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Ketamine and its preservative, benzethonium chloride, both inhibit human recombinant alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors in Xenopus oocytes.

ABSTRACT: 1. Ketamine is a dissociative anaesthetic that is formulated as Ketalar, which contains the preservative benzethonium chloride (BCl). We have studied the effects of pure racemic ketamine, the preservative BCl and the Ketalar mixture on human neuronal nicotinic acetylcholine receptors (nAChRs) composed of the alpha7 subunit or alpha4 and beta2 subunits expressed in Xenopus laevis oocytes. 2. Ketamine inhibited responses to 1 mM acetylcholine (ACh) in both the human alpha7 and alpha4beta2 nAChRs, with IC(50) values of 20 and 50 microM respectively. Inhibition of the alpha7 nAChRs occurred within a clinically relevant concentration range, while inhibition of the alpha4beta2 nAChR was observed only at higher concentrations. The Ketalar formulation inhibited nAChR function more effectively than was expected given its ketamine concentration. The surprising increased inhibitory potency of Ketalar compared with pure ketamine appeared to be due to the activity of BCl, which inhibited both alpha7 (IC(50) value of 122 nM) and alpha4beta2 (IC(50) value of 49 nM) nAChRs at concentrations present in the clinical formulation of Ketalar. 3. Ketamine is a noncompetitive inhibitor at both the alpha7 and alpha4beta2 nAChR. In contrast, BCl causes a parallel shift in the ACh dose-response curve at the alpha7 nAChR suggesting competitive inhibition. Ketamine causes both voltage-dependent and use-dependent inhibition, only in the alpha4beta2 nAChR. 4. Since alpha7 nAChRs are likely to be inhibited during clinical use of Ketalar, the actions of ketamine and BCl on this receptor subtype may play a role in the profound analgesia, amnesia, immobility and/or autonomic modulation produced by this anaesthetic.

SUBMITTER: Coates KM

PROVIDER: S-EPMC1573008 | biostudies-literature | 2001 Oct

REPOSITORIES: biostudies-literature

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Ketamine and its preservative, benzethonium chloride, both inhibit human recombinant alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors in Xenopus oocytes.

Coates K M KM Flood P P

British journal of pharmacology 20011001 4

1. Ketamine is a dissociative anaesthetic that is formulated as Ketalar, which contains the preservative benzethonium chloride (BCl). We have studied the effects of pure racemic ketamine, the preservative BCl and the Ketalar mixture on human neuronal nicotinic acetylcholine receptors (nAChRs) composed of the alpha7 subunit or alpha4 and beta2 subunits expressed in Xenopus laevis oocytes. 2. Ketamine inhibited responses to 1 mM acetylcholine (ACh) in both the human alpha7 and alpha4beta2 nAChRs, ...[more]

PMID: 11606328

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Project description:The neuronal alpha4beta2 nicotinic acetylcholine receptor (nAChR) is one of the most widely expressed nAChR subtypes in the brain. Its subunits have high sequence identity (54 and 46% for alpha4 and beta2, respectively) with alpha and beta subunits in Torpedo nAChR. Using the known structure of the Torpedo nAChR as a template, the closed-channel structure of the alpha4beta2 nAChR was constructed through homology modeling. Normal-mode analysis was performed on this closed structure and the resulting lowest frequency mode was applied to it for a "twist-to-open" motion, which increased the minimum pore radius from 2.7 to 3.4 A and generated an open-channel model. Nicotine could bind to the predicted agonist binding sites in the open-channel model but not in the closed one. Both models were subsequently equilibrated in a ternary lipid mixture via extensive molecular dynamics (MD) simulations. Over the course of 11 ns MD simulations, the open channel remained open with filled water, but the closed channel showed a much lower water density at its hydrophobic gate comprised of residues alpha4-V259 and alpha4-L263 and their homologous residues in the beta2 subunits. Brownian dynamics simulations of Na+ permeation through the open channel demonstrated a current-voltage relationship that was consistent with experimental data on the conducting state of alpha4beta2 nAChR. Besides establishment of the well-equilibrated closed- and open-channel alpha4beta2 structural models, the MD simulations on these models provided valuable insights into critical factors that potentially modulate channel gating. Rotation and tilting of TM2 helices led to changes in orientations of pore-lining residue side chains. Without concerted movement, the reorientation of one or two hydrophobic side chains could be enough for channel opening. The closed- and open-channel structures exhibited distinct patterns of electrostatic interactions at the interface of extracellular and transmembrane domains that might regulate the signal propagation of agonist binding to channel opening. A potential prominent role of the beta2 subunit in channel gating was also elucidated in the study.

Project description:We and others have shown that one of the mechanisms of growth regulation of small cell lung cancer cell lines and cultured pulmonary neuroendocrine cells is by the binding of agonists to the alpha7 neuronal nicotinic acetylcholine receptor. In addition, we have shown that the nicotine-derived carcinogenic nitrosamine, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a high affinity agonist for the alpha7 nicotinic acetylcholine receptor. In the present study, our goal was to determine the extent of alpha7 mRNA and protein expression in the human lung.Experiments were done using reverse transcription polymerase chain reaction (RT-PCR), a nuclease protection assay and western blotting using membrane proteins.We detected mRNA for the neuronal nicotinic acetylcholine receptor alpha7 receptor in seven small cell lung cancer (SCLC) cell lines, in two pulmonary adenocarcinoma cell lines, in cultured normal human small airway epithelial cells (SAEC), one carcinoid cell line, three squamous cell lines and tissue samples from nine patients with various types of lung cancer. A nuclease protection assay showed prominent levels of alpha7 in the NCI-H82 SCLC cell line while alpha7 was not detected in SAEC, suggesting that alpha7 mRNA levels may be higher in SCLC compared to normal cells. Using a specific antibody to the alpha7 nicotinic receptor, protein expression of alpha7 was determined. All SCLC cell lines except NCI-H187 expressed protein for the alpha7 receptor. In the non-SCLC cells and normal cells that express the alpha7 nAChR mRNA, only in SAEC, A549 and NCI-H226 was expression of the alpha7 nicotinic receptor protein shown. When NCI-H69 SCLC cell line was exposed to 100 pm NNK, protein expression of the alpha7 receptor was increased at 60 and 150 min.Expression of mRNA for the neuronal nicotinic acetylcholine receptor alpha7 seems to be ubiquitously expressed in all human lung cancer cell lines tested (except for NCI-H441) as well as normal lung cells. The alpha7 nicotinic receptor protein is expressed in fewer cell lines, and the tobacco carcinogen NNK increases alpha7 nicotinic receptor protein levels.

Project description:ObjectiveThe central nervous system can regulate peripheral inflammation, but the efferent neuronal routes and the mediators remain poorly defined. One candidate is the cholinergic pathway, which releases acetylcholine (ACh). This neurotransmitter can bind to the alpha7 cholinergic receptor (alpha7R) expressed by nonneuronal cells and reduce inflammation. To test this possibility, we evaluated the expression of alpha7R and its potential role as a target in rheumatoid arthritis (RA).MethodsThe expression of alpha7R in human synovium and fibroblast-like synoviocytes (FLS) was determined using immunohistochemical, Western blot, and quantitative polymerase chain reaction (PCR) analyses. The effects of ACh in vitro were determined in interleukin-1 (IL-1)-stimulated FLS using immunoassays for protein, quantitative PCR for messenger RNA (mRNA), luciferase reporter constructs for IL-6 and NF-kappaB promoter activity, and electrophoretic mobility shift assays. Expression of alpha7R was knocked down with small interfering RNA (siRNA) or was inhibited with the selective alpha7R antagonist methyllycaconitine (MLA).ResultsProtein and mRNA for alpha7R were demonstrated in RA and osteoarthritis synovium and cultured synoviocytes. Expression in synovium was mainly in the intimal lining. ACh significantly reduced the production of IL-6, CXCL8, CCL2, CCL3, CCL5, and granulocyte colony-stimulating factor by IL-1-stimulated FLS. This effect was blocked by the alpha7R antagonist MLA or by using alpha7R siRNA to knock down receptor expression. The selective alpha7R agonist PNU-282,987 decreased the production of IL-6 by IL-1-stimulated FLS. ACh did not reduce IL-6 transcription, but it decreased IL-6 mRNA half-life and reduced IL-6 mRNA steady-state levels.ConclusionThe alpha7 receptor is expressed in the synovium and by synoviocytes. Receptor ligation inhibits cytokine expression in FLS through a posttranscriptional mechanism. Therefore, alpha7R is a potential therapeutic target for inflammatory diseases.

Dataset Information

Ketamine and its preservative, benzethonium chloride, both inhibit human recombinant alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors in Xenopus oocytes.

Publications

Ketamine and its preservative, benzethonium chloride, both inhibit human recombinant alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors in Xenopus oocytes.

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