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Binding of [(3)H]acetylcholine to a particulate fraction of Torpedo electroplax was measured by equilibrium dialysis. Two high-affinity sites present on phospholipoproteins bound acetylcholine reversibly, and binding was blocked by nicotinic drugs. Characteristics of this binding suggest that these phospholipoproteins may be acetylcholine receptors.
The effect of pinealectomy, superior cervical ganglionectomy and melatonin replacement on diurnal variations in submaxillary lymph node ornithine decarboxylase activity, tyrosine hydroxylase activity and [3H]choline conversion to [3H]acetylcholine were examined in rats subjected to pinealectomy, bilateral superior cervical ganglionectomy or their respective sham-operations, and treated with Freund's complete adjuvant or its vehicle. In both immunized and nonimmunized sham-operated rats, significant diurnal variations in ornithine decarboxylase activity were detectable, with a maximum at 13.00 h (vehicle) or at 17.00 h (Freund's adjuvant). In rats subjected to pinealectomy, ornithine decarboxylase activity decreased by about half, still exhibiting significant diurnal variations with a maximum at 13.00 h. Abolition of circadian rhythmicity and depression of ornithine decarboxylase activity to about one third of controls were found in submaxillary lymph nodes of bilaterally superior cervical ganglionectomized rats. Administration of melatonin (30 micrograms/animal) in the late evening during 11 days counteracted the depressed levels and suppressed the amplitude of diurnal rhythmicity of ornithine decarboxylase in pinealectomized or bilaterally superior cervical ganglionectomized rats, as well as augmented enzyme activity in sham-operated controls. The amplitude and mean levels of 24-hour rhythms in submaxillary lymph node tyrosine hydroxylase activity and [3H]choline conversion to acetylcholine (that attained their maxima at 21.00-1.00 and 13.00-17.00 h, respectively) decreased significantly after pinealectomy, these effects being significantly counteracted by melatonin injection. Melatonin augmented tyrosine hydroxylase activity and acetylcholine synthesis in sham-pinealectomized rats. The results are compatible with the view that the pineal gland plays a role in circadian changes of immune responsiveness in lymphoid tissue via an immunopotentiating effect of melatonin on lymph node cell proliferation.
BACKGROUND: Nicotine is a psychoactive drug presenting a diverse array of biological activities, some positive, such as enhancement of cognitive performances, others negative, such as addiction liability. Ligands that discriminate between the different isotypes of nicotinic acetylcholine receptors (nAChRs) could present improved pharmacology and toxicity profile. RESULTS: Based on the recent crystal structure of a soluble acetylcholine binding protein from snails, we have built atomic models of acetylcholine and nicotine bound to the pocket of four different human nAChR subtypes. The structures of the docked ligands correlate with available biochemical data, and reveal that the determinants for isotype selectivity are relying essentially on four residues, providing diversity of the ligand binding pocket both in terms of Van der Waals boundary, and electrostatic potential. We used our models to screen in silico a large compound database and identify a new ligand candidate that could display subtype selectivity. CONCLUSION: The nAChR-agonist models should be useful for the design of nAChR agonists with diverse specificity profiles.
A series of eighteen consecutive overlapping synthetic peptides, of uniform size and overlaps, which encompass the entire extracellular part (residues 1-210) of the Torpedo californica acetylcholine receptor alpha chain were examined in vitro for their ability to stimulate lymph node cells from acetylcholine receptor-primed C57BL/6 (H-2b), C3H/He (H-2k), SWR (H-2q) and SJL (H-2s) mice. The recognition sites (T sites) by acetylcholine receptor-primed lymph node cells from these mouse strains resided within six regions on the extracellular part of the alpha chain. Three of the regions recognized by T cells coincided with regions recognized by antibodies (i.e. B cells) and one of these three regions also coincided with an alpha-neurotoxin-binding region. It is noteworthy that, in addition to sites recognized by both T and B cells, the protein has at least two sites which are recognized exclusively by T cells and to which no detectable antibody responses are directed.
The local and endogenous nicotinic neuronal transmissions of dopaminergic neurons in the substantia nigra were confirmed electrophysiologically using a slice-patch technique. After identifying dopaminergic neurons based on their electrophysiological characteristics, miniature postsynaptic inward currents were recorded in the presence of atropine (a muscarinic acetylcholine receptor antagonist), bicuculline (a GABA receptor antagonist) and L-glutamic acid diethyl ester (GDEE) (a non-selective glutamate receptor antagonist). Under conditions that eliminated muscarinic, GABAergic and glutamatergic synaptic transmissions, we found miniature currents that were inhibited by the specific neuronal nicotinic receptor antagonists, dihydro-beta-erythroidine (DHbetaE) and/or methyllycaconitine (MLA) (selective alpha4beta2 and/or alpha7 nicotinic acetylcholine receptor antagonists, respectively). Under the same extracellular conditions, local stimulations in the vicinity of a target neuron evoked excitatory postsynaptic inward currents (EPSCs). These EPSCs were elicited in an extracellular Ca(2+) dependent manner and were also blocked by DHbetaE and/or MLA. These results suggest that dopaminergic neurons in the substantia nigra receive excitatory cholinergic inputs that are mediated via at least two types of postsynaptic nicotinic receptors, namely alpha7 and alpha4beta2 subtypes.
This study sought primarily to locate the acetylcholine (ACh) binding site in the vesicular acetylcholine transporter (VAChT). The design of the study also allowed us to locate residues linked to (a) the binding site for the allosteric inhibitor vesamicol and (b) the rates of the two transmembrane reorientation steps of a transport cycle. In more characterized proteins, ACh is known to be bound in part through cation-pi solvation by tryptophan, tyrosine, and phenylalanine residues. Each of 11 highly conserved W, Y, and F residues in putative transmembrane domains (TMDs) of rat VAChT was mutated to A and a different aromatic residue to test for loss of cation-pi solvation. Mutated VAChTs were expressed in PC12(A123.7) cells and characterized with the goal of determining whether mutations widely perturbed structure. The thermodynamic affinity for ACh was determined by displacement of trace [(3)H]-(-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol) with ACh, and Michaelis-Menten parameters were determined for [(3)H]ACh transport. Expression levels were determined with [(3)H]vesamicol saturation curves and Western blots, and they were used to normalize V(max) values. "Microscopic" parameters for individual binding and rate steps in the transport cycle were calculated on the basis of a published kinetics model. All mutants were expressed adequately, were properly glycosylated, and bound ACh and vesamicol. Subcellular mistargeting was shown not to be responsible for poor transport by some mutants. Mutation of residue W331, which lies in the beginning of TMD VIII proximal to the vesicular lumen, produced 5- and 9-fold decreased ACh affinities and no change in other parameters. This residue is a good candidate for cation-pi solvation of bound ACh. Mutation of four other residues decreased the ACh affinity up to 6-fold and also affected microscopic rate constants. The roles of these residues in ACh binding and transport thus are complex. Nine mutations allowed us to resolve the ACh and vesamicol binding sites from each other. Other mutations affected only the rates of the transmembrane reorientation steps, and four mutations increased the rate of one or the other. Two mutations increased the value of K(M) up to 5-fold as a result of rate effects with no ACh affinity effect. The results demonstrate that analysis of microscopic kinetics is required for the correct interpretation of mutational effects in VAChT. Results also are discussed in terms of recently determined three-dimensional structures for other transporters in the major facilitator superfamily.
The characteristics of ATP-dependent transport of acetylcholine (ACh) in homogenates of pheochromocytoma (PC-12) cells stably transfected with the human vesicular acetylcholine transporter (VAChT) cDNA are described. The human VAChT protein was abundantly expressed in this line and appeared as a diffuse band with a molecular mass of approximately 75 kDa on Western blots. Vesicular [3H]ACh accumulation increased approximately 20 times over levels attained by the endogenous rat VAChT, expressed at low levels in control PC-12 cells. The transport of [3H]ACh by human VAChT was dependent upon the addition of exogenous ATP at 37 degrees C. Uptake was abolished by low temperature (4 degrees C), the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (2.5 microM) and bafilomycin A1 (1 microM), a specific inhibitor of the vesicular H+-ATPase. The kinetics of [3H]ACh uptake by human VAChT were saturable, exhibiting an apparent Km of 0.97 +/- 0.1 mM and Vmax of 0.58 +/- 0.04 nmol/min/mg. Maximal steady-state levels of vesicular [3H]ACh accumulation were directly proportional to the concentration of substrate present in the medium with saturation occurring at approximately 4 mM. Uptake was stereospecifically inhibited by L-vesamicol with an IC50 of 14.7 +/- 1.5 nM. The apparent affinity (Kd) of [3H]vesamicol for human VAChT was 4.1 +/- 0.5 nM, and the Bmax was 8.9 +/- 0.6 pmol/mg. The turnover (Vmax/Bmax) of the human VAChT was approximately 65/min. This expression system should prove useful for the structure/function analysis of VAChT.
Aberrations in cortical cholinergic transmission have been hypothesized to mediate the development and manifestation of psychotic cognition. Based primarily on hypotheses about mesolimbic dopaminergic hyperactivity in schizophrenia, the actions of antipsychotic drugs, the trans-synaptic regulation of the excitability of basal forebrain corticopetal cholinergic neurons, and the role of cortical cholinergic inputs in attentional functions, we hypothesized that persistent disinhibition of cortical cholinergic inputs mediates the fundamental cognitive dysfunctions which form the basis for the development of positive symptoms in schizophrenia. In contrast to this hypothesis, Perry and Perry (1995), based on evidence from hallucinating patients with Lewy Body Dementia (LBD), concluded that the extensive loss of cortical acetylcholine allows irrelevant information to enter "conscious awareness" and thus hallucinations to emerge. The discussion of these contrasting hypotheses highlights the need for more dynamic and precise theories describing the cognitive variables and neuronal processes which contribute to the development and manifestation of psychotic cognition. While the hypothesis that a disinhibited cholinergic system mediates the evolution of psychotic symptoms corresponds more convincingly with current theories about the cognitive functions of cortical cholinergic inputs, both hypotheses stress the critical role of cortical acetylcholine in the highest levels of cognitive functioning.
The blood dwelling stages of schistosomes have acetylcholinesterase (AChE) and nicotinic-like acetylcholine receptors (nAChR) on their teguments. Both AChE and nAChR are concentrated on the dorsal surface of the adult male, a major surface for nutrient uptake for the worm pair. Exposure of tegumental AChE and nAChR to acetylcholine (ACh), the natural ligand of these molecules, has a consequence for the transporting function of this membrane in some schistosome species. The rate of glucose uptake in vitro by Schistosoma haematobium and Schistosoma bovis adult worm pairs was enhanced by approximately 60% at blood concentrations of ACh. Schistosoma mansoni did not show a similar response. The specificity of the ACh interaction with nAChR and AChE was shown by ablation of the effect with specific antagonists of nAChR (d-tubocurarine and alpha-bungarotoxin) and an inhibitor of AChE (BW284C51). The primary effect occurs on the tegument since alpha-bungarotoxin and BW284C51 do not penetrate the schistosome tegument. The species differences in reliance on this mechanism are consistent with their relative sensitivities to the AChE inhibitory drug, metrifonate.
1.20 mM Cs+ ions reduce background current and decrease drastically the K+ depletion process, probably as a consequence of the reduction of iK1. The background current-voltage relationship becomes linear. 2. 20 mM Cs+ ions completely abolish the current induced by acetylcholine. 3. The possibility that the K+ current induced by acetylcholine is due to an increase of iK1 is discussed.
Lithium chloride administered acutely or chronically to guinea-pigs had no effect on brain level of acetylcholine or on peripheral release of acetylcholine from longitudinal muscle of the ileum. The results suggest differences between in vitro and in vivo action of lithium.
The nicotinic acetylcholine receptor (AChR) is a heteropentameric, ligand-gated ion channel at the neuromuscular junction, where it is responsible for signal transduction between the motorneuron and the muscle. Point mutations in the subunits of the receptor change the channel's electrophysiological properties and underlie inherited forms of muscle weakness, the congenital myasthenic syndromes. One point mutation (P121L) has been identified in the epsilon-subunit of patients suffering from the fast-channel congenital myasthenic syndrome, which is evoked by reduced AChR openings. We introduced the P121L mutation into all murine AChR subunits and performed electrophysiological studies in Xenopus laevis oocytes. The P121L mutation in the epsilon-subunit of the adult mouse AChR affected ligand binding and channel gating in a manner similar to that described for human AChR. At equivalent positions in the alpha- and beta-subunits, the mutation caused only minor electrophysiological changes. Mutation of the delta-subunit had similar, but less pronounced functional consequences compared to epsilonP121L, reflecting the asymmetry of the acetylcholine binding sites and the dominant effect of the alpha-epsilon site on channel opening.
The nicotinic acetylcholine receptor regulates the ion permeability of the postsynaptic membrane. This report presents evidence that the transmitter binding site and the ion channel may be located on distinct subunits. By hybridisation of receptor complexes, in which the transmitter binding site was blocked with complexes in which the ion channel was irreversibly inhibited, we reconstituted active acetylcholine receptor complexes. The reconstituted system was similar to the native receptor in its ability to regulate the ion permeability of lipid vesicles in response to nicotinic cholinergic effectors.
A molecular model for the exobilayer portion of the alpha-subunit of the acetylcholine receptor is presented. Binding sites for an acetylcholine and non-competitive antagonists are indicated.
The importance of depolarization induced hydrolysis of cytoplasmic acetylcholine (ACh) in providing choline for the veratridine-and high K+-induced release of acetylcholine was studied in mouse forebrain minces. Results indicated that a loss of hydrolyzable cytoplasmic ACh prior to depolarization reduced the amount of ACh released by veratridine but not the amount released by high K+. The reduction in the veratridine-induced release of ACh did not occur during the first 5 min of incubation. Loss of vesicular ACh prior to depolarization reduced both the veratridine- and K+-induced release of ACh during the first 5 min of incubation. Blockade of extra-cellular choline transport by hemicholinium (HC-3) did not affect the veratridine-induced release of ACh during a 10 min incubation period unless the cytoplasmic pool of ACh had first been depleted and was unavailable as a source of choline. In contrast, HC-3 reduced the K+-induced release of ACh from brain tissue with normal stores of cytoplasmic ACh. These results indicate that both depolarizing agents primarily stimulate the release of preformed ACh from a vesicular fraction during the first 5 min of mince incubation. Thereafter, they both stimulate the release of newly synthesized ACh, however, they differ in one important respect. The principal source of choline for the veratridine-induced release of newly synthesized ACh appears to be the cytoplasmic pool of ACh, whereas the major source of choline for the K+-induced release of newly synthesized ACh appears to be extracellular choline.
The effect of the acetylcholine (ACh) transport blocker 2-(4-phenylpiperidino) cyclohexanol (AH5183) on the subcellular storage and release of acetylcholine was studied in mouse forebrain. Results indicated that AH5183 reduced the amount of ACh released from mouse forebrain minces by high K+ and veratridine over the identical concentration range as it inhibits the active transport of ACh into synaptic vesicles isolated from the electric organ of Torpedo. However, AH5183 did not block the K+- or veratridine-induced reduction of cytoplasmic (S3) ACh. Also, it did not block the loss of vesicular (P3) ACh caused by these depolarizing agents. It did, however, cause a disappearance of nerve ending ACh which was partially matched by a selective gain in the choline content of the P3 fraction. When minces of mouse forebrain were pretreated in high K+ to deplete the S3 and P3 fractions of their ACh content and then subsequently incubated in normal Krebs with [14C]choline, AH5183, at a concentration which reduces ACh release by 50%, did not affect the repletion of P3 stores with newly synthesized [14C]ACh. At somewhat higher concentrations, however, AH5183 reduced the amount of [14C]ACh in the P3 fraction without affecting the amount of [14C]ACh in the S3 fraction. At these concentrations it did not inhibit extracellular choline transport or ChAT activity. These results suggest that AH5183 may reduce the amount of ACh released from central cholinergic nerve terminals in response to depolarization through a combination of effects: (1) it may facilitate the breakdown or loss of ACh stored in the vesicular fraction; (2) it may also block the transport of newly synthesized ACh into the vesicular fraction.
The process of acetylcholine receptor (AChR) desensitization in the presence of transmitter was evaluated for human myoballs and medullo-blastoma cells. The cell under investigation was placed in one of two parallel streams of solution ejected from a double-barrelled pipette. Ultrafast (less than 50 ms) application of acetylcholine (ACh) in a specific concentration was accomplished by a shift of the cell into the other stream. ACh-induced current was measured in the whole-cell mode of patch clamping. Parameters for AChR desensitization were almost identical for the two types of human cells. Desensitization could be described by two time constants. With an ACh concentration of 3 microM, the fast time constant, tau f, was about 0.4 s and the slow time constant, tau s, was about 3.6 s. When the ACh concentration was raised to 10 microM, desensitization became faster. We also measured desensitization of the AChRs in rat myoballs and compared the values to our results from human cells. With 3 microM ACh, tau f in rat myoballs was 2.3 s and tau s was 12.7 s. Thus, rat AChRs desensitize more slowly than human AChRs. However, desensitization times for rat AChRs obtained in our experiments are much faster than published values. Therefore, ultrafast solution change would seem to be requisite for correct assessment of the desensitization process.
Nicotinic acetylcholine receptor (nAChR) purified from rat brains by cholinergic ligand affinity chromatography was characterized. Monoclonal antibody 299, which binds an acetylcholine (ACh) binding subunit termed alpha 4, depleted more than 85% of [3H]ACh binding activity of the purified preparation. A number of cholinergic agonists strongly inhibited [3H]ACh binding to the purified nAChR, whereas potential antagonists were less effective than the agonists. These results show that most of the purified nAChR contains alpha 4 subunit and the pharmacological properties are preserved upon purification.