α-Bungarotoxin blocks the nicotinic receptor mediated increase in cell number in a neuroendocrine cell line

Exposure of H69 small cell lung carcinoma cells to nicotinic agonists resulted in a significant increase (up to 100%) in cell number after 6 to 12 days. The effect of nicotine (10⁻⁸ M to 10⁻⁴ M) was both dose and time dependent as was that of another nicotinic agonist cytisine (10⁻⁶ M to 10⁻⁴ M). Interstingly, both the nicotine and cytisine induced increases in H69 cell number were blocked by α-bungarotoxin, as well as d-tubocurarine a nicotinic blocker which appears to interact with most nicotinic receptors. These results suggest that the nicotine induced increase in cell number is mediated through an interaction at the nicotinic α-bungarotoxin receptor. This idea is further supported by experiments which show (1) that H69 cells possess high affinity α-bungarotoxin sites (K_d = 25 nM, B_max = 10.4 fmol/10⁶ cells) with the characteristics of a nicotinic α-bungarotoxin receptor and (2) that the potencies of nicotinic receptor ligands in the α-bungarotoxin binding assay were similar to those observed in the functional studies. Northern analysis showed that mRNA for α7, a putative nicotinic α-bungarotoxin binding subunit, and for α5 were present in H69 cells. The present data provide further evidence that nicotine increases cell number in small cell lung carcinoma and are the first to show that this effect is mediated through an interaction at the nicotinic α-bungarotoxin receptor population. These results suggest that the α-bungarotoxin site may be involved in modulating proliferative responses in neuroendocrine derived SCLC cells.     

Distribution of an α-bungarotoxin-binding cholinergic nicotinic receptor in rat brain

Cholinergic nicotinic receptors in rat brain were demonstrated by the use of the potent nicotinic antagonist [¹²⁵I]α-bungarotoxin ([¹²⁵I]α-Btx). Biochemical studies on binding of [¹²⁵I]α-Btx to rat hippocampal homogenates revealed saturable binding sites which are protected by nicotine, d-tubocurarine and acetylcholine but not by atropine or oxotremorine. The hippocampus and hypothalamus displayed relatively high [¹²⁵I]α-Btx specific binding whereas the cerebellum was devoid of specific binding. Other regions displayed intermediate binding levels. Analysis of the regional distribution of [¹²⁵I]α-Btx binding by autoradiography of frontal brain sections revealed high labeling in the hippocampus, hypothalamic supraoptic, suprachiasmatic and periventricular nuclei, ventral lateral geniculate and the mesencephalic dorsal tegmental nucleus. It is suggested that the limbic forebrain and midbrain structures as well as sensory nuclei are the main nicotinic cholinoceptive structures in the brain.     

Is there acetylcholine receptor in human thymus?

Recent studies have suggested that thymus tissue of the calf may bear nicotinic acetylcholine receptors. The presence of similar receptors in thymus tissue of man could thereby serve as a source of antigen for the production of antibody to the acetylcholine receptor in patients with myasthenia gravis. In the present experiments, human thymus tissue was examined for the presence of acetylcholine receptor. Both [¹²⁵I]α-bungarotoxin binding and antiserum to the human acetylcholine receptor were used in tests for acetylcholine receptor in thymus glands from normal individuals and from patients with myasthenia gravis. Neither normal nor myasthenic thymus tissue were found to posses the [¹²⁵I]α-bungarotoxin binding or the antigenic properties of the acetylcholine receptor.     

Search for nicotinic acetylcholine receptors on human leukocytes: absence of alpha-bungarotoxin binding in studies of healthy individuals and myasthenia gravis patients

To investigate whether nicotinic acetylcholine receptors might be present on blood mononuclear cells we studied the binding [125I]alpha-bungarotoxin to mononuclear cells from three normal controls and seven myasthenia gravis patients. The medulloblastoma cell line, TE671, which expresses a functional nicotinic acetylcholine receptor having pharmacological properties similar to that of skeletal muscle receptor, was used as a positive control for alpha-bungarotoxin binding. None of the mononuclear cell samples studied exhibited specific binding of alpha-bungarotoxin.     

A critical evaluation of the use of toxins fromDendro- aspis viridis to block nicotinic responses at central and ganglionic synapses

Previous work by other investigators has shown that toxins prepared fromDendroaspis viridis venom block cholinergic transmission at the neuromuscular junction, as well as nicotinic transmission in frog spinal cord and in snail neurons. This suggested that these ligands may be useful for studying nicotinic receptors in the central nervous system. Thus,Dendroasis viridis venom was fractionated into its toxin components. Only one of the fractions possessed activity as assessed by: (1) inhibition of α-bungarotoxin (α-BGT) binding at the neuromuscular junction (25% at 50 μg toxin/ml) or (2) inhibition of the ventral root-dorsal root potential (VR-DRP), a nicotinic response in frog spinal cord. However, in the spinal cord preparation, in addition to this blockade of the nicotinic pathway, convulsant activity and an increase in the amplitude of other root potentials was observed. Binding experiments using [¹²⁵I]den- drotoxin demonstrated that the labeled compound bound to central nervous tissue such as brain or spinal cord; this was not displaced by nicotine (10⁻⁴ M) ord-tubocurarine (10^-4 M), a nicotinic antagonist, indicating either non-specific binding or binding to a non-nicotinic receptor. These results thus suggest that toxins fromDendroaspis viridis venom are not suitable ligands for central nicotinic receptors. In addition, as experiments also demonstrated that the dendrotoxins did not block cholinergic transmission in frog sympathetic ganglia, it contraindicates their use at ganglionic nicotinic receptors.     

A new mechanism revealed for the action of antibodies on acetylcholine receptor function

Rabbits and sheep were inoculated with acetylcholine (ACh) receptor protein from Torpedo electric organ. Immunoglobulins (IgGs) were purified from the sera, and their effects on binding of ligands to Torpedo membranes as well as ACh receptor-induced ²²Na⁺ flux in these membranes were determined. Immune rabbit IgGs inhibited binding of [³H]ACh and [¹²⁵I]α-bungarotoxin to the ACh receptor sites but not the binding of [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) to the receptor's ionic channel site. On the other hand, immune sheep IgGs did not inhibit binding of any of these three ligands. Yet, the two IgGs from rabbit and sheep inhibited the ACh receptor-induced ²²Na⁺ flux. The initial rate of binding of [³H]H₁₂-HTX to the membranes was potentiated by carbamylcholine, which reflected the allosteric effect of the receptor site on the ionic channel site. Both rabbit and sheep IgGs inhibited this potentiation. It is suggested that these immune sheep sera contain IgGs which inhibit ACh receptor function by means of interfering with the agonist-induced conformational changes in the receptor-channel complex. Immune rabbit IgGs inhibit receptor function possibly by a similar mechanism but, in addition, by inhibiting [³H]ACh binding to the receptor. Antibodies such as those found in the sheep sera would inhibit receptor function by blocking its conformational changes, while leaving the α-BGT binding site unoccupied.     

Is there an endogenous bungarotoxin-like molecule in the vertebrate central nervous system?

It has been postulated that there is an endogenous bungarotoxin-like ligand in rat brain which can inhibit the binding of α-bungarotoxin (α-BGT) to its target nicotinic acetylcholine (ACh) receptor. We have examined this further by testing the ability of rat and chick brain and spinal cord extracts to inhibit the binding of α-BGT to ACh receptors in cultured chick and rat myotubes. We find no evidence for inhibition by any of these extracts, and thus cannot support the hypothesis of an endogenous α-BGT-like ligand.     

Allosteric modification of α-bungarotoxin binding by the ‘calcium channel antagonist’ verapamil

The putative calcium channel antagonist verapamil has been shown to affect a variety of other ion channels as well as neurotransmitter receptors. We report that verapamil reversibly inhibits binding of the nicotinic receptor probe ¹²⁵I-α-bungarotoxin to membranes from rat brain, Torpedo californica, the rat pheochromocytoma cell line PC12 and the human medulloblastoma cell line TE671, with K_i values of 24.0, 90.5, 165.4 and 869.6 μM, respectively. These effects are calcium independent and insensitive to a variety of organic and inorganic calcium channel antagonists. While Hill coefficients suggest mechanistic differences for verapamil action in the tissues examined, derivative graphical analysis demonstrates a common mechanism of heterotypic, allosteric inhibition. Verapamil may be useful as a probe to elucidate aspects of receptor-effector coupling.     

Enhancement of [H]dopamine release and its [H]metabolites in rat striatum by nicotinic drugs

Recent evidence has identified directly muscarinic acetylcholine receptor (m-ACh R) and nicotinic acetylcholine receptor (n-ACh R) in the brain utilizing receptor binding assay. Several studies suggest that release of dopamine (DA) in the striatum is regulated by presynaptic receptors present on dopaminergic terminals. In the present study, the effects of cholinergic drugs on [³H]DA release were examined using micropunched tissue and synaptosomes obtained from rat striatum. ACh (5 × 10⁻⁴M) significantly increased spontaneous [³H]DA release, and the overflow was partially inhibited by d-tubocurarine (1 mM) but not atropine. Nicotine, lobeline, coniine and spartein, nicotinic agonists, significantly increased spontaneous and 25 mM K⁺ evoked [³H]DA release almost in a dose-dependent manner. In contrast, oxotremorine (2 × 10⁻⁴M), muscarinic agonist, did not any change in [³H]DA efflux. Furthermore, the metabolites of [³H]DA were separated by column chromatography. The main metabolite of [³H]DA in the spontaneous release from rat striatal synaptosomes was [³H]DOPAC (3,4-dihydroxyphenylacetic acid). Lobeline (5 × 10⁻⁵M) accelerated the outflow of [³H]DOPAC and [³H]OMDA metabolites (O-methylated and deaminated metabolites).These results could give rise to the suggestion that there was n-ACh R on the dopaminergic nerve terminals in the striatum and n-ACh R might have related to a directly excitatory effect on the DA release.     

Comparison of the regional expression of nicotinic acetylcholine receptor α7 mRNA and [125I]-α-bungarotoxin binding in human postmortem brain

Neuronal nicotinic acetylcholine receptors are expressed in the human central nervous system. A specific subtype of this receptor family, the α7 nicotinic acetylcholine receptor, is thought to be the principal α-bungarotoxin (αBTX)-binding protein in mammalian brain. Although the expression of this receptor subtype has been characterized in rat, no study has specifically compared the expression of both the α7 gene and the localization of BTX binding sites in human brain. Expression of α7 mRNA and receptor protein in human postmortem brain tissue was examined by in situ hybridization and [¹²⁵I]-α-bungarotoxin autoradiography, respectively, with particular emphasis on regions associated with sensory processing. Regions with high levels of both α7 gene expression and [¹²⁵I]-αBTX binding include the nucleus reticularis of the thalamus, the lateral and medial geniculate bodies, the basilar pontine nucleus, the horizontal limb of the diagonal band of Broca, the nucleus basalis of Meynert, and the inferior olivary nucleus. High-to-moderate levels of α7 probe hybridization were also seen in the hippocampus and the cerebral cortex; however, there was a reduced or variable degree of [¹²⁵I]-αBTX binding in these regions compared with the level of probe hybridization. In most brain regions, [¹²⁵I]-αBTX binding was localized to neuronal cell bodies similar in morphology to those that exhibited α7 hybridization, suggesting that the high-affinity [¹²⁵I]-αBTX binding sites in the human brain are likely to be principally composed of α7 receptor subtypes. J. Comp. Neurol. 387:385–398, 1997. © 1997 Wiley-Liss, Inc.     

Cholinergic transmission regulates extrajunctional acetylcholine receptors

To determine the role of ACh transmission in the regulation of extrajunctional ACh receptors, we compared the effect of postsynaptic cholinergic blockade with that of surgical denervation. Blockade of ACh transmission was produced in the soleus muscles of rats by continuous local infusion of α-bungarotoxin, delivered by implantable osmotic pumps. Extrajunctional ACh receptors were measured by an ¹²⁵I-α-BuTx binding method. Our results showed an increase of extrajunctional ACh receptors quantitatively equivalent to that resulting from surgical denervation. This denervation-like effect is attributed to elimination of (i) impulse-dependent ACh transmission (which normally triggers muscle usage), and (ii) spontaneous quantal and nonquantal ACh transmission. The influence of the nerve in regulating extrajunctional ACh receptors appears to be due to the sum of these forms of ACh transmission.     

A comparison of nerve transection and chronic application of β-bungarotoxin on acetylcholine receptor distribution and other nerve-muscle properties

β-Bungarotoxin (β-BuTX), a snake venom neurotoxin which acts presynaptically to inhibit acetylcholine (ACh) release at the neuromuscular junction, was applied to the rat phrenic nerve-diaphragm muscle preparation to determine its effectiveness to mimic denervation. The distribution of junctional and extrajunctional ACh receptors on the muscle were assayed biochemically by [¹²⁵I]α-bungarotoxin ([¹²⁵I]α-BuTX) binding and electrophysiologically by iontophoretic application of ACh. Spontaneous transmitter release and muscle membrane potential were measured under conditions of denervation, β-BuTX treatment, and bee venom phospholipase A₂ exposure. Within 7 days after treatment with a single dose (5μg/kg) of enzymatically active β-BuTX, extrajunctional [¹²⁵I]α-BuTX binding increased fivefold, and there was a decrease in miniature end-plate potential (MEPP) frequency and in resting membrane potential (RMP) to values less than those of control muscles but greater than those of denervated muscles. The same dose of enzymatically inactive β-BuTX or snake venom phospholipase A₂ was without effect, but a fivefold greater dose of enzymatically inactive β-BuTX resulted in changes in extrajunctional binding and RMP similar to those of muscles exposed to the enzymatically active toxin. However, unlike muscles treated with active toxin, those treated with inactive toxin had MEPP frequencies similar to control muscles and exhibited contraction elicited by phrenic nerve stimulation. These results taken together indicate that the nerve exerts a trophic influence on muscle independent of ACh and muscle activity.     

3-[2,4-Dimethoxybenzylidene]anabaseine (DMXB) selectively activates rat α7 receptors and improves memory-related behaviors in a mecamylamine-sensitive manner

The α7 nicotinic receptor agonist 3-[2,4-dimethoxybenzylidene]anabaseine (DMXB; GTS-21) was investigated for its ability to: (1) activate a variety of nicotinic receptor subtypes in Xenopus oocytes; (2) improve passive avoidance and spatial Morris water task performances in mecamylamine-sensitive manners in bilaterally nucleus basalis lesioned rats; and (3) elevate high-affinity [³H]acetylcholine (ACh) and high-affinity α-[¹²⁵I]bungarotoxin binding in rat neocortex following 2 weeks of daily injections. DMXB (100 μM) activated α7 homo-oligomeric receptors, without significant activity at α2-, α3- and α4-containing subtypes. Mecamylamine blocked rat α7 receptors weakly if co-administered with agonist, but much more potently when pre-applied. Bilateral ibotenic acid lesions of the nucleus basalis interfered with passive avoidance and spatial memory-related behaviors. DMXB (0.5 mg/kg, i.p.) improved passive avoidance behavior in lesioned animals in a mecamylamine-sensitive manner. DMXB (0.5 mg/kg 15 min before each session) also improved performance in the training and probe components of the Morris water task. DMXB-induced improvement in the probe component but not the training phase was mecamylamine-sensitive. [³H]ACh binding was elevated after 14 days of daily i.p. injections with 0.2 mg/kg nicotine but not after 1 mg/kg DMXB. Neither drug elevated high-affinity α-[¹²⁵I]bungarorotoxin binding over this interval.     

Interactions of anti-nicotinic acetylcholine receptor antibodies at α-bungarotoxin binding sites across species and tissues

Two antisera prepared against the nicotinic acetylcholine receptor (nAcChoR) from Electrophorus exhibit comparable ability to inhibit high-affinity α-bungarotoxin binding to membrane fractions from rat brain or muscle, PC12 or TE671 cells, or Torpedo electric tissue. Only one of several monoclonal antibodies raised against nAcChoR from Torpedo inhibits toxin binding to membranes from rat brain or muscle or TE671 cells, but is considerably more potent as an inhibitor of toxin binding to Torpedo nAcChoR. These results indicate that some antibodies prepared against nAcChoR from electric tissue recognize epitopes near the high-affinity toxin binding sites. Some of these toxin binding site epitopes are preserved across species and tissues. The positive outcome of this study supports the continued use of toxin as a probe for at least a subset of mammalian neuronal nAcChoR.     

α-Bungarotoxin binding sites on sensory neurones and their axonal transport in sensory afferents

The autoradiographic localization of [¹²⁵I]α-bungarotoxin binding sites on primary sensory fibres was investigated. Nicotinic α-bungarotoxin binding sites were localized to a small sub-population of large dorsal root ganglion cells in the rat, monkey, cat and human dorsal root ganglia. Ligation of the sciatic nerve or dorsal root in the rat resulted in an anterograde accumulation of binding sites proximal to the dorsal root ganglion, and a small retrograde accumulation. Unilateral dorsal root section in the rat produced a loss of toxin binding sites mainly within lamina III of the dorsal horn. These results suggest that nicotinic α-bungarotoxin binding sites manufactured in large dorsal root ganglion cell bodies are transported both centrally to the spinal cord and also peripherally.     

Localization of α-bungarotoxin binding sites to the goldfish retinotectal projection

The optic tectum of the goldfishCarassius auratus is a rich source of α-bungarotoxin (α-Btx) binding protein. In order to determine whether some fraction of these receptors is present at retinotectal synapses, we have compared the histological distribution of receptors revealed by the use of [¹²⁵Iα-Btx radioautography to the distribution of optic nerve terminals revealed by the use of cobalt and horseradish peroxidase (HRP) techniques. The majority of α-Btx binding is concentrated in those tectal layers containing primary retinotectal synapses. The same layers contain high concentrations of acetylcholinesterase (AChE), revealed histochemically. Following enucleation of one eye, there is a loss of α-Btx binding in the contralateral tectum, observed both by radioautography and by a quantitative binding assay of α-Btx binding. Approximately 40% of the α-Btx binding sites are lost within two weeks following enucleation. By contrast, no significant change in AChE activity could be demonstrated up to 6 months enucleation. These results are discussed in light of recent studies which show that the α-Btx binding protein and the nicotinic acetylcholine receptor are probably identical in goldfish tectum. We conclude that the 3 main classes of retinal ganglion cells projecting to the goldfish tectum are nicotinic cholinergic and that little or no postdenervation hypersensitivity due to receptor proliferation occurs in tectal neurons following denervation of the retinal input.     

α-Bungarotoxin binding to a high molecular weight component from lower vertebrate brain identified on dodecyl sulfate protein-blots

The binding of [¹²⁵I]iodo-α-bungarotoxin ([¹²⁵]α-BuTX) to the dissociated α-subunit of Torpedo acetylcholine receptor (AChR) can be readily demonstrated in a modified ‘protein-blot’ analysis utilizing electrophoretically transferred, dissociated subunits immobilized onto positively charged nylon membranes which are then incubated directly with [¹²⁵I]α-BuTX. We report here the use of the protein-blotting technique to detect the α-BuTX binding site present in the central nervous system of lower vertebrates and to characterize some of the physicochemical properties of the toxin binding site. High molecular weight (M200,000 and 120,000) α-BuTX-binding components can be readily demonstrated in avian and fish brain extracts upon protein-blotting with [¹²⁵I]α-BuTX following lithium dodecyl sulfate PAGE. Neither extensive reduction with dithiothreitol nor prior reduction followed by alkylation with iodoacetamide alter the mobility of the CNS-derived BuTX-binding sites. In contrast to our findings with Torpedo AChR or muscle AChR derived from a number of different species, no binding is observed in the molecular weight range of the α-subunit (M_r= 40,000) nor is any binding at any molecular weight observed in similar fractions prepared from adult, mammalian (rat, guinea pig) brain using this technique. These results demonstrated the existence in lower vertebrate brain of a BuTX binding site comparable in size to the AChR oligomeric complex of electric organ and muscle. They also suggest, however, striking structural differences between muscle AChR and the central neuronal BuTX-binding complex as well as a considerable difference between the neuronal BuTX-binding sites derived from lower and higher vertebrate brain.     

Acetylcholine receptor in myasthenia gravis: Increased affinity for α-bungarotoxin

Studies of the binding of ¹²⁵I-labeled α-bungarotoxin to myasthenic motor end-plates have been interpreted as showing a decrease in the number of acetylcholine (ACh) receptors at these end-plates. Equilibrium binding studies of ¹²⁵I-tagged α-bungarotoxin to detergent-extracted ACh receptors from normal and myasthenic intercostal muscle were carried out to determine whether the reduced toxin binding previously reported could be due to a reduced affinity of myasthenic receptors for α-bungarotoxin rather than to a decreased number of receptors. Our results show increased rather than decreased affinity of myasthenic receptors for α-bungarotoxin and also suggest that the number of ACh receptors is indeed reduced. The presence of a change in binding affinity, in addition to the reduced number of ACh receptors, suggests the presence of membrane changes that may contribute to the pathogenesis of myasthenia gravis.     

α-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with α7 subunits and up-regulation by chronic nicotine treatment

High density neuronal cultures from rat E18 hippocampus and cortex have been characterised with respect to cholinergic binding sites. No specific binding of [³H]nicotine or [³H]cyttine to live cells in situ was detected, although the limit for detection was estimated to be 30 fmol/mg protein. Muscarinic binding sites labelled with [³H]QNB were present at a density of 0.75 pmol/mg protein. [¹²⁵I]α-Bungarotoxin (αBgt) bound to hippocampal cultures with a B_max of 128 fmol/mg protein and a K_d of 0.6 nM; cortical cultures expressed five times fewer [¹²⁵I]α-Bgt binding sites. Fluorescence cytochemistry with rhodamine-α-Bgt indicated that 95% of hippocampal neurons were labelled, compared with only 36% of cortical neurons. Average densities of 4 × 10⁴ and 2 × 10⁴ binding sites/cell were calculated for hippocampal and cortical cultures, respectively. Double labelling experiments with mAb307 (which recognises the rat α7 nicotinic receptor subunit) and rhodamine-α-Bgt gave coincident labelling patterns, supporting the correlation between the α7 subunit and Bgt-sensitive neuronal nicotinic receptor. Treatment of hippocampal cultures with 10 μM nicotine for 14 days elicited a 40% increase in the numbers of [¹²⁵I]α-Bgt binding sites, mimicking the up-regulation observed in vivo studies. Primary cultures offer a useful in in vitro system for investigating the expression and regulation of brain α-Bgt-sensitive receptors.     

α7-Nicotinic receptor expression and the anatomical organization of hippocampal interneurons

C3H and DBA/2 mice differ in their hippocampal inhibitory function, as measured by the inhibitory gating of pyramidal neuron response to repeated auditory stimulation. This functional difference appears to be related to differences in expression of the α7 nicotinic cholinergic receptor, which may be generally expressed by interneurons. This study examines the relationship between genetic variation in α7 receptor subunit expression and GABAergic interneuron distribution in various regions and layers of the hippocampus in the two mouse strains. Subpopulations of hippocampal interneurons in both mouse strains were found to bind [¹²⁵I]α-bungarotoxin. However, the distribution of the [¹²⁵I]α-bungarotoxin-positive hippocampal interneurons was significantly different between C3H and DBA/2 mice. In region CA1, and to a lesser extent in region CA3, DBA/2 mice had increased numbers of [¹²⁵I]α-bungarotoxin-positive neurons in stratum lacunosum-moleculare and decreased numbers in stratum oriens. Similar differences in GABAergic neuron distribution were observed in region CA1 in the two strains. C3H/DBA/2 F1 animals were backcrossed to the C3H parental strain for six generations, with selection for either the DBA/2 or C3H allelic variant of the α7 receptor gene. The distribution of [¹²⁵I]α-bungarotoxin labeling closely resembled the DBA/2 parental phenotype in animals retaining the DBA/2 allele of the α7 gene. These data suggest that the α7 receptor gene locus may influence the anatomical organization of at least a subset of hippocampal interneurons by an as yet unidentified mechanism. This difference in interneuron anatomy may also contribute to functional differences in inhibitory sensory gating between the two strains.