A Kinetic Model for the Action of Xylocaine on Receptors for Acetylcholine

A kinetic scheme postulating the rapid formation of a partially active acetylcholine-receptor-drug complex from Xylocaine (or a derivative) and the active acetylcholine-receptor complex can account for the effects of Xylocaine and its derivatives at the neuromuscular junction. Transmembrane currents generated by an analogue computer programmed according to the scheme can exactly match end plate currents produced by nerve stimulation in the presence of the drugs. The scheme also accounts for the qualitatively different effects of the drugs on the end plate potential and on responses to iontophoretically applied acetylcholine. The analysis presented is consistent with very rapid reactions between acetylcholine and receptors, characterized by rate coefficients in the range 10⁴ to 10⁶ sec^-1. It is based on the hypothesis that the activation of receptors by acetylcholine changes the structure of the receptors and thus their affinity for Xylocaine. The analysis does not require pharmacological separability of sodium and potassium conductances during the end plate current.     

Monovalent ion effects on acetylcholine receptor from Torpedo californica

The effects of the five Group I monovalent ions, Li, Na, K, Rb, and Cs, on [³H]acetylcholine binding to Triton X-100 solubilized acetylcholine receptor from Torpedo californica electroplax were examined. Acetylcholine binding was not greatly affected by Li or Na, but was inhibited by the other ions in the order Cs > Rb > K. The inhibition by K appeared to occur by a mechanism identical to that for d-tubocurarine inhibition of acetylcholine binding.     

On Some Structural Analogies between Acetylcholinesterase and the Macromolecular Receptor of Acetylcholine

Several properties of the enzyme acetylcholinesterase (AChE) isolated in vitro are compared with those of the membrane receptor(s) of acetylcholine expressed by the in vivo electrical response of the electroplax membrane. AChE strongly binds in vitro effectors of the electroplax: agonists e.g., decamethonium or antagonists, e.g., d-tubocurarine and flaxedil. It also reacts covalently with an affinity labeling reagent of the acetylcholine receptor site(s) in vivo (TDF). Two classes of sites on AChE molecule account for the binding of these quaternary nitrogen containing compounds: (1) the anionic site of the active center and (2) noncatalytic "peripheral anionic centers" located outside the active center. A disulfide bond breaking agent, dithiothreitol (DTT) alters in a parallel manner the reaction of AChE and the excitable membrane of the electroplax to TDF. The irreversibility of TDF action is lost in both cases, after exposure to DTT. Both AChE and the acetylcholine receptor thus contain disulfide bonds—they are closely related but not necessarily identical proteins.     

Effect of cholinergic ligands and local anesthetics on acetylcholine receptor enriched membrane preparations from Torpedo californica electroplax.

Pyrene was introduced in acetylcholine receptor (AcChR)-rich membrane preparations of Torpedo californica electroplax. The lifetime of the singlet excited state of pyrene was used to probe the properties of the hydrocarbon regions of the lipid bilayer as well as the possible perturbing effects of cholinomimetic agents on this region. After excitation with a single 15-ns pulse with a Q-switched ruby laser, the lifetime of the pyrene singlet excited state in the membranes was 200 ns. In desensitized membranes the pyrene fluorescence lifetimes remained unchanged when the cholinergic ligands carbamylcholine, d-tubocurarine, decamethonium, and hexamethonium, as well as α-bungarotoxin, were present. By contrast, the lifetime was shortened when local anesthetics were present. In sensitized membranes no changes in the pyrene lifetimes were detected when the membranes were converted from their resting state to a carbamylcholine-induced “desensitized state.” Water-soluble fluorescence quenchers affected the lifetime of pyrene in membranes. The second order rate constants for the pyrene-quencher interaction were used to detect changes in fluidity and/or membrane lipid accessibility to quenchers induced by ligands or anesthetics. No changes were detected in the quenching constants of nitromethane or Tl⁺ in the presence of cholinergic agents (with the exception of d-tubocurarine); on the other hand, a marked decrease in Tl⁺ accessibility was induced by the anesthetics procaine and tetracaine. Fluorescene dynamics measurements indicate that the hydrocarbon core of the bulk lipid in electroplax is not significantly affected by binding cholinergic ligands to membranebound AcChR. However, the hydrophobic region of the membrane is perturbed by both local anesthetics and one cholinergic ligand, d-tubocurarine. Pyrene was also incorporated into lipid vesicles prepared from T. californica electroplax lipids. The fluorescence lifetimes and quenching values of these lifetimes yielded results similar to those obtained with both sensitized and “desensitized” membrane preparations. The d-tubocurarine effect on the Tl⁺ quenching of the pyrene probe is ascribed to direct interaction of d-tubocurarine with the lipids. These findings favor a mechanism in which perturbation of the hydrophobic (lipid) environment of the AcChR in membranes by local anesthetics and even d-tubocurarine may influence the receptor conversion: sensitized state ? desensitized state.     

Alteration of Acetylcholine Penetration into, and Effects on, Venom-Treated Squid Axons by Physostigmine and Related Compounds

Choline and neostigmine markedly antagonize the effect of acetylcholine (ACh) on the action potential of the venom-treated squid axon, although they themselves have no effect on conduction. Physostigmine also antagonizes the blocking action of ACh at a concentration well below that which has any effect on conduction. In contrast, d-tubocurarine (curare) increases the effect of ACh on the action potential. Choline, neostigmine, and physostigmine markedly decrease the penetration of C¹⁴-labeled ACh into the axoplasm of the squid axon. Curare, in contrast, increases the penetration of ACh, whereas dimethylcurare gives variable results. The results provide an explanation why physostigmine and neostigmine do not influence the action of ACh on axonal conduction in a way similar to that observed at the junction. The additive effect of curare and ACh on the action potential may be due either to the greater rate of penetration of ACh or to an additive effect of the two compounds on the receptor, or to a combination of both factors.     

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.     

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.     

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.     

Two binding sites in acetylcholine receptor from Torpedo marmorata electroplax

The sensitivity of acetylcholine receptor to eleven cholinergic drugs, phospholipase A, heat and pH provided evidence that the so-called high-affinity binding (K_d for acetylcholine 11 nm in 1% Triton) and low-affinity binding (K_d 562 nm) were related to two distinct binding sites. The low-affinity binding site was less sensitive to heat and several of the cholinergic drugs, but was a little more sensitive to bungarotoxin than the high-affinity site. Zinc (0.4 mm) and EDTA (10 mm) abolished acetylcholine binding to both sites; the EDTA inhibition was time-dependent.     

Cholinergic receptors and catecholamine secretion from adrenal chromaffin cells of the toad

1. The effects of cholinergic drugs on catecholamine (CA) secretion from adrenal chromaffin tissue of the toad were studied.2. CA secretion was induced by ACh or nicotine, but not by muscarine.3. Hexamethonium inhibited the CA release evoked by ACh or nicotine, while d-tubocurarine only affected the nicotinic response. Atropine did not prevent the secretory response.4. Muscarine abolished the secretion induced by the agonists, this effect being prevented by atropine or gallamine, but not by pirenzepine.5. In conclusion, CA secretion in the toad is stimulated by activation of nicotinic receptors. Inhibitory muscarinic receptors are present, most likely of type M₂, which may play a regulatory function.     

The effects of electrical inactivity and denervation on the distribution of acetylcholine receptors in developing rat muscle

Explants of thoracic body wall from rat embryos, including intercostal muscles, ribs, and the adjacent segments of spinal cord, were maintained in organ culture. Nerve-muscle differentiation proceeded in culture with a pattern and time course similar to that of the same synapses developing in utero. To understand further the factors that regulate acetylcholine sensitivity in developing rat myotubes, we studied the effects of electrical inactivity and denervation on the distribution of acetylcholine receptors. When muscle and spinal cord were explanted at 15 days of gestation, prior to the appearance of junctional receptor clusters, intact nerve terminals were required to initiate receptor aggregation at the site of nerve-muscle junction. Electrical activity was not necessary for induction of these primary junctional clusters. Inactivity resulted, however, in the appearance of secondary multiple receptor clusters at random sites along the fibers. In the presence of tetrodotoxin, the electrically inactive nerve terminals sprouted; this was accompanied by the enlargement of the junctional receptor clusters, at the end plate, but there was no correlation between nerve sprouting and the location of extrajunctional receptor aggregates. Later in development, at a time when the junctional receptors are metabolically more stable, terminal sprouting failed to induce the increase in size of junctional receptor aggregates.     

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.     

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for α-bungarotoxin. Using radiolabeled ligand binding techniques, we show that the binding of [³H]-(±)-epibatidine is heterogeneous and is characterized by two classes of binding sites with equilibrium dissociation constants of about 15 nM and 1 μM. These classes of sites exist in approximately equal numbers and all [³H]-(±)-epibatidine binding is competitively displaced by acetylcholine, suberyldicholine and d-tubocurarine. These results provide further evidence for the complexity of agonist binding to the nAChR and underscore the difficulties in determining simple relationships between site occupancy and functional responses.     

Presynaptic inhibitory effects of rocuronium and SZ1677 on [3H]acetylcholine release from the mouse hemidiaphragm preparation

It has been shown that nondepolarizing muscle relaxants may have effects on nicotinic acetylcholine receptors (nAChRs) other than those located on the skeletal muscle: some of them possess inhibitory effects on neuronal nAChRs [Anesth. Analg. 59 (1980) 935; Trends Pharmacol. Sci. 9 (1988) 16; Pharmacol. Ther. 73 (1997) 75]. It was shown that, e.g. (+)-tubocurarine and pancuronium are able to inhibit ACh release from the axon terminals of hemidiaphragm preparations and produce tetanic fade indicating their presynaptic effect. In this study rocuronium, a nondepolarizing steroidal muscle relaxant with shorter onset of action, and SZ1677 [1-(3-hydroxy-17β-acetyloxy)-2β-(1.4-dioxa-8-azaspiro-[4,5]-dec-8-yl)-(5-androstane-16β-yl)-1-(2-propenyl) pyrrolidinium bromide], a short-acting muscle relaxant [Ann. New York Acad. Sci. 757 (1995b) 84] inhibited the release of ACh in response to axonal stimulation, while -bungarotoxin failed to reduce the stimulation evoked release of ACh and did not produce tetanic fade. These results indicate that in addition to their postsynaptic effect, rocuronium and SZ1677 have presynaptic inhibitory effects on neuronal nAChRs at the neuromuscular junction. The finding that -bungarotoxin does not inhibit the release and does not produce tetanic fade indicates that it possesses affinity only for the postsynaptic muscle nAChRs.     

Acetylcholine induces vasodilation and prostacyclin synthesis in rat lungs

Acetylcholine causes pulmonary vasodilation, but its mechanism of action is unclear. We hypothesized that acetylcholine-induced pulmonary vasodilation might be associated with prostacyclin formation. Therefore, we used isolated rat lungs perfused with a recirculating cell- and plasma-free physiological salt solution to study the effect of acetylcholine infusion on pulmonary perfusion pressure, vascular responsiveness and lung prostacyclin production. Acetylcholine (20 ug infused over 1 minute) caused immediate vasodilation during ongoing hypoxic vasoconstriction and prolonged depression of subsequent hypoxic and angiotensin II-induced vasoconstrictions. Both effects of acetylcholine were abolished by atropine pretreatment. The prolonged acetylcholine effect, but not the immediate response, was blocked by meclofenamate, an inhibitor of cyclooxygenase. The prolonged effect, but not the immediate response, of acetylcholine was associated with an increase in perfusate 6-keto-PGF_1α concentration. The acetylcholine stimulated increase in 6-keto-PGF_1α production was inhibited by meclofenamate and by atropine. Thus, blockade of prostacyclin production corresponded with blockade of the prolonged acetylcholine effect. In conclusion, acetylcholine caused in isolated rat lungs an immediate vasodilation and a prolonged, time-dependent depression of vascular responsiveness. Whereas both acetylcholine effects were under muscarinic receptor control, only the prolonged effect depended on the cyclooxygenase pathway and, presumably, protacyclin synthesis.     

A comparative analysis of the contracture responses induced by acetylcholine and choline in the twitch and tonic fibers of frog skeletal muscles

A comparative analysis of the contractile responses induced by acetylcholine and replacement of the external Na⁺ ions with choline ions in the isolated twitch and tonic fibers of frog skeletal muscles was performed. The effects of extracellular Ca²⁺ concentration and several pharmacological agents modulating the activity of various systems maintaining Ca²⁺ level in the myoplasm (dantrolene, cresol, d-tubocurarine, and tetrodotoxin) were studied. It has been found that a voltage-dependent Ca²⁺ release from the sarcoplasmic reticulum depot is the main mechanism inducing the acetylcholine contracture in the fibers of both types. However, the twitch and tonic fibers differ in the properties of the α-isoform and(or) the ratio of α- to β-isoforms of ryanodine-sensitive channels. In the fibers of both types, the replacement of over 25% of Na⁺ ions with choline induces long-term contracture responses, which are also mediated by activation of acetylcholine receptors. It is assumed that an additional mechanism—accumulation of choline ions in the myoplasm and their direct action on the ryanodine-sensitive channels—is involved in the development of such contractile responses.     

Differentiation of chick embryo myoblasts is transiently sensitive to functional denervation

Clonal analysis of myoblast differentiation has been used to assess effects of denervation on developing skeletal muscle: chick embryo legs denervated by spinal cord cautery yield reduced proportions of clonable myoblasts (P. H. Bonner, 1978, Develop. Biol., 66, 207–219). The present work examines the effects on clonable myoblasts of functional denervation by d-tubocurarine. Curare treatment during the third or fourth days of embryonic development had no effect on clonable myoblasts later in development, treatment during the fifth or sixth days resulted in reduced proportions of clonable myoblasts, and treatment during the eighth or ninth days again had no effect. Clonal analysis of treated and control embryo leg muscle cells was performed between Days 10 and 18. Embryos were also permanently denervated by spinal cord cautery late in the sixth day. These embryos showed no effect of denervation on clonable myoblast proportion. It is concluded that the differentiation of skeletal muscle myoblasts is affected by interference with normal nerve-muscle relationships only during a “window” of sensitivity and that this “window” extends approximately from Hamburger and Hamilton stage 27 to stage 30.     

Acetylcholine induces vasodilation and prostacyclin synthesis in rat lungs

Acetylcholine causes pulmonary vasodilation, but its mechanism of action is unclear. We hypothesized that acetylcholine-induced pulmonary vasodilation might be associated with prostacyclin formation. Therefore, we used isolated rat lungs perfused with a recirculating cell- and plasma-free physiological salt solution to study the effect of acetylcholine infusion on pulmonary perfusion pressure, vascular responsiveness and lung prostacyclin production. Acetylcholine (20 micrograms infused over 1 minute) caused immediate vasodilation during ongoing hypoxic vasoconstriction and prolonged depression of subsequent hypoxic and angiotensin II-induced vasoconstrictions. Both effects of acetylcholine were abolished by atropine pretreatment. The prolonged acetylcholine effect, but not the immediate response, was blocked by meclofenamate, an inhibitor of cyclooxygenase. The prolonged effect, but not the immediate response, of acetylcholine was associated with an increase in perfusate 6-keto-PGF1 alpha concentration. The acetylcholine stimulated increase in 6-keto-PGF1 alpha production was inhibited by meclofenamate and by atropine. Thus, blockade of prostacyclin production corresponded with blockade of the prolonged acetylcholine effect. In conclusion, acetylcholine caused in isolated rat lungs an immediate vasodilation and a prolonged, time-dependent depression of vascular responsiveness. Whereas both acetylcholine effects were under muscarinic receptor control, only the prolonged effect depended on the cyclooxygenase pathway and, presumably, prostacyclin synthesis.     

Characterization of acetylcholine receptor isolated from Torpedo californica electroplax through the use of an easily removable detergent, β-d-octylglucopyranoside

Non-ionic detergents used for the solubilization and purification of acetylcholine receptor from Torpedo californica electroplax may remain tightly bound to this protein. The presence of detergent greatly hinders spectrophotometric and hydrodynamic studies of the receptor protein. β-d-Octylglucopyranoside, however, is found to be effective in solubilizing the receptor from electroplax membranes with minimal interference in the characterization of the protein. The acetylcholine receptor purified from either octylglucopyranoside or Triton X-100-solubilized extracts exhibits identical amino acid compositions, α-Bungarotoxin and (+)-tubocurarine binding parameters, and subunit distributions in SDS-polyacrylamide gels. The use of octylglucopyranoside allows for the assignment of a molar absorptivity for the purified receptor at 280 nm of approx. 530 000 $\text{M}{}^{\mathrm{?1}}\text{·}\text{cm}{}^{\mathrm{?1}}$. Additionally, successful reconstitution of octylglucopyranoside-extracted acetylcholine receptor into functional membrane vesicles has recently been achieved (Gonzales-Ros, J.M., Paraschos, A. and Martinez-Carrion, M. (1980) Proc.Natl. Acad. Sci. U.S.A. 77, 1796–1799).Removal of octylglucopyranoside by dialysis does not alter the specific toxin and antagonist binding ability of the receptor or its solubility at low protein concentrations. Sedimentation profiles of the purified acetylcholine receptor in sucrose density gradients reveal several components. Sedimentation coefficients obtained for the slowest sedimenting species agree with previously reported molecular weight values. Additionally, the different sedimenting forms exhibit distinctive behavior in isoelectric focusing gels. Our results suggest that both the concentration and type of detergent greatly influence the physicochemical behavior of the receptor protein.     

Pre- and post-synaptic actions of memantine at cholinergic central synapse of Achatina fulica

The effects of memantine, an anticonvulsant, on (1) postsynaptic currents (PSC), (2) the responses to microperfused acetylcholine (ACh) and (3) Ca-currents were studied at voltage-clamped identified Lp10 neuron of Achatina fulica. Memantine (5.58 μM) evoked PSCs; however, it did not affect responses of Lp 10 neuron to microperfused ACh. PSCs evoked by memantine were blocked by d-tubocurarine (d-Tc) and removed in low Ca²⁺-high Mg²⁺ solutions. Memantine did not evoke PSCs if the same neurone was physically isolated. The PSCs evoked by memantine and microperfusion of ACh opened the same Cl-currents and both currents were blocked by d-Tc. Memantine increased the amplitude of the voltage-activated Ca-current at the neurones neighborhood to Lp10 neuron by 43±7%. Flunarizine (10 μM), a Ca channel antagonist, decreased 66±5% of the amplitude of Ca current and it also prevented memantine-induced PSCs. The amplitude of responses to microperfused ACh was not affected by flunarizine. These results suggest that memantine triggers the release of ACh at this synapse and this effect of memantine seems to be secondary to memantine-induced increase of Ca²⁺ entry through voltage activated Ca channels.