α-Bungarotoxin Binding in House Fly Heads and Torpedo Electroplax

Abstract: House fly heads contain a site that binds α-bungarotoxin with high affinity. It is present at about 23 pmol/g of heads and binds α-bungarotoxin (labeled with [³H]pyridoxamine phosphate) reversibly with a K _d of 6 nM. The effects of 48 drugs have been compared on the α-bungarotoxin binding sites of house fly and Torpedo. The pharmacology of the house fly site is similar to that previously reported for neuronal α-bungarotoxin binding sites in both vertebrates and invertebrates and is distinguishable from that of the classic nicotinic neuromuscular acetylcholine receptor, as exemplified by that of Torpedo electroplax. Differences between the house fly site and Torpedo include higher affinities of the Torpedo receptor for decamethonium, hexamethonium, carbamylcholine, and acetyl-β-methylcholine, but lower affinities for nicotine, atropine, and dihydro-β-erythroidine.     

Nicotinic cholinergic receptor in brain detected by binding of α-[3H]bungarotoxin

α-[³H]Bungarotoxin was prepared by catalytic reduction of iodinated α-bungarotoxin with tritium gas. Crude mitochondrial fraction from rat cerebral cortex bound 40 · 10^?15 ?60 · 10^?15 moles of α-[³H]bungarotoxin per mg of protein. This binding was reduced by 50% in the presence of approx. 10^?6 M d-tubocurarine or nicotine, 10^?5 M acetylcholine, 10^?4 M carbamylcholine or decamethonium or 10^?3 M atropine. Hexamethonium and eserine were the least effective of the drugs tested. Crude mitochondrial fraction was separated into myelin, nerve endings, and mitochondria. The highest binding of toxin per mg of protein was found in nerve endings, as well as the greatest nhibition of toxin binding of d-tubocurarine. Binding of α-[³H]bungarotoxin to membranes obtained by osmotic shock of the crude mitochondrial fraction indicates that the receptor for the toxin is membrane bound. ¹²⁵I-Labeled α-bungarotoxin, prepared with Na ¹²⁵I and chloramine T, was highly specific for the acetylcholine receptor in diaphragm, however, it was less specific and less reliable than α-[³H]bungarotoxin in brain. We conclude that a nicotinic cholinergic receptor exists in brain, and that α-[³H]bungarotoxin is a suitable probe for this receptor.     

Studies of nicotinic acetylcholine receptor protein from rat brain

Specific binding of ¹²⁵I-labeled α-bungarotoxin to a 34 800 × g pellet of a whole rat brain homogenate has been obtained at levels 2 pmol toxin per g of whole brain with a K_d of 8·10^?9 M. Binding is reduced 90% by 10^?5 M (+)- tubocurarine chloride and 10^?4 M nicotine, whereas concentrations of 10^?4 M choline chloride, atropine sulfate and eserine sulfate have essentially no effect on toxin binding. These results compare closely with those obtained from binding studies with ¹²⁵I-labeled α-bungarotoxin and soluble acetylcholine receptor protein preparations form Torpedo nobiliana; suggesting that this mammalian receptor protein is nicotinic in character.Extraction of the 34 800 × g pellet with 1% Emulphogene yields a soluble fraction with specifically binds ¹²⁵I-labeled α-bungarotoxin with a K_d of 5·10^?9 M. Nicotine and α-bungarotoxin at concentrations of 10^?5 M abolish toxin- receptor complex formation and carbachol and (+)-tubocurarine chloride reduce complex formation 35–40% at similar concentrations. Eserine sulfate, atropine sulfate, decamethonium, and pilocarpine had no effect on complex formation at concentrations of 10^?5 M.     

Catecholamine secretion by hamster adrenal cells.

Abstract— Suspensions of isolated adrenal cells were prepared by digesting hamster adrenal glands with collagenase, and the secretion of catecholamine from these cells was studied. Acetylcholine (ACh) produces a dose-dependent increase in catecholamine secretion; half-maximal secretion is produced by 3 μm -ACh, and maximal secretion by 100 μm -ACh. The cholinergic receptor in these cells appears to be nicotinic, since catecholamine secretion is stimulated by the nicotinic agonists nicotine and dimeth-ylphenylpiperaziniurn, but not by the muscarinic agonists pilocarpine or oxotremorine. ACh-induced catecholamine secretion is inhibited by hexamethonium, tubocurarine, and atropine, but is not inhibited by α-bungarotoxin. ACh-induced catecholamine secretion is dependent upon the presence of extracellular Ca²⁺, and appears to occur by exocytosis, since the release of catecholamine is accompanied by the release of dopamine β-monooxygenase, but not of lactate dehydrogenase. These biochemical studies complement the morphological evidence for exocytosis in hamster adrenal glands, and indicate that catecholamine secretion from hamster chromaffin cells is similar to that from chromaffin cells of other species.     

A characterization of alpha-bungarotoxin binding in the brain of the horseshoe crab, Limulus polyphemus

The binding of ¹²⁵I-labeled α-bungarotoxin to membrane fragments prepared from Limulus brain tissue has been investigated. Toxin binding approaches saturation in the range of 30 to 40 nm, with maximum binding of 2 to 6 pmol/mg of protein. The saturation kinetics and the rate of displacement of bound toxin are consistent with multiple toxin binding sites. Pharmacological studies show that binding is inhibited by both cholinergic agonists and antagonists, I₅₀′s for inhibition by d-tubocurarine, nicotine, decamethonium, carbachol, and atropine are 2 × 10^?6, 7 × 10^?6, 2 × 10^?5, 6 × 10^?4, and 3 × 10^?4m, respectively. Nicotinic ligands inhibited binding much more effectively than muscarinic ligands. Toxin binding activity was solubilized with Triton X-100. Velocity sedimentation analysis of the solubilized activity revealed three separate components. Seventy to eighty percent of the binding activity had a sedimentation coefficient of 8.6 S. The remaining activity was composed of two components with sedimentation coefficients of 15.1 and 17 S.     

Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax

The acetylcholine receptor of Torpedo electroplax is purified by affinity adsorption using cobra toxin (Naja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 mm benzoquinonium produces a protein that binds α-[¹²⁵I]bungarotoxin but not [³H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 m carbamylcholine produces an acetylcholine receptor protein that binds [³H]acetylcholine, [³H]decamethonium, [³H]nicotine, [¹⁴C]dimethyl-d-tubocurarine, and α-[¹²⁵I]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine receptor (as measured by acetylcholine binding) averaging 69.2 ± 14.6%. The purity of the isolated acetylcholine receptor protein is estimated to be at best 87% as judged by disc gel electrophoresis and electrofocusing.The purified acetylcholine receptor binds 7.8 nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (K_D = 0.02 μM) and 5.1 nmoles a lower affinity (K_D = 1.97 μM. If the protein concentration used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations up to 10 μm. Binding of acetylcholine was totally inhibited by α-bungarotoxin or cobra toxin and was partially blocked by four nicotinic drugs, but not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.     

Saturable (3H)cocaine binding in central nervous system of mouse

(³H)Cocaine was bound saturably to mouse brain membrane preparations, with a dissociation constant (K_d) of 0.6 μM and a maximal binding capacity of 3 pmol/mg of membrane protein. Binding was virtually maximal at 2°C, was sodium-insensitive, and was distributed rather uniformly throughout the brain. No, or only slight, displacing activities were observed for the neuro-transmitters norepinephrine, dopamine, acetylcholine, serotonin, and GABA, and for nicotine (nicotinic cholinergic receptor agonist), tubocurarine (nicotinic cholinergic receptor antagonist), morphine (opiate receptor agonist), and naloxone (opiate receptor antagonist). The cocaine analogs WIN 35,065-2 and WIN 35,428, which have enhanced stimulatory potency as compared with cocaine and only 15% of its local anesthetic activity, had affinities for the binding site similar to the affinity of cocaine itself. Displacing activities between 1 and 2 orders of magnitude weaker than those of cocaine itself were displayed by the local anesthetics, d-amphetamine, decamethonium, and atropine.     

CHARACTERIZATION OF AN α-BUNGAROTOXIN BINDING COMPONENT FROM DROSOPHILA MELANOGASTER

Abstract— We describe an α-bungarotoxin binding component from Dromphila melanoyaster that has the properties expected of an acetylcholine receptor. Toxin binding to a paniculate form of this component has been shown to be proportional to amount of extract, to be saturable and to be destroyed by heat. Localization studies using ¹²⁵I-α-bungarotoxin binding to frozen sections has shown toxin binding to be restricted to synaptic areas of the Drosophila CNS. We have also shown that this toxinbinding component can be treated with Triton X-100 without significantly altering its toxin-binding and pharmacological specificity. The ability of preincubation with cholinergic ligands to block labeled α-bungarotoxin binding to both particulate and detergent treated extracts has been studied. The nicotinic agents nicotine, d-tubocurarine, and acetylcholine are the most effective blocking agents. All of the muscarinic agents tested and the nicotinic agent decamethonium were less effective than acetylcholine in preventing α-bungarotoxin binding.     

α-Bungarotoxin binding in the central nervous system of Limulus polyphemus

The binding of ¹²⁵I-labeled α-bungarotoxin in the central nervous system of the horseshoe crab, Limulus polyphemus, was investigated. Comparative binding studies in various tissues of L. polyphemus demonstrated a selective association of the toxin with nervous tissues. The greatest enrichment of toxin binding in subcellular fractions of brain tissue was observed in a fraction enriched in mitochondria and acetylcholinesterase-containing membranes. Autoradiographic studies revealed the localization of α-bungarotoxin binding to the longitudinal connectives and neuropile regions of the abdominal ganglia. Three toxin binding components with approximate sedimentation coefficients of 9 S, 15.4 S and 17.4 S were present in solubilized extracts of brain tissue. ¹²⁵I-labeled α-bungarotoxin binding to these components was inhibited 72%, 47%, 9% and 0% by 10 μM concentrations of (+)-tubocurarine, nicotine, scopolamine and pilocarpine, respectively. The apparent formation of the 15.4 S and 17.4 S proteins from the 9 S protein was obtained. The 15.4 S and 17.4 S components are suggested as aggregates of the 9 S protein. This 9 S protein is proposed as an acetylcholine receptor from the central nervous system of L. polyphemus.     

Studies of nicotinic acetylcholine receptor protein from rat brain: II. Partial purification

The pharmacological specificity of the binding of ¹²⁵I-labeled α-bungarotoxin to a 1% Emulphogene BC-720 extract of a rat brain particulate fraction has been investigated. The extract contains a component which possesses the binding characteristics of a nicotinic acetylcholine receptor protein. The crude soluble acetylcholine receptor protein was purified by affinity chromatography utilizing the α-neurotoxin of Naja naja siamensis as ligand and 1.0 M carbamylcholine chloride as eluant. A single, batch-wise, affinity chromatography procedure yields an average purification of 510-fold. When this purified material is treated a second time by affinity chromatography, purification as high as 12 600-fold has been obtained. Binding of ¹²⁵I-labeled α-bungarotoxin to this purified acetylcholine receptor protein is saturable with a K_d of 1·10^?8 M. Nicotine and acetylcholine iodide at concentrations of 10^?5 M inhibit ¹²⁵I-labeled toxin-acetylcholine receptor protein complex formation by 41 and 61% respectively. At 10^?4 M, carbamylcholine chloride and (+)-tubocurarine chloride give respectively 52 and 82% inhibition. Eserine sulfate and atropine sulfate have no effect on complex formation at a concentration of 10^?4 M. These data support the isolation of partially purified nicotinic acetylcholine receptor protein.     

Change in the intrinsic fluorescence of acetylcholine receptor purified from Narke japonica upon binding with cholinergic ligands

Effects of various cholinergic ligands on the intrinsic fluorescence of acetylcholine receptor purified from the electric organ of Narke japonica were investigated. Binding with acetylcholine decreased the fluorescence by 7–8%, and that with carbamylcholine by 4–5% at 20 °C. Decamethonium and d-tubocurarine did not affect significantly the fluorescence intensity, while hexamethonium enhanced it. These changes were completely inhibited by preincubation of the receptor with α-bungarotoxin, which indicated that the observed intrinsic fluorescence change was due to the specific binding of each ligand. Data of the quenching experiment using iodide ion as an extrinsic quencher suggested the occurrence of the conformational change in the receptor upon binding with various cholinergic ligands. Considering these results together with those on intrinsic fluorescence change, conformational change provoked by binding with acetylcholine or carbamylcholine seems to differ from that provoked by binding with other cholinergic ligands examined.     

Antibodies to nicotinic acetylcholine receptors used to probe the structural and functional relationships between brain α-bungarotoxin binding sites and nicotinic receptors

Antibodies against peripheral nicotinic acetylcholine receptors (nAChR) were used to determine the proportion of brain α-bungarotoxin binding sites that are immunologically related to the peripheral nAChR. The α-bungarotoxin binding component partially purified from rat brain was labelled with [¹²⁵I]α-bungarotoxin and reacted with increasing concentrations of rabbit anti(nAChR) antisera. At least 75% of the brain protein could be immunoprecipitated by rabbit anti(rat muscle junctional nAChR) antiserum (M) whereas an antiserum against Torpedo nAChR (J) was without effect and clearly failed to cross-react with the brain component. Both antisera precipitated 100% of [¹²⁵I]α-bungarotoxin-labelled nAChR from Torpedo marmorata. The lower precipitation of the brain protein was not a consequence of [¹²⁵I]α-bungarotoxin dissociating during the precipitation. We conclude that the majority of α-bungarotoxin binding sites in brain are clearly recognised by the crossreacting antiserum.Release of [³H]dopamine from striatal synaptosomes could be elicited by nicotine in a dose-dependent manner and the response was prevented by the ganglionic blocker mecamylamine, although antagonism by α-bungarotoxin was less clearcut. Preincubation of the synaptosomes with antiserum M resulted in a statistically significant decrease in the [³H]dopamine response to nicotine at all agonist concentrations tested. Antiserum J, however, had no consistent effect on the response. Thus the actions of the antisera parallel their ability to recognise the brain α-bungarotoxin binding component. We conclude that the cholinergic regulation of dopamine release is in part mediated through a nAChR that is immunologically related to the nAChR of the neuromuscular junction and to the α-bungarotoxin binding component that can be isolated from rat brain.     

Interaction between calcium and ligand-binding sites of the purified acetylcholine receptor studied by use of a fluorescent lanthanide

The acetylcholine receptor isolated from $\text{Torpedo ocellata}$ binds about 10 moles of a fluorescent lanthanide, terbium, per mole α-bungarotoxin-binding site, a process which is accompanied by a fluorescence enhancement (λexcitation 295 nm, λemission 546 nm) which allows detection of receptor-Tb³⁺ complexes at μM concentrations. In presence of calcium two types of terbium-binding site are revealed, both with terbium dissociation constants of 18 ± 0.5 μM. About 60% of the sites bind calcium with an apparent dissociation constant of 1.1 ± 0.1 mM. Sites which interact with calcium also interact with activators of neural transmission, carbamylcholine and decamethonium, but not with the inhibitors, d-tubocurarine and α-bungarotoxin. Whether the displacement of calcium by chemical mediators is directly responsible for activator-induced changes in ion permeability of neural membranes is an important question raised by our experiments. The results show that fluorescent lanthanides can be an important tool in such studies.     

Inhibition of α-bungarotoxin binding to acetylcholine receptors by antisera from animals with experimental autoimmune myasthenia gravis

Conditions are described for an assay that allows the percent inhibition of α-bungarotoxin binding to acetylcholine receptors by antisera and monovalent antigen-binding fragments of antibody molecules (Fab) to be determined. Anti-Torpedo californica acetylcholine-receptor antisera, prepared in New Zealand White rabbits and Lewis rats, were tested for the ability to inhibit [¹²⁵I]-α-bungarotoxin binding to membrane-associated and detergent-solubilized T californica acetylcholine receptors. Similar inhibition studies were performed using rabbit antisera and antigen-binding fragments prepared against each of the four acetylcholine receptor subunits. Antisera and antigen-binding fragments prepared against intact receptor could inhibit a maximum of 50% of the α-bungarotoxin binding to solubilized receptor. The results using monovalent antigen-binding fragments indicated that the inhibition was not due to antibody-mediated aggregation of receptor molecules. Rabbits and rats immunized with receptor denatured by sodium dodecyl sulfate all produced antisera that could bind to nondenatured receptor, but none of these animals developed experimental autoimmune myasthenia gravis. These results suggest that the antigenic determinants present on acetylcholine receptors responsible for induction of experimental auto-immune myasthenia gravis are lost with sodium dodecyl sulfate denaturation. A strong correlation was also observed between the presence of experimental autoimmune myasthenia gravis in rats and rabbits and the ability of the antisera from these animals to inhibit 50% of α-bungarotoxin binding to solubilized acetylcholine receptors.     

Functional expression of the α7 and α4-containing nicotinic acetylcholine receptors on the neonatal rat carotid body

The carotid bodies (CBs) are chemosensory organs that respond to hypoxemia with transmitter neurosecretion, leading to a respiratory reflex response. It has been proposed that acetylcholine is a key regulator of transmitter release through activation of presynaptic nicotinic acetylcholine receptors (nAChRs). In the present work, we studied the identity of such nAChRs and their contribution to catecholamine release from CBs. Neonatal rat CBs were placed in a recording chamber for electrochemical recordings or disassociated for voltage-clamp studies on isolated cells. Fast nicotine superfusion increases catecholamine release from intact CBs. This response was diminished reversibly by the non-selective nAChR blocker hexamethonium, by the selective α7 blocker α-bungarotoxin and by the α4-containing nAChR blocker erysodine. In isolated CB cells the nAChR agonists nicotine, acetylcholine and cytisine all evoke inward currents with similar potencies. The nicotine-evoked current was fully blocked by mecamylamine and partially inhibited by α-bungarotoxin or erysodine. However, the combination of both α-bungarotoxin an erysodine failed to suppress this response. Immunodetection studies confirm the presence of α7 and α4 subunits in isolated dopaminergic CB cells. Our results show that activation of α7 and/or α4-containing nAChR subtypes have the ability to regulate catecholamine release from intact CB due to activation of fast inward currents expressed in chemoreceptor cells. Therefore, our results suggest that both nAChR subtypes contribute to the cholinergic nicotinic regulation of catecholamine signaling in the carotid body system.     

Nicotinic acetylcholine receptor is involved in acetylcholine regulating stomatal movement

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Autoradiographic localization of strychnine-sensitive glycine receptor in bovine adrenal medulla

The presence of an uptake system and a functional glycine receptor in adrenal medulla chromaffin cells was investigated using an autoradiographic technique in adrenal gland slices. Specific³[H]-glycine binding was observed in both adrenal cortex and medulla slices, while only specific binding of [³H]strychnine was seen only in chromaffin cells and was not associated with cortical cells. [³H]Glycine binding sites in the cortex are apparently different from those of [³H]strychnine binding sites in the medulla since excess strychnine does not displace [³H]glycine from adrenal cortex but does so from medulla. This difference supports biochemical evidence for glycine transport into medulla cells and glycine receptor sites on the chromaffin cell membrane.     

Characterization of polypeptide neurotoxins from the venom of Bungarus caeruleus

The venom of the krait Bungarus caeruleus has been fractionated into several components. Two of the basic components were highly toxic to mice and had significant levels of phospholipase A activity. These components appear to be similar in their action to the presynaptic neurotoxin β-bungarotoxin. Two other components were toxic to mice and also reduced the rate of α-bungarotoxin binding to the purified acetylcholine receptor: These components appear to be postsynaptic neurotoxins similar to α-bungarotoxin. Two acidic components displayed A-type phospholipase activity and perturbed the carbamylcholine binding properties of acetylcholine receptor-rich membrane preparations.     

Sperm motility and calcium transport: a neurochemically controlled process

Elucidation of the functional significance of the presence of acetylcholinesterase in spermatozoa was deferred until considerably after the initial observations that eserine increased flagellar beat frequency in $\text{Mytilus}$ sperm (54). Discovered about forty years ago during surveys of seminal plasma constituents (2), the enzyme was later found to be associated with the particulate fraction (4). Even though characterized as a “true” acetylcholinesterase in the sperm flagellum (3), interpretations of the role of acetylcholine as a factor in motile processes remained speculative until a wide variety of cholinergic agents was found to affect sperm cell performance (32). Those agents which inhibit acetylcholine synthesis or block cholinergic receptors generally elicit a monophasic, inhibitory, dose-dependent response on sea urchin sperm swimming speed. These include hemicholinium, curare, α-bungarotoxin and decamethonium. Cholinomimetic substances and anticholinesterases (acetylcholine and nicotine; eserine, diisopropylfluorophosphate and neostigmine) act biphasically to increase the sperm swim rate at concentrations of 1 micromolar or less while higher concentrations slow or stop the forward motion of the sperm cells. Procaine (7), which competes with Ca²⁺ for cell surface binding sites, initially stimulates then completely inhinits sperm cell progression. Other substances which interfere with the cells′ access to Ca²⁺ or with the transport of Ca²⁺ into and within the cell adversely affect spermatozoan function. A conceptual model has been proposed, a) to relate the uptake of Ca²⁺ to cholinergic regulation of ionophoric channels through the sperm cell plasma membrane and b) for this Ca²⁺ to trigger release of sequestered calcium for coupling of excitation to flagellar wave propagation.     

A stereoselective pentobarbital binding site in cholinergic membranes from Torpedocalifornica

Acetylcholine receptor-rich membranes from $\text{Torpedo}\text{californica}$ contain a binding site for [¹⁴C] pentobarbital which has a dissociation constant of 210 ± 24 μM and 1.4 ± 0.18 sites per acetylcholine site. (+) pentobarbital competes for this site three times more effectively than (?) pentobarbital. Cholinergic ligands decrease [¹⁴C] pentobarbital binding and this effect is blocked by pre-incubation with α-bungarotoxin. Pentobarbital decreases [³H] acetylcholine binding non-competitively with an apparent dissociation constant similar to the dissociation constant for [¹⁴C] pentobarbital binding. Thus, the pentobarbital and acetylcholine binding sites appear to interact with each other allosterically.