6.2 micromol/kg.IP) in normal mice. However, GTS-21 did improve learning performance of monkeys in the delayed matching-to-sample task (32-130 nmol/kg.i.m.).