Muscarinic Acetylcholine Receptor Antagonist Research Articles

The human muscarinic acetylcholine receptor antagonist, Dicyclomine targets signal transduction genes and inhibits the virulence factors in the human pathogen, Candida albicans.

Dicyclomine is a human muscarinic acetylcholine receptor antagonist used for the treatment of abdominal cramps. We are reporting here that dicyclomine can inhibit the in vitro growth and virulence factors of the human pathogen Candida albicans very effectively. Dicyclomine inhibited adhesion, early biofilm, mature biofilm, and planktonic growth. Yeast to hyphal form transition of C. albicans in various inducer media such as serum, proline, glucose, and N-acetylglucosamine was inhibited. Dicyclomine also could kill C. albicans cells within 15 min of exposure. Dicyclomine appears to inhibit the yeast to hyphal conversion by affecting signal transduction pathway. The expression of selected genes associated with yeast to hyphal form transition in serum in presence of dicyclomine was studied using real-time polymerase chain reaction (RtPCR). The RtPCR analysis showed that dicyclomine targets both cAMP pathway as well as MAPK cascade. Eight genes were upregulated. Out of these, three major upregulated genes were Bcy1, Tup1, and Mig1. Dicyclomine downregulated Ume6, Ece1, and Pde2 genes which are involved in cAMP signaling pathway and also downregulated the DNA binding protein gene, Rfg1. Dicyclomine significantly upregulated the master negative regulator of hyphal formation, Tup1. Based on this study we suggest that the muscarinic acetylcholine receptor antagonist, dicyclomine could be repositioned as a potential anti-Candida albicans as well as anti-virulence agent.

Acute and chronic modulation of striatal endocannabinoid-mediated plasticity by nicotine.

The endocannabinoid (eCB) system modulates several phenomena related to addictive behaviors, and drug‐induced changes in eCB signaling have been postulated to be important mediators of physiological and pathological reward‐related synaptic plasticity. Here, we studied eCB‐mediated long‐term depression (eCB‐LTD) in the dorsolateral striatum, a brain region critical for acquisition of habitual and automatic behavior. We report that nicotine differentially affects ex vivo eCB signaling depending on previous exposure in vivo. In the nicotine‐naïve brain, nicotine facilitates eCB‐signaling and LTD, whereas tolerance develops to this facilitating effect after subchronic exposure in vivo. In the end, a progressive impairment of eCB‐induced LTD is established after protracted withdrawal from nicotine. Endocannabinoid‐LTD is reinstated 6 months after the last drug injection, but a brief period of nicotine re‐exposure is sufficient to yet again impair eCB‐signaling. LTD induced by the cannabinoid 1 receptor agonist WIN55,212‐2 is not affected, suggesting that nicotine modulates eCB production or release. Nicotine‐induced facilitation of eCB‐LTD is occluded by the dopamine D2 receptor agonist quinpirole, and by the muscarinic acetylcholine receptor antagonist scopolamine. In addition, the same compounds restore eCB‐LTD during protracted withdrawal. Nicotine may thus modulate eCB‐signaling by affecting dopaminergic and cholinergic neurotransmission in a long‐lasting manner. Overall, the data presented here suggest that nicotine facilitates eCB‐LTD in the initial phase, which putatively could promote neurophysiological and behavioral adaptations to the drug. Protracted withdrawal, however, impairs eCB‐LTD, which may influence or affect the ability to maintain cessation.

Intermittent Stimulation of the Nucleus Basalis of Meynert Improves Working Memory in Adult Monkeys

Dysfunction in the brain's cholinergic systems results in working memory and executive function impairments. The source of acetylcholine for the neocortex is the Nucleus Basalis of Meynert. We tested whether electrical activation of this nucleus would improve executive function in young adult monkeys. Continuous stimulation led to consistent performance impairments. In contrast, intermittent stimulation led to supranormal working memory performance. Optimal performance was achieved with delivery of 200 microsecond biphasic pulses at a frequency of 60 pulses per second, for 20 seconds every minute. Effects onset and offset within three minutes. Pharmacological experiments suggested both intermittent and continuous stimulation effects depended on cholinergic activation. Donepezil, a cholinesterase inhibitor, improved performance to supranormal levels in animals already impaired by continuous electrical stimulation. The same treatment did not impact performance while animals received intermittent stimulation. Administration of either nicotinic or muscarinic acetylcholine receptor antagonists prevented intermittent stimulation from altering performance. In the months after stimulation testing began, animal performance, measured in sessions without stimulation, improved. Over these months, the working memory delay at which animals achieved 80% correct performance increased three to fivefold. Our results show that cholinergic activation by deep brain stimulation enhances working memory performance and is a candidate treatment for losses in executive function in aging and Alzheimer's Disease.

Counteracting desensitization of human α7-nicotinic acetylcholine receptors with bispyridinium compounds as an approach against organophosphorus poisoning

Irreversible inhibition of acetylcholinesterase (AChE) resulting in accumulation of acetylcholine and overstimulation of muscarinic and nicotinic receptors accounts for the acute toxicity of organophosphorus compounds (OP). Accordingly, the mainstay pharmacotherapy against poisoning by OP comprises the competitive muscarinic acetylcholine receptor antagonist atropine to treat muscarinic effects and, in addition, oximes to reactivate inhibited AChE. A therapeutic gap still remains in the treatment of desensitized nicotinic acetylcholine receptors following OP exposure. Hereby, nicotinic effects result in paralysis of the central and peripheral respiratory system if untreated. Thus, these receptors pose an essential target for therapeutic indication to address these life-threatening nicotinic symptoms of the cholinergic crisis. Identification of ligands regulating dynamic transitions between functional states by binding to modulatory sites appears to be a promising strategy for therapeutic intervention. In this patch clamp study, the ability of differently substituted bispyridinium non-oximes to “resensitize” i.e. to recover the activity of desensitized human homomeric α7-type nAChRs stably transfected in CHO cells was investigated and compared to the already described α7-specific positive allosteric modulator PNU-120596. The structures of these bispyridinium analogues were based on the lead structure of the tert-butyl-substituted bispyridinium propane MB327, which has been shown to have a positive therapeutic effect due to a non-competitive antagonistic action at muscle-type nAChRs in vivo and has been found to have a positive allosteric activity at neuronal receptors in vitro. Prior to test compounds, desensitization of hα7-nAChRs was verified by applying an excess of nicotine revealing activation at low, and desensitization at high concentrations. Thereby, desensitization could be reduced by modulation with PNU-120596. Desensitization was further verified by dose-response profiles of agonists, carbamoylcholine and epibatidine in the absence and presence of PNU-120596. Although less pronounced than PNU-120596 and the lead structure MB327, bispyridinium compounds, particularly those substituted at position 3 and 4, resensitized the nicotine desensitized hα7-nAChRs in a concentration-dependent manner and prolonged the mean channel open time. In summary, identification of more potent compounds able to restore nAChR function in OP intoxication is needed for development of a putative efficient antidote.

New Insight Into the Mechanisms of Fast-Acting Antidepressants: What We Learn From Scopolamine

SEE CORRESPONDING ARTICLE ON PAGE 29 Activity-Dependent Brain-Derived Neurotrophic Factor Release Is Required for the Rapid Antidepressant Actions of ScopolamineBiological PsychiatryVol. 83Issue 1PreviewBrain-derived neurotrophic factor (BDNF) plays a key role in the pathophysiology and treatment of depression. Recent clinical studies demonstrate that scopolamine, a nonselective muscarinic acetylcholine receptor antagonist, produces rapid antidepressant effects in patients with depression. Rodent studies demonstrate that scopolamine increases glutamate transmission and synaptogenesis in the medial prefrontal cortex (mPFC). Here we tested the hypothesis that activity-dependent BDNF release within the mPFC is necessary for the antidepressant actions of scopolamine. Full-Text PDF

Establishing zebrafish as a model to study the anxiolytic effects of scopolamine

Scopolamine (hyoscine) is a muscarinic acetylcholine receptor antagonist that has traditionally been used to treat motion sickness in humans. However, studies investigating depressed and bipolar populations have found that scopolamine is also effective at reducing depression and anxiety symptoms. The potential anxiety-reducing (anxiolytic) effects of scopolamine could have great clinical implications for humans; however, rats and mice administered scopolamine showed increased anxiety in standard behavioural tests. This is in direct contrast to findings in humans, and complicates studies to elucidate the specific mechanisms of scopolamine action. The aim of this study was to assess the suitability of zebrafish as a model system to test anxiety-like compounds using scopolamine. Similar to humans, scopolamine acted as an anxiolytic in individual behavioural tests (novel approach test and novel tank diving test). The anxiolytic effect of scopolamine was dose dependent and biphasic, reaching maximum effect at 800 µM. Scopolamine (800 µM) also had an anxiolytic effect in a group behavioural test, as it significantly decreased their tendency to shoal. These results establish zebrafish as a model organism for studying the anxiolytic effects of scopolamine, its mechanisms of action and side effects.

Heterodimerization of Dibenzodiazepinone-Type Muscarinic Acetylcholine Receptor Ligands Leads to Increased M2R Affinity and Selectivity

In search for selective ligands for the muscarinic acetylcholine receptor (MR) subtype M2, the dimeric ligand approach, that is combining two pharmacophores in one and the same molecule, was pursued. Different types (agonists, antagonists, orthosteric, and allosteric) of monomeric MR ligands were combined by various linkers with a dibenzodiazepinone-type MR antagonist, affording five types of heterodimeric compounds (“DIBA-xanomeline,” “DIBA-TBPB,” “DIBA-77-LH-28-1,” “DIBA-propantheline,” and “DIBA-4-DAMP”), which showed high M2R affinities (pKi > 8.3). The heterodimeric ligand UR-SK75 (46) exhibited the highest M2R affinity and selectivity [pKi (M1R–M5R): 8.84, 10.14, 7.88, 8.59, and 7.47]. Two tritium-labeled dimeric derivatives (“DIBA-xanomeline”-type: [3H]UR-SK71 ([3H]44) and “DIBA-TBPB”-type: [3H]UR-SK59 ([3H]64)) were prepared to investigate their binding modes at hM2R. Saturation-binding experiments showed that these compounds address the orthosteric binding site of the M2R. The investigation of the effect of various allosteric MR modulators [gallamine (13), W84 (14), and LY2119620 (15)] on the equilibrium (13–15) or saturation (14) binding of [3H]64 suggested a competitive mechanism between [3H]64 and the investigated allosteric ligands, and consequently a dualsteric binding mode of 64 at the M2R.

Hijiki and sodium arsenite stimulate growth of human colorectal adenocarcinoma cells through ERK1/2 activation

Hijiki seaweed (Hijikia fusiformes) contains high levels of inorganic arsenic, a known carcinogen. However, scientific reports on carcinogenic risks associated with the consumption of this seaweed are limited. This study investigated the effects of seaweed extracts contaminated with arsenic on two colorectal cancer cell lines. Two seaweed extracts, including Hijiki and red seaweed, induced H508 but not HT29 cell proliferation. Growth induction of H508 cells after treatments with Hijiki and sodium arsenite at concentrations equivalent to arsenic found in Hijiki was observed by both MTT and BrdU assays. Hijiki and sodium arsenite induced epidermal growth factor receptor (EGFR) and ERK1/2 activations. AG1478, an EGFR inhibitor, decreased the activation of EGFR and ERK1/2 induced by Hijiki and sodium arsenite. U0126, an ERK1/2 upstream inhibitor, and atropine, a muscarinic acetylcholine receptor (mAChR) antagonist, but not AG1478 completely inhibited the proliferative effect of Hijiki. Altogether, the results suggest that the presence of arsenic in seaweed may partly contribute to the proliferation of colorectal cancer cells. EGFR-dependent, and -independent ERK1/2 signaling pathways, and mAChR may be involved in the growth stimulation by Hijiki. These results raise concern regarding the potential colorectal cancer risks from regular consumption of Hijiki containing high contents of inorganic arsenic.

Role of ventrolateral orbital cortex muscarinic and nicotinic receptors in modulation of capsaicin-induced orofacial pain-related behaviors in rats

Acetylcholine, as a major neurotransmitter, mediates many brain functions such as pain. This study was aimed to investigate the effects of microinjection of muscarinic and nicotinic acetylcholine receptor antagonists and agonists into the ventrolateral orbital cortex (VLOC) on capsaicin-induced orofacial nociception and subsequent hyperalgesia. The right side of VLOC was surgically implanted with a guide cannula in anaesthetized rats. Orofacial pain-related behaviors were induced by subcutaneous injection of a capsaicin solution (1.5µg/20µl) into the left vibrissa pad. The time spent face rubbing with ipsilateral forepaw and general behavior were recorded for 10min, and then mechanical hyperalgesia was determined using von Frey filaments at 15, 30, 45 and 60min post-capsaicin injection. Alone intra-VLOC microinjection of atropine (a muscarinic acetylcholine receptor antagonist) and mecamylamine (a nicotinic acetylcholine receptor antagonist) at a similar dose of 200ng/site did not alter nocifensive behavior and hyperalgesia. Microinjection of oxotremorine (a muscarinic acetylcholine receptor agonist) at doses of 50 and 100ng/site and epibatidine (a nicotinic acetylcholine receptor agonist) at doses of 12.5, 25, 50 and 100ng/site into the VLOC suppressed pain-related behaviors. Prior microinjections of 200ng/site atropine and mecamylamine (200ng/site) prevented oxotremorine (100ng/site)-, and epibatidine (100ng/site)-induced antinociception, respectively. None of the above-mentioned chemicals changed general behavior. These results showed that the VLOC muscarinic and nicotinic acetylcholine receptors might be involved in modulation of orofacial nociception and hypersensitivity.

The brain is required for normal muscle and nerve patterning during early Xenopus development

Possible roles of brain-derived signals in the regulation of embryogenesis are unknown. Here we use an amputation assay in Xenopus laevis to show that absence of brain alters subsequent muscle and peripheral nerve patterning during early development. The muscle phenotype can be rescued by an antagonist of muscarinic acetylcholine receptors. The observed defects occur at considerable distances from the head, suggesting that the brain provides long-range cues for other tissue systems during development. The presence of brain also protects embryos from otherwise-teratogenic agents. Overexpression of a hyperpolarization-activated cyclic nucleotide-gated ion channel rescues the muscle phenotype and the neural mispatterning that occur in brainless embryos, even when expressed far from the muscle or neural cells that mispattern. We identify a previously undescribed developmental role for the brain and reveal a non-local input into the control of early morphogenesis that is mediated by neurotransmitters and ion channel activity.

Muscarinic receptor signaling contributes to atypical antipsychotic drug reversal of the phencyclidine-induced deficit in novel object recognition in rats.

Enhancement of cholinergic function via muscarinic acetylcholine receptor M1 agonism improves cognition in some schizophrenia patients. Most atypical antipsychotic drugs, including clozapine and its active metabolite, N-desmethylclozapine, and lurasidone, enhance the release of acetylcholine in key brain regions involved in cognition (e.g. hippocampus). We determined the effect of muscarinic acetylcholine receptor M1 stimulation on novel object recognition and its contribution to the ability of atypical antipsychotic drugs to reverse the novel object recognition deficit in rats withdrawn from subchronic phencyclidine, a rodent model of cognitive impairment in schizophrenia. In control rats, the non-specific muscarinic acetylcholine receptor antagonist, scopolamine, and the M1 selective antagonist, VU0255035, induced a novel object recognition deficit, which was reversed by the M1 agonist, AC260584. Scopolamine fully blocked the effect of clozapine and N-desmethylclozapine, but not lurasidone, to restore novel object recognition in subchronic phencyclidine-treated rats. VU0255035 also blocked these effects of clozapine and N-desmethylclozapine, but not lurasidone; however, the blockade was not as complete as that achieved with scopolamine. Furthermore, subchronic phencyclidine increased hippocampal M1 mRNA expression. These data suggest that M1 agonism is required for clozapine and N-desmethylclozapine to ameliorate the phencyclidine-induced deficit in novel object recognition, additional evidence that M1 agonism is a potential target for treating cognitive impairment in schizophrenia.

L-Ornithine and l-lysine stimulate gastrointestinal motility via transient receptor potential vanilloid 1.

The gastrointestinal (GI) tract senses and responds to intraluminal nutrients and these interactions often affect GI functions. We found that, among basic amino acids, l-ornithine (Orn) and l-lysine (Lys) stimulated but l-arginine (Arg) suppressed GI motility after oral administration (24 mmol/kg) in mice (Orn and Lys, 14.3 and 26.4% promotion; Arg, 7.7% suppression). We investigated the mechanism of the action of Orn and Lys on GI motility. Orn-induced promotion of small intestinal transit was significantly inhibited (p<0.05) by oral administration of capsazepine, a transient receptor potential vanilloid 1 (TRPV1) antagonist. Moreover, the stimulatory effect of Orn and Lys was abolished in TRPV1-knockout mice. In TRPV1-transfected HEK293 cells, Orn and Lys (10 mM) evoked Ca2+ influx, which was blocked by ruthenium red, a TRP channel antagonist. These results suggest that Orn and Lys promote GI motility via activation of TRPV1. The GI motility stimulation by Orn and Lys was also blocked by atropine, a muscarinic acetylcholine receptor (mAChR) antagonist, or NG -nitro-l-arginine methyl ester, a nitric oxide (NO) synthase inhibitor. Orally administered Orn and Lys stimulate GI motility via TRPV1, mAChR and NO synthase in mice.

Network oscillatory activity driven by context memory processing is differently regulated by glutamatergic and cholinergic neurotransmission

Memory retrieval requires coordinated intra- and inter-regional activity in networks of brain structures. Dysfunction of these networks and memory impairment are seen in many psychiatric disorders, but relatively little is known about how memory retrieval and memory failure are represented at the level of local and regional oscillatory activity. To address this question, we measured local field potentials (LFPs) from mice as they explored a novel context, retrieved memories for contextual fear conditioning, and after administration of two amnestic agents: the NMDA receptor antagonist MK-801 and muscarinic acetylcholine receptor antagonist scopolamine (SCOP). LFPs were simultaneously recorded from retrosplenial cortex (RSC), dorsal hippocampus (DH), and anterior cingulate cortex (ACC), which are involved in processing contextual memories, and analyzed for changes in intra-regional power and inter-regional peak coherence of oscillations across multiple frequency bands. Context encoding and memory retrieval sessions yielded similar patterns of changes across all three structures, including decreased delta power and increased theta peak coherence. Baseline effects of MK-801 and SCOP were primarily targeted to gamma oscillations, but in opposite directions. Both drugs also blocked memory retrieval, as indicated by reduced freezing when mice were returned to the conditioning context, but this common behavioral impairment was only associated with power and peak coherence disruptions after MK-801 treatment. These findings point to neural signatures for memory impairment, whose underlying mechanisms may serve as therapeutic targets for related psychiatric disorders.

EEG machine learning for accurate detection of cholinergic intervention and Alzheimer\u2019s disease

Monitoring effects of disease or therapeutic intervention on brain function is increasingly important for clinical trials, albeit hampered by inter-individual variability and subtle effects. Here, we apply complementary biomarker algorithms to electroencephalography (EEG) recordings to capture the brain’s multi-faceted signature of disease or pharmacological intervention and use machine learning to improve classification performance. Using data from healthy subjects receiving scopolamine we developed an index of the muscarinic acetylcholine receptor antagonist (mAChR) consisting of 14 EEG biomarkers. This mAChR index yielded higher classification performance than any single EEG biomarker with cross-validated accuracy, sensitivity, specificity and precision ranging from 88–92%. The mAChR index also discriminated healthy elderly from patients with Alzheimer’s disease (AD); however, an index optimized for AD pathophysiology provided a better classification. We conclude that integrating multiple EEG biomarkers can enhance the accuracy of identifying disease or drug interventions, which is essential for clinical trials.

Precise spike timing dynamics of hippocampal place cell activity sensitive to cholinergic disruption.

New memory formation depends on both the hippocampus and modulatory effects of acetylcholine. The mechanism by which acetylcholine levels in the hippocampus enable new encoding remains poorly understood. Here, we tested the hypothesis that cholinergic modulation supports memory formation by leading to structured spike timing in the hippocampus. Specifically, we tested if phase precession in dorsal CA1 was reduced under the influence of a systemic cholinergic antagonist. Unit and field potential were recorded from the dorsal CA1 of rats as they completed laps on a circular track for food rewards before and during the influence of the systemically administered acetylcholine muscarinic receptor antagonist scopolamine. We found that scopolamine significantly reduced phase precession of spiking relative to the field theta, and that this was due to a decrease in the frequency of the spiking rhythmicity. We also found that the correlation between position and theta phase was significantly reduced. This effect was not due to changes in spatial tuning as tuning remained stable for those cells analyzed. Similarly, it was not due to changes in lap‐to‐lap reliability of spiking onset or offset relative to either position or phase as the reliability did not decrease following scopolamine administration. These findings support the hypothesis that memory impairments that follow muscarinic blockade are the result of degraded spike timing in the hippocampus.

Activity-Dependent Brain-Derived Neurotrophic Factor Release Is Required for the Rapid Antidepressant Actions of Scopolamine

BackgroundBrain-derived neurotrophic factor (BDNF) plays a key role in the pathophysiology and treatment of depression. Recent clinical studies demonstrate that scopolamine, a nonselective muscarinic acetylcholine receptor antagonist, produces rapid antidepressant effects in patients with depression. Rodent studies demonstrate that scopolamine increases glutamate transmission and synaptogenesis in the medial prefrontal cortex (mPFC). Here we tested the hypothesis that activity-dependent BDNF release within the mPFC is necessary for the antidepressant actions of scopolamine. MethodsBehavioral effects of scopolamine were assessed in BDNF Val/Met knock-in mice, in which BDNF processing and release are impaired. In addition, intra-mPFC infusion of a BDNF-neutralizing antibody was performed to test the necessity of BDNF release in driving scopolamine-induced behavioral responses. Further in vivo and in vitro experiments were performed to delineate BDNF-dependent mechanisms underlying the effects of scopolamine. ResultsWe found that BDNF Met/Met mice have attenuated responses to scopolamine and that anti-BDNF antibody infusions into the mPFC prevented the antidepressant-like behavioral effects of scopolamine. In vitro experiments show that scopolamine rapidly stimulates BDNF release and tropomyosin receptor kinase B–extracellular signal-regulated kinase signaling. Moreover, these effects require alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor activation and are blocked by neuronal silencing. Importantly, pretreatment with verapamil prevented scopolamine-induced behavioral responses and BDNF–tropomyosin receptor kinase B signaling, suggesting that these effects are dependent on activation of voltage-dependent calcium channels. ConclusionsThe results identify an essential role for activity-dependent BDNF release in the rapid antidepressant effects of scopolamine. Attenuation of responses in BDNF Met mice indicates that patients with the Met allele may be less responsive to scopolamine.

Novel muscarinic acetylcholine receptor hybrid ligands embedding quinuclidine and 1,4-dioxane fragments

To obtain novel muscarinic acetylcholine receptor (mAChR) antagonists, the enantiomers of the hybrid compounds 3–5, in which the quinuclidin-3-yloxy fragment of solifenacin and the 6,6-diphenyl-1,4-dioxane-2-yl moiety of 2 linked by an ester or ether spacer were embedded in the same chemical entity, were prepared and evaluated for their affinity at the five mAChR subtypes (M1-M5). Stereochemistry and the nature of the linker between the quinuclidine moiety and the 1,4-dioxane nucleus play an important role on the affinities of the compounds. The presence of an ether bridge confers higher affinities for all mAChR subtypes to the ligand. Interestingly, the ether enantiomer (R,S)-5 shows the highest affinity at all mAChR subtypes with pKi values similar to that of solifenacin at M3 and higher at the other subtypes. Unlike solifenacin, it shows a preference for M1 mAChR subtype with respect to the other subtypes. This compound, lacking a permanent positive charge on the nitrogen atom, can be a useful tool for the pharmacological study of mAChRs in the central nervous system.

Sodium tanshinone IIA sulfonate stimulated Cl- secretion in mouse trachea.

Sodium tanshinone IIA sulfonate (STS) is a derivate of tanshinone IIA, a lipophilic compound in Salvia miltiorrhiza. This study aimed to investigate the effect of STS on ion transport in mouse tracheal epithelium and the mechanisms underlying it. Short-circuit current (Isc) was measured to evaluate the effect of STS on transepithelial ion transport. Intracellular Ca2+ imaging was performed to observe intracellular Ca2+ concentration ([Ca2+]i) changes induced by STS in primary cultured mouse tracheal epithelial cells. Results showed that the apical application of STS at mouse trachea elicited an increase of Isc, which was abrogated by atropine, an antagonist of muscarinic acetylcholine receptor (mAChR). By removing ambient Cl− or applying blockers of Ca2+-activated Cl− channel (CaCC), the response of STS-induced Isc was suppressed. Moreover, STS elevated the [Ca2+]i in mouse tracheal epithelial cells. As a result, STS stimulated Cl− secretion in mouse tracheal epithelium via CaCC in an mAChR-dependent way. Due to the critical role of Cl− secretion in airway hydration, our findings suggested that STS may be used to ameliorate the airway dehydration symptom in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD).

The T-type calcium channel enhancer SAK3 inhibits neuronal death following transient brain ischemia via nicotinic acetylcholine receptor stimulation

The T-type calcium channel enhancer SAK3 (ethyl 8'-methyl-2′,4-dioxo-2-(piperidin-1-yl)-2′H-spiro[cyclopentane-1,3'-imidazo[1,2-a]pyridin]-2-ene-3-carboxylate) promotes acetylcholine (ACh) release in mouse hippocampus, enhancing cognitive function. Here, we tested SAK3 neuroprotective activity in the context of transient brain ischemia using a 20-min bilateral common carotid arteries occlusion (BCCAO) mouse model. Mice were administered with SAK3 (0.1, 0.5 or 1.0 mg/kg, p.o.) 24 h after BCCAO ischemia. Oral SAK3 (0.5 or 1.0 mg/kg/day, p.o.) administration significantly blocked loss of hippocampal CA1 neurons and memory deficits seen in BCCAO mice. Treatment with α7 nicotinic ACh receptor (nAChR)-selective inhibitor methyllycaconitine (MLA: 6.0 mg/kg/day, i.p.) significantly antagonized both neuroprotection and improvement in memory promoted by SAK3 (0.5 mg/kg/day, p.o.). Acute SAK3 (0.5 mg/kg, p.o.) administration significantly enhanced protein kinase B (Akt) phosphorylation levels in CA1 of control and BCCAO mice. Importantly, treatment of control and BCCAO mice with the non-selective nAChR antagonist mecamylamine (MEC: 1.0 mg/kg, i.p.) or the α7-selective nAChR antagonist MLA (6.0 mg/kg, i.p.), but not the M1 muscarinic ACh receptor (mAChR) antagonist pirenzepine (PZ: 10 mg/kg, i.p.), blocked enhanced Akt activity elicited by SAK3 (0.5 mg/kg, p.o.). We also confirmed that decreased phosphorylated Akt immunoreactivities were rescued by SAK3 (0.5 mg/kg, p.o.) administration in NeuN-positive CA1 neurons of BCCAO mice, an effect blocked by MLA (6.0 mg/kg, i.p.). Finally, we observed α7 nAChR and phosphorylated Akt expression in CA1 pyramidal neurons. We conclude that the T-type calcium channel enhancer SAK3 is neuroprotective in the context of brain ischemia by stimulating nicotinic cholinergic neurotransmission.

Antagonism of muscarinic acetylcholine receptors in medial prefrontal cortex disrupts the context preexposure facilitation effect

Cholinergic function plays a role in a variant of context fear conditioning known as the context preexposure facilitation effect (CPFE; Robinson-Drummer, Dokovna, Heroux, & Stanton, 2016). In the CPFE, acquisition of a context representation, the context-shock association, and expression of context fear occur across successive phases, usually 24h apart. Systemic administration of scopolamine, a muscarinic acetylcholine receptor antagonist, prior to each phase (context preexposure, immediate-shock training, and testing) disrupts the CPFE in juvenile rats (Robinson-Drummer et al., 2016). Dorsal hippocampal (dHPC) cholinergic function contributes significantly to this effect, as local infusion of scopolamine into the dHPC prior to any individual phase of the CPFE produces a disruption identical to systemic administration (Robinson-Drummer et al., 2016). The current experiment extended these findings to another forebrain region implicated in the CPFE, the medial prefrontal cortex (mPFC). Adolescent rats received bilateral infusions of scopolamine (35μg/side) or PBS 10min before all three phases of the CPFE or only prior to a single phase. Intra-mPFC administration of scopolamine prior to all three phases significantly impaired fear conditioning suggesting that mPFC cholinergic function is necessary for successful CPFE performance. Analyses of the individual infusion days revealed a significant impairment of the CPFE when infusions occurred prior to preexposure or training (i.e. immediate footshock) but not prior to testing. In total, these findings suggests a role of mPFC cholinergic function in the acquisition and/or consolidation of a contextual representation and the context-shock association but not in retrieval or expression of fear memory. Implications for mPFC involvement in contextual fear conditioning and neurological dysfunction following neonatal alcohol exposure are discussed.