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We examined the effects of two drugs, AH5183 and cetiedil, demonstrated to be potent inhibitors of acetylcholine (ACh) transport by isolated synaptic vesicles on cholinergic functions in Torpedo synaptosomes. AH5183 exhibited a high specificity toward vesicular ACh transport, whereas cetiedil was shown to inhibit both high-affinity choline uptake and vesicular ACh transport. Choline acetyltransferase was not affected by either drug. High external choline concentrations permitted us to overcome cetiedil inhibition of high-affinity choline transport, and thus synthesis of [14C]ACh in treated preparations was similar to that in controls. We then tested evoked ACh release in drug-treated synaptosomes under conditions where ACh translocation into the vesicles was blocked. We observed that ACh release was impaired only in cetiedil-treated preparations; synaptosomes treated with AH5183 behaved like the controls. Thus, this comparative study on isolated nerve endings allowed us to dissociate two steps in drug action: upstream, where both AH5183 and cetiedil are efficient blockers of the vesicular ACh translocation, and downstream, where only cetiedil is able to block the ACh release process.
To determine whether intraventricular administration of nerve growth factor alters presynaptic cholinergic function in the intact hippocampus or following partial lesions of the fimbria, we investigated the effects of recombinant human nerve growth factor treatment on [3H]acetylcholine synthesis and release by hippocampal slices following various treatment regimens. For chronic nerve growth factor treatment, 1 microgram of recombinant human nerve growth factor was injected intraventricularly every second day. Lesions reduced [3H]acetylcholine synthesis (by 48%) and spontaneous and evoked [3H]acetylcholine release by 35 and 61%, respectively. Chronic nerve growth factor treatment over three weeks elevated [3H]acetylcholine synthesis (by 39%) and spontaneous and evoked [3H]acetylcholine release by 27 and 64%, respectively, over values in lesioned hippocampi of animals treated with a control protein (cytochrome c). The nerve growth factor-induced enhancement of presynaptic cholinergic function persisted for three weeks following the termination of nerve growth factor administration. Furthermore, chronic (nine-week) treatment with nerve growth factor increased [3H]acetylcholine by 118% over values in lesioned hippocampi of animals treated with cytochrome c. These findings indicate that chronic treatment with recombinant human nerve growth factor increases the capacity of hippocampal cholinergic neurons surviving a partial fimbrial transection to synthesize, store and release acetylcholine. Application of recombinant human nerve growth factor during the initial weeks after lesioning was necessary to product significant elevations in acetylcholine synthesis, since chronic recombinant human nerve growth factor treatment after delays of three or more weeks were ineffective. Furthermore, chronic nerve growth factor treatment failed to stimulate acetylcholine synthesis and release in intact hippocampal cholinergic systems. Single intraventricular injections of recombinant human nerve growth factor at the time of lesioning resulted in a small decrease in acetylcholine synthesis which, however, was not accompanied by a change in the rate of evoked acetylcholine release from cholinergic neurons surviving the lesion. The study indicates that chronic or repeated administration of nerve growth factor during the onset of degenerative events is necessary for the stimulation of presynaptic cholinergic function in the hippocampus of adult rats with partial fimbrial transections.
The synthesis and release of non-neuronal acetylcholine, a widely expressed signaling molecule, were investigated in the human placenta. This tissue is free of cholinergic neurons, i.e. a contamination of neuronal acetylcholine can be excluded. The villus showed a choline acetyltransferase (ChAT) activity of 0.65 nmol/mg protein per h and contained 500 nmol acetylcholine/g dry weight. In the absence of cholinesterase inhibitors the release of acetylcholine from isolated villus pieces amounted to 1.3 nmol/g wet weight per 10 min corresponding to a fractional release rate of 0.13% per min. The following substances did not significantly modify the release of acetylcholine: oxotremorine (1 microM), scopolamine (1 microM), (+)-tubocurarine (30 microM), forskolin (30 microM), ouabain (10 microM), 4alpha-phorbol 12,13-didecanoate (1 microM) and tetrodotoxin (1 microM). Removal of extracellular calcium, phorbol 12,13-dibutyrate (1 microM) and colchicine (100 microM) reduced the acetylcholine release between 30% and 50%. High potassium chloride (54 mM and 108 mM) increased the acetylcholine release slightly (by about 30%). A concentration of 10 microM nicotine was ineffective, but 100 microM nicotine enhanced acetylcholine release gradually over a 50-min period without desensitization of the response. The facilitatory effect of nicotine was prevented by 30 microM (+)-tubocurarine. Inhibitors of cholinesterase (physostigmine, neostigmine; 3 microM) facilitated the efflux of acetylcholine about sixfold, and a combination of both (+)-tubocurarine (30 microM) and scopolamine (1 microM) halved the enhancing effect. In conclusion, release mechanisms differ between non-neuronal and neuronal acetylcholine. Facilitatory nicotine receptors are present which are activated by applied nicotine or by blocking cholinesterase. Thus, cholinesterase inhibitors increase assayed acetylcholine by two mechanisms, protection of hydrolysis and stimulation of facilitatory nicotine receptors.
Studies of the binding of [3H]acetylcholine to receptor-rich membranes of Torpedo californica electric organ under conditions that normally lead to a state of equilibrium did not give rise to equilibrium binding curves. Instead, the acetylcholine receptor was found to develop very long lived metastable states resulting in hysteresis in binding. Under conditions where the concentration of free [3H]acetylcholine is both less than 0.1 microM and smaller or comparable to the total receptor concentration, the degree of binding of acetylcholine depends on the rate, i.e., the mode, of increasing the acetylcholine concentration (rapid mixing vs. dialysis). The equilibrium positive cooperativity in high-affinity acetylcholine binding previously inferred from the data is deceiving; the curvature in Scatchard representations is a consequence of long-lived nonequilibrium distributions between high-affinity and lower affinity receptor conformers. By manipulation of the experimental conditions, true equilibrium binding, resulting in a linear Scatchard binding curve, was obtained and yielded the apparent equilibrium constant, K = 5 +/- 1 nM at 4 degrees C. The stoichiometry of the high-affinity site associated with this K value was found to be one acetylcholine per receptor monomer (Mr 250 000) when carefully standardized [3H]acetylcholine analyzed for both radiopurity and acetylcholine concentration was used. While our fresh membrane fragments prepared in the presence of 4 mM Ca2+ revealed up to twice as many 125I-alpha-bungarotoxin sites in 0.1% nonionic detergent relative to those assayed in the absence of detergent, nonionic detergent treatment of membrane fragments did not result in any change in total available acetylcholine binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
By using both a 3 to 4 ms quenched-86Rb+ flux assay and native acetylcholine receptor (AChR) rich electroplaque vesicles on which 50-60% of acetylcholine activation sites were blocked with alpha-BTX, we determined apparent rates of agonist-induced inactivation in AChR from Torpedo under conditions where measured flux response was directly proportional to initial 86Rb+ influx rate. Inactivation kinetics with acetylcholine in both the activating range (10 microM-10 mM) and the self-inhibiting range (15-100 mM) were measured at 4 degrees C. In the presence of 10 microM-1 mM acetylcholine, inactivation is characterized by a single exponential rate constant, kd (fast desensitization). Plots of kd vs. acetylcholine concentration display maximum kds [kd(max)] of 6.6-8.0 s-1, half-maximal kd at 102 +/- 16 microM, and a Hill coefficient of 1.6 +/- 0.3, closely paralleling the initial ion flux response of AChR. Thus, fast desensitization probably occurs from a doubly-liganded preopen state or the open channel state. In the self-inhibiting acetylcholine concentration range, inactivation is biphasic. A "rapid inactivation" phase is complete within 30 ms, followed by fast desensitization at a rate close to kd(max). Both the rate and extent of rapid inactivation increase with acetylcholine concentration, indicating that acetylcholine binds to its self-inhibition site with apparent kon approximately equal to 10(3) M-1s-1 and koff approximately equal to 40 s-1. This slow kon suggests either hindered access to the inhibitory allosteric site or that a fast binding step is followed by a slower conformational change leading to channel inhibition. Overall, our data suggest that acetylcholine binds preferentially to its inhibitory site when the receptor is in the open-channel conformation and that fast desensitization can occur from all multiple-liganded states.
We describe the responses of single units in the awake (24 cells) or urethane-anesthetized (37 cells) rat somatosensory cortex during repeated iontophoretic pulses (1.0 s, 85 nA) of acetylcholine, both before and after systemic treatment with the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (i.p., 0.3-0.5 LD50). The time-course of the response to acetylcholine pulses differed among cortical neurons but was characteristic for a given cell. Different time-courses included monophasic excitatory or inhibitory responses, biphasic (excitatory-inhibitory, inhibitory-excitatory, excitatory-excitatory, and inhibitory-inhibitory), and triphasic (excitatory-excitatory-inhibitory, inhibitory-inhibitory-excitatory, and inhibitory-excitatory-inhibitory) responses. Although the sign and time-course of the individual responses remained consistent, their magnitude fluctuated across time; most cells exhibited either an initial increase or decrease in response magnitude followed by oscillations in magnitude that diminished with time, gradually approaching the original size. The time-course of the characteristic response to an acetylcholine pulse appeared to determine direction and rate of change in response magnitude with successive pulses of acetylcholine. Diisopropylfluorophosphate treatment, given 1 h after beginning repeated acetylcholine pulses, often resulted in a gradual increase in spontaneous activity to a slightly higher but stable level. Superimposed on this change in background activity, the oscillations in the response amplitude reappeared and then subsided in a pattern similar to the decay seen prior to diisopropylfluorophosphate treatment. Our results suggest that dynamic, homeostatic mechanisms control neuronal excitability by adjusting the balance between excitatory and inhibitory influences within the cortical circuitry and that these mechanisms are engaged by prolonged increases in extracellular acetylcholine levels caused by repeated pulses of acetylcholine and by acetylcholinesterase inhibition. However, this ability of neurons in the cortical neuronal network to rapidly adjust to changes in extracellular levels of acetylcholine questions the potential efficacy of therapeutic treatments designed to increase ambient levels of acetylcholine as a treatment for Alzheimer's disease or to enhance mechanisms of learning and memory.
1. The exchangeability with added radioactive acetylcholine of the acetylcholine in isolated presynaptic nerve terminals (synaptosomes) and isolated synaptic vesicles was studied by a Sephadex-column method. 2. A substantial proportion of the synaptosomal acetylcholine is exchangeable with added radioactive acetylcholine. It is liberated by hypo-osmotic shock and ultrasonic treatment, and behaves as though it occupies the cytoplasmic compartment of synaptosomes. 3. Methods of isolating vesicles from hypo-osmotically ruptured synaptosomes in optimum yield are discussed. 4. The acetylcholine of synaptic vesicles isolated on a sucrose density gradient is released by hypo-osmotic conditions, suggesting that it is enclosed by a semi-permeable membrane; however, it is not easily released by ultrasonic treatment. 5. Added radioactive acetylcholine does not exchange with vesicular acetylcholine under a variety of different conditions. These include addition of ATP and Mg(2+), and pre-loading of the synaptosome with radioactive acetylcholine before hypo-osmotic rupture. This failure to exchange is discussed in terms of the possible storage mechanism of vesicular acetylcholine.
In order to obtain quantitative data on the relation between the conformation of acetylcholine and its interaction with biologically significant proteins, a series of acetylcholine analogues with absorption bands in the region 200-300 nm have been synthesized or obtained commercially. Each of these compounds were assayed to measure its activity as an ion channel activator of the nicotinic acetylcholine receptor protein (AChR). In addition, the suitability of some of these compounds as substrates for hydrolysis by acetylcholine esterase (AChE) was determined. One of these analogues, dimethylthionocarbamylcholine (DMTC-Ch), has the ester carbonyl oxygen replaced by a thionyl sulfur. DMTC-Ch has been found to be quite active as an ion channel activator when bound to AChR and was found to react with the enzyme AChE as a suicide substrate. It forms a thionoester of the serine at the AChE active site by an ester exchange reaction that releases the choline as the first product. However, the second or acid product is not released even at pH 7.5 over a period of days. This acetylcholine analog has an absorption band at about 240 nm and exhibits very strong ultraviolet resonance Raman (UVRR) spectra using 239 nm excitation from a frequency modified Nd:YAG laser. This technique allows observation of both conformational changes of the ligand molecule that result in frequency changes as well as changes in the excited state electronic structure that results in changes in the relative intensity of the Raman bands. The time dependence of the UVRR spectrum of the ligand upon binding to both AChE and AChR has been studied from 0.1 msec to minutes. Some time dependence in the conformation of DMTC-Ch upon binding to AChE has been found for very short (0.1-0.5 msec) times. However, no change in the conformation of this neurotransmitter analog is found in the available time range upon binding to AChR. From these data it is concluded that a previous suggestion that acetylcholine has a conformational change upon binding to AChR may be incorrect since the solution behavior of the carbamyl cholines and acetylcholine are similar. Even if acetylcholine does change conformation upont binding to AChR, it is unlikely that such a conformational change plays a significant role in channel activation. We present strong evidence that acetylcholine and its analogues can be active in a variety of conformations.(ABSTRACT TRUNCATED AT 400 WORDS)
A prolongation of the ventricular effective refractory period in response to cholinergic agonists or vagal stimulation has been demonstrated in a number of in vivo animal models. However, exposure of isolated myocardial tissues obtained from these hearts to as much as 10(-4) M acetylcholine has been shown to produce essentially no change in action potential duration or effective refractory period. The discrepancy between the in vivo and in vitro findings generally has been explained on the basis of accentuated antagonism, whereby parasympathetic agonists exert their influence through antagonism of the effects of beta-adrenergic tone in vivo. The fact that acetylcholine exerts little if any direct effect on the electrical activity of ventricular myocardium, although well accepted, is based exclusively on studies performed using endocardial preparations. Our recent demonstration of major electrophysiological differences between canine ventricular endocardium and epicardium prompted us to examine the effects of acetylcholine and the role of accentuated antagonism in these two tissue types. Using standard microelectrode techniques, we show that acetylcholine (10(-7)-10(-5) M) has little if any effect in canine ventricular endocardium but a pronounced effect to either prolong or markedly abbreviate action potential duration and effective refractory period in epicardium. These effects of acetylcholine on epicardium are attended by an accentuation of the spike and dome morphology of the action potential, are readily reversed with atropine, fail to appear when epicardium is pretreated with the transient outward current blocker 4-aminopyridine, are accentuated in the presence of isoproterenol (10(-7) to 5 x 10(-6) M), and persist in the presence of propranolol. Isoproterenol-induced abbreviation of action potential duration and effective refractory period is also shown to be more pronounced in epicardium than in endocardium; equimolar concentrations of acetylcholine completely antagonize the effects of isoproterenol in endocardium and epicardium. We conclude that acetylcholine exerts important direct effects on the electrical response of canine ventricular myocardium, which are accentuated in the presence of beta-adrenergic agonists. Our findings suggest the differential response of epicardium and endocardium to acetylcholine is due to the presence of a transient outward current-mediated spike and dome morphology in the epicardial action potential. Finally, the data suggest that acetylcholine may exert antiarrhythmic as well as arrhythmogenic effects through its actions to alter conduction and refractoriness.
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The nicotinic acetylcholine receptor is a prototype ligand-gated ion channel that mediates signal transduction in the neuromuscular junctions and other cholinergic synapses. The molecular basis for the energetics of ligand binding and unbinding is critical to our understanding of the pharmacology of this class of receptors. Here, we used steered molecular dynamics to investigate the unbinding of acetylcholine from the ligand-binding domain of human alpha7 nicotinic acetylcholine receptor along four different predetermined pathways. Pulling forces were found to correlate well with interactions between acetylcholine and residues in the binding site during the unbinding process. From multiple trajectories along these unbinding pathways, we calculated the potentials of mean force for acetylcholine unbinding. Four available methods based on Jarzynski's equality were used and compared for their efficiencies. The most probable pathway was identified to be along a direction approximately parallel to the membrane. The derived binding energy for acetylcholine was in good agreement with that derived from the experimental binding constant for acetylcholine binding protein, but significantly higher than that for the complete human alpha7 nicotinic acetylcholine receptor. In addition, it is likely that several intermediate states exist along the unbinding pathways.
Two kinetic models are introduced which predict amplitudes and time-courses of endplate currents and miniature endplate currents at neuromuscular junctions, at both normal and acetylcholinesterase-inhibited endplates. Appropriate differential rate equations reflecting interactions of acetylcholine with acetylcholine receptor and with esterase, diffusion of acetylcholine both within and from the synaptic cleft, and cooperativity between receptor site occupancy and ion channel opening are solved. Acetylcholine release into the cleft is assumed to be instantaneous. The simpler homogeneous reaction space model accurately predicts decay phase time constants are inaccurate. The two-reaction space model predicts amplitudes and time constants within a factor of two of those observed experimentally. The simulations indicate that the amplitudes and time-courses are primarily determined by the chemical reaction rates that characterize acetylcholine interactions with receptor and esterase and that these interactions occur under nonequilibrium conditions. Approximately 50% of the total ion channels in the initial reaction space are predicted to be opened at the peak endplate current. The cooperative opening of ion channels by acetylcholine requires that acetylcholine be introduced into the cleft in discrete, concentrated elements. Virtually all the open channels are confined to the initial reaction space, although acetylcholine-bound receptor sites can be much more widely distributed.
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.
Equilibrium binding studies performed with fresh membrane fragments from Torpedo marmorata reveal a low affinity for [3H]acetylcholine with an equilibrium dissociation constant in the micromolar range and no indication of cooperative interactions. The low binding affinity is an artifact caused by the presence of endogenous acetylcholine and is not related to the active conformation of the receptor. Endogenous acetylcholine is identified by its interaction with acetylcholine esterase and choline kinase. It is present in presynaptic vesicles as shown in electron micrographs. Leakage of these synaptosomes is of the order of 300 pmol acetylcholine per g tissue as determined by means of binding studies performed with [3H]acetylcholine. In the absence of endogenous acetylcholine, equilibrium binding studies show a high affinity for [3H]acetylcholine and a slight cooperativity of sites (K1D = 30nM; K2D = 10nM). The addition of detergents, local anesthetics or alcohols to a further increase in affinity and to a decrease in cooperativity (K1D = 11nM; K2D = 5nM). No low-affinity binding can be detected in the micromolar range.
Sera from 75 patients with Myasthenia gravis were tested for acetylcholine receptor antibodies using acetylcholine receptors from human skeletal muscle. From the crude Triton x-100 extract, which has so far been used for antibody tracing, a pure acetylcholine receptor preparation was obtained by affinity chromatography using alpha-Najatoxin-Sepharose 4B. When the purified 125J-alpha-Bungarotoxin-acetylcholine receptor complex was applied in a radioimmunoassay 80% of the Myasthenia gravis patients had acetylcholine receptor antibodies in contrast to none of the tested control persons. Inspite of using a pure acetylcholine receptor preparation, no clear-cut correlation was found between the amount of serum acetylcholine receptor antibodies and the clinical stage of the disease. When individual antibody titration curves were established, different reaction patterns were observed indicating either different antibody specificities in regard to antigenic determinants on the receptor molecule or differences in the antibody affinity.
Evidence for a voltage-dependent regulatory (inhibitory) site on the nicotinic acetylcholine receptor to which acetylcholine binds was obtained in membrane vesicles prepared from the Torpedo californica electric organ. Two rate coefficients, JA and alpha, which pertain to the receptor-controlled ion flux, were measured. A 1000-fold concentration range of acetylcholine was used in a transmembrane voltage (Vm) range from 0 to -48 mV under a voltage-clamped condition at pH 7.4, 1 degrees C. The following observations were made. (i) At low acetylcholine concentrations, the value of JA, the rate coefficient for ion translocation by the active (nondesensitized) state of the receptor, increased with increasing concentration. (ii) JA decreased at high acetylcholine concentrations. (iii) In contrast, alpha, the rate coefficient for receptor desensitization, did not show such a decrease. (iv) When the transmembrane potential of the vesicle membrane was changed to more negative values, the value of KR (the dissociation constant for binding of acetylcholine to the regulatory site) decreased by a factor of approximately 9 for a 25 mV change in Vm, while KI (the dissociation constant for binding of acetylcholine to the receptor site that controls channel opening) did not show such a change and has a value of 80 microM. When Vm is -48 mV, KR has a value of 8 microM. (v) The effect of a transmembrane voltage on the regulatory site was reversible and occurred within the time resolution (5 ms) of the quench-flow technique used in the measurements.(ABSTRACT TRUNCATED AT 250 WORDS)