Human Acetylcholine Research Articles

Anthelmintic paraherquamides are cholinergic antagonists in gastrointestinal nematodes and mammals.

Oxindole alkaloids in the paraherquamide/marcfortine family exhibit broad-spectrum anthelmintic activity that includes drug-resistant strains of nematodes. Paraherquamide (PHQ), 2-deoxoparaherquamide (2DPHQ), and close structural analogs of these compounds rapidly induce flaccid paralysis in parasitic nematodes in vitro, without affecting adenosine triphosphate (ATP) levels. The mechanism of action of this anthelmintic class was investigated using muscle tension and microelectrode recording techniques in isolated body wall segments of Ascaris suum. None of the compounds altered A. suum muscle tension or membrane potential. However, PHQ blocked (when applied before) or reversed (when applied after) depolarizing contractions induced by acetylcholine (ACh) and the nicotinic agonists levamisole and morantel. These effects were mimicked by the nicotinic ganglionic blocker mecamylamine, suggesting that the anthelmintic activity of PHQ and marcfortines is due to blockade of cholinergic neuromuscular transmission. The effects of these compounds were also examined on subtypes of human nicotinic ACh receptors expressed in mammalian cells with a Ca2+ flux assay. 2DPHQ blocked nicotinic stimulation of cells expressing alpha3 ganglionic (IC50 approximately 9 microm) and muscle-type (IC50 approximately 3 microm) nicotinic cholinergic receptors, but was inactive at 100 microm vs. the alpha7 CNS subtype. PHQ anthelmintics are nicotinic cholinergic antagonists in both nematodes and mammals, and this mechanism appears to underlie both their efficacy and toxicity.

The structures of the human neuronal nicotinic acetylcholine receptor beta2- and alpha3-subunit genes (CHRNB2 and CHRNA3).

The alpha4-subunit gene (CHRNA4) of the neuronal nicotinic acetylcholine receptor (nAChR) subunit family has recently been identified in two families as the gene responsible for autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), a rare monogenic idiopathic epilepsy. As a result of this finding, other subunits of the neuronal nAChR gene family are being considered as candidate genes for ADNFLE in families not linked to CHRNA4 and for other idiopathic epilepsies. Alpha4-subunits often assemble together with beta2-subunits (gene symbol CHRNB2) to build heteromeric nAChRs. The gene encoding another abundant AChR subunit, the alpha3-subunit gene (CHRNA3), is present with those encoding two other subunits, CHRNB4 and CHRNA5, in a gene cluster whose functional role is still unclear. Here we provide the information on the genomic structures of both the CHRNB2 and the CHRNA3 genes that is necessary for comprehensive mutational analyses, and we refine the genomic assignment of CHRNB2 on chromosome 1.

Human neuronal nicotinic acetylcholine receptors (nAChRs) may participate in the sexual dimorphism functionality of the brain

Human neuronal nicotinic acetylcholine receptors (nAChRs) may participate in the sexual dimorphism functionality of the brain

21 Effects of atracurium, a neuromuscular blocking agent, on reconstituted human neuronal nicotinic acetylcholine receptors

21 Effects of atracurium, a neuromuscular blocking agent, on reconstituted human neuronal nicotinic acetylcholine receptors

Acetylcholine synthesis and muscarinic receptor subtype mRNA expression in T-lymphocytes

We used a sensitive and specific radioimmunoassay for acetylcholine (ACh), and detected significant amounts of ACh in the blood of various mammals, including humans. About 60% of human blood ACh was localized in mononuclear leukocytes. Human leukemic T-cell Unes, used as T-lymphocyte models, contained both ACh and choline acetyltransferase (ChAT) activity. Furthermore, ChAT mRNA and protein were detected in the T-cell line MOLT-3. Phytonemagglutinin, a T-cell activator, increased both synthesis and release of ACh by MOLT-3 cells. Muscarinic receptor subtype mRNA expression was confirmed in various T-cell lines. These findings indicate that ACh synthesized by ChAT in T-lymphocytes acts on the muscarinic receptors on lymphocytes in automne and/or paracrine pathways and suggest that ACh in blood functions as a modulator of T-cell-dependent immune responses.

6] Functional identification of vesicular monoamine and acetylcholine transporters

Active transport of monoamines by vesicular monoamine transporter (VMAT1) and VMAT2 is performed in permeabilized fibroblasts transiently expressing these proteins by the recombinant T7 vaccinia virus system. Active transport of acetylcholine (ACh) by human vesicular ACh transporter (VAChT) is performed in postnuclear homogenates containing secretory vesicles from stably transfected neuroendocrine PC-12 cells. Neurotransmission depends on the regulated release of transmitter molecules, such as the biogenic amines and ACh. This requires the packaging of these molecules into the specialized secretory vesicles of neurons and neuroendocrine cells, a process mediated by specific vesicular transporters. Active transport of neurotransmitters into secretory organelles requires the presence of a transmembrane H + electrochemical gradient, established and maintained by a vacuolar-type H + -ATPase and a vesicular transporter molecule, which catalyzes the exchange of H + ions for neurotransmitter. A characteristic feature of the VMATs is their broad range of substrate specificity. The chapter describes the development of active transport and binding assay in permeabilized cells.

Short Report on DNA Marker at Candidate Locus: Dinucleotide polymorphism in the first intron of the human neuronal nicotinic acetylcholine receptor α4 subunit gene (CHRNA4)

Short Report on DNA Marker at Candidate Locus: Dinucleotide polymorphism in the first intron of the human neuronal nicotinic acetylcholine receptor α4 subunit gene (CHRNA4)

Effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic acetylcholine receptors on transmembrane potentials and currents in guinea pig ventricular myocytes.

The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K+ current and decreased the inward basal ICa significantly. The reversal potential of both carbachol- and antibody-induced extra currents were close to -80 mV, being in proximity to the calculated Ek of -90 mV. A beta-adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward ICa and outward Ik simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated ICa but not the isoprenaline-stimulated Ik. The antibody- or carbachol-induced outward K+ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated ICa were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events.

Endothelial prostanoids involved in the relaxation produced by acetylcholine in the human pulmonary artery.

In ring preparations of human pulmonary artery contracted with noradrenaline (NA), the application of acetylcholine (ACh) enhanced the tension, and withdrawal produced a large relaxation which was sustained for about 10 min and required over 20 min for recovery; the latter relaxation appeared only in the endothelium-intact preparation. Indomethacin increased the amplitude of NA contractions, changed the ACh-induced contraction to relaxation, and inhibited the ACh-induced sustained relaxation. Nitroarginine increased the amplitude of NA and ACh-induced contractions, with no significant change in the ACh-induced sustained relaxation. These effects of indomethacin and nitroarginine were observed only in the endothelium-intact preparations. In NA-contracted preparations, exogenously applied prostaglandin I2 (PGI2) produced relaxation. Thus, in human pulmonary arteries, NA and ACh activities release vasodilator prostanoids and nitroarginine-sensitive EDRF from the endothelium, and initiate direct contractile actions to smooth muscle. The prostanoid-induced relaxation is sustained for a long time after the withdrawal of ACh stimulation and can be mimicked by exogenously-applied PGI2.

Down-regulation of the G-proteins Gqα and G11α by transfected human M3 muscarinic acetylcholine receptors in Chinese hamster ovary cells is independent of receptor down-regulation

Chinese hamster ovary cells stably transfected with human M3 muscarinic acetylcholine receptors show a 40-50% reduction in the immunoreactive G-proteins Gq alpha and G11 alpha when stimulated with the cholinergic agonist carbachol. This effect is seen after 9 h, is maximal after 24 h, and occurs over a range of carbachol concentrations that activate phosphoinositide hydrolysis in these cells. The effect is specific for Gq alpha family proteins as Gs alpha was slightly increased after carbachol treatment and G13 alpha was unchanged. Using a urea gel system, we were able to resolve Gq alpha and G11 alpha, both of which were down-regulated by carbachol. An M3 receptor mutant, with C-terminal threonines changed to alanines as described previously, binds ligand and activates phosphoinositide hydrolysis normally but is not down-regulated in response to carbachol. This receptor, however, induces Gq alpha/G11 alpha down-regulation similarly to wild-type M3 receptors, indicating that G-protein down-regulation is not directly coupled to receptor down-regulation. Thus down-regulation of Gq alpha and G11 alpha may contribute to heterologous desensitization particularly at longer times of agonist exposure.

Detection of a CfoI polymorphism within exon 5 of the human neuronal nicotinic acetylcholine receptor alpha 4 subunit gene (CHRNA4).

The gene coding for the alpha 4 subunit of the neuronal nicotinic acetylcholine receptor (CHRNA4) has been recently mapped in the candidate region for benign familial neonatal convulsions (BFNC) on chromosome 20q13.2-q13.3. The region is only partially covered with polymorphic markers, and so far no PCR-based polymorphisms have been described in the critical region for BFNC. We now report the first polymorphic marker in the coding region of CHRNA4. The new marker, which is detected by PCR, will be useful for evaluation of the role of CHRNA4 as a candidate gene for BFNC. It will further enable the investigation of this important brain-specific gene in association studies with different types of epileptic diseases and other neurological disorders.

Effects of phospholipids on agonist-dependent phosphorylation of human muscarinic acetylcholine receptors by G protein-coupled receptor kinases: An alternate mode to kinase activation?

Effects of phospholipids on agonist-dependent phosphorylation of human muscarinic acetylcholine receptors by G protein-coupled receptor kinases: An alternate mode to kinase activation?

Role of endothelium in the human uterine arteries during normal menstrual cycle.

1. The present experiments were designed to investigate the role of endothelium in the human uterine arteries during the normal menstrual cycle. 2. Acetylcholine (ACh) produced a concentration-dependent relaxation response during the higher level of plasma 17 beta-oestradiol (E2) (follicular and luteal phases, E2 = 131.9 +/- 15.9 pg ml-1, n = 13; group I). However, the agent did not produce a definite relaxation, but produced a slight contraction during the ovulatory and menstruation phases (E2 = 19.8 +/- 2.9 pg mg-1, n = 5; group II). During the follicular and luteal phases (E2 = 181.1 +/- 9.0 pg ml-1, n = 6), ACh produced a slight contraction, but not relaxation in 6 cases (group III). Relaxation in response to A23187 in group II was not different from that in group I, while it was significantly (P < 0.05 and P < 0.005) reduced in group III. Sodium nitroprusside (SNP)-induced relaxation was similar in the three groups. 3. Correlation between the maximum response to ACh and the plasma E2 was highly significant (gamma = 0.8142, P < 0.001) in 18 cases of groups I and II, but not in all 24 cases including group III (gamma = 0.1183, NS). 4. Relaxations in response to ACh in group I or A23187 in all groups were abolished after removal of the endothelium. In group I, ACh- and A23187-induced relaxations were greatly inhibited by methylene blue or NG-nitro-L-arginine (L-NOARG) and partially inhibited by indomethacin. None of these treatments except for methylene blue modified the SNP-induced relaxation, which was significantly inhibited by methylene blue.5. The A23187-induced relaxation was hardly affected by methylene blue or L-NOARG in group III,but was partially inhibited by these agents in group II. The effect of indomethacin in inhibiting the A23187 induced-relaxation was most potent (58.9%) in group III and least (16.9%) in group I.6. There were no histological changes in 14 cases out of 18 (groups I and II), but very slight intimal thickening was observed in 4 cases in group I. On the other hand, severe intimal thickening was observed in all 6 cases in group III.7. These results indicate that, in human uterine artery strips, ACh and A23187 cause endothelium dependent relaxations, which are mediated mainly through EDRF/NO in group I, mainly prostacyclin(PGI2) in group III, or both in group II. It is suggested that lack of the production/release of EDRF/NO and/or of interaction between EDRF/NO and PGI2 might play a role in the formation of intimal thickening in human uterine arteries.

Single channel properties of human muscle nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes

Single channel properties of human muscle nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes

Human muscle acetylcholine receptor reactive T and B lymphocytes in myasthenia gravis.

Human muscle acetylcholine receptor reactive T and B lymphocytes in myasthenia gravis.

T cell stimulatory and myasthenogenic regions in human skeletal muscle acetylcholine receptor (AChR) α subunit

T cell stimulatory and myasthenogenic regions in human skeletal muscle acetylcholine receptor (AChR) α subunit

Assignment of the human nicotinic acetylcholine receptor genes: the alpha and delta subunit genes to chromosome 2 and the beta subunit gene to chromosome 17

The chromosomal assignments of the genes coding for the alpha, beta and delta subunits of the human nicotinic acetylcholine receptor have been determined from a panel of somatic cell hybrids and by direct in situ hybridization. The results localize CHRNA to 2q24-2q32. CHRNB to 17p11-17p12, and CHRND to chromosome 2q33-2qter.

Isolation and expression of cDNAS for the human muscle nicotinic acetylcholine receptor

Isolation and expression of cDNAS for the human muscle nicotinic acetylcholine receptor

TE671 cell and solubilized human muscle acetylcholine receptors: Similarities and differences in immunological properties

TE671 cell and solubilized human muscle acetylcholine receptors: Similarities and differences in immunological properties

Functional properties of human skeletal muscle acetylcholine receptors expressed by the TE671 cell line.

Functional properties of acetylcholine receptors from intact TE671 human medulloblastoma cells were examined using tracer ion flux, ligand competition against 125I-labeled alpha-bungarotoxin binding, and single channel recording measurements. 125I-Labeled alpha-bungarotoxin binds to surface receptors with the forward rate constant 1.8 X 10(5) M-1 s-1 and dissociates with the rate constant 4.6 X 10(-5) s-1, at 21 degrees C; the apparent dissociation constant is 2.6 X 10(-10) M. alpha-Bungarotoxin binds to at least two sites/receptor, but blocks agonist-induced 22Na+ uptake when bound to only one site. The reversible antagonists, dimethyl-d-tubocurarine and gallamine, occupy two sites which exhibit nearly equivalent affinities, but block agonist-induced uptake by occupying only one site. Strong agonists activate rapid sodium uptake with relatively low affinity, but desensitize with a much higher affinity; among agonists, the ratio of low to high affinity dissociation constants ranges from 1600 to 4000. By using the estimated dissociation constants, the allosteric model of Monod, Wyman, and Changeux (MWC) can be fitted to the concentration dependencies of both steady-state agonist occupancy and desensitization. The fitting analysis discloses an allosteric constant of 3 X 10(-5), which is the ratio of activatable to desensitized receptors in the absence of agonist. The rate of recovery from desensitization can exceed the rate of onset of desensitization elicited by low concentrations of agonist, further supporting the general MWC framework. Single channel recordings show that the channel opening probability is greater than 0.7 at high agonist concentrations. Favorable channel opening is shown to only slightly oppose strong desensitization.