The effects of alcohols and diols at the nicotinic acetylcholine receptor of the neuromuscular junction

A series of straight chain aliphatic alcohols from ethanol to octanol were tested at voltage-clamped frog endplates. In the presence of high concentrations of ethanol (greater than 1 M) the individual current responses to ionophoretic pulses of ethanol were reduced in amplitude and the dose-response curve for acetylcholine was shifted to the right. All the alcohols tested had this effect and their potency increased with the length of the carbon chain. The results were interpreted to indicate that as the molecular weight of the alcohol increased, its potency as a channel blocker also increased. The diol derivative of ethanol, which is ethylene glycol (ethanediol), was totally inactive up to 400 mM. However, 1,3-propanediol was a more potent blocker than propanol. After dose-response curves were carried out in high doses of ethanol and propanediol, the number of receptors was found to be permanently reduced. This effect could be due to irreversible denaturation of the receptor and therefore reversible denaturation could account for some of the reversible blocking effects caused by such drugs. An additional effect on the receptor was observed in that low concentrations of ethanol and propanol reduced the apparent dissociation constant for acetylcholine, thus increasing the amplitude of individual responses and shifting the dose-response curve to the left.     

Lead modulation of the neuronal nicotinic acetylcholine receptor in PC12 cells

Lead is known to modulate several ligand- and voltage-gated ion channels, including the nicotinic acetylcholine receptor (AChR) channel. We examined the effects of lead on the nicotinic AChR in rat clonal phaeochromocytoma PC12 cells using whole-cell and single-channel patch-clamp techniques to clarify the detailed mechanism of action. Lead suppressed acetylcholine-induced currents in a dose-dependent manner with an EC₅₀ value of 37 μM and a Hill coefficient of 0.82. At the single-channel level, 1–10 μM lead shortened the opening and burst durations, and increased the duration of mean closed time. The open probability was significantly decreased by lead. These changes of single-channel kinetics result in a significant decrease in the total charge carried through the open AChR channels explaining the suppressive effect of lead on acetylcholine-induced whole-cell currents. © 1997 Elsevier Science B.V. All rights reserved.     

Qualitative and quantitative changes in acetylcholine receptor distribution at the neuromuscular junction following free muscle transfer

The qualitative and quantitative changes in acetylcholine receptor distribution were studied in the gracilis muscle of the Wistar rat following free neurovascular transfer. Even at 30 weeks after transfer, the morphology of the neuromuscular junction failed to return to the presurgical state. The number of acetylcholine receptors at the reinnervated neuromuscular junction also remained lower than the control. The persistent weakness following free neurovascular muscle transfer may be attributed to these qualitative and quantitative changes at the neuromuscular junction.     

Direct actions of anticholinesterases on the neuronal nicotinic acetylcholine receptor channels

Recent studies have suggested that anticholinesterases including organophosphates and carbamates act directly on the nicotinic acetylcholine receptor (AChR) channel. We performed whole-cell and single-channel patch-clamp experiments to elucidate the mechanism of action of anticholinesterases on the nicotinic AChR in rat clonal phaeochromocytoma (PC12) cells. Neostigmine and carbaryl showed a biphasic effect; enhancement and suppression of carbachol-induced whole-cell currents. The currents induced by 100 μM carbachol was enhanced by the first co-application with 10 or 100 μM neostigmine, and the current was eventually suppressed below the control level during repeated co-applications. The decay phase of current was accelerated by neostigmine. Carbaryl at 0.1 μM greatly potentiated the carbachol-induced current, and at higher concentrations (0.3–3 μM), current was suppressed. In single-channel experiments, these compounds increased the short closures or gaps during channel opening without changing the single-channel conductance. Mean open time and burst duration were decreased in the presence of neostigmine and carbaryl. These results indicate that neostigmine and carbaryl directly block the nicotinic AChR channel.     

Epibatidine, a nicotinic acetylcholine receptor agonist, inhibits the capsaicin response in dorsal root ganglion neurons

In patch-clamp recordings from small-medium diameter dorsal root ganglion neurons in culture, (+/-)-epibatidine (1 microM) was able to inhibit the capsaicin response (IC(50)=0.32 microM) in neurons where there was no detectable direct nicotinic response. Thus, (+/-)-epibatidine may inhibit the vanilloid receptor in a manner that is not dependent upon nicotinic current activation, representing another mechanism by which such ligands could modulate vanilloid receptor signaling.     

Calcium channel blocker influences the density of alpha-actinin labeling at the rat neuromuscular junction

Alpha-actinin is a muscle protein located along the Z-disc. Incubation of frog muscle with the calcium ionophore, A23187, can decrease the immunogold labelling of alpha-actinin. Pyridostigmine (PYR) is an inhibitor of acetylcholinesterase, which causes disruption of Z-discs only in the region of the motor endplate. This is probably due to excess influx of calcium ions, leading to activation of proteases. Pretreating animals with the calcium channel blocker diltiazem can significantly reduce damage to the Z-discs at the motor endplate caused by PYR. It was of interest to determine whether the distribution of alpha-actinin had been altered following PYR administration and whether diltiazem could prevent those changes. There was less alpha-actinin labelling at the motor endplate compared to away from this region for all treatment groups. Animals administered diltiazem showed less labelling compared to PYR, but with no disruption of Z-discs at the motor endplate following diltiazem. Pretreatment with diltiazem reduced the incidence of Z-disc damage, but the degree of alpha-actinin labeling at the endplate was less than that seen with diltiazem alone. The greater effect seen at the endplate implies that neuromuscular activity is an important factor. The drugs may be causing a reduction in alpha-actinin labelling by different mechanisms.     

Involvement of neuronal nicotinic acetylcholine receptors in the regulation of mediator release in the neuromuscular synapses of mice

We demonstrated that low doses of nicotine (10 nM) decreased the quantum content of the end plate potentials during short-term burst activity of the mouse neuromuscular synapse. The inhibitory effects of nicotine were calcium-dependent and could be inhibited by α-cobratoxin and apamin. These facts suggested that the presynaptic neuronal nicotinic acetylcholine receptors and calcium-activated potassium channels were involved in nicotine effects. The long-term bursts, like nicotine, decreased the quantum content of the end plate potentials and this decrease could be prevented by α-cobratoxin (2 nM). Original Russian Text ? V.V. Fedorin, O.P. Balezina, 2008, published in Neirokhimiya, 2008, Vol. 25, Nos. 1–2, pp. 99–104.     

精神分裂症伴发2型糖尿病与烟碱型乙酰胆碱受体基因多态性关联研究

目的在中国汉族精神分裂症患者中探讨烟碱型乙酰胆碱受体基因多态性与2型糖尿病共病的关联。方法采用Taq Man荧光探针基因分型技术对346例伴发2型糖尿病的中国汉族精神分裂症患者和360例不伴糖尿病的精神分裂症患者的α3、α4、α7、α5烟碱型乙酰胆碱受体(neuronal nicotinic acetylcholine receptor,n Ach R)基因即CHRNA3(rs1317286)、CHRNA4(rs1044396)、CHRNA7(rs6494212)及CHRNA5(rs16969968、rs684513)多态位点进行基因分型,比较等位基因频率和基因型频率,并进一步进行基因-基因交互作用分析。结果单位点分析显示男性患者rs6494212位点的等位基因分布和基因型分布在两组间差异均有统计学意义(P0.05);rs1317286、rs1044396、rs16969968、rs684513位点的基因型分布和等位基因分布均无明显差异(P均0.05)。CHRNA5基因的两个单核苷酸多态位点(single nucleotide polymorphisms,SNPs)组成的单体型与两组疾病共病关联无统计学意义(P0.05)。进一步基因交互作用分析显示rs131726、rs1044396、rs6494212及rs684513这4个位点的联合作用模式可能与两组疾病共病相关(P=0.002)。结论 CHRNA7(rs6494212)可能是中国汉族男性精神分裂症患者患2型糖尿病的易感基因。rs1317286、rs1044396、rs6494212及rs684513位点的联合作用可能与精神分裂症共病2型糖尿病相关。     

Agonist and blocking effects of choline at the neuromuscular junction

The effects of choline chloride were studied at the voltage-clamped frog neuromuscular junction by measuring miniature endplate currents and equilibrium dose response curves for acetylcholine applied by microionophoresis. Choline reduced the amplitude and shortened the time constant of miniature endplate currents in a dose dependent manner. Dose response curves carried out in the presence of low doses of choline (200 microM) were shifted to the right and the apparent dissociation constant for ACh was increased without affecting the Hill coefficient or the maximum conductance at the endplate. Higher doses of choline shifted the curve even further to the right but reduced the Hill coefficient and maximum conductance. Choline ionophoretic dose response curves were carried out but the conductance response was only about 1% of the response to comparable concentrations of ACh. In the presence of ethanol, which reduces the agonist dissociation constant, choline responses were increased and the dose response curve analysis revealed that the efficacy of choline was about 17% in comparison to ACh. Similar effects were measured at rat endplates. Rat nerve-muscle preparations were used to investigate the effects of choline upon neuromuscular transmission.     

Expression of nicotinic acetylcholine receptor mRNA in the adult rat peripheral vestibular system

The mRNA expression of the neuronal nicotinic acetylcholine receptor subunits was determined in adult rat vestibular end-organs and in Scarpa's ganglion (SCG) by in situ hybridization with [³⁵S]riboprobes. Neurons in the SCG expressed the α4–7 and β2–3 mRNAs, but not α3 or β4 mRNAs. Not all SCG neurons expressed every mRNA found in SCG. The α6 and β2–3 riboprobes labeled all neurons, but α4, α5, and α7 mRNAs were selectively expressed in one or more subpopulations of SCG neurons. Vestibular sensory hair cells, in contrast, expressed only a9 mRNA.     

Reduction of glutamate responses by caroverine at the crayfish neuromuscular junction

Caroverine depressed the glutamate current induced by ionophoretically applied glutamate, but affected the neurally evoked excitatory junctional responses less. Caroverine caused more marked reduction of a conditioned glutamate current. The results obtained suggest that caroverine is an open channel blocker for glutamate.     

Ibogaine acts at the nicotinic acetylcholine receptor to inhibit catecholamine release

In an effort to determine mechanisms of action of the putative anti-addictive agent ibogaine, we have measured its effects on catecholamine release in a model neuronal system, cultured bovine chromaffin cells. Various modes of stimulating catecholamine release were used including nicotinic ACh receptor activation, membrane depolarization with elevated K⁺ and Na⁺ channel activation with veratridine. In addition, because ibogaine has been reported to interact with kappa opioid receptors, we tested whether kappa receptor antagonists could reverse ibogaine's effects on catecholamine release. Ibogaine, at low concentration (<10 μM) was found to selectively inhibit nicotinic receptor-mediated catecholamine release, while having no significant effect on release evoked by either veratridine or membrane depolarization with elevated K⁺. The inhibitory actions of ibogaine and the kappa agonists were not reversed by preincubation with the opioid antagonists nor-binaltorphimine or naltrexone, suggesting that these inhibitory effects are not mediated by the kappa opioid receptor. The effects of low dose (10 μM) ibogaine were rapidly reversible, while the inhibitory effects of higher ibogaine doses persisted for at least 19 h following ibogaine washout. The results provide evidence for a mechanism of action ibogaine at the nicotinic ACh receptor. The results are consistent with a model in which the initial high transient brain concentrations (100 μM) of ibogaine act at multiple cellular sites and then have a selective action at the nicotinic ACh receptor cation channel following its metabolism to lower brain concentrations. The present findings are relevant to potential anti-addictive actions of ibogaine and to the development of drugs to combat nicotine addiction.     

Presynaptic localization of ω-conotoxin-sensitive calcium channels at the frog neuromuscular junction

By the use of anti-ω-CTx antibodies in indirect immunofluorescence we demonstrated the presence of ω-CTx binding sites in the presynaptic compartment of frog nerve-muscle preparations. The images we obtained indicate that ω-CTx-sensitive channels are clustered at discrete sites corresponding in distribution to active zones.     

Acetylcholine receptor distribution and synapse elimination at the developing neuromuscular junction of mdx mice

The pattern of innervation of the vertebrate neuromuscular junction is established during early development, when junctions go from multiple to single innervation in the phenomenon of synapse elimination, suggesting that changes at the molecular level in the postsynaptic cell lead to the removal of nerve terminals. The mdx mouse is deficient in dystrophin and associated proteins that are part of the postsynaptic cytoskeleton. We used rhodamine-alpha-bungarotoxin and anti-neurofilament IgG-FITC to stain acetylcholine receptors and nerve terminals of the sternomastoid muscle during postnatal development in mdx and control C57BL/10 mice. Using fluorescence confocal microscopy, we observed that, 7 days after birth, 86.7% of the endplates of mdx mice were monoinnervated (n = 200) compared with 41.4% in control mice (n = 200). By the end of the second postnatal week, all endplates were innervated singly (100% mdx and 94.7% controls, n = 200 per group). These results show that dystrophic fibers achieve single innervation earlier, perhaps because dystrophin or a normal cytoskeletal complex is implicated in this phenomenon.     

Regulation of acetylcholine release by muscarinic receptors at the mouse neuromuscular junction depends on the activity of acetylcholinesterase

Muscarinic acetylcholine receptors (mAChRs) play an important role in regulating the release of acetylcholine (ACh) in various tissues. We used subtype-specific antibodies and a fluorescent-labelled muscarinic toxin to demonstrate that mammalian neuromuscular junction expresses mAChR subtypes M1 to M4, and that localization of all subtypes is highly restricted to the innervated part of the muscle. To elucidate the roles of the mAChR subtypes regulating ACh release, we measured the mean quantal content of endplate potentials in isolated mouse phrenic--hemidiaphragm preparations in which release was reduced by a low Ca2+/high Mg2+ medium. Muscarine decreased evoked ACh release in normal junctions but, depending on the concentration, reduced or increased transmitter release in collagen Q-deficient junctions completely lacking acetylcholinesterase (AChE). Both effects were also seen in normal junctions when AChE was inhibited by various doses of fasciculin-2. Block of mAChRs by atropine had no effect on evoked release at normal junctions, but decreased release at junctions lacking AChE. The muscarine-elicited depression of ACh release in normal junctions was completely abolished by pertussis toxin or methoctramine pretreatment, but was not affected by muscarinic toxin MT-3, thus indicating the involvement of the M2 mAChR. The muscarine-induced increase of ACh release in AChE-deficient junctions was not affected by pertussis toxin, but was completely blocked by MT-7, a specific M1 mAChR antagonist. Our results show that the M1 and M2 mAChRs have opposite presynaptic functions in modulating quantal ACh release, and that regulation of release by the two receptor subtypes depends on the functional state of AChE at the neuromuscular junction.     

Depression of spontaneous and lonophore-induced transmitter release by ruthenium red at the neuromuscular junction

The effects on spontaneous and ionophore-induced transmitter release of the inorganic dye, ruthenium red (RuR), a known inhibitor of calcium binding sites, were observed at the frog sartorius neuromuscular junction using intracellular recording techniques. Both crude and purified RuR, at concentrations of 1 and 5 μM depressed or blocked spontaneous release of acetylcholine (ACh) and reduced postsynaptic sensitivity to ACh, the crude dye being more potent than the pure. Pretreatment of muscles with RuR prevented the catastrophic reaction of junctions to 100 μMX537A ionophore. Increased levels of Ca²⁺ restored spontaneous transmitter release to control levels after depression or blockade by RuR. It was concluded that RuR blocks a critical membrane-bound binding site for calcium which is necessary for quantal release of transmitter.     

Restoring long-term potentiation impaired by amyloid-beta oligomers: Comparison of an acetylcholinesterase inhibitior and selective neuronal nicotinic receptor agonists

As nicotinic acetylcholine receptor (nAChR) agonists directly address cholinergic neurotransmission with potential impact on glutamatergic function, they are considered as potential new symptomatic treatment options for Alzheimer's disease compared to the indirectly operating acetylcholinesterase inhibitors such as the current gold standard donepezil. In order to evaluate the therapeutic value of nAChR activation to ameliorate cognitive dysfunction, a direct comparison between α4β2, α7 nAChR agonists, and donepezil was performed on the level of an ex vivo experimental model of impaired memory formation. First, we demonstrated that amyloid beta (Aβ)₄₂ oligomers, which are believed to be the synaptotoxic Aβ-species causally involved in the pathophysiology of Alzheimer's disease, have a detrimental effect on long-term potentiation (LTP) in the CA1 region of rat hippocampal slices, a widely used cellular model of learning and memory. Second, we investigated the potential of donepezil, the α4β2 nAChR agonist TC-1827 and the α7 nAChR partial agonist SSR180711 to reverse Aβ₄₂ oligomer induced LTP impairment. Donepezil showed only a slight reversal of Aβ₄₂ oligomer induced impairment of early LTP, and had no effect on Aβ₄₂ oligomer induced impairment of late LTP. The same was demonstrated for the α4β2 nAChR agonist TC-1827. In contrast, the α7 nAChR partial agonist SSR180711 completely rescued early as well as late LTP impaired by Aβ₄₂ oligomers. As activating α7 nAChRs was found to be most efficacious in restoring Aβ₄₂ oligomer induced LTP deficits, targeting α7 nAChRs might represent a powerful alternative approach for symptomatic treatment of AD.     

Selective changes in the levels of nicotinic acetylcholine receptor protein and of corresponding mRNA species in the brains of patients with Parkinson's disease

Reductions in the number of neuronal nicotinic acetylcholine receptors (nAChRs) have been shown to occur in connection with Parkinson's disease (PD), but it is still unclear which subtype of this receptor is affected. In the present study we examined various nAChR subtypes employing ligand binding, as well as levels of subunit protein and mRNA in the brains of PD patients and age-matched controls. Binding of [3H]epibatidine and levels of alpha3 mRNA in the caudate nucleus and temporal cortex, but not in the hippocampus were significantly decreased in the PD brain. The level of the alpha3 protein subunit was significantly reduced in all these brain regions but there was no change in the level of alpha4. The level of the beta2 protein subunit in the temporal cortex and hippocampus and the beta2 mRNA in the temporal cortex was lowered. Both the levels of the alpha7 subunit protein and [125I]alpha-bungarotoxin binding were significantly increased in the temporal cortex of PD patients whereas the alpha7 mRNA level was unchanged. These findings reveal selective losses of the alpha3- and beta2-containing nAChRs and an increase in the alpha7 nAChRs that might be related to the pathogenesis of PD.     

Agonist and inhibitory effects of pyridostigmine at the neuromuscular junction

When all of the AChE at the endplate is irreversibly inhibited by phospholine iodide the ionophoretically induced ACh endplate currents are increased more than 10-fold in amplitude. The reversible AChE inhibitor pyridostigmine only increases the current to about half this value because its effects are obscured by receptor blocking. It was found that pyridostigmine can activate the receptor ion channels when released by ionophoresis at the endplate, thus suggesting that agonist-like desensitization could contribute to the blocking effects.