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PubMed1982

Identification of a cDNA clone coding for the acetylcholine binding subunit of Torpedo marmorata acetylcholine receptor.

A recombinant DNA plasmid has been constructed that contains sequences of the gene coding for the acetylcholine binding subunit (alpha-subunit, 40 000 daltons) of Torpedo marmorata acetylcholine receptor protein (AChR). Polyadenylated RNA purified from Torpedo electric organ was used to construct a cDNA library. The AChR alpha-subunit cDNA clone was then identified by a two-step screening of 700 recombinant clones. As AChR is present in Torpedo electric organ but not in Torpedo liver or spleen, differential screening led to the selection of 12 clones specific for the electric organ. We then tested the ability of cDNA inserts to hybridize alpha-subunit mRNA specifically, as judged by cell-free translation and immunoprecipitation. The insert from one clone, p alpha-1, selectively hybridized with a mRNA species which elicited the synthesis of a 38 000 mol. wt. polypeptide. This polypeptide was precipitated by: (1) a rabbit serum raised against purified denatured alpha-subunit (the pure alpha-subunit displaced the complex); and (2) a rat monoclonal antibody specific for the denatured alpha-subunit. It was thus identified as a precursor of the alpha chain. Blot hybridization analysis of polyadenylated RNA from Torpedo electric organ with the p alpha-1 probe revealed a major species of 2.0 kb, which thus contains approximately 800 non-coding nucleotides.

Animals↗
PubMed1973

Studies on purified eel acetylcholine receptor and anti-acetylcholine receptor antibody.

Rabbit antiserum against purified Electrophorus electricus acetylcholine receptor is studied using an immunoprecipitin assay to measure either antibody titer or concentration of toxin-binding sites in solubilized receptor preparations. This antiserum, unlike control serum, blocks the electrophysiological response of the electroplax to carbamylcholine. Toxin (alpha-neurotoxin, Naja naja) and several cholinergic ligands produce partial inhibition of the reaction of antiserum with purified acetylcholine receptor. Evidence is presented that some of the toxin-binding sites on receptor, purified by affinity chromatography on toxin-agarose conjugates, are occluded by toxin. In addition, evidence is presented that antireceptor antiserum will cause precipitation of more toxin-binding sites present in an initial extract than in purified receptor preparations.

Acetates↗
PubMed2005

Crystal structure of acetylcholine-binding protein from Bulinus truncatus reveals the conserved structural scaffold and sites of variation in nicotinic acetylcholine receptors.

The crystal structure of acetylcholine-binding protein (AChBP) from the mollusk Lymnaea stagnalis is the established model for the ligand binding domains of the ligand-gated ion channel family, which includes nicotinic acetylcholine, 5-hydroxytryptamine (5-HT3), gamma-aminobutyric acid (GABA), types A and C, and glycine receptors. Here we present the crystal structure of a remote homolog, AChBP from Bulinus truncatus, which reveals both the conserved structural scaffold and the sites of variation in this receptor family. These include rigid body movements of loops that are close to the transmembrane interface in the receptors and changes in the intermonomer contacts, which alter the pentamer stability drastically. Structural, pharmacological and mutational analysis of both AChBPs shows how 3 amino acid changes in the binding site contribute to a 5-10-fold difference in affinity for nicotinic ligands. Comparison of these structures will be valuable for improving structure-function studies of ligand-gated ion channel receptors, including signal transduction, homology modeling, and drug design.

Amino Acid Sequence↗
PubMed2006

Acetylcholine-binding proteins: functional and structural homologs of nicotinic acetylcholine receptors.

Acetylcholine-binding protein (AChBP) is a water-soluble protein released from molluscan glial cells and modulates ACh-mediated synaptic transmission. Acetylcholine-binding protein (AChBP) is a water-soluble homolog of the ligand-binding domain of nicotinic receptors and other members of the pharmaceutically important family of pentameric ligand-gated ion channels (LGICs), GABAA, GABAC, 5-HT3 serotonin, and glycine receptors. The crystal structure of AChBP from Lymnaea stagnalis has become an established model for the extracellular domain of the pentameric LGICs, and homology models have been generated to analyze receptor-ligand interactions. AChBP has pharmacological properties similar to the homomeric alpha7 subtype of nicotinic ACh receptors (nAChRs), with relatively weak affinity for ACh and a 10-fold higher affinity for nicotine.

Animals↗
PubMed1977

Acetylcholine receptors and myasthenia gravis: the effect of antibodies to eel acetylcholine receptors on eel electric organ cells.

Antisera to acetylcholine receptors purified from Electrophorus electricus were tested for their ability to bind to receptors on electric organ cells, block the depolarizing response of the cells to carbamylcholine, and inhibit binding of 125-I-alpha-bungarotoxin to the cells. It was found that although antibodies could bind to most of the receptors, resulting in substantial inhibition of the depolarizing response, binding of 125-I-alpha-bungarotoxin to the cells was only slightly inhibited. This was consistent with the observation that these antibodies did not compete for the toxin binding site on detergent-solubilized receptor. These results suggest that the antibodies inhibited receptor activity primarily by interfering with the ionophore of the receptor or its regulation by the acetylcholine binding site. However, the possibilities could not be completely eliminated that blockage of a small fraction of the binding sites caused a large inhibition of the depolarizing response or that bound antibody allosterically reduced binding affinity for carbamylcholine without completely inhibiting toxin binding.

Animals↗
PubMed1997

[Effect of acetylcholine receptor antibody derived from myasthenia gravis on the rat central neuronal nicotinic acetylcholine receptor].

Immunoreaction between acetylcholine receptor antibody (AChRab) derived from myasthenia gravis (MG) and neuronal nicotinic acetylcholine receptor (AChR) and the action of AChRab on rat brain stem auditory transmission were investigated by immunocytochemistry and brain stem auditory evoked potential (BAEP) in a search for abnormalities indicating central nervous system (CNS) involvement in this disorder. 25 microliters of AChRab were injected into the ventricular system of rats once every 48 hours for periods of up to a week BAEP examination was taken before and after injection of AChRab. Immunocytochemical investigation of the rat brain sections provided evidence that the neuronal nicotinic AChR like immunoreactivity is widely distributed throughout the rat CNS such as cerebral cortex, hippocampus, motor neurons in brain stem, auditory nucleus cerebellar cortex, etc. Analysis of the immunocytochemical results showed that the distribution property of AChRab in rat CNS correlated well with known neuronal nicotinic AChR subtypes e.g., alpha 7 and alpha 4. BAEP inter peak latency was significantly prolonged in rats after injection of AChRab as compared with the control rats and the rats prior to injection of AChRab. The results indicated that cerebroventricular administration of AChRab not only cause dysfunction of brain stem auditory transmission but also leads to development of experimental autoimmune MG symptoms. It is concluded that AChRab influences the rat brain stem auditory transmission and it is suggested that the pathogenic effect of AChRab is not only limited at neuromuscular junction, but also involves the central neuronal nicotinic AChR.

Animals↗
PubMed1998

Kinetic parameters for the vesicular acetylcholine transporter: two protons are exchanged for one acetylcholine.

The vesicular acetylcholine transporter (VAChT) mediates ACh storage in synaptic vesicles by exchanging cytoplasmic ACh with vesicular protons. This study sought to determine the stoichiometry of exchange by analysis of ligand binding and transport kinetics. The effects of different pH values inside and outside, external ACh concentrations, and electrical potential gradients on ACh transport by vesicles isolated from the electric organ of Torpedo were determined using a pH-jump protocol. The equilibrium binding of a high-affinity analogue of ACh is inhibited by protonation with a pKa of 7.4 +/- 0.3. A two-proton model fits the transport data much better than a one-proton model does, and uptake increases at more positive internal electrical potential, as expected for the two-proton model. Thus, the results support the two-proton model. The transport cycle begins with binding of external ACh to outwardly oriented site 2 (KACho = 20 mM) and protonation of inwardly oriented site 1 (pKa1 = 4.73 +/- 0.05). Loaded VAChT reorients quickly (73 000 min-1) and releases ACh to the inside (KAChi = 44 000 mM) and the proton to the outside. Unloaded, internally oriented site 2 binds a proton (pKa2 = 7.0), after which VAChT reorients (150 +/- 20 min-1) in the rate-limiting step and releases the proton to the outside to complete the cycle. Rate constants for the reverse direction also were estimated. Two protons provide a thermodynamic driving force beyond that utilized in vivo, which suggests that vesicular filling is regulated. Other phenomena related to VAChT, namely the time required to fill synaptic vesicles, the fractional orientation of the ACh binding site toward cytoplasm, orientational lifetimes, and the rate of nonquantal release of ACh from cholinergic nerve terminals, were computer-simulated, and the results are compared with physiological observations.

Acetylcholine↗
PubMed2000

(-)-Spiro[1-azabicyclo[2.2.2]octane-3,5'-oxazolidin-2'-one], a conformationally restricted analogue of acetylcholine, is a highly selective full agonist at the alpha 7 nicotinic acetylcholine receptor.

Neuronal nicotinic acetylcholine receptors are members of the ligand-gated ion channel receptor superfamily and may play important roles in modulating neurotransmission, cognition, sensory gating, and anxiety. Because of its distribution and abundance in the CNS, the alpha 7 nicotinic receptor is a strong candidate to be involved in some of these functions. In this paper we describe the synthesis and in vitro profile of AR-R17779, (-)-spiro[1-azabicyclo[2.2. 2]octane-3,5'-oxazolidin-2'-one] (4a), a potent full agonist at the rat alpha 7 nicotinic receptor, which is highly selective for the rat alpha 7 nicotinic receptor over the alpha 4 beta 2 subtype. Preliminary SAR of AR-R17779 presented here indicate that there is little scope for modification of this rigid molecule as even minor changes result in significant loss of the alpha 7 nicotinic receptor affinity.

Acetylcholine↗
PubMed1999

Evoked acetylcholine release by immortalized brain endothelial cells genetically modified to express choline acetyltransferase and/or the vesicular acetylcholine transporter.

Immortalized rat brain endothelial RBE4 cells do not express choline acetyltransferase (ChAT), but they do express an endogenous machinery that enables them to release specifically acetylcholine (ACh) on calcium entry when they have been passively loaded with the neurotransmitter. Indeed, we have previously reported that these cells do not release glutamate or GABA after loading with these transmitters. The present study was set up to engineer stable cell lines producing ACh by transfecting them with an expression vector construct containing the rat ChAT. ChAT transfectants expressed a high level of ChAT activity and accumulated endogenous ACh. We examined evoked ACh release from RBE4 cells using two parallel approaches. First, Ca2+-dependent ACh release induced by a calcium ionophore was followed with a chemiluminescent procedure. We showed that ChAT-transfected cells released the transmitter they had synthesized and accumulated in the presence of an esterase inhibitor. Second, ACh released on an electrical depolarization was detected in real time by a whole-cell voltage-clamped Xenopus myocyte in contact with the cell. Whether cells synthesized ACh or whether they were passively loaded with ACh, electrical stimulation elicited the release of ACh quanta detected as inward synaptic-like currents in the myocyte. Repetitive stimulation elicited a continuous train of responses of decreasing amplitudes, with rare failures. Amplitude analysis showed that the currents peaked at preferential levels, as if they were multiples of an elementary component. Furthermore, we selected an RBE4 transgenic clone exhibiting a high level of ChAT activity to introduce the Torpedo vesicular ACh transporter (VAChT) gene. However, as the expression of ChAT was inactivated in stable VAChT transfectants, the potential influence of VAChT on evoked ACh release could only be studied on cells passively loaded with ACh. VAChT expression modified the pattern of ACh delivery on repetitive electrical stimulation. Stimulation trains evoked several groups of responses interrupted by many failures. The total amount of released ACh and the mean quantal size were not modified. As brain endothelial cells are known as suitable cellular vectors for delivering gene products to the brain, the present results suggest that RBE4 cells genetically modified to produce ACh and intrinsically able to support evoked ACh release may provide a useful tool for improving altered cholinergic function in the CNS.

Acetylcholine↗
PubMed1999

Nerve growth factor increases the synthesis and release of acetylcholine and the expression of vesicular acetylcholine transporter in primary cultured rat embryonic septal cells.

Acetylcholine (ACh) is synthesized by choline acetyltransferase (ChAT) in the cytoplasm of cholinergic nerve terminals and transported into synaptic vesicles by vesicular ACh transporter (VAChT). The genes encoding ChAT and VAChT are colocalized within the genome, and their products are known to be coregulated by various neurotrophic factors. In the present study, nerve growth factor (NGF; 100 ng/ml) was shown to enhance expression of VAChT and ChAT mRNA in primary cultured rat embryonic septal cells. By using a radioimmunoassay, we also found that NGF increased both neuronal content and spontaneous release of ACh, which were first detected on day 2 of culture and time-dependently increased up to day 10. Stimulated release of ACh elicited by high K+ (50 mM KCl) was also significantly greater in NGF-treated cells than in control cells. NGF enhanced immunoreactivity to antiserum against VAChT, indicating that the augmented responses were due to, at least in part, increased expression of VAChT protein. In contrast, the numbers of immunocytochemically positive cells were unaffected. Thus, NGF appears to augment ACh synthesis, its transport into synaptic vesicles, and its subsequent release. The data also suggest that NGF facilitates growth and development of cholinergic neurons, but not their survival.

Acetylcholine↗
PubMed2003

Influence of acetylcholine levels on the binding of a SPECT nicotinic acetylcholine receptor ligand [123I]5-I-A-85380.

Although in vitro theory indicates that ligand binding is sensitive to competition with neurotransmitters, only some imaging ligands have shown such competition in vivo. The purpose of this study was to determine whether increases in acetylcholine (ACh) levels induced by an acetylcholinesterase inhibitor, physostigmine, inhibit in vivo binding of [(123)I]5-iodo-3-(2(S)-2-azetidinyl-methoxy) pyridine (5-I-A-85380), a single photon emission computed tomography ligand for the high-affinity type nicotinic ACh receptor (nAChR). Baboons were used for seven studies with a bolus plus constant infusion equilibrium paradigm. After achieving equilibrium at 5 h, physostigmine (0.02 (n = 1), 0.067 (n = 3), and 0.2 (n = 3) mg/kg) was administered intravenously and data were acquired for up to 8 h. To confirm equilibrium conditions, [(123)I]5-I-A-85380 plasma levels were measured in four studies, including all studies with 0.2 mg/kg physostigmine. Prior to physostigmine administration, thalamic activities were stable, with changes of 1.1%/h or less, except in one study with a gradual increase of 4.2%/h. Thalamic activities were decreased by 15% in one study with 0.067 mg/kg and 14-17% in all studies with 0.2 mg/kg physostigmine administration (P = 0.009). In these studies with 0.2 mg/kg physostigmine administration, [(123)I]5-I-A-85380 plasma levels showed a transient or a sustained increase after physostigmine administration that would have increased thalamic activities. These results suggest that elevated ACh levels induced by physostigmine can effectively compete in vivo with [(123)I]5-I-A-85380 binding at nAChRs. However, decreased thalamic activities could have been caused by other mechanisms, including internalization of the receptor with an associated decreased affinity for radioligand.

Acetylcholine↗
PubMed1994

Effects of supplemental choline on extracellular acetylcholine in the nucleus accumbens during normal behavior and pharmacological acetylcholine depletion.

Brain microdialysis was used to determine whether systemic or local application of choline would modify the extracellular concentration of acetylcholine (ACh) in the nucleus accumbens (NAc) of freely moving rats. Supplemental choline given intraperitoneally or into the NAc of normal rats did not increase extracellular ACh. When local ACh interneurons in the NAc were treated pharmacologically to deplete the intracellular stores of ACh, then systemic choline (80 mg/kg) was an effective treatment. Specifically, 1) blockade of the high-affinity choline transporter with hemicholinium-3 (HC-3) to reduce ACh synthesis caused a decrease in extracellular ACh, but choline supplementation restored ACh toward its normal level in the NAc. 2) Local bicuculline treatment released ACh to the point of depletion, but systemic choline or locally infused choline helped maintain normal ACh levels. These results suggest that choline supplementation might be useful in preventing depletion of ACh in the nucleus accumbens during pathological conditions.

Acetylcholine↗
PubMed1997

Nitric oxide and peroxynitrite affect differently acetylcholine release, choline acetyltransferase activity, synthesis, and compartmentation of newly formed acetylcholine in Torpedo marmorata synaptosomes.

Recent reports proposed that nitric oxide was a modulator of cholinergic transmission. Here, we examined the role of NO on cholinergic metabolism in a model of the peripheral cholinergic nervous synapse: synaptosomes from Torpedo electric organ. The presence of NO synthase was immunodetected in the cell bodies, in the nerve ending area of nerve-electroplate tissue and in the electroplates. Exogenous source of NO was provided from SIN1, a donor of NO and O2-., and an end-derivative peroxynitrite (ONOO-). SIN1 increased calcium-dependent acetylcholine (ACh) release induced by KCl depolarization or a calcium ionophore A23187. The formation of ONOO- was continuously followed by a new chemiluminescent assay. The addition of superoxide dismutase, that decreases the formation of ONOO-, did not impair the stimulation of ACh release, suggesting that NO itself was the main stimulating agent. When the endogenous source of NO was blocked by proadifen, an inhibitor of cytochrome P450 activity of NO synthase, both KCl- and A23187-induced ACh release were abolished; nevertheless, the inhibitor Ng-monomethyl-L-arginine did not modify ACh release when applied in a short time duration of action. Both NO synthase inhibitors reduced the synthesis of ACh from the radioactive precursor acetate and its incorporation into synaptic vesicles as did ONOO- chemically synthesized or formed from SIN1. In addition, choline acetyltransferase activity was strongly inhibited by ONOO- and SIN1 but not by the NO donors SNAP and SNP or, by NO synthase inhibitors. Altogether these results indicate that NO and ONOO modulate presynaptic cholinergic metabolism in the micromolar range, NO (up to 100 microM) being a stimulating agent of ACh release and ONOO- being an inhibitor of ACh synthesis and choline acetyltransferase activity.

Acetylcholine↗
PubMed1978

Effects of chronic lead exposure on levels of acetylcholine and choline and on acetylcholine turnover rate in rat brain areas in vivo.

Rats were exposed to lead acetate from birth, and were killed at the age of 44--51 days for analysis of levels and turnover rates of acetylcholine (ACh). Steady-state levels of ACh were not altered in midbrain, cortex, hippocampus, or striatum of lead-exposed rats. Similarly, no changes in choline (Ch) concentrations were found in cortex, hippocampus, or striatum. In the midbrain, however, a 30% reduction in Ch levels was observed. Changes in specific activity of Ch and ACh were measured as a function of time in selected brain areas of rats infused with a radio-labeled precursor of Ch. Specific activities of ACh were not altered. Ch specific activities were, however, significantly elevated in all brain areas examined, as compared with age-matched control rats. The in vivo ACh turnover rate in cortex, hippocampus, and striatum was diminished by 35%, 54%, 51%, and 33%, respectively. These findings provide direct evidence for an inhibitory effect of lead exposure from birth on central cholinergic function in vivo. Since a significant reduction of body weight was found in those animals treated with lead acetate, the alteration of central cholinergic function may partially be attributed to malnutrition observed in the lead-exposed animals.

Acetylcholine↗
PubMed1995

Acetylcholine and central respiratory control: perturbations of acetylcholine synthesis in the isolated brainstem of the neonatal rat.

The brainstem neurochemical processes which support spontaneous ventilation are not known. Cholinergic transmission may play an important role. If this is true, perturbations in acetylcholine (ACh) turnover should alter ventilatory output in a predictable manner. Using the isolated superfused brainstem-spinal axis from the neonatal rat, the effects of modifiers of ACh synthesis on spontaneous C-4 (phrenic) output were determined. 3-Bromopyruvate and hydroxycitrate, inhibitors of acetyl-CoA (substrate for ACh synthesis) formation, caused depression of the C-4 output in a dose-dependent manner when added to the superfusate. Triethylcholine, a false-transmitter generating choline analog, caused a similar depression. Citrate, a cytosolic precursor to acetyl-CoA formation, caused stimulation of C-4 (phrenic) output. The stimulatory effects of citrate were blocked by the muscarinic cholinergic blocker, atropine. These findings are consistent with the view that the ACh synthetic pathway provides a continuous and important input to the normal brainstem elements that support ventilation.

Acetylcholine↗
PubMed1986

A study of the contractile response to acetylcholine in human ileal and detrusor muscle: origin of the low efficacy of acetylcholine.

In Krebs solution (3.36 mM Ca2+), the maximal contractile response of human ileal and urinary bladder detrusor muscle to acetylcholine (ACh) was 40-60% that to carbachol (CCh). The maximum response to ACh was reached at a bath concentration of about 1 microM and was maintained throughout a range extending to 100 microM. In the presence of neostigmine (0.1 microM), the maximum response to ACh reached the level of that of CCh. However, bioassay of bath concentrations of ACh at various points of the maximal response in the absence of neostigmine revealed only slight to insignificant diminution of the applied concentration of ACh. Joint application of ACh and CCh generated a dose-response profile consistent with a model of competitive antagonism between a full agonist (CCh) and a partial one (ACh). Also, choline (100 microM) reduced the maximum response to ACh in the presence of neostigmine and that to CCh to 60-80% of control. These observations are consistent with a mechanism whereby intact cholinesterase together with its substrate ACh and possibly a breakdown product of ACh constitute a filter or diffusional barrier regulating the flow of agonist from the enzyme compartment to the receptor compartment.

Acetylcholine↗
PubMed1982

Effects of inhibitors of acetylcholine synthesis on brain acetylcholine and survival in soman-intoxicated animals.

The effects of hemicholinium-3 (HC-3) or 4-(l-naphthylvinyl)pyridine (4-NVP) alone and together with cholinolytics and/or cholinesterase inhibitors on brain acetylcholine (ACh) levels and survival were studied. Intracerebroventricular (ICVT) injection of 10 micrograms HC-3 280 min before euthanasia by microwave irradiation reduced rat cerebral ACh levels from 28.4 to 5.4 nmoles ACh/g wet tissue. In rats pretreated with HC-3 alone or with other pretreatment drugs prior to giving up to 2.7 LD50 of soman, iv, cerebral ACh levels increased very little, but in animals not receiving HC-3, brain ACh levels increased to 67.1 nmoles. Treatment of unpoisoned rats with 4-NVP resulted in a significant (26%) reduction in ACh. The inclusion of atropine with 4-NVP caused sign-free doses of physostigmine to produce toxic signs in rabbits and did not enhance the efficacy of carbamate pretreatment against soman. Pretreatment of rabbits with pyridostigmine and atropine methyl nitrate (AMN) failed to provide any protection against soman, but when HC-3, ICVT, was included with those drugs, the protective ratio (PR), against soman was increased excess ACh is a primary lesion in organophosphorus anticholinesterase intoxication and that the central nervous system is quite sensitive to excesses of ACh.

Acetylcholine↗
PubMed1983

Effect of parasympathetic denervation on acetylcholine levels in the rat parotid gland. Is there an extraneuronal pool of acetylcholine?

Parasympathetic denervation of the rat parotid gland by avulsion of the auriculotemporal nerve caused a marked and lasting decrease in gland weight. Parasympathectomy did not change the levels of choline in the gland but decreased by 60% the levels of acetylcholine (ACh) ten days after surgery and 65% at 28 days. It is puzzling that relatively high levels of ACh remained after parasympathetic denervation. The presence of additional cholinergic nerves that innervate the gland, or pass through it en route to other structures may account for some of the remaining ACh. Also, Schwann cells from denervated nerves might have contributed to some of the ACh. The existence of an extraneuronal source of ACh is considered.

Acetylcholine↗