subecholine and Choline

Tissue engineering and regenerative medicine is envisaged as the future option for esophageal replacement; however, engineering of a functional esophagus is impeded by the limited understanding of the anatomical complexity of this dynamic muscular organ. The aim of this study was to characterize the function of native esophageal tissue and determine differences in functional response to stimulation between anatomical sites.. The in-vitro response of guinea pig esophageal preparations, from various anatomical sites, to muscle agonists was investigated. Esophageal strips were exposed to bethanechol, an agonist of muscarinic receptors located on smooth muscle, and suberyldicholine, an agonist of nicotinic receptors located on striated muscle, within a Schuler organ bath, to determine the contractile response on the various segments of the esophagus.. The esophagus responded in a reliable and consistent manner to agonist stimulation. Bethanechol dose response curves were constructed with doses of 10 to 300 μM. The average maximal contractions to bethanechol exposure were 4.51, 4.80, 5.55, and 9.15 mN for upper, upper middle, lower middle, and lower esophageal segments, respectively. Responses to singular stimulation with 30 μM suberyldicholine in the presence of tetrodotoxin (100 μM) gave average contractions of 1.07, 0.84, 2.60, and 3.02 mN for upper, upper middle, lower middle, and lower esophageal segments, respectively. Bethanechol and suberyldicholine-induced responses were greater in the lower esophagus in comparison to the upper esophageal segments.. These findings pave the way for the use of an in-vitro bethanechol and suberyldicholine-induced contraction model for future assessment of engineered esophageal tissue.

Topics: Animals; Bethanechol; Choline; Dose-Response Relationship, Drug; Esophagus; Female; Guinea Pigs; Male; Muscle Contraction; Tetrodotoxin; Tissue Engineering

The interactions of a series of bisholine esters [(CH3)3N+CH(2)CH2OCO-(CH2)n-COOCH2CH2N+(CH3)3] with the Torpedo nicotinic acetylcholine receptor have been investigated. In equilibrium binding studies, [3H]-suberyldicholine (n=6) binds to an equivalent number of sites as [3H]-acetylcholine and with similar affinity (KD approximately 15 nM). In competition studies, all bischoline esters examined displaced both radioligands in an apparently simple competitive manner. Estimated dissociation constants (KI) showed clear chain length dependence. Short chain molecules (n6) had high affinity similar to suberyldicholine. Functional responses were measured by either rapid flux techniques using Torpedo membrane vesicles or voltage-clamp analyses of recombinant receptors expressed in Xenopus oocytes. Both approaches revealed that suberyldicholine (EC50 approximately 3.4 microM) is 14-25-fold more potent than acetylcholine. However, suberyldicholine elicited only about 45% of the maximum response of the natural ligand, i.e., it is a partial agonist. The potency of this bischoline series increased with chain length. Whereas the shorter ligands (nor=4) had similar (or higher) potency to suberyldicholine. Ligand efficacy had an approximately bell-shaped dependence on chain length and compounds where nor=8 were very poor partial agonists. Based on estimates of interonium distances, we suggest that bisquaternary ligands can interact with multiple binding sites on the nAChR and, depending on the conformational state of the receptor, these sites are 15-20A apart.

Topics: Acetylcholine; Animals; Binding, Competitive; Choline; Ligands; Nicotinic Agonists; Protein Conformation; Receptors, Nicotinic; Structure-Activity Relationship; Thenoyltrifluoroacetone; Torpedo

Suberyldicholine, a bisquaternary compound, is a potent nicotinic acetylcholine receptor agonist. Previously, we suggested that at least some of the unusual binding properties of this ligand may be a consequence of its ability to cross-link two binding "subsites" within each of the high-affinity agonist binding domains [Dunn, S. M. J., and Raftery, M. A. (1997) Biochemistry 36, 3846-3853]. Tryptophan 86 of the alpha subunit has previously been implicated in the binding of agonist to this receptor. However, on the basis of the crystal structure of a homologous acetylcholine binding protein, this residue is predicted to lie 15-20 A from the high-affinity site, i.e., a distance that approximates the interonium distance of suberyldicholine. Tryptophan 86 was mutated to either an alanine or a phenylalanine, and the mutated subunit was coexpressed with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Although the alanine mutation resulted in a loss of receptor expression, the alphaW86F mutant receptor was expressed on the oocyte surface, albeit with a much reduced efficiency. Acetylcholine-evoked currents of the alphaW86F receptor were not significantly different from those of the wild type with respect to the concentration dependence of channel activation, receptor desensitization, or d-tubocurarine inhibition. In contrast, the EC(50) for suberyldicholine-mediated activation of the alphaW86F receptor was increased by approximately 500-fold. Furthermore, suberyldicholine-evoked currents in the mutant receptor did not desensitize and were insensitive to block by d-tubocurarine. Thus, tryptophan 86 of the Torpedo receptor alpha subunit may be part of a subsite for recognition of suberyldicholine and other bisquaternary ligands.

Topics: Acetylcholine; Amino Acid Substitution; Animals; Choline; Cholinergic Agents; Female; Gene Expression; Membrane Potentials; Nicotinic Antagonists; Oocytes; Protein Binding; Protein Subunits; Receptors, Nicotinic; Torpedo; Tryptophan; Tubocurarine; Xenopus

The temporal fine structure of single channel currents was studied to obtain information on how agonists open nicotinic acetylcholine receptor channels. Currents were recorded from mouse myoballs with quartz pipettes in the on-cell mode of the patch-clamp technique. With 62 kHz filter cut-off and root mean square (r.m.s.) noise levels as low as 1.45 pA at 200 mV hyperpolarization, events down to 6 micros duration could be resolved with negligible error rate. Three types of openings with mean durations of 750 micros, 89 micros and 4 micros were identified with 0.1-10 microM suberyldicholine (SubCh). The relative frequencies of the three types of openings were 84% for long, 5% for medium and 11% for short openings with 1 microM SubCh. Stability plots and single channel current amplitude comparisons suggest that the three types of openings arise from a homogenous channel population. Above 10 microM SubCh, the three types of openings could not be discerned because channel openings occurred too closely spaced and open channels were increasingly blocked. Three types of openings can be generated with a mechanistic receptor model with two unequal binding sites, short and medium openings arising from one or the other monoliganded state, and long openings from the fully liganded state of the receptor. Maximum likelihood fitting of the rate constants of this model directly to the sequence of observed open and shut times accurately predicted the main physiological properties of the receptors with 0.1 microM SubCh. However, fitting recordings with 0.1-10 microM SubCh simultaneously revealed that this model cannot reproduce the weak influence of SubCh concentration on the proportions of the three types of openings. Therefore we conclude that short and medium openings are unlikely to arise preferentially from one or the other monoliganded state of nicotinic acetylcholine receptor channels.

Topics: Animals; Animals, Newborn; Cells, Cultured; Choline; Cholinergic Agents; Computer Simulation; Dose-Response Relationship, Drug; Ion Channel Gating; Kinetics; Membrane Potentials; Mice; Models, Biological; Muscle Fibers, Skeletal; Nicotinic Agonists; Receptors, Nicotinic

We describe an optical technique using total internal reflection fluorescence (TIRF) microscopy to obtain simultaneous and independent recordings from numerous ion channels via imaging of single-channel Ca2+ flux. Muscle nicotinic acetylcholine (ACh) receptors made up of alphabetagammadelta subunits were expressed in Xenopus oocytes, and single channel Ca2+ fluorescence transients (SCCaFTs) were imaged using a fast (500 fps) electron-multiplied c.c.d. camera with fluo-4 as the indicator. Consistent with their arising through openings of individual nicotinic channels, SCCaFTs were seen only when a nicotinic agonist was present in the bathing solution, were blocked by curare, and increased in frequency as roughly the second power of [ACh]. Their fluorescence amplitudes varied linearly with membrane potential and extrapolated to zero at about +60 mV. The rise and fall times of fluorescence were as fast as 2 ms, providing a kinetic resolution adequate to characterize channel gating kinetics; which showed mean open times of 7.9 and 15.8 ms when activated, respectively, by ACh or suberyldicholine. Simultaneous records were obtained from >400 channels in the imaging field, and we devised a novel "channel chip" representation to depict the resultant large dataset as a single image. The positions of SCCaFTs remained fixed (<100 nm displacement) over tens of seconds, indicating that the nicotinic receptor/channels are anchored in the oocyte membrane; and the spatial distribution of channels appeared random without evidence of clustering. Our results extend single-channel TIRFM imaging to ligand-gated channels that display only partial permeability to Ca2+, and demonstrate an order-of-magnitude improvement in kinetic resolution. We believe that functional single-channel imaging opens a new approach to ion channel study, having particular advantages over patch-clamp recording in that it is massively parallel, and provides high-resolution spatial information that is inaccessible by electrophysiological techniques.

Topics: Acetylcholine; Animals; Calcium; Cells, Cultured; Choline; Cloning, Molecular; Dose-Response Relationship, Drug; Fluorescent Dyes; Interferometry; Ion Channel Gating; Kinetics; Membrane Potentials; Microscopy, Fluorescence; Nicotinic Agonists; Nicotinic Antagonists; Oocytes; Optics and Photonics; Patch-Clamp Techniques; Receptors, Nicotinic; Video Recording; Xenopus laevis

Acetylcholine (ACh) activates two types of chloride conductances in Aplysia neurons that can be distinguished by their kinetics and pharmacology. One is a rapidly desensitizing current that is blocked by alpha-conotoxin-ImI and the other is a sustained current that is insensitive to the toxin. These currents are differentially expressed in Aplysia neurons. We report here that neurons that respond to ACh with a sustained chloride conductance also generate 8-lipoxygenase metabolites. The sustained chloride conductance and the activation of 8-lipoxygenase have similar pharmacological profiles. Both are stimulated by suberyldicholine and nicotine, and both are inhibited by alpha-bungarotoxin. Like the sustained chloride conductance, the activation of 8-lipoxygenase is not blocked by alpha-conotoxin-ImI. In spite of the similarities between the metabolic and electrophysiological responses, the generation of 8-lipoxygenase metabolites does not appear to depend on the ion current since an influx of chloride ions is neither necessary nor sufficient for the formation of the lipid metabolites. In addition, the application of pertussis toxin blocked the ACh-activated release of arachidonic acid and the subsequent production of 8-lipoxygenase metabolites, yet the ACh-induced activation of the chloride conductance is not dependent on a G protein. Our results are consistent with the idea that the nicotinic ACh receptor that activates the sustained chloride conductance can, independent of the chloride ion influx, initiate lipid messenger synthesis.

Topics: Acetylcholine; Animals; Aplysia; Arachidonate Lipoxygenases; Arachidonic Acids; Arecoline; Atropine; Bungarotoxins; Chloride Channels; Chlorides; Choline; Cholinergic Antagonists; Conotoxins; Depression, Chemical; Enzyme Activation; Ganglia, Invertebrate; Hexamethonium; Hydroxyeicosatetraenoic Acids; Ion Channel Gating; Ion Transport; Nerve Tissue Proteins; Neurons; Nicotine; Nicotinic Agonists; Pertussis Toxin; Receptor Cross-Talk; Receptors, Nicotinic; Tetraethylammonium; Tubocurarine; Virulence Factors, Bordetella

Isoflurane increases the apparent agonist affinity of ligand-gated ion channels. This action reflects a reduction in the receptor's agonist dissociation constant and/or the preopen/open channel state equilibrium. To evaluate the effect of isoflurane on each of these kinetic constants in the nicotinic acetylcholine receptor, the authors analyzed isoflurane's actions on (1) the binding of the fluorescent agonist Dns-C6-Cho to the nicotinic acetylcholine receptor's agonist self-inhibition site and (2) the desensitization kinetics induced by the binding of the weak partial agonist suberyldicholine.. The dissociation constant for Dns-C6-Cho binding to the self-inhibitory site was determined using stopped-flow fluorescence spectroscopy. The values of the kinetic constants for agonist binding, channel gating, and desensitization were determined by modeling the suberyldicholine concentration-dependence of the apparent rate of desensitization.. Isoflurane did not significantly alter the dissociation constant for Dns-C6-Cho binding to the self-inhibitory site even at a concentration as high as 1.5 mM, the highest concentration studied. At this concentration, isoflurane substantially reduced the dissociation constant for suberyldicholine binding to its channel opening site by 97% from 17 +/- 5 microM to 0.5 +/- 0.2 microM, whereas the preopen/open channel state equilibrium was reduced only from 19.1 to 5 +/- 1.. Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor primarily by reducing the agonist dissociation constant of the site responsible for channel opening rather than altering channel gating kinetics.

Topics: Algorithms; Anesthetics, Inhalation; Animals; Choline; Cholinergic Agents; Dansyl Compounds; Fluorescent Dyes; In Vitro Techniques; Ion Channel Gating; Isoflurane; Kinetics; Membranes; Nicotinic Agonists; Quaternary Ammonium Compounds; Receptors, Nicotinic; Spectrometry, Fluorescence; Torpedo

Acetylcholine, nicotine, a selective agonist of N-cholinoreceptors suberildicholine dibromide, as well as a selective agonist of M-cholinoreceptors 5-methylfurmethide inhibited spike discharges in a dose-dependent manner up to a complete ceasing of the firing in cholinoreceptors situated on the identified neurone TAN of African giant snail Achatina fulica. M-cholinoblocker metamizylum completely prevented the inhibitory effect of methylfurmethide. Central cholinoblocker aetherophen completely prevented the inhibitory effect of suberildicholine dibromide. Metamizylum or aetherophen used alone were only able to decrease the inhibitory effect of acetylcholine, whereas a mixture of these agents suppressed completely the acetylcholine-induced inhibition. The findings suggest that, on the TAN membrane, nicotinic and muscarinic cholinoreceptors co-exist and function in one and the same direction.

Topics: Acetylcholine; Animals; Choline; Cholinergic Agents; Dose-Response Relationship, Drug; Ganglia, Invertebrate; In Vitro Techniques; Membrane Potentials; Microelectrodes; Motor Neurons; Muscarine; Nicotine; Nicotinic Agonists; Parasympathomimetics; Receptors, Cholinergic; Snails

Two anionic residues in the nicotinic acetylcholine receptor, Asp-152 in the alpha-subunit and Asp-174 in the gamma-subunit or the corresponding Asp-180 in the delta-subunit, are presumed to reside near the two agonist binding sites at the alphagamma and alphadelta subunit interfaces of the receptor and have been implicated in electrostatic attraction of cationic ligands. Through site-directed mutagenesis and analysis of state changes in the receptor elicited by agonists, we have distinguished the roles of anionic residues in conferring ligand specificity and ligand-induced state changes. alphaAsp-152 affects agonist and antagonist affinity similarly, whereas gammaAsp-174 and deltaAsp-180 primarily affect agonist affinity. Combining charge neutralization on the alpha subunit with that on the gamma and delta subunits shows an additivity in free energy changes for carbamylcholine and d-tubocurarine, suggesting independent contributions of these residues to stabilizing the bound ligands. Since both aromatic and anionic residues stabilize cationic ligands, we substituted tyrosines (Y) for the aspartyl residues. While the substitution, alphaD152Y, reduced the affinities for agonists and antagonists, the gammaD174Y/deltaD180Y mutations reduced the affinity for agonist binding, but surprisingly enhanced the affinity for d-tubocurarine. To ascertain whether selective changes in agonist binding stem from the capacity of agonists to form the desensitized state of the receptor, carbamylcholine binding was measured in the presence of an allosteric inhibitor, proadifen. Mutant nAChRs carrying alphaD152Q or gammaD174N/deltaD180N show similar reductions in dissociation constants for the desensitized compared with activable receptor state and a similar proadifen concentration dependence. Hence, these mutations influence ligand recognition rather than the capacity of the receptor to desensitize. By contrast, the alphaD200Q mutation diminishes the ratio of dissociation constants for two states and requires higher proadifen concentrations to induce desensitization. Thus, the contributions of alphaAsp-152, gamma/deltaAsp-174/180, and alphaAsp-200 in stabilizing ligand binding can be distinguished by the interactions between agonists and allosteric inhibitors.

Topics: Amino Acid Sequence; Binding Sites; Carbachol; Cell Line; Choline; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Nicotinic Agonists; Nicotinic Antagonists; Proadifen; Receptors, Nicotinic; Sequence Alignment; Tubocurarine; Tyrosine

Ionotropic, nicotinic receptors have previously been shown to mediate both inhibitory (Cl-dependent) and excitatory (cationic) cholinergic responses in Aplysia neurons. We have used fast perfusion methods of agonist and antagonist application to reevaluate the effects on these receptors of a wide variety of cholinergic compounds, including a number of recently isolated and/or synthesized alpha toxins [alpha-conotoxin (alphaCTx)] from Conus snails. These toxins have been shown in previous studies to discriminate between the many types of nicotinic receptors now known to be expressed in vertebrate muscle, neuroendocrine, and neuronal cells. One of these toxins (alphaCTx ImI from the worm-eating snail Conus imperialis) revealed that two kinetically and pharmacologically distinct elements underlie the ACh-induced Cl-dependent response in Aplysia neurons: one element is a rapidly desensitizing current that is blocked by the toxin; the other is a slowly desensitizing current that is unaffected by the toxin. The two kinetically defined elements were also found to be differentially sensitive to different agonists. Finally, the proportion of the rapidly desensitizing element to the sustained element was found to be cell-specific. These observations led to the conclusion that two distinct nicotinic receptors mediate Cl currents in Aplysia neurons. The receptor mediating the rapidly desensitizing Cl-dependent response shows a strong pharmacological resemblance to the vertebrate alpha-bungarotoxin-sensitive, alpha7-containing receptor, which is permeable to calcium and mediates a rapidly desensitizing excitatory response.

Topics: Acetylcholine; Aconitine; Alkaloids; alpha7 Nicotinic Acetylcholine Receptor; Animals; Aplysia; Azocines; Bungarotoxins; Cations; Chlorides; Choline; Cholinergic Antagonists; Conotoxins; Dihydro-beta-Erythroidine; Electric Conductivity; Glycine Agents; Insecticides; Ion Channel Gating; Mollusk Venoms; Nicotinic Agonists; Nicotinic Antagonists; Oligopeptides; Patch-Clamp Techniques; Peptides; Quinolizines; Receptors, Nicotinic; Strychnine; Vertebrates

Examination of the kinetics of dissociation of [3H]acetylcholine and [3H]suberyldicholine from the membrane-bound acetylcholine receptor from Torpedo californica has revealed complexities in the high-affinity binding of nicotinic agonists. Each agonist binds to two high-affinity sites per receptor with an equilibrium dissociation constant of approximately 15 nM. When dissociation of [3H]acetylcholine from the receptor complex was triggered by dilution, dissociation occurred as a monophasic process with an apparent rate of 0.023 +/- 0.010 s(-1). However, when micromolar concentrations of unlabeled agonists (acetylcholine, carbamylcholine or suberyldicholine) were included in the dilution buffer this rate increased about 5-fold. This accelerating effect occurred even when the two high-affinity sites were initially saturated with the radioligand. This suggested the presence of an additional site (or subsite) for agonist with affinity in the micromolar range. However, at concentrations of 0-20 microM, no additional sites for [3H]acetylcholine were detected at equilibrium. To explain these results, we propose that each high-affinity site is made up of two subsites, A and B, which are mutually exclusive at equilibrium. With [3H]acetylcholine initially occupying site A, occupancy of site B by unlabeled ligand reduces the affinity for site A and accelerates the dissociation of the radioligand. Studies of dissociation of [3H]suberyldicholine, a large bis-quaternary agonist, provide some clue as to the possible physical nature of these subsites. Whereas its dissociation rate was similar to that of [3H]acetylcholine (0.028 +/- 0.012 s(-1)), this rate was only marginally, if at all, affected by the presence of unlabeled ligands. These results, in addition to those presented in the accompanying manuscript, lead to the proposal that [3H]suberyldicholine is able to cross-link the two subsites or at least sterically occlude the second site.

Topics: Acetylcholine; Alkaloids; Animals; Binding Sites; Binding, Competitive; Bungarotoxins; Carbachol; Cell Membrane; Choline; Cholinergic Agonists; Dihydro-beta-Erythroidine; Kinetics; Ligands; Membrane Proteins; Protein Binding; Receptors, Cholinergic; Torpedo; Tubocurarine

The binding of suberyldicholine to membrane-bound Torpedo acetylcholine receptor has been monitored by fluorescence changes of covalently bound 5-iodoacetamidosalicylic acid (IAS). At equilibrium, suberyldicholine binds to two high-affinity binding sites (Kd approximately 20 nM). Kinetic experiments reveal that there is rapid formation of an initial complex (Kd approximately 2 microM) which undergoes sequential fast (k(app) approximately 1 s(-1)) and slow (k(app) approximately 0.05 s(-1)) conformational changes. These kinetics differ from those reported for other agonists [Blanchard, S. G., Dunn, S. M. J., & Raftery, M. A. (1982) Biochemistry 24, 6258-6264] in that, for suberyldicholine, there is no evidence for a second pathway involving the binding of an additional agonist molecule. These results, considered together with the observed dissociation kinetics (accompanying manuscript), suggest that each high-affinity site for acetylcholine is made up of two subsites, which suberyldicholine is able to bridge, thus occluding the binding of a second ligand. The kinetic mechanism for acetylcholine binding has been re-examined to accommodate the complexities of the [3H]-acetylcholine dissociation kinetics and the observation that, at equilibrium, no more than two occupied binding sites are detected [accompanying manuscript: Dunn, S. M. J., & Raftery, M. A. (1997) Biochemistry 36, 3846-3853]. It is suggested that, for each acetylcholine binding site, a second ligand is able to bind but that the ternary complex is transient since one of the two bound ligands again dissociates in the formation of the equilibrium mono-liganded complex. To further probe the physical nature of the two subsites, the binding of a series of bis-quaternary suberyldicholine analogues, (CH3)3N+CH2CH2OCO(CH2)n-COOCH2CH2N+(CH3)3, to IAS-labeled receptor preparations has been examined. Analogues in which n < 5 behave like acetylcholine, i.e., a second ligand binding pathway is observed, but longer ligands (n = 5-10) act like suberyldicholine and may be long enough to cross-link the sites.

Topics: Acetamides; Acetylcholine; Animals; Binding Sites; Cell Membrane; Choline; Cholinergic Agonists; Fluorescence; Fluorescent Dyes; Kinetics; Membrane Proteins; Protein Binding; Protein Conformation; Receptors, Cholinergic; Salicylates; Torpedo

1. This work continues our examination of the electrophysiology and contractions of single fibers dissociated from a widely studied molluscan muscle, the accessory radula closer (ARC) muscle of Aplysia californica, aimed at understanding its excitation-contraction mechanisms and their modulation. 2. Extensive previous work has characterized a number of basal ion currents present in the fibers and effects of transmitters and peptide cotransmitters that modulate ARC-muscle contractions in vivo. Here we use current clamp, voltage clamp, and contraction measurements to examine the actions of acetylcholine (ACh), the transmitter that induces the contractions. 3. As in the whole ARC muscle, ACh depolarizes unclamped fibers maximally to about -25 mV where, no matter how much ACh is applied, the depolarization saturates. 4. The underlying ACh-activated current is in fact the sum of two quite distinct components, IACh,cat and IACh,Cl. 5. IACh,cat is itself a mixed current carried by cations (physiologically mainly by Na+, but to a significant degree also by Ca2+), reverses near +20 mV, rectifies inwardly, exhibits prominent voltage-dependent kinetics of activation with hyperpolarization, and is selectively blocked by hexamethonium. 6. In contrast, IACh,Cl is carried by Cl-, reverses near -60 mV, exhibits little rectification or voltage-dependent kinetics, is activated selectively by suberyldicholine, and is blocked by alpha-bungarotoxin. 7. Both currents activate fast when ACh is applied, desensitize relatively slowly in its presence, then deactivate fast. Both currents are activated at similar ACh concentrations, half-maximally at approximately 10 microM. Both currents also are activated by carbachol and propionylcholine and blocked by d-tubocurarine, bicuculline and paraoxon. Picrotoxin and atropine block IACh,cat better, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and anthracene 9-carboxylic acid IACh,Cl better. 8. The two currents are virtually identical to ACh-activated cationic (Na) and Cl currents that are ubiquitous in molluscan neurons. As has been proposed for the neuronal currents, IACh,cat resembles vertebrate neuronal nicotinic ACh-receptor (nAChR) currents, whereas IACh,Cl resembles vertebrate skeletal muscle nAChR currents. 9. Functionally, we believe that IACh,cat serves primarily to depolarize the ARC muscle to open voltage-activated L-type Ca channels, allow Ca2+ in

Topics: Acetylcholine; Animals; Aplysia; Atropine; Bungarotoxins; Cations; Chloride Channels; Choline; Hexamethonium; Ion Channels; Membrane Potentials; Muscle Contraction; Neuromuscular Junction; Tubocurarine

Following a complete transection of the spinal cord at the Th12-L1, an augmented chemosensitivity of the nuclear bag and nuclear chain fibres was observed. The supersensitivity of the intrafusal fibres to subecholine was higher than to acetylcholine. The supersensitivity of the intrafusal muscle fibres against the background of intact anatomical projections suggests that the motor activity rather than axonal transport plays the leading role in regulation of the cholinergic sensitivity of intrafusal muscle fibres.

Topics: Acetylcholine; Animals; Cats; Choline; Decerebrate State; Motor Activity; Muscle Spindles; Muscle, Skeletal; Receptors, Cholinergic; Spinal Cord

Fluorescence spectroscopy was used to begin to localize the agonist inhibitory binding site on the nicotinic acetylcholine receptor (AcChR) from Torpedo californica. High concentrations of three cholinergic agonists, suberyldicholine (SubCh), acetylcholine (AcCh), and carbamylcholine (CCh), differentially inhibited the binding of two noncompetitive inhibitors (NCIs), quinacrine and ethidium, which bind at distinctly different loci on the desensitized AcChR at zero membrane potential. The agonist-induced inhibition of quinacrine binding occurred at significantly lower (17-fold) concentrations than the inhibition of ethidium binding. Schild plots of SubCh inhibition of ethidium and quinacrine binding showed the competitive nature of the agonist inhibition of the binding of these two NCIs. The quenching constants for short-range quenching of receptor-bound quinacrine and ethidium fluorescence by spin-labeled acetylcholine were about the same as their inhibition constants for agonist-induced displacement of AcChR-bound quinacrine and ethidium. The results demonstrate that agonists can directly bind to both the quinacrine and the ethidium binding sites, albeit at different agonist concentrations. Because the agonist-induced displacement of receptor-bound quinacrine occurs at significantly lower concentrations than the displacement of ethidium, the quinacrine binding site is more likely than the ethidium binding site to form part of the agonist inhibitory binding site.

Topics: Acetylcholine; Amphibian Venoms; Animals; Binding Sites; Binding, Competitive; Carbachol; Choline; Cholinergic Agonists; Cholinergic Antagonists; Ethidium; Quinacrine; Receptors, Cholinergic; Torpedo

1. The aim of these experiments was to determine the ability of the muscle-type nicotinic acetylcholine receptor (AChR) stably expressed in quail fibroblasts (QF18 cells) to elevate intracellular calcium ([Ca2+]i) upon activation. Ratiometric confocal microscopy, with the calcium-sensitive fluorescent dye Indo-1 was used. 2. Application of the nicotine agonist, suberyldicholine (SDC), to the transfected QF18 cells caused an increase in [Ca2+]i. Control [Ca2+]i levels in QF18 cells were found to be 164 +/- 22 nM (mean +/- s.e. mean; n = 40 cells) rising to 600 +/- 81 nM on addition of SDC (10 microM; n = 15 cells), whereas no increase in [Ca2+]i was seen in non-transfected control QT6 fibroblasts (before: 128 +/- 9 nM, n = 40; after; 113 +/- 13 nM, n = 15). 3. The increase in [Ca2+]i caused by application of SDC was dose-dependent, with an EC50 value of 12.7 +/- 5.9 microM (n = 14). 4. The responses to SDC in QF18 cells were blocked by prior application of alpha-bungarotoxin (200 nM), by the addition of Ca2+ (100 microM), by removal of Na+ ions from the extracellular solution, or by the voltage-sensitive calcium channel blockers nifedipine and omega-conotoxin, which act with IC50 values of 100 nM and 100 pM respectively. 5. We conclude that activation of the nicotinic AChRs leads to a Na(+)-dependent depolarization and hence activation of endogenous voltage-sensitive Ca2+ channels in the plasma membrane and an increase in [Ca2+]i. There is no significant entry of Ca2+ through the nicotinic receptor itself.

Topics: Animals; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Choline; Fibroblasts; Microscopy, Confocal; Nicotinic Agonists; Nifedipine; omega-Conotoxin GVIA; Peptides; Quail; Reproducibility of Results; Signal Transduction; Transfection

Oxotremorine methiodide (oxotremorine-M) is the quaternary amine derivative of oxotremorine and is known to be a potent and oft-reported pure, muscarinic receptor agonist. We report here, for the first time, that oxotremorine-M also has strong nicotinic actions at the single channel level. Although previous reports have suggested that oxotremorine-M has mixed cholinergic properties, its nicotinic actions have only been reported in systems which contain both muscarinic and nicotinic receptors, or in skeletal neuromuscular systems where the site of action of oxotremorine-M may have been ambiguous. We tested the possibility that oxotremorine-M is a nicotinic receptor agonist by examining the responses of single nicotinic acetylcholine receptors in primary cultures of myocytes from skeletal myotomes of Xenopus larvae. Myotomal myocytes are known to express the nicotinic acetylcholine receptor and no evidence exists that muscarinic receptors are expressed in these progenitors of the skeletal musculature. Furthermore, because we used aneural myocyte cultures, the effects of oxotremorine-M cannot be attributed to action on presynaptic receptors. Using cell-attached patches, we compared the responses of the nicotinic acetylcholine receptors to suberyldicholine and oxotremorine-M. Our results show that (1) both agonists activate the receptor channel in nanomolar concentrations; (2) the mean channel open-time is significantly smaller in oxotremorine-M; and (3) activation of the nicotinic acetylcholine receptor by oxotremorine-M is accompanied by a large percentage of short openings and a high frequency of event flickering. We conclude that oxotremorine-M is a mixed function agonist, showing partial blocking behavior, which effectively activates pure nicotinic acetylcholine receptors.

Topics: Animals; Cells, Cultured; Choline; Ion Channels; Membrane Potentials; Muscle, Skeletal; Oxotremorine; Receptors, Nicotinic; Xenopus laevis

Acetylcholine (ACh) is the major neurotransmitter released from the efferent fibers in the cochlea onto the outer hair cells (OHCs). The type of ACh receptor on OHCs and the events subsequent to receptor activation are unclear. Therefore we studied the effect of agonists and antagonists of the ACh receptor on isolated OHCs from the guinea pig. OHCs were recorded from in whole cell voltage and current clamp configuration. ACh induced an increase in outward K+ current (IACh) which hyperpolarized the OHCs. No desensitization to ACh application was observed. Cs+ replaced K+ in carrying the IACh. The IACh is Ca(2+)-dependent, time and voltage sensitive, and different from the IKCa induced by depolarization of the membrane potential. When tested at 100 microM, several agonists also induced outward current responses (acetylcholine > suberyldicholine > or = carbachol > DMPP) whereas nicotine, cytisine and muscarine did not. The IACh response to 10 microM ACh was blocked by low concentrations of traditional and non-traditional-nicotinic antagonists (strychnine > curare > bicuculline > alpha-bungarotoxin > thimethaphan) and by higher concentrations of muscarinic antagonists (atropine > 4-DAMP > AF-DX 116 > pirenzepine). Pharmacologically, the ACh receptor on OHCs is nicotinic.

Topics: Acetylcholine; Alkaloids; Animals; Atropine; Azocines; Bungarotoxins; Carbachol; Choline; Cholinergic Antagonists; Curare; Dimethylphenylpiperazinium Iodide; Guinea Pigs; Hair Cells, Auditory, Outer; In Vitro Techniques; Membrane Potentials; Nicotine; Piperidines; Pirenzepine; Quinolizines; Receptors, Cholinergic; Strychnine; Trimethaphan

Ethanol is known to cause a leftward shift of the acetylcholine concentration-response curve for channel opening of the nicotinic acetylcholine receptor (nAcChoR). However, it remains uncertain whether the mechanism underlying ethanol's effect is an increase in the binding affinity of the agonist to the receptor or an increase in the open/closed equilibrium for those receptors occupied by agonist. In the present study, this question was resolved by measuring the efflux of 86Rb+ over 9 msec from Torpedo vesicles after rapid mixing with the partial agonist suberyldicholine with or without ethanol as appropriate. Suberyldicholine's concentration-response curve is bell-shaped. Two actions underlie this bell-shaped curve, namely activation at low concentration (apparent dissociation constant for activation, Ka = 38 microM) and self-inhibition at higher concentration (apparent dissociation constant for inhibition, Kb = 9 mM), but the overlap of these two actions only reduces the maximum observable flux by 20%. Increasing ethanol concentration from 0 to 0.9 M causes: a linear increase in the maximum response of the nAcChoR to suberyldicholine from 5 to 80% of the maximum induced by acetylcholine, a moderate increase in Ka, and no change in Kb. Analysis of our results using the sequential two-site binding model revealed that the main action of ethanol on nAcChoR was to increase the fraction of occupied receptors that open. The equilibrium constant describing this effect changed by 8-fold at anesthetic concentrations. Ethanol also decreased the affinity of suberyldicholine for its self-inhibition site by a comparable amount, suggesting that its main action is to stabilize the open state. In addition, ethanol caused a small increase in suberyldicholine's affinity for the agonist site.

Topics: Animals; Choline; Ethanol; In Vitro Techniques; Receptors, Nicotinic; Rubidium; Torpedo

The action of some nicotinic acetylcholine receptor agonists was re-examined on the surface field potentials (N-waves) evoked by electrical stimulation of the lateral olfactory tract in guinea-pig olfactory cortical brain slices. Bath superfusion of nicotine or the nicotinic stimulants dimethylphenylpiperazinium (DMPP), lobeline, cytisine, tetramethylammonium or suberyldicholine (up to 100 microM) had little or no effect on the extracellular N-wave amplitude, or the membrane potential, input resistance or excitability of olfactory neurones recorded intracellularly. In contrast, the muscarinic agonists, carbachol or oxotremorine-M consistently depressed the field in a reversible dose-dependent manner. Interestingly, in the presence of the ganglionic stimulants DMPP (n = 6 slices) or lobeline (n = 5 slices) (10-50 microM), the effects of carbachol or oxotremorine-M were antagonized in a weak competitive-type manner (pA2 values = 5.58 and 5.63 respectively, estimated from Schild plots, constrained to unity slope). This anti-muscarinic action was unaffected by d-tubocurarine or hexamethonium. Nicotine, cytisine, tetramethylammonium and suberyldicholine showed much weaker and inconsistent carbachol-blocking effects. Combination of DMPP with atropine produced dose ratio shifts close to those predicted for a common-site interaction of two competitive antagonists. In conclusion, consistent pre- or postsynaptic nicotinic agonist actions could not be detected in olfactory cortex slices; however, some ganglionic nicotinic agonists were shown to exhibit significant anti-muscarinic effects on this preparation. We suggest this action might be due to a direct atropine-like mechanism.

Topics: Alkaloids; Animals; Azocines; Carbachol; Choline; Dimethylphenylpiperazinium Iodide; Electric Stimulation; Ganglionic Stimulants; Guinea Pigs; In Vitro Techniques; Lobeline; Olfactory Bulb; Oxotremorine; Parasympatholytics; Quaternary Ammonium Compounds; Quinolizines

We studied desensitization of nicotinic acetylcholine (ACh) receptor channels in the clonal BC3H-1 cell line. We measured the current response to rapid perfusion of outside-out patches with 1 microM to 5 mM ACh, carbamylcholine and suberyldicholine. After binding to the receptors and opening the ion channels, all agonists induce a rapid, concentration-dependent decay of channel activity. The time constant of the current decay ranged from several seconds at low agonist concentrations to about 50 ms at saturating concentrations. The decay rate at saturating concentrations was independent of voltage. The ratio of steady-state to peak current ranged from 0.5 at low agonist concentrations to 0.02 or less at high concentrations. The rate of recovery from desensitization after removal of agonist was also measured. For ACh, the recovery time constant was 320 ms; recovery from desensitization by carbamylcholine was twice as fast. A linear kinetic results. The data are consistent with a cyclic model, although, it is not possible to uniquely determine all of the rate constants in this scheme. The results are compared with competitive binding and single channel studies of desensitization in BC3H-1 cells.

Topics: Carbachol; Cells, Cultured; Choline; Receptors, Cholinergic

1. Single-ion-channel recording has been used to estimate the equilibrium concentration-response relationship for several acetylcholine analogues. The response, corrected for desensitization, was taken as the probability of a channel being open during clusters of openings that were separated by desensitized periods. 2. All agonists were able to block the channels which they themselves opened. Carbachol, suberyldicholine and the sulphonium analogue of acetylcholine were all found to be efficacious agonists in the sense that the results indicate that all of them, in sufficiently high concentration, would be able to open 90% or more of channels if it were not for channel block. 3. In the case of suberyldicholine the results are much as predicted by the interpretation of the fine structure of channel openings at low agonist concentrations. 4. The maximum probability of opening that could be obtained with decamethonium and with phenyltrimethylammonium was low (below 4%), and it was not possible to distinguish whether this was wholly a result of the powerful (relative to activation potency) channel-blocking action of these agonists, or whether it was to some extent attributable to their being genuine partial agonists. 5. The results suggest that, for a range of agonists, differences in equilibrium potency are usually more strongly influenced by affinity for binding to the resting state of the receptor than by ability to activate the receptor once bound, though in the case of suxamethonium (relative to acetylcholine) the contributions of each factor are similar.

Topics: Acetylcholine; Animals; Carbachol; Choline; Decamethonium Compounds; In Vitro Techniques; Ion Channels; Motor Endplate; Quaternary Ammonium Compounds; Rana temporaria; Tetraethylammonium; Tetraethylammonium Compounds

1. Limnaea stagnalis neurones have been used to study the functioning of membrane receptor-channel complexes. The experiments were performed using a fixed membrane potential (E) and the intracellular perfusion technique. The cells employed responded to acetylcholine (ACh) by changing only their Cl- conductance. 2. ACh-induced currents, their fluctuations and relaxations resulting from a jump of E were studied. 3. The following facts have been established based on analysis of ACh currents, their fluctuations and relaxations: (1) the characteristic time of the exponential decay of the autocorrelation function, tau N, is in the range of 15-20 ms; (2) the characteristic relaxation time, tau R, equals 50-60 ms (ACh concentration = 0.25 microM, desensitization is not observed); (3) E does not exert any functionally significant effect upon tau N or tau R which could have governed the non-linearity of the membrane voltage-current characteristic; (4) variation of ACh concentration from 0.25 to 1 microM has a significant effect on tau R but not on tau N; (5) lowering of the ACh solution temperature from 22 to 8.5 degrees C results in a 20% increase of the ACh current, a 3- to 4-fold decrease of the single-channel conductance (gamma), a 20% increase in tau N and a 3- to 4-fold increase in tau R. 4. The suberylcholine (SCh)-induced membrane current has approximately the same value as the ACh-induced current at equal concentrations of ACh and SCh (0.25 microM); the tau N and gamma values were also quite close, but tau R was 2.3 times lower for SCh than for ACh. 5. An essentially two-stage scheme of functioning of membrane receptor-channel complexes is proposed. The scheme has two distinguishable and measurable stages and involves five closed states and one open state; it offers an explanation for our experimental data as well as the results of other workers.

Topics: Acetylcholine; Animals; Chloride Channels; Choline; Electric Conductivity; Ion Channels; Kinetics; Membrane Potentials; Membrane Proteins; Models, Biological; Models, Theoretical; Mollusca; Neurons; Receptors, Cholinergic

The effects of acetylcholine, subecholine and succinylcholine on the afferent activity of the m.EDL muscle spindles were compared before and after suppression of axonal transport by colchicine (20 nM, 30 min). The colchicine increased cholinergic sensitivity of nuclear-bag fibres: i. a. administration of cholinergic substances at concentrations tenfold lower than in control animals, produced a sharp enhancement of afferent activity of the muscle spindles. The increase of chemosensitivity was maximal to Ach and less obvious for subecholine and succinylcholine. No increase of activity could be produced by cholinomimetics in afferents originating from nuclear-chain fibres either in control, or after suppression of axonal transport.

Topics: Acetylcholine; Afferent Pathways; Animals; Axonal Transport; Cats; Choline; Colchicine; Dose-Response Relationship, Drug; Muscle Spindles; Succinylcholine

Patch-clamp technique in cell-attached configuration was applied to investigate ion permeability induced by suberyldicholine in the neurons of freshwater mollusc Planorbarius corneus. The inward currents through single channels were registered at patch potentials 50-100 mV more negative than resting potential of the cell. When the patch pipette contained suberyldicholine (5 mol/l), single channel currents were recorded which grouped in bursts and bursts of openings could themselves be grouped together in clusters of bursts. The desensitisation was not too pronounced: usually we were able to observe single-channel currents for 20-30 minutes. The unit conductance gamma was found to be about 10 pS.

Topics: Animals; Chlorides; Choline; In Vitro Techniques; Ion Channels; Mollusca; Neurons; Receptors, Cholinergic

1. Nicotinic acetylcholine receptors (nAChR)4 from BC3H1 cells (which express a skeletal muscle-type receptor) and from Torpedo californica electric organ were expressed in Xenopus laevis oocytes and studied with a voltage-clamp technique. 2. We found that bath application of ATP in the micromolar to millimolar range increased the ACh-elicited current in both muscle and electrocyte receptors. The effect of ATP increased with successive applications. This "use-dependent" increase in potentiation was Ca2+ dependent, while the potentiation itself was not. 3. Four other nucleotides were tested on muscle nAChR: ADP, AMP, adenosine, and GTP. Of these, only ADP was a potentiator, but its effect was not use dependent. Neither ATP nor ADP affected the resting potential of the oocyte membrane. 4. ADP potentiated the response to suberyldicholine and nicotine, as well as ACh. 5. Finally, ADP reversed the phencyclidine-induced block of ACh currents in oocytes expressing muscle nAChR.

Topics: Acetylcholine; Adenine Nucleotides; Animals; Calcium; Choline; Drug Interactions; Mice; Nicotine; Oocytes; Phencyclidine; Receptors, Nicotinic; Recombinant Proteins; Torpedo; Xenopus laevis

Topics: Adenosine; Adenosine Triphosphate; Animals; Choline; Dipyridamole; Electric Stimulation; Ileum; In Vitro Techniques; Muscle Relaxation; Myenteric Plexus; Neurons; Purinergic Antagonists; Rats; Receptors, Purinergic

In human myotubes cultured from biopsies of normal subjects and dystrophic patients we investigated, with the patch-clamp technique, the activation properties of the nicotinic acetylcholine receptor (AChoR) in the presence of acetylcholine and suberyldicholine. The single-channel conductance and the lifetime of the openings were not found to differ. In contrast, the average frequency of openings was about four times higher in Duchenne muscular dystrophy (DMD) myotubes in the presence of equal amounts of acetylcholine, but not of suberyldicholine. The most reasonable conclusion from this observation is that the behaviour of the AChoR is not altered in DMD cells but that there is a greater average concentration of ACho molecules present around AChoRs. This leads to the tentative conclusion that the activity of the enzyme acetylcholinesterase (AChoE) is impaired by some unknown mechanism in the dystrophic myotube.

Topics: Acetylcholine; Cells, Cultured; Child; Child, Preschool; Choline; Electric Conductivity; Humans; Ion Channels; Kinetics; Membrane Potentials; Muscles; Muscular Dystrophies; Receptors, Nicotinic; Reference Values

The effect of preliminary injection of the inhibitor of protein synthesis actinomycin D on development of extrajunctional cholinergic receptors in denervated intrafusal muscle in cats, was investigated. Chronic motor denervation of the soleus muscle (3-5 days after denervation) caused supersensitivity of intrafusal muscle fibres to acetylcholine (Ah), subecholine (SCh) and succinylcholine (Sh). Injections of actinomycin D prevented the supersensitivity, the latter being dependent on the synthesis of new cholinoreceptors and their incorporation in the extrajunctional region of intrafusal fibres.

Topics: Acetylcholine; Animals; Cats; Choline; Dactinomycin; Denervation; Muscle Spindles; Receptors, Cholinergic; Succinylcholine; Time Factors

Agonist concentration-response relationships at nicotinic postsynaptic receptors were established by measuring 86Rb+ efflux from acetylcholine receptor rich native Torpedo membrane vesicles under three different conditions: integrated net ion efflux (in 10 s) from untreated vesicles, integrated net efflux from vesicles in which most acetylcholine sites were irreversibly blocked with alpha-bungarotoxin, and initial rates of efflux (5-100 ms) from vesicles that were partially blocked with alpha-bungarotoxin. Exposure to acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, or (-)-nicotine over 10(8)-fold concentration ranges results in bell-shaped ion flux response curves due to stimulation of acetylcholine receptor channel opening at low concentrations and inhibition of channel function at 60-2000 times higher concentrations. Concentrations of agonists that inhibit their own maximum 86Rb+ efflux by 50% (KB values) are 110, 211, 3.0, 39, and 8.9 mM, respectively, for the agonists listed above. For acetylcholine and carbamylcholine, KB values determined from both 10-s and 15-ms efflux measurements are the same, indicating that the rate of agonist-induced desensitization increases to maximum at concentrations lower than those causing self-inhibition. For all partial and full agonists studied, Hill coefficients for self-inhibition are close to 1.0. Concentrations of agonists up to 8 times KB did not change the order parameter reported by a spin-labeled fatty acid incorporated in Torpedo membranes. We conclude that agonist self-inhibition cannot be attributed to a general nonspecific membrane perturbation. Instead, these results are consistent with a saturable site of action either at the lipid-protein interface or on the acetylcholine receptor protein itself.

Topics: Acetylcholine; Animals; Carbachol; Choline; Electric Organ; Electron Spin Resonance Spectroscopy; Isoflurophate; Kinetics; Nicotine; Quaternary Ammonium Compounds; Receptors, Nicotinic; Rubidium; Synaptic Membranes; Torpedo

The patch-clamp technique was used to examine the activation of single acetylcholine receptor channels of clonal BC3H-1 mouse muscle cells. Single-channel currents were activated by low concentrations of the strong agonists acetylcholine (ACh, 50-100 nM), carbamylcholine (1-2 microM), and suberyldicholine (30-50 nM). At low agonist concentrations channel openings occur as isolated short-duration openings and as bursts of longer duration openings separated by brief closed periods. Two distinct types of brief closed periods separate long duration openings: brief closures (mean duration, 50 microseconds) and intermediate closures (mean duration, 0.5-1.0 ms). The kinetic properties of intermediate closures depend on the agonist, suggesting that they reflect receptor reopening from the closed state leading to the open state. Properties of brief closures, in contrast, are independent of the agonist, indicating that they result from an additional closed state leading away from the pathway producing the open state. A receptor activation scheme is proposed which accounts for the observed closed states, and transition rate estimates are presented for steps within the proposed scheme. The channel opening rate, beta, differs several-fold for the agonists studied (200-1400 s-1) and is comparable to the dissociation rate, k-2 (900 s-1). The dissociation rate is similar for the three agonists studied. The channel closing rate, alpha, is much slower than the opening rate (20-60 s-1). The probability is high that a doubly liganded channel is in the open state and depends on the agonist (0.75-0.97). Beta increases and alpha decreases at more negative membrane potentials, whereas k-2 shows little potential dependence.

Topics: Acetylcholine; Action Potentials; Animals; Carbachol; Choline; Clone Cells; Ion Channels; Kinetics; Mathematics; Mice; Receptors, Cholinergic; Time Factors

Three nicotinic agonists, suberyldicholine, acetylcholine and carbachol, have been investigated by single channel recording at the endplates of adult frog muscle fibres. All three agonists can block the channels that they open. Suberyldicholine is the most potent blocker; it has an equilibrium constant for binding to the open channel of about 6 microM and blockages last for about 5 ms on average, at -105 mV. A plot of the mean number of blockages per unit open time against concentration ('blockage frequency plot') suggests that suberyldicholine does not produce long-lived blocked states such as might occur, for example, if it could be trapped within a shut channel. The characteristics of the 'blockage frequency plot' are analysed in Appendix 2. Block by acetylcholine and carbachol has much lower affinity (the equilibrium constants being a few millimolar for both), and blockages are much briefer, so that blockage appears to produce noisy single channel currents of reduced amplitude. A method based on the spectral density of the excess 'open' channel noise has been used to investigate the rate of blocking and unblocking. The basis of this method is discussed in Appendix 1. It is estimated that the mean duration of a blockage is about 18 microseconds for acetylcholine and 9 microseconds for carbachol.

Topics: Acetylcholine; Animals; Carbachol; Choline; Dose-Response Relationship, Drug; Electrophysiology; Ion Channels; Kinetics; Mathematics; Membrane Potentials; Neuromuscular Junction; Rana temporaria

The fine structure of ion-channel activations by junctional nicotinic receptors in adult frog muscle fibres has been investigated. The agonists used were acetylcholine (ACh), carbachol (CCh), suberyldicholine (SubCh) and decan-1,10-dicarboxylic acid dicholine ester (DecCh). Individual activations (bursts) were interrupted by short closed periods; the distribution of their durations showed a major fast component ('short gaps') and a minor slower component ('intermediate gaps'). The mean duration of both short and intermediate gaps was dependent on the nature of the agonist. For short gaps the mean durations (microseconds) were: ACh, 20; SubCh, 43; DecCh, 71; CCh, 13. The mean number of short gaps per burst were: ACh, 1.9; SubCh, 4.1; DecCh, 2.0. The mean number of short gaps per burst, and the mean number per unit open time, were dependent on the nature of the agonist, but showed little dependence on agonist concentration or membrane potential for ACh, SubCh and DecCh. The short gaps in CCh increased in frequency with agonist concentration and were mainly produced by channel blockages by CCh itself. Partially open channels (subconductance states) were clearly resolved rarely (0.4% of gaps within bursts) but regularly. Conductances of 18% (most commonly) and 71% of the main value were found. However, most short gaps were probably full closures. The distribution of burst lengths had two components. The faster component represented mainly isolated short openings that were much more common at low agonist concentrations. The slower component represented bursts of longer openings. Except at very low concentrations more than 85% of activations were of this type, which corresponds to the 'channel lifetime' found by noise analysis. The frequency of channel openings increased slightly with hyperpolarization. The short gaps during activations were little affected when (a) the [H+]o or [Ca2+]o were reduced to 1/10th of normal, (b) when extracellular Ca2+ was replaced by Mg2+, (c) when the [Cl-]i was raised or (d) when, in one experiment on an isolated inside-out patch, the normal intracellular constituents were replaced by KCl. Reduction of [Ca2+]O to 1/10 of normal increased the single-channel conductance by 50%, and considerably increased the number of intermediate gaps. No temporal asymmetry was detectable in the bursts of openings. Positive correlations were found between the lengths of successive apparent open times at low SubCh concentrations, but no correlation

Topics: Acetylcholine; Action Potentials; Animals; Calcium; Carbachol; Chlorides; Choline; Dicarboxylic Acids; Hydrogen-Ion Concentration; In Vitro Techniques; Ion Channels; Motor Endplate; Neuromuscular Junction; Rana temporaria; Time Factors

Photoaffinity labeling of membrane-bound nicotinic acetylcholine receptor from Torpedo marmorata electric tissue with the ion-channel blocker [3H]TPMP+ reveals various functional states of the receptor protein if labeling is performed with ms time resolution. In the resting and in the activated state most of the label is incorporated into the alpha-polypeptide chains of the receptor complex. When equilibrated with agonists and antagonists, predominantly the delta-polypeptide chain (and to a lesser extent the beta-chain) reacts with the photolabel. Reactivity of the delta-chain increases after exposure to cholinergic effectors with a half-life slower than the kinetics of receptor activation or rapid desensitization. Agonists and antagonists stimulate photolabelling of the delta-chain with different kinetics. For acetylcholine, carbamoylcholine and suberyldicholine the half-life of the reactivity increases is 400 - 500 ms; for the antagonists hexamethonium, d-tubocurarine and flaxedil it is about 10 s. The latter slow kinetics are also observed when the receptor is preequilibrated with agonists or antagonists prior to mixing with [3H]TPMP+ and starting the photoreaction. We conclude that time-resolved photoaffinity labeling can convalently mark protein structures involved in receptor functions. Of special interest is the observation that antagonists also induce a conformational change in the receptor protein.

Topics: Acetylcholine; Affinity Labels; Animals; Carbachol; Chemical Phenomena; Chemistry; Choline; Electric Organ; Gallamine Triethiodide; Hexamethonium; Hexamethonium Compounds; In Vitro Techniques; Ion Channels; Kinetics; Lasers; Onium Compounds; Photochemistry; Protein Conformation; Receptors, Cholinergic; Torpedo; Trityl Compounds; Tubocurarine

In acute experiments on cats, an isolated portion of the small intestine was vascularly perfused with arterial blood by means of the constant volume perfusion pump. Contractile activity of the ileal and/or jejunal segment was estimated by means of isometric tension. Activation of the myenteric cholinergic neurons as the result of the infusion of nicotinic cholinoreceptor agonist subecholine or electrical stimulation of the vagal efferents was followed by increased contractile response of intestinal smooth muscle to serotonin (2.5 X 10(-8) or 5.0 X 10(-9) mol). The same effect was observed after increasing endogenous acetylcholine concentration induced with application of anticholinesterase drug prozerin. The data obtained suggested that the modulating effect of acetylcholine might be of a certain physiological importance.

Topics: Animals; Cats; Choline; Cholinergic Fibers; Drug Interactions; Gastrointestinal Motility; In Vitro Techniques; Intestine, Small; Neostigmine; Receptors, Cholinergic; Serotonin; Vagus Nerve

We studied activation of the nicotinic acetylcholine (ACh) receptor on cells of a mouse clonal muscle cell line (BC3H1). We analyzed single-channel currents through outside-out patches elicited with various concentrations of acetylcholine (ACh), carbamylcholine (Carb) and suberyldicholine (Sub). Our goal is to determine a likely reaction scheme for receptor activation by agonist and to determine values of rate constants for transitions in that scheme. Over a wide range of agonist concentrations the open-time duration histograms are not described by single exponential functions, but are well-described by the sum of two exponentials, a brief-duration and a long-duration component. At high concentration, channel openings occur in groups and these groups contain an excess number of brief openings. We conclude that there are two open states of the ACh receptor with different mean open times and that a single receptor may open to either open state. The concentration dependence of the numbers of brief and long openings indicates that brief openings do not result from the opening of channels of receptors which have only one agonist molecule bound to them. Closed-time duration histograms exhibit a major brief component at low concentrations. We have used the method proposed by Colquhoun and Sakmann (1981) to analyze these brief closings and to extract estimates for the rates of channel opening (beta) and agonist dissociation (k-2). We find that this estimate of beta does not predict our closed-time histograms at high agonist concentration (ACh: 30-300 microM; Carb: 300-1,000 microM). We conclude that brief closings at low agonist concentrations do not result solely from transitions between the doubly-liganded open and the doubly-liganded closed states. Instead, we postulate the existence of a second closed-channel state coupled to the open state.

Topics: Acetylcholine; Animals; Carbachol; Cell Line; Choline; Clone Cells; Ion Channels; Kinetics; Mathematics; Mice; Models, Biological; Muscles; Receptors, Nicotinic

We have examined the effects of high concentrations of cholinergic agonists on currents through single acetylcholine receptor (AChR) channels on clonal BC3H1 cells. We find that raised concentrations of acetylcholine (ACh; above 300 microM) or carbamylcholine (Carb; above 1,000 microM) produce a voltage- and concentration-dependent reduction in the mean single-channel current. Raised concentrations of suberyldicholine (Sub; above 3 microM) produce a voltage- and concentration-dependent increase in the number of brief duration low-conductance interruptions of open-channel currents. These observations can be quantitatively described by a model in which agonist molecules enter and transiently occlude the ion-channel of the AChR.

Topics: Acetylcholine; Animals; Carbachol; Cell Line; Choline; Clone Cells; Electric Conductivity; Membrane Potentials; Mice; Receptors, Cholinergic

Does the acetylcholine receptor have a specific regulatory (inhibitory) site for the natural receptor ligand acetylcholine? This paper deals with this question. The inhibition of acetylcholine-receptor function by diverse organic cations including local anesthetics such as procaine has been well documented. Evidence indicates that these compounds are noncompetitive inhibitors, enter the open-channel form of the receptor, and block it and that the extent of this blockage depends on the transmembrane voltage of the cell. Recently we reported that in the electroplax of Electrophorus electricus the receptor-controlled transmembrane ion flux is inhibited by acetylcholine in a voltage-dependent, noncompetitive manner. We report here that the Torpedo californica receptor also has an inhibitory site for acetylcholine. The question of whether acetylcholine, which is an organic cation, binds to the same site as other organic cations such as the noncompetitive inhibitor procaine is important and is addressed. The results reported here of chemical kinetic investigations, with receptor-rich E. electricus and T. californica membrane vesicles, indicate that the inhibition of receptor function by acetylcholine and by a local anesthetic, procaine, involves two different receptor sites. The existence of a specific inhibitory site for the natural receptor-ligand acetylcholine suggests that this site can play an important role in the modulation of receptor function and in the regulation of transmission of signals between cells.

Topics: Acetylcholine; Animals; Binding Sites; Choline; In Vitro Techniques; Kinetics; Procaine; Receptors, Cholinergic; Torpedo

The channel of the purified acetylcholine receptor from Torpedo californica electric organ reconstituted in lipid vesicles was assayed by direct electrical recording using patch-clamp pipets. High-resistance seals were obtained by gentle suction of vesicles into the pipet or after the formation of lipid bilayers from monolayers at the tip of the pipet. Single-channel currents were activated by three cholinergic ligands: acetylcholine, carbamylcholine, and suberyldicholine. The single-channel conductance, gamma, was 40 +/- 5 pS in 0.5 M NaCl, irrespective of the agonist used. The distributions of channel open times were fitted by a sum of two exponentials. The lifetimes of the two exponential components were a factor of 2 longer for suberyldicholine than for acetylcholine or carbamylcholine. At desensitizing concentrations of agonists the single events appeared in paroxysms of channel activity followed by quiescent periods. These results suggest that the full cycle of solubilization, purification, and reconstitution of this membrane receptor can be achieved without impairment of channel function.

Topics: Acetylcholine; Animals; Carbachol; Choline; Electric Conductivity; Electric Organ; Ion Channels; Lipid Bilayers; Methods; Receptors, Cholinergic; Torpedo

The efficacy of acetylcholine, carbachol and suberyldicholine at the frog neuromuscular junction was estimated by single channel recording. The probability of channel opening at high concentration was found to be greater than 0.9 for each, thus showing (a) that their efficacy is high and (b) that the opening rate for the fully-liganded channel is higher than previously thought.

Topics: Acetylcholine; Animals; Carbachol; Choline; Electrophysiology; Ion Channels; Neuromuscular Junction; Rana temporaria

Furosemide (2.10(-4) to 1.10(-3) g/ml) was shown to prevent the increase of chloride conductance induced in isolated neurons of freshwater mollusc Planorbarius corneus by ionophoretic application of acetylcholine, suberyldicholine or gamma-aminobutyric acid. Furosemide caused no shift in the reversal potential of the chloride-dependent responses if the experiments were carried out employing microelectrode filled with potassium sulphate. When potassium chloride-filled microelectrodes were used, the reversal potential became less negative in the presence of furosemide. This effect seems to be due to the blocking of the active transport of chloride by furosemide. Neither sodium-dependent, nor potassium-dependent responses induced by cholinoreceptor activation were influenced by furosemide. Furosemide was found to diminish both the amplitude and the rate of chloride-dependent responses. The lack of selectivity with respect to acetylcholine or gamma-aminobutyric acid suggests that furosemide blocks the chloride channels of chemoceptive membrane which are probably common to acetylcholine and gamma-aminobutyric acid and does not affect their receptors.

Topics: Acetylcholine; Animals; Cell Membrane Permeability; Chlorides; Choline; Furosemide; GABA Antagonists; Ganglia; In Vitro Techniques; Ion Channels; Neurons; Snails

A voltage-clamp study was made of some properties of the non-synaptic hyperpolarization-activated Cl- conductance recently described in Aplysia neurones loaded with Cl- ions (Chesnoy-Marchais, 1982). The experiments were performed on an identified family of neurones, which present cholinergic responses allowing an easy measurement of the equilibrium potentials of Cl- (ECl) and K+ ions (EK). The Cl- selectivity of the hyperpolarization-activated conductance was deduced from four observations: (1) the extrapolated reversal potential of the hyperpolarization-activated current, Er, was close to the reversal potential of the cholinergic Cl- response, which is the equilibrium potential for Cl- ions, ECl. (2) Modifications of the intracellular or extracellular Cl- concentration induced changes of the reversal potential Er. (3) A prolonged and intense activation of the current lowered the intracellular Cl- concentration. (4) The current persisted after complete substitution of intracellular and extracellular cations by CS+ ions, as well as after replacement of extracellular Na+ ions by Tris. The steady-state Cl- conductance (gss) increases steeply with hyperpolarization. The kinetics of activation and deactivation are exponential and are characterized by the same voltage-dependent time constant (tau), of the order of a few seconds or fractions of seconds. The curves gss(V) and tau (V) can both be fitted by a two-state model in which the rate constants are exponential functions of the membrane potential (e-fold change for 12-16 mV). The Cl- current is much more affected by changes of the intracellular Cl- concentration than predicted simply from the change in Cl- driving force. Both the conductance and the time constant of activation are strongly modified. Modifications of the extracellular Cl- concentration do not always alter the amplitude of the hyperpolarization-activated Cl- current, but systematically affect its kinetics. The hyperpolarization-activated current is abolished after prolonged exposure of the cell to an artificial sea water where NO3- ions replace Cl- ions, as well as after intracellular injections of NO3- ions. Increasing the external pH shifts the gss(V) and tau (V) curves to the left. Lowering the external pH has reverse but less pronounced effects. In cells which were not loaded with Cl- ions and did not present the hyperpolarization-activated Cl- current, this current could be detected if the hyperpolarizing jump was preceded by short depol

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Aplysia; Cesium; Chlorides; Choline; In Vitro Techniques; Isethionic Acid; Mannitol; Membrane Potentials; Neurons; Nitrates; Nystatin

Single cholinergic receptor channel currents activated by curare were recorded in tissue-cultured embryonic rat muscle, directly confirming curare's action as a weak cholinergic agonist. In embryonic muscle, curare, in addition to its classical action as a competitive cholinergic antagonist, produces small sustained depolarizations which can be blocked by alpha-bungarotoxin. The single-channel events are of short duration but otherwise exhibit the major features observed with other cholinergic agonists. The single-channel events are blocked by alpha-bungarotoxin. Two values of unit conductance, 30.4 +/- 3.5 pS and 47 +/- 6 pS, were measured in cells from different cultures. Histograms of open-state duration are well fit by a distribution which is a sum of two exponentials, with time constants of 0.33 +/- 0.08 msec for the fast component and 1.84 +/- 0.43 msec for the slow component.

Topics: Action Potentials; Animals; Bungarotoxins; Carbachol; Choline; Culture Techniques; Curare; Drug Interactions; Embryo, Mammalian; Muscles; Neuromuscular Depolarizing Agents; Rats; Receptors, Cholinergic; Succinylcholine

Suberyldicholine-induced ion translocation in the millisecond time region in acetylcholine receptor rich membrane vesicles prepared from the electric organ of Electrophorus electricus was investigated in eel Ringer's solution, pH 7.0, 1 degree C. A quench-flow technique with a time resolution of 5 ms was used to measure the transmembrane flux of a radioactive tracer ion (86Rb+). JA, the rate coefficient for ion flux mediated by the active form of the receptor, and alpha, the rate coefficient for the inactivation of the ion flux, increase with increasing suberyldicholine concentrations and reach a plateau value at about 15 microM. At higher suberyldicholine concentrations (greater than 50 microM), a concentration-dependent decrease in the ion flux rate was observed without a corresponding decrease in the rate of receptor inactivation. This regulatory effect was not observed with acetylcholine or carbamoylcholine. The minimal kinetic scheme previously presented for acetylcholine and carbamoylcholine, modified by the inclusion of an additional regulatory ligand-binding site for suberyldicholine and characterized by a single dissociation constant, KR, is consistent with the results obtained over a 10 000-fold concentration range of this ligand. Rate and equilibrium constants pertaining to this scheme were elucidated. Suberyldicholine binds to the regulatory site (KR = 500 microM) approximately 100-fold less well than to its activating sites, and the binding to the regulatory site has no effect on the inactivation (desensitization) rate coefficient alpha [alpha(max) = 5.7 s-1], which is comparable to that observed with acetylcholine. The maximum influx rate coefficient [JA(max) = 18.5 s-1] is approximately twice that obtained when carbamoylcholine is the activating ligand and somewhat higher than when acetylcholine is used.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding Sites; Biological Transport; Choline; Dose-Response Relationship, Drug; Electric Organ; Electrophorus; Mathematics; Receptors, Cholinergic; Rubidium

Single cholinergic channel currents were recorded in adult human muscle tissue culture. The agonists suberyldicholine and carbamylcholine produce channels with the same conductance as channels produced by acetylcholine but with different closing kinetics. The antagonist tubocurarine, alone or mixed with suberyldicholine, activates channels which close very rapidly. For agonist-activated channels, the distribution of open state lifetimes shows deviations from the usual single exponential form. An excess of short duration openings indicates the presence of an additional faster kinetic process. The lifetime distribution data can be interpreted in terms of varying proportions of slow and fast components which are present in a ratio determined by curve-fitting the appropriate two-exponential function to observed open time distributions. This ratio shows great variability in muscle from older cultures, but the fast and slow time constants are relatively constant. The observation of double exponential open time distributions indicates that the mechanism of channel closing is more complicated than earlier evidence indicated.

Topics: Acetylcholine; Adult; Carbachol; Cells, Cultured; Choline; Electric Conductivity; Humans; Ion Channels; Muscles; Receptors, Cholinergic

Topics: Acetylcholine; Animals; Carbachol; Cell Membrane; Choline; Electrophorus; Ion Channels; Kinetics; Potassium; Receptors, Cholinergic; Sodium

Current responses to acetylcholine (ACh) and to suberyldicholine (D-6) applied from the double-barrelled ionophoretic micropipette were studied in two identified neurones (LPed-2 and LPed-3) isolated from the left ganglion of pulmonate mollusc, Planorbarius corneus. Experiments made with K2SO4-filled microelectrodes show that in LPed-2 neurone two kinds of cholinoreceptors are involved in the rapid phase of ACh response one of which induces chloride conductance and the other, sodium conductance. The Cl-dependent component can be separated from the cationic one by C-6 whereas the cationic component can be separated from the Cl--dependent one by furosemide. Cl- conductance can be induced selectively by D-6. In the LPed-3 neurone only Cl- conductance increases during rapid phase of ACh response. The reversal potential of Cl--dependent responses was found to be more negative than the resting potential in experiments made with K2SO4-filled microelectrodes but less negative than the resting potential in the case of KCl-filled microelectrodes. This difference seems to be due to the artificial increase of intracellular chloride concentration.

Topics: Acetylcholine; Animals; Chlorides; Choline; Electric Conductivity; Furosemide; Kinetics; Membrane Potentials; Mollusca; Neurons; Receptors, Cholinergic; Receptors, Nicotinic; Sodium

Topics: Animals; Choline; Dicarboxylic Acids; Electric Conductivity; Ion Channels; Kinetics; Membrane Potentials; Rana temporaria; Receptors, Cholinergic

1. The end-plate region in surface fibres of rat diaphragm was located by the use of polarizing filters. 2. Carbachol (100 microM) produced depolarization at the end-plate to -55 mV, as shown by continuous recording, with some indication of spontaneous recovery in the presence of the drug. The miniature end-plate potentials disappeared and remained absent. 3. By repeated sampling it was found that the resting potential at the end-plate had largely recovered after 45 min in the presence of carbachol. Individual fibres showed much variation in the rate of recovery, and in some fibres the repolarization was rapid. 4. In the absence of K, carbachol produced depolarization at the end-plate without significant recovery, as shown by repeated sampling. 5. When muscles were exposed to ouabain (100 microM) in addition to carbachol the end-plate remained depolarized without recovery for 60 min. The effect of ouabain was reversible: withdrawal of ouabain (in the presence of carbachol) led to substantial recovery. 6. Suberyldicholine (100 microM) gave results which were similar to those produced by carbachol. 7. It was inferred that the spontaneous recovery of membrane potential in the presence of carbachol and of suberyldicholine is a process which is sensitive to K and to ouabain.

Topics: Animals; Carbachol; Choline; Diaphragm; Dicarboxylic Acids; In Vitro Techniques; Membrane Potentials; Motor Endplate; Muscles; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Ouabain; Potassium; Rats

Topics: Acetylcholine; Acetylthiocholine; Animals; Anura; Carbachol; Choline; Cold Temperature; Dicarboxylic Acids; Ion Channels; Motor Endplate; Neuromuscular Junction; Parasympathomimetics; Receptors, Cholinergic

The effect of pretreatment with agonists of various chemical structure on acetylcholine action at isolated Limnaea stagnalis neurones was studied. Threshold concentrations of monoquaternary agonists or dicholinic ester of adipinic acid increase and inhibit the responses to low and high acetylcholine concentrations respectively. In contrast, suberyldicholine and compound IEM-1054 (containing two charged ammonium groups in a molecule with an internitrogen distance of 2.0-2.2 nm) decrease the action of any acetylcholine concentration. The results are discussed in terms of the cooperativity model of acetylcholine receptors (AChR) and the hypothesis about regular arrangement of AChRs in the membrane, the so called 'C-16 structure'.

Topics: Acetylcholine; Animals; Cell Membrane; Choline; Dicarboxylic Acids; Dose-Response Relationship, Drug; Neurons; Piperazines; Receptors, Cholinergic; Snails

Topics: Animals; Anura; Choline; Dicarboxylic Acids; Feedback; In Vitro Techniques; Membrane Potentials; Neuromuscular Junction; Synapses; Synaptic Membranes; Synaptic Transmission; Time Factors

1. The effect of step changes in membrane potential on the end-plate conductance change produced by bath-applied suberyldicholine was studied in voltage-clamped frog muscle fibres. 2. The suberyldicholine-induced conductance increased exponentially from its previous equilibrium level to a new equilibrium level following a step hyperpolarization. 3. For low suberyldicholine concentrations the time constant of this relaxation was independent of the concentration. 4. For low suberyldicholine concentrations the voltage dependence of equilibrium conductance and relaxation time constants was identical. 5. Bungarotoxin pretreatment did not affect the responses beyond a simple reduction in their amplitude. 6. The conductance evoked by high suberyldicholine concentrations was less voltage-sensitive than that evoked by low concentrations. 7. A new model for explaining noise and relaxation data is proposed. This postulates rate-limiting binding steps followed by a voltage-dependent isomerization.

Topics: Animals; Anura; Bungarotoxins; Choline; Dicarboxylic Acids; Dose-Response Relationship, Drug; Electric Conductivity; In Vitro Techniques; Kinetics; Membrane Potentials; Models, Neurological; Motor Endplate; Muscle Contraction; Muscle Relaxation; Neostigmine; Rana esculenta; Rana temporaria

1. An increased uptake of labelled sodium was found in the end-plate region of rat diaphragm following brief exposure to solution containing (24)Na plus carbachol (100 muM), with a wash in inactive saline. Tetrodotoxin (0.1 muM) was also present. Comparable results were obtained with decamethonium and suberyldicholine.2. With carbachol (100 muM) the influx of labelled sodium at the end-plate region was increased by a factor of at least three as compared with that at the end of the fibres.3. After entry the labelled sodium spread along the fibres with an apparent diffusion coefficient which was half that expected in the external solution.4. The dose-response curve for the effect of carbachol gave a half-maximal value of 72 muM.5. In muscles depolarized by potassium methyl sulphate the effect of carbachol on the entry of sodium was reduced although demonstrable.6. The entry of labelled sodium at the end-plate was maintained during prolonged exposure to carbachol (100 muM) or decamethonium (100 muM).7. The rate of entry of (24)Na obtained with carbachol, after corrections for the wash, was estimated as 1.5 x 10(3) ions channel(-1) sec(-1), measured over a period of 15 sec.8. Labelled decamethonium and labelled carbachol also accumulated at the end-plate region. After extrapolation to allow for the effects of the wash the entry of decamethonium when expressed as a clearance (pl. mg(-1) sec(-1)) was comparable to that of sodium, as expected if decamethonium and sodium enter through the same channels.

Topics: Animals; Biological Transport; Carbachol; Choline; Decamethonium Compounds; Diaphragm; Dicarboxylic Acids; Dose-Response Relationship, Drug; In Vitro Techniques; Motor Endplate; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Potassium; Rats; Sodium; Tetrodotoxin

Topics: Acetylcholine; Animals; Binding, Competitive; Choline; Dicarboxylic Acids; Electric Organ; Fishes; Kinetics; Receptors, Cholinergic; Spectrometry, Fluorescence

Membrane currents from voltage-clamped frog muscle fibers were recorded during iontophoretic application of steady doses of carbachol, acetylcholine, and suberyldicholine to the endplate region. In the presence of these drugs, an exponentially relaxing current was observed after step changes of membrane potential. The time constant of relaxation was found to be voltage-dependent. It was equal to the time constant obtained from the autocorrelation function of drug-induced conductance fluctuations measured under similar conditions. Analysis of instantaneous current at the on- and offsets of voltageclamp pulses showed that there is no shift in equilibrium potential during the pulses.

Topics: Acetylcholine; Animals; Anura; Carbachol; Choline; Dicarboxylic Acids; Electric Conductivity; Iontophoresis; Mathematics; Membrane Potentials; Neuromuscular Junction; Rana esculenta

Topics: Acetylcholine; Animals; Atropine; Caprylates; Cats; Choline; Cholinesterase Inhibitors; Cholinesterases; Dicarboxylic Acids; Electric Stimulation; Hydrolysis; Muscle Contraction; Muscles; Nitrobenzenes; Organophosphonates; Organophosphorus Compounds