The Relationship between Muscarinic and Cannabinoid Receptors in Neuronal Excitability and Epilepsy: A Review.

Background: Of the seventy million people who suffer from epilepsy, 40 percent of them become resistant to more than one antiepileptic medication and have a higher chance of death. While the classical definition of epilepsy was due to the imbalance between excitatory glutamatergic and inhibitory γ-aminobutyric acid (GABA)-ergic signalling, substantial evidence implicates muscarinic receptors in the regulation of neural excitability. Summary: Cannabinoids have shown to reduce seizure activity and neuronal excitability in several epileptic models through the activation of muscarinic receptors with drugs which modulate their activity. Cannabinoids also have been effective in reducing antiepileptic activity in pharmaco-resistant individuals; however, the mechanism of its effects in temporal lobe epilepsy is not clear. Key Messages: This review seeks to elucidate the relationship between muscarinic and cannabinoid receptors in epilepsy and neural excitability.

Intracellular Signaling Pathways Involved in the Regulation of Gene Expression by Pilocarpine.

OBJECTIVES: Pilocarpine is commonly used clinically to treat dry mouth. The long-term administration of pilocarpine reportedly improves salivary secretion more effectively than short-term administration. Therefore, we hypothesized that pilocarpine alters gene expression in salivary glands via muscarinic receptor stimulation. This study aimed to investigate the effects of pilocarpine use on gene expression mediated by mitogen-activated protein kinase (MAPK) activity. METHODS: The effects of pilocarpine on gene expression were investigated in rats and human salivary gland (HSY) cells using several inhibitors of intracellular signaling pathways. Gene expression in the rat submandibular gland and HSY cells was determined using reverse transcription-quantitative polymerase chain reaction analysis of total RNA. RESULTS: In animal experiments, at 7 days after pilocarpine stimulation, Ctgf and Sgk1 expressions were increased in the submandibular gland. In cell culture experiments, pilocarpine increased Ctgf expression in HSY cells. The mitogen-activated protein kinase kinase inhibitor trametinib, the Src inhibitor PP2, and the muscarinic acetylcholine receptor antagonist atropine suppressed the effect of pilocarpine on gene expression. CONCLUSIONS: Pilocarpine enhances Ctgf and Sgk1 expressions by activating Src-mediated MAPK activity. Although further studies are required to fully understand the roles of Ctgf and Sgk1, changes in gene expression may play an important role in improving salivary secretions.

Bile acids differentially regulate longitudinal smooth muscle contractility in everted mouse ileum.

Bile acids regulate gastrointestinal motility by mechanisms that are poorly understood. Standard isolated tissue bath assays might not recapitulate in vivo physiology if contractile responses to certain bile acids require direct application to the intestinal mucosa. We sought to determine the feasibility of quantifying longitudinal smooth muscle contractile responses to bile acids from intact segments of everted mouse ileum. Ileum from adult female C57BL/6J mice was isolated, gently everted over a notched metal rod, and mounted in tissue baths. Individual bile acids and agonists of bile acid receptors were added to the baths, and longitudinal smooth muscle contractile responses were quantified by isometric force transduction. Ursodeoxycholic acid robustly increased contractile responses in a dose-dependent manner. Deoxycholic acid stimulated contractility at low doses but inhibited contractility at high doses. Chenodeoxycholic acid, glycocholic acid, and lithocholic acid did not alter contractility. The dose-dependent increase in contractility resulting from the application of ursodeoxycholic acid was recapitulated by INT-777, an agonist of the Takeda G protein-coupled receptor 5 (TGR5), and by cevimeline, a muscarinic acetylcholine receptor agonist. Agonists to the nuclear receptors farnesoid X receptor, glucocorticoid receptor, pregnane X receptor, vitamin D receptor, and to the plasma membrane epidermal growth factor receptor did not modify baseline contractile patterns. These results demonstrate that gentle eversion of intact mouse ileum facilitates the quantification of longitudinal smooth muscle contractile responses to individual bile acids. Prokinetic effects of ursodeoxycholic acid and low-dose deoxycholic acid are replicated by agonists to TGR5 and muscarinic acetylcholine receptors.

[Retracted] MicroRNA­30e inhibits adhesion, migration, invasion and cell cycle progression of prostate cancer cells via inhibition of the activation of the MAPK signaling pathway by downregulating CHRM3.

Following the publication of the above article, a concerned reader drew to the Editor's attention that certain of the Transwell invasion assay data shown in Fig. 7B on p. 451 were strikingly similar to data that had appeared in Fig. 3D in a previously published paper written by different authors at a different research institute, which had been received at the journal Cancer Letters at around the same time, and which has subsequently been retracted [Gu J, Wang Y, Wang X, Zhou D, Shao C, Zhou M and He Z: Downregulation of lncRNA GAS5 confers tamoxifen resistance by activating miR­222 in breast cancer. Cancer Lett 434: 1­10, 2018]. In addition, there were potentially anomalous features associated with the western blot and cell cycle data in this paper.  In view of the fact that certain of the data in the above article were also submitted to a different journal within the space of a few days, the Editor of International Journal of Oncology has decided that this paper should be retracted from the publication. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Oncology 54: 443­454, 2019; DOI: 10.3892/ijo.2018.4647].

Cholinergic modulation in the vertebrate auditory pathway.

Acetylcholine (ACh) is a prevalent neurotransmitter throughout the nervous system. In the brain, ACh is widely regarded as a potent neuromodulator. In neurons, ACh signals are conferred through a variety of receptors that influence a broad range of neurophysiological phenomena such as transmitter release or membrane excitability. In sensory circuitry, ACh modifies neural responses to stimuli and coordinates the activity of neurons across multiple levels of processing. These factors enable individual neurons or entire circuits to rapidly adapt to the dynamics of complex sensory stimuli, underscoring an essential role for ACh in sensory processing. In the auditory system, histological evidence shows that acetylcholine receptors (AChRs) are expressed at virtually every level of the ascending auditory pathway. Despite its apparent ubiquity in auditory circuitry, investigation of the roles of this cholinergic network has been mainly focused on the inner ear or forebrain structures, while less attention has been directed at regions between the cochlear nuclei and midbrain. In this review, we highlight what is known about cholinergic function throughout the auditory system from the ear to the cortex, but with a particular emphasis on brainstem and midbrain auditory centers. We will focus on receptor expression, mechanisms of modulation, and the functional implications of ACh for sound processing, with the broad goal of providing an overview of a newly emerging view of impactful cholinergic modulation throughout the auditory pathway.

Transcutaneous neuromodulation versus oxybutynin for neurogenic detrusor overactivity in persons with spinal cord injury: A randomized, investigator blinded, parallel group, non-inferiority controlled trial.

STUDY DESIGN: This study is a randomized, investigator-blinded, controlled trial with a non-inferiority design. OBJECTIVE: To investigate the effectiveness of neuromodulation by transcutaneous electrical stimulation of the somatic afferent nerves of the foot in neurogenic detrusor overactivity (NDO) in persons with spinal cord injury (SCI) and compare its effectiveness with oral oxybutynin. SETTING: The study was conducted in a rehabilitation in-patient ward of a tertiary care hospital. METHODS: Twenty-nine persons with SCI with NDO, either sex, aged 18 years and above were randomized into two groups, one group receiving oral oxybutynin (5 mg thrice a day for two weeks) and the other transcutaneous electrical stimulation (5 Hz, 200 µs pulse, biphasic, amplitude up to 60 mA, 30 min/day for two weeks). Bladder capacity was evaluated by clinical bladder evaluation (i.e. bladder capacity measured by adding leak volume, voiding volume if any, and post-void residue using a catheter) and cystometric bladder capacity by one-channel cystometry. Maximum cystometric pressure was evaluated by one-channel water cystometry. Data were analyzed with Fisher's Exact, t-test, and Wilcoxon rank sum tests. RESULTS: Bladder capacity improved significantly in the oxybutynin and neuromodulation groups as measured by one-channel water cystometry (136 ml vs. 120.57 ml) and clinical evaluation (138.93 ml vs. 112 ml). The increase in the neuromodulation group achieved the pre-decided non-inferiority margin of 30 ml over the oxybutynin group when measured by one-channel water cystometry but not by clinical evaluation. Maximum cystometric pressure did not significantly improve in either group when compared with the baseline. CONCLUSION: Transcutaneous neuromodulation and oxybutynin effectively increased bladder capacity in persons with SCI with NDO. Neuromodulation by once-a-day transcutaneous electrical stimulation was non-inferior to thrice-a-day oxybutynin when evaluated by one-channel water cystometry.Trial registration: Clinical Trials Registry India identifier: CTRI/2018/05/013735.

Contribution of transient receptor potential vanilloid 1 (TRPV1) channel to cholinergic contraction of rat bladder smooth muscle.

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-permeable ion channel that is gated by the pungent constituent of red chili pepper, capsaicin, and by related chemicals from the group of vanilloids, in addition to noxious heat. It is expressed mostly in sensory neurons to act as a detector of painful stimuli produced by pungent chemicals and high temperatures. Although TRPV1 is also found outside the sensory nervous system, its expression and function in the bladder detrusor smooth muscle (DSM) remain controversial. Here, by using Ca2+ imaging and patch clamp on isolated rat DSM cells, in addition to tensiometry on multicellular DSM strips, we show that TRPV1 is expressed functionally in only a fraction of DSM cells, in which it acts as an endoplasmic reticulum Ca2+-release channel responsible for the capsaicin-activated [Ca2+]i rise. Carbachol-stimulated contractions of multicellular DSM strips contain a TRPV1-dependent component, which is negligible in the circular DSM but reaches ≤50% in the longitudinal DSM. Activation of TRPV1 in rat DSM during muscarinic cholinergic stimulation is ensured by phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists. Immunofluorescence detection of TRPV1 protein in bladder sections and isolated DSM cells confirmed both its preferential expression in the longitudinal DSM sublayer and its targeting to the endoplasmic reticulum. We conclude that TRPV1 is an essential contributor to the cholinergic contraction of bladder longitudinal DSM, which might be important for producing spatial and/or temporal anisotropy of bladder wall deformation in different regions during parasympathetic stimulation. KEY POINTS: The transient receptor potential vanilloid 1 (TRPV1) heat/capsaicin receptor/channel is localized in the endoplasmic reticulum membrane of detrusor smooth muscle (DSM) cells of the rat bladder, operating as a calcium-release channel. Isolated DSM cells are separated into two nearly equal groups, within which the cells either show or do not show TRPV1-dependent [Ca2+]i rise. Carbachol-stimulated, muscarinic ACh receptor-mediated contractions of multicellular DSM strips contain a TRPV1-dependent component. This component is negligible in the circular DSM but reaches ≤50% in longitudinal DSM. Activation of TRPV1 in rat DSM during cholinergic stimulation involves phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists.

Co-stimulation of muscarinic M1 and M4 acetylcholine receptors prevents later cocaine reinforcement in male and female mice, but not place-conditioning.

Acute stimulation of M1 or M4 muscarinic cholinergic receptors reduces cocaine abuse-related effects in mice and rats. The combined activation of these receptor subtypes produces synergistic effects on some behavioural endpoints in mice. M1 and M1 + M4 receptor stimulation in a cocaine vs. food choice assay in rats and microdialysis in rats showed delayed and lasting "anticocaine effects". Here, we tested whether these putative lasting neuroplastic changes are sufficient to occlude the reinforcing effects of cocaine at the behavioural level in mice. Mice were pre-treated with the M1 receptor partial agonist VU0364572, M4 receptor positive allosteric modulator VU0152100, or VU0364572 + VU0152100 two weeks prior to acquisition of cocaine intravenous self-administration (IVSA). Male C57BL/6JRj mice received vehicle, VU0364572, VU0152100, or VU0364572 + VU0152100. Female mice were tested with two VU0364572 + VU0152100 dose combinations or vehicle. To attribute potential effects to either reduced rewarding effects or increased aversion to cocaine, we tested VU0364572 alone and VU0364572 + VU0152100 in acquisition of cocaine-conditioned place preference (CPP) in male mice using an unbiased design. The acquisition of cocaine IVSA was drastically reduced and/or slowed in male and female mice receiving VU0364572 + VU0152100, but not either drug alone. Food-maintained operant behaviour was unaffected, indicating that the treatment effects were cocaine-specific. No treatment altered the acquisition of cocaine-CPP, neither in the post-test, nor in a challenge 14 days later. The cocaine IVSA findings confirm unusual long-lasting "anticocaine" effects of muscarinic M1 + M4 receptor stimulation. Thus, in mice, simultaneous stimulation of both receptor subtypes seems to produce potential neuroplastic changes that yield lasting effects.

Voltage dependence of M2 muscarinic receptor antagonists and allosteric modulators.

Muscarinic receptors are G protein-coupled receptors (GPCRs) that play a role in various physiological functions. Previous studies have shown that these receptors, along with other GPCRs, are voltage-sensitive; both their affinity toward agonists and their activation are regulated by membrane potential. To our knowledge, whether the effect of antagonists on these receptors is voltage-dependent has not yet been studied. In this study, we used Xenopus oocytes expressing the M2 muscarinic receptor (M2R) to investigate this question. Our results indicate that the potencies of two M2R antagonists, atropine and scopolamine, are voltage-dependent; they are more effective at resting potential than under depolarization. In contrast, the M2R antagonist AF-DX 386 did not exhibit voltage-dependent potency.Furthermore, we discovered that the voltage dependence of M2R activation by acetylcholine remains unchanged in the presence of two allosteric modulators, the negative modulator gallamine and the positive modulator LY2119620. These findings enhance our understanding of GPCRs' voltage dependence and may have pharmacological implications.

Scopolamine infusion in the basolateral amygdala after saccharin intake induces conditioned taste avoidance in rats.

RATIONALE: Muscarinic receptor activity in the basolateral amygdala (BLA) is known to be involved in plasticity mechanisms that underlie emotional learning. The BLA is involved in the Attenuation of Neophobia, an incidental taste learning task in which a novel taste becomes familiar and recognized as safe. OBJECTIVE: Here we assessed the role of muscarinic receptor activity in the BLA in incidental taste learning. METHODS: Young adult male Wistar rats were bilaterally implanted with cannulas aimed at BLA. After recovery, rats were randomly assigned to either vehicle or muscarinic antagonist group, for each experiment. We tested the effect of specific and non-specific muscarinic antagonists administered either 1) 20 min before novel taste presentation; 2) immediately after novel taste presentation; 3) immediately after retrieval (the second taste presentation on Day 5 -S2-) or immediately after the fifth taste presentation on Day 8 (S5). RESULTS: Non-specific muscarinic receptor antagonist scopolamine infused prior to novel taste, while not affecting novel taste preference, abolished AN, i.e., the increased preference observed in control animals on the second presentation. When administered after taste consumption, intra-BLA scopolamine not only prevented AN but caused a steep decrease in the taste preference on the second presentation. This scopolamine-induced taste avoidance was not dependent on taste novelty, nor did it generalize to another novel taste. Targeting putative postsynaptic muscarinic receptors with specific M1 or M3 antagonists appeared to produce a partial taste avoidance, while M2 antagonism had no effect. CONCLUSION: These data suggest that if a salient gustatory experience is followed by muscarinic receptors antagonism in the BLA, it will be strongly and persistently avoided in the future. The study also shows that scopolamine is not just an amnesic drug, and its cognitive effects may be highly dependent on the task and the structure involved.

Protective effects of bioactive components targeting ß2-adrenergic receptors and muscarinic-3 acetylcholine receptor in Zhisou San on ovalbumin-induced allergic asthma.

One promising approach to overcome drug resistance in asthma treatments involves dual-target therapy, specifically targeting the ß2 adrenergic receptor (ß2-AR) and muscarinic-3 acetylcholine receptor (M3R). This study investigated the anti-asthma effects and dual-target mechanisms of glycyrrhizic acid, hesperidin, and platycodin D (GHP) from Zhisou San. GHP administration effectively attenuated OVA-induced inflammatory infiltration and overproduction of mucus in asthmatic mice. Additionally, GHP treatment significantly suppressed M3R and promoted ß2-AR activation, resulting in the relaxation of tracheal smooth muscle. These findings concluded that GHP mitigated asthma by targeting ß2-AR and M3R to ameliorate airway inflammation and modulate airway smooth muscle relaxation.

G protein-coupled receptors related to autoimmunity in postural orthostatic tachycardia syndrome.

Postural orthostatic tachycardia syndrome (POTS) is characterized by exaggerated orthostatic tachycardia in the absence of orthostatic hypotension. The pathophysiology of POTS may involve hypovolemia, autonomic neuropathy, a hyperadrenergic state, and cardiovascular deconditioning, any of which can co-occur in the same patient. Furthermore, there is growing evidence of the role of autoimmunity in a subset of POTS cases. In recent years, investigators have described an increased rate of autoimmune comorbidities as evidenced by the finding of several types of neural receptor autoantibody and non-specific autoimmune marker in patients with POTS. In particular, the association of the disease with several types of anti-G protein-coupled receptor (GPCR) antibodies and POTS has frequently been noted. A previous study reported that autoantibodies to muscarinic AChRs may play an important role in POTS with persistent, gastrointestinal symptoms. To date, POTS is recognized as one of the sequelae of coronavirus disease 2019 (COVID-19) and its frequency and pathogenesis are still largely unknown. Multiple autoantibody types occur in COVID-related, autonomic disorders, suggesting the presence of autoimmune pathology in these disorders. Herein, we review the association of anti-GPCR autoantibodies with disorders of the autonomic nervous system, in particular POTS, and provide a new perspective for understanding POTS-related autoimmunity.

[COPD inhaled therapy narrative review]. / Revisión narrativa de la terapia inhalatoria en la EPOC.

The basis of COPD maintenance treatment is the long-acting bronchodilators and the inhaled corticosteroids. Faced with the recent modifications in the clinical practice guidelines, we have carried out a review of studies that contrast the various therapeutic alternatives and pharmacological agents within each category, with the fundamental purpose of shedding light on which of these options prove to be more effective. Triple therapy stands out as essential in poorly controlled patients or with an eosinophilic phenotype, surpassing dual therapy. However, among the combinations of LAMA/LABA or LAMA/LABA/IC, no drug is observed to be superior in the reviewed evidence. Although triple therapies include corticosteroids, there does not appear to be a significant increase in side effects or pneumonia. Regarding monotherapy with LAMA, no significant differences are seen between the drugs, but in dual therapy with LABA/IC, the budesonide/formoterol combination seems to offer better control than fluticasone/salmeterol.

Sex differences in cholinergic signaling affect functional outcomes for theta-gamma coordination in hippocampal subcircuits following experimental febrile status epilepticus.

OBJECTIVE: Sex determines cognitive outcome in animal models of early life seizure, where males exhibit impaired hippocampal-dependent learning and memory compared with females. The physiological underpinnings of this sex effect are unclear. Cholinergic signaling is essential for the generation of hippocampal oscillations, and supplementation of cholinergic precursors prior to status epilepticus in immature male rats prevents subsequent memory deficits. We hypothesized that there are sex differences in acetylcholine circuits and their response to experimental febrile status epilepticus (eFSE). METHODS: eFSE was induced in male and female rat pups. We transversed the hippocampus of postnatal day >60 control (CTL) and eFSE rats with a 64-channel laminar silicon probe to assay cholinergic-dependent theta oscillations under urethane anesthesia. Local field potential properties were compared during (1) baseline sensory stimulation, (2) pharmacological stimulation via acetylcholine reuptake blockade, and (3) sensory stimulation after muscarinic acetylcholine receptor block (atropine). RESULTS: In all groups, a baseline tail pinch could elicit theta oscillations via corticohippocampal synaptic input. Following atropine, a tail pinch response could no longer be elicited in CTL male, CTL female, or eFSE female rats. In contrast, induced slow theta power in eFSE males after atropine was not decreased to spontaneous levels. Analysis of oscillation bandwidths revealed sex differences in acetylcholine modulation of theta frequency and slow gamma frequency and power. This study also identified significant effects of both sex and eFSE on baseline theta-gamma comodulation, indicating a loss of coupling in eFSE males and a potential gain of function in eFSE females. SIGNIFICANCE: There are differences in cholinergic modulation of theta and gamma signal coordination between male and female rats. These differences may underlie worse cognitive outcomes in males following eFSE. Promoting the efficacy of muscarinic acetylcholine signaling prior to or following early life seizures could elucidate a mechanism for the temporal discoordination of neural signals within and between hippocampus and neocortex and provide a novel therapeutic approach for improving cognitive outcomes.

LAMA improves tissue oxygenation more than LABA in patients with COPD.

The effect of bronchodilators is mainly assessed with forced expiratory volume in 1 s (FEV1) in chronic obstructive pulmonary disease (COPD). Their impact on oxygenation and lung periphery is less known. Our objective was to compare the action of long-acting ß2-agonists (LABA-olodaterol) and muscarinic antagonists (LAMA-tiotropium) on tissue oxygenation in COPD, considering their impact on proximal and peripheral ventilation as well as lung perfusion. FEV1, Helium slope (SHe) from a single-breath washout test (SHe decreases reflecting a peripheral ventilation improvement), frequency dependence of resistance (R5-R19), area under reactance (AX), lung capillary blood volume (Vc) from double diffusion (DLNO/DLCO), and transcutaneous oxygenation (TcO2) were measured before and 2 h post-LABA (day 1) and LAMA (day 3) in 30 patients with COPD (FEV1 54 ± 18% pred; GOLD A 31%/B 48%/E 21%) after 5-7 days of washout, respectively. We found that TcO2 increased more (P = 0.03) after LAMA (11 ± 12% from baseline, P < 001) compared with LABA (4 ± 11%, P = 0.06) despite a lower FEV1 increase (P = 0.03) and similar SHe (P = 0.98), AX (P = 0.63), and R5-R19 decreases (P = 0.37). TcO2 and SHe changes were negatively correlated (r = -0.47, P = 0.01) after LABA, not after LAMA (r = 0.10, P = 0.65). DLNO/DLCO decreased and Vc increased after LAMA (P = 0.04; P = 0.01, respectively) but not after LABA (P = 0.53; P = 0.24). In conclusion, LAMA significantly improved tissue oxygenation in patients with COPD, while only a trend was observed with LABA. The mechanisms involved may differ between both drugs: LABA increased peripheral ventilation, whereas LAMA increased lung capillary blood volume. Should oxygenation differences persist over time, LAMA could arguably become the first therapeutic choice in COPD.NEW & NOTEWORTHY Long-acting muscarinic antagonists (LAMAs) significantly improved tissue oxygenation in patients with COPD, while only a trend was observed with ß2-agonists (LABAs). The mechanisms involved may differ between drugs: increased peripheral ventilation for LABA and likely lung capillary blood volume for LAMA. This could argue for LAMA as the first therapeutic choice in COPD.

Development of simultaneous determination of dopamine 2, histamine 1, and muscarinic acetylcholine receptor occupancies by antipsychotics using liquid chromatography with tandem mass spectrometry.

Receptor occupancy is an indicator of antipsychotic efficacy and safety. It is desirable to simultaneously determine the occupancy of multiple brain receptors as an indicator of the efficacy and central side effects of antipsychotics because many of these drugs have binding affinities for various receptors, such as dopamine 2 (D2), histamine 1 (H1), and muscarinic acetylcholine (mACh) receptors. The purpose of this study was to develop a method for the simultaneous measurement of multiple receptor occupancies in the brain by the simultaneous quantification of unlabeled tracer levels using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Rats were pre-administered with a vehicle, displacer, or olanzapine, and mixed solutions of raclopride, doxepin, and 3-quinuclidinyl benzilate (3-QNB) were administered (3, 10, and 30 µg/kg). The brain tissue and plasma tracer concentrations were quantified 45 min later using LC-MS/MS, and the binding potential was calculated. The highest binding potential was observed at 3 µg/kg raclopride, 10 µg/kg doxepin, and 30 µg/kg 3-QNB. Tracer-specific binding at these optimal tracer doses in the cerebral cortex was markedly reduced by pre-administration of displacers. D2, H1, and mACh receptor occupancy by olanzapine increased in a dose-dependent manner, reaching 70-95%, 19-43%, and 12-45%, respectively, at an olanzapine dose range of 3-10 mg/kg. These results suggest that simultaneous determination of in vivo D2, H1, and mACh receptor occupancy is possible using LC-MS/MS.

Berberine inhibits intracellular Ca2+ signals in mouse pancreatic acinar cells through M3 muscarinic receptors: Novel target, mechanism, and implication.

Berberine, a natural isoquinoline alkaloid, exhibits a variety of pharmacological effects, but the pharmacological targets and mechanisms remain elusive. Here, we report a novel finding that berberine inhibits acetylcholine (ACh)-induced intracellular Ca2+ oscillations, mediated through an inhibition of the muscarinic subtype 3 (M3) receptor. Patch-clamp recordings and confocal Ca2+ imaging were applied to acute dissociated pancreatic acinar cells prepared from CD1 mice to examine the effects of berberine on ACh-induced Ca2+ oscillations. Whole-cell patch-clamp recordings showed that berberine (from 0.1 to 10 µM) reduced ACh-induced Ca2+ oscillations in a concentration-dependent manner, and this inhibition also depended on ACh concentrations. The inhibitory effect of berberine neither occurred in intracellular targets nor extracellular cholecystokinin (CCK) receptors, chloride (Cl-) channels, and store-operated Ca2+ channels. Together, the results demonstrate that berberine directly inhibits the muscarinic M3 receptors, further confirmed by evidence of the interaction between berberine and M3 receptors in pancreatic acinar cells.

Two-point immobilization of M3 muscarinic receptor: a method for recognizing receptor antagonists in natural products.

In the investigation of active ingredients from natural products, current technologies relying on drug-target affinity recognition analysis face significant challenges. This is primarily due to their limited specificity and inability to provide downstream pharmacodynamic information, such as agonistic or antagonistic activity. In this study, a two-point method was developed by immobilizing M3 acetylcholine receptor (M3R) through the combination of the conformation-specific peptide BJ-PRO-13a and the HaloTag trap system. We systematically assessed the specificity of the immobilized M3R using known M3R antagonists (pirenzepine and atropine) and agonists (cevimeline and pilocarpine). By frontal analysis and nonlinear chromatography, the performance of immobilized M3R was evaluated in terms of binding kinetics and thermodynamics of four drugs to the immobilized M3R. Additionally, we successfully identified two M3R antagonists within an extract from Daturae Flos (DF), specifically hyoscyamine and scopolamine. Our findings demonstrate that this immobilization method effectively captures receptor-ligand binding interactions and can discern receptor agonists from antagonists. This innovation enhances the efficiency of receptor chromatography to determine binding-affinity in the development of new drugs, offering promise for the screening and characterization of active compounds, particularly within complex natural products.

The New Horizon of Antipsychotics beyond the Classic Dopaminergic Hypothesis-The Case of the Xanomeline-Trospium Combination: A Systematic Review.

Although the dopamine hypothesis of schizophrenia explains the effects of all the available antipsychotics in clinical use, there is an increasing need for developing new drugs for the treatment of the positive, negative, and cognitive symptoms of chronic psychoses. Xanomeline-trospium (KarXT) is a drug combination that is based on the essential role played by acetylcholine in the regulation of cognitive processes and the interactions between this neurotransmitter and other signaling pathways in the central nervous system, with a potential role in the onset of schizophrenia, Alzheimer's disease, and substance use disorders. A systematic literature review that included four electronic databases (PubMed, Cochrane, Clarivate/Web of Science, and Google Scholar) and the US National Library of Medicine database for clinical trials detected twenty-one sources referring to fourteen studies focused on KarXT, out of which only four have available results. Based on the results of these trials, the short-term efficacy and tolerability of xanomeline-trospium are good, but more data are needed before this drug combination may be recommended for clinical use. However, on a theoretical level, the exploration of KarXT is useful for increasing the interest of researchers in finding new, non-dopaminergic, antipsychotics that could be used either as monotherapy or as add-on drugs.

Voltage Sensors Embedded in G Protein-Coupled Receptors.

Some signaling processes mediated by G protein-coupled receptors (GPCRs) are modulated by membrane potential. In recent years, increasing evidence that GPCRs are intrinsically voltage-dependent has accumulated. A recent publication challenged the view that voltage sensors are embedded in muscarinic receptors. Herein, we briefly discuss the evidence that supports the notion that GPCRs themselves are voltage-sensitive proteins and an alternative mechanism that suggests that voltage-gated sodium channels are the voltage-sensing molecules involved in such processes.