Selective α7 nicotinic acetylcholine receptor agonists worsen disease in experimental colitis

Background and purpose:

In various models vagus nerve activation has been shown to ameliorate intestinal inflammation, via nicotinic acetylcholine receptors (nAChRs) expressed on immune cells. As the α7 nAChR has been put forward to mediate this effect, we studied the effect of nicotine and two selective α7 nAChR agonists (AR-{"type":"entrez-nucleotide","attrs":{"text":"R17779","term_id":"771389","term_text":"R17779"}}R17779, (-)-spiro[1-azabicyclo[2.2.2] octane-3,5′-oxazolidin-2′-one and GSK1345038A) on disease severity in two mouse models of experimental colitis.

Experimental approach:

Colitis was induced by administration of 1.5% dextran sodium sulphate (DSS) in drinking water or 2 mg 2,4,6-trinitrobenzene sulphonic acid (TNBS) intrarectally. Nicotine (0.25 and 2.50 µmol·kg⁻¹), AR-{"type":"entrez-nucleotide","attrs":{"text":"R17779","term_id":"771389","term_text":"R17779"}}R17779 (0.6–30 µmol·kg⁻¹) or GSK1345038A (6–120 µmol·kg⁻¹) was administered daily by i.p. injection. After 7 (DSS) or 5 (TNBS) days clinical parameters and colonic inflammation were scored.

Key results:

Nicotine and both α7 nAChR agonists reduced the activation of NF-κB and pro-inflammatory cytokines in whole blood and macrophage cultures. In DSS colitis, nicotine treatment reduced colonic cytokine production, but failed to reduce disease parameters. Reciprocally, treatment with AR-{"type":"entrez-nucleotide","attrs":{"text":"R17779","term_id":"771389","term_text":"R17779"}}R17779 or GSK1345038A worsened disease and led to increased colonic pro-inflammatory cytokine levels in DSS colitis. The highest doses of GSK1345038A (120 µmol·kg⁻¹) and AR-{"type":"entrez-nucleotide","attrs":{"text":"R17779","term_id":"771389","term_text":"R17779"}}R17779 (30 µmol·kg⁻¹) ameliorated clinical parameters, without affecting colonic inflammation. Neither agonist ameliorated TNBS-induced colitis.

Conclusions and implications:

Although nicotine reduced cytokine responses in vitro, both selective α7 nAChR agonists worsened the effects of DSS-induced colitis or were ineffective in those of TNBS-induced colitis. Our data indicate the need for caution in evaluating α7 nAChR as a drug target in colitis.     

Combined administration of anisodamine and neostigmine produces anti-shock effects: involvement of α7 nicotinic acetylcholine receptors

Aim:

To evaluate the anti-effects of anisodamine and neostigmine in animal models of endotoxic and hemorrhagic shock.

Methods:

Kunming mice were injected with lipopolysaccharide (LPS 30 mg/kg, ip) to induce endotoxic shock. Anisodamine (12.5, 25, and 50 mg/kg, ip) and neostigmine (12.5, 25, and 50 μg/kg, ip) were administered immediately after LPS injection. Survival rate was monitored, and the serum levels of TNF-α and IL-1β were analyzed using ELISA assays. The effects of anisodamine and neostigmine were also examined in α7 nicotinic acetylcholine receptor (α7 nAChR) knockout mice with endotoxic shock and in Beagle dogs with hemorrhagic shock.

Results:

In mice with experimental endotoxemia, combined administration of anisodamine and neostigmine significantly increased the survival rate and decreased the serum levels of inflammatory cytokines, as compared to those produced by either drug alone. The anti-shock effect of combined anisodamine and neostigmine was abolished in α7 nAChR knockout mice. On the other hand, intravenous injection of the combined anisodamine and neostigmine, or the selective α7 nAChR agonist PNU282987 exerted similar anti-shock effects in dogs with hemorrhagic shock.

Conclusion:

The results demonstrate that combined administration of anisodamine and neostigmine produces significant anti-shock effects, which involves activation of α7 nAChRs.     

Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin

α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of Bungarus multicinctus snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant α-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged α-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of α-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged α-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic α-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent α-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the α-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged α-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors.     

Functional characterization of a mutated chicken α7 nicotinic acetylcholine receptor subunit with a leucine residue inserted in transmembrane domain 2

1. Site-directed mutagenesis was used to create an altered form of the chicken α₇ nicotinic acetylcholine (ACh) receptor subunit (α₇x61) in which a leucine residue was inserted between residues Leu9′ and Ser10′ in transmembrane domain 2. The properties of α₇x61 receptors are distinct from those of the wild-type receptor.
2. Oocytes expressing wild-type α₇ receptors responded to 10 μM nicotine with rapid inward currents that desensitized with a time-constant of 710±409 ms (mean±s.e.mean, n=5). However in α₇x61 receptors 10 μM nicotine resulted in slower onset inward currents that desensitized with a time-constant of 5684±3403 ms (mean±s.e.mean, n=4). No significant difference in the apparent affinity of nicotine or acetylcholine between mutant and wild-type receptors was observed. Dihydro-β-erythroidine (DHβE) acted as an antagonist on both receptors.
3. Molecular modelling of the α₇x61 receptor channel pore formed by a bundle of M2 α-helices suggested that three of the channel lining residues would be altered by the leucine insertion i.e.; Ser10′ would be replaced by the leucine insertion, Val13′ and Phe14′ would be replaced, by Thr and Val, respectively.
4. When present in the LEV-1 nicotinic ACh receptor subunit from Caenorhabditis elegans the same alteration conferred resistance to levamisole anthelmintic drug. Levamisole blocked responses to nicotine of wild-type and α₇x61 receptors. However, block was more dependent on membrane potential for the α₇x61 receptors.
5. We conclude that the leucine insertion in transmembrane domain 2 has the unusual effect of slowing desensitization without altering apparent agonist affinity.