Muscarinic Receptor Subtypes Research Articles

The expression level of muscarinic M1 receptor subtypes in different regions of rat brain

Amac: Travma sonrasi stres bozuklugu (TSSB) yasami tehdit eden travma, asiri uyarilma, flashbackler ve kabuslar ile karakterizedir. TSSB arastirmalari, travmatik bir deneyimin beyinin davranis ve islevleri uzerinde uzun sureli bozulma etkilerine yol actigini anlamanin zorluguyla karsi karsiyadir. Herhangi bir spesifik ajanin etkinligini destekleyecek uygun kanit olmadiginda, TSSB’nin farmakoterapisinde bircok farmakolojik ajan kullanilmaktadir. Olumsuz bir deneyimin geri cagrilmasinda M1 muskarinik reseptor alt tiplerinin onemli rol oynayabilecegi ongorulmektedir. Bu arastirmanin amaci, TSSB’de kronik fluoksetin (FLU) (2.5 mg/ gun; i.p) tedavisinin hem davranissal hem de molekuler etkinligini ve farmakoterapinin sicanlarda M1 muskarinik reseptor alt tipi ekspresyonu uzerine muhtemel etkisini arastirmaktir. Gerec ve Yontem: Deney tasariminda tum gruplar rastgele olarak secilerek Kontrol, Stres ve Tedavi gruplari olusturulmustur. Kronik FLU tedavisinin etkileri, sican hipokampus ve frontal korteksinde, M1 reseptorlerinin ekspresyon seviyeleri acisindan degerlendirilmistir. Bulgular: Sicanlar deneyin son gununde (30.Gun) travma hatirlaticilara maruz kaldiklarinda, stres gruplarindaki anksiyete indekslerinin kontrol grubuna gore belirgin bir sekilde arttigi gozlemlenmistir (P<0.001). Ayrica, kronik FLU tedavisinin stress gruplarinda anksiyete degerlerini dusurerek geriye dondurdugu gozlemlenmistir (P<0.001). Sonuc: Bu calismada, stresin kaygi benzeri davranislari arttirarak sican beyin hipokampus ve frontal korteksinde M1 ekspresyon seviyesini azalttigi gozlemlenmistir. Dusuk doz kronik FLU tedavisi ile bu etkilerin geri dondurulebilecegi ongorulmektedir.

Selective inhibition of M5 muscarinic acetylcholine receptors attenuates cocaine self-administration in rats.

Cocaine use disorder (CUD) remains a debilitating health problem in the United States for which there are no Food and Drug Administration-approved treatment options. Accumulating anatomical and electrophysiological evidence indicates that the muscarinic acetylcholine receptor (mAChR) subtype 5 (M5 ) plays a critical role in the regulation of the mesolimbic dopaminergic reward circuitry, a major site of action for cocaine and other psychostimulants. In addition, M5 knockout mice exhibit reduced cocaine self-administration behaviors with no differences in sugar pellet-maintained responding relative to wild-type mice. These findings suggest that selective inhibition of M5 mAChR may provide a novel pharmacological approach for targeting CUD. Recently, we reported the synthesis and characterization of ML375, a selective negative allosteric modulator (NAM) for the rat and human M5 mAChR with optimized pharmacokinetic properties for systemic dosing in rodents. In the present study, male Sprague-Dawley rats were trained to self-administer intravenous cocaine (0.1-0.75mg/kg/infusion) under a 10-response fixed ratio or a progressive ratio schedule of reinforcement. Under both schedules of reinforcement, ML375 produced dose-related reductions in cocaine self-administration. ML375 also modestly reduced sugar pellet-maintained responding on the 10-response, fixed ratio schedule but had no effect under a progressive ratio schedule of reinforcement. Further, ML375 did not affect general motor output as assessed by a rotarod test. Collectively, these results provide the first demonstration that selective inhibition of M5 using the M5 NAM ML375 can attenuate both the reinforcing effects and the relative strength of cocaine and suggest that M5 NAMs may represent a promising, novel treatment approach for CUD.

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.

An Approach to Discovering Novel Muscarinic M1 Receptor Positive Allosteric Modulators with Potent Cognitive Improvement and Minimized Gastrointestinal Dysfunction

Activation of muscarinic M1 receptor (M1R) is a promising approach for improving cognitive impairment in Alzheimer's disease. However, an M1R-selective positive allosteric modulator (PAM), benzyl quinolone carboxylic acid (BQCA), at 30 mg/kg, induced diarrhea in wild-type mice, but not in M1R knockout mice. Moreover, BQCA (0.1-1000 nM) augmented electric field stimulation (EFS)-induced ileum contraction in an in vitro Magnus assay. Thus, we decided to establish a drug-screening strategy to discover novel M1 PAMs producing potent cognitive improvement with minimized gastrointestinal (GI) dysfunction. We assessed PAM parameters of various M1 PAMs with ≥100-fold selectivity over other muscarinic receptor subtypes by using in vitro binding and functional analysis. Evaluation of these M1 PAMs in the Magnus assay revealed a significant correlation between percentage of ileum contractions at 1 μM and their α-value, a PAM parameter associated with the binding cooperativity between acetylcholine and M1 PAM. M1 PAMs with lower α-value showed lower impact on EFS-induced ileum contraction. Next, we characterized in vivo profiles of two M1 PAMs: compound A (log α = 1.18) and compound B (log α = 3.30). Compound A, at 30 mg/kg, significantly improved scopolamine-induced cognitive deficits without prominent signs of diarrhea at up to 1000 mg/kg in mice. In contrast, compound B, at 10 mg/kg, showed both significant improvement of scopolamine-induced cognitive deficits and severe diarrhea. Thus, fine adjustment of the α-values could be a key to discovering M1 PAMs yielding potent cognitive improvement with a lower risk of GI effects.

Abstract P184: Muscarinic Receptor Modulators’ Effect on Cilia Structure and Nitric Oxide Release

Primary endothelial cilia are microtubule-based organelles that act as mechano-sensors to help detect, and respond to changes in the extracellular environment. One of these responses includes its ability to detect fluid shear stress and translating that signal into a biochemical process to synthesize nitric oxide, an endogenous vasodilator. Abnormal cilia structure and function has been known to cause a myriad of human disorders, including polycystic kidney disease and hypertension. It is also well documented that muscarinic acetylcholine receptors subtype M1 also plays a key role in producing a vasodilation response in the vasculature. However, nothing is known about the relationship between primary endothelial cilia and the M1 receptor. To further explore the relationship between AChM1R and the mechanosensory function of primary cilia, the effects of muscarinic modulators on cilia length and function in wild-type, and mechano-insensitive cilia mutant endothelial cells, Pkd1 – / – (dysfunctional cilia) and Tg737 orpk/orpk (cilia-less) were examined. We show that AChM1R localizes to primary endothelial cilia. AChM1R activation lead to the significant increase cilia length in endothelial cells treated with CDD102A, an AChM1R agonist (1.21 ± 0.07 vs. 2.33 ± 0.04 for wild-type, 1.02 ± 0.01 vs. 1.44 ± 0.01 for Pkd1 – / – , and 0.024 ± 0.005 vs. 0.3 ± 0.004 for Tg737 orpk/orpk ) compared to non-treated cells. Treating endothelial cells with pirenzepine, an AChM1R antagonist, led to a significant decrease of cilia length (1.34 ± 0.02 vs. 1.15 ± 0.01 in wild-type, and 1.16 ± 0.01 vs. 0.71 ± 0.01 in Pkd1 – / – ) compared to non-treated cells. Treatment with CDD102A also significantly upregulated the expression of AChM1R, and phosphorylated eNOS. Our data clearly suggested that modulating cilia sensory function could have a positive influence on nitric oxide generation and blood pressure regulation.

Recovery of hippocampal functions and modulation of muscarinic response by electroacupuncture in young diabetic rats

The muscarinic receptor response to acetylcholine regulates the hippocampal-related learning, memory, neural plasticity and the production and processing of the pro-nerve growth factor (proNGF) by hippocampal cells. The development and progression of diabetes generate a mild cognitive impairment reducing the functions of the septo-hippocampal cholinergic circuitry, depressing neural plasticity and inducing proNGF accumulation in the brain. Here we demonstrate, in a rat model of early type-1 diabetes, that a physical therapy, the electroacupuncture, counteracts the diabetes-induced deleterious effects on hippocampal physiology by ameliorating hippocampal-related memory functions; recovering the impaired long-term potentiation at the dentate gyrus (DG-LTP) and the lowered expression of the vesicular glutamate transporter 1; normalizing the activity-dependent release of proNGF in diabetic rat hippocampus. Electroacupuncture exerted its therapeutic effects by regulating the expression and activity of M1- and M2-acetylcholine muscarinic receptors subtypes in the dentate gyrus of hippocampus. Our results suggest that a physical therapy based on repetitive sensory stimulation could promote hippocampal neural activity, neuronal metabolism and functions, and conceivably improve the diabetes-induced cognitive impairment. Our data can support the setup of therapeutic protocols based on a better integration between physical therapies and pharmacology for the cure of diabetes-associated neurodegeneration and possibly for Alzheimer’s disease.

Muscarinic receptors in adrenal chromaffin cells: physiological role and regulation of ion channels

Adrenal medullary chromaffin cells in mammals are innervated by sympathetic preganglionic nerve fibers, as are sympathetic ganglion neurons. Acetylcholine in the ganglion neurons is well established as mediating fast and slow excitatory postsynaptic potentials through nicotinic and muscarinic acetylcholine receptors (AChRs), respectively. The role of muscarinic AChRs during neuronal transmission in chromaffin cells varies among different mammals. Furthermore, the ion channel mechanisms associated with the muscarinic AChR-mediated increase in excitability of chromaffin cells are complicated and different from the excitation of ganglion neurons, which has been ascribed to the inhibition of M-type K+ channels. In this review, we focus on muscarinic receptor-mediated excitation in rodent and guinea pig chromaffin cells, in particular, on the role of muscarinic receptors in neuronal transmission, the muscarinic receptor subtypes involved in excitation and secretion, and the muscarinic regulation of ion channels including TWIK-related acid-sensitive K+ channels. Finally, we discuss prospectively the future of muscarinic receptor research in adrenal chromaffin cells.

Cognitive enhancement and antipsychotic-like activity following repeated dosing with the selective M4 PAM VU0467154

Although selective activation of the M1 muscarinic acetylcholine receptor (mAChR) subtype has been shown to improve cognitive function in animal models of neuropsychiatric disorders, recent evidence suggests that enhancing M4 mAChR function can also improve memory performance. Positive allosteric modulators (PAMs) targeting the M4 mAChR subtype have shown therapeutic potential for the treatment of multiple symptoms observed in schizophrenia, including positive and cognitive symptoms when assessed in acute preclinical dosing paradigms. Since the cholinergic system has been implicated in multiple stages of learning and memory, we evaluated the effects of repeated dosing with the highly selective M4 PAM VU0467154 on either acquisition and/or consolidation of learning and memory when dosed alone or after pharmacologic challenge with the N-methyl-d-aspartate subtype of glutamate receptors (NMDAR) antagonist MK-801. MK-801 challenge represents a well-documented preclinical model of NMDAR hypofunction that is thought to underlie some of the positive and cognitive symptoms observed in schizophrenia. In wildtype mice, 10-day, once-daily dosing of VU0467154 either prior to, or immediately after daily testing enhanced the rate of learning in a touchscreen visual pairwise discrimination task; these effects were absent in M4 mAChR knockout mice. Following a similar 10-day, once-daily dosing regimen of VU0467154, we also observed 1) improved acquisition of memory in a cue-mediated conditioned freezing paradigm, 2) attenuation of MK-801-induced disruptions in the acquisition of memory in a context-mediated conditioned freezing paradigm and 3) reversal of MK-801-induced hyperlocomotion. Comparable efficacy and plasma and brain concentrations of VU0467154 were observed after repeated dosing as those previously reported with an acute, single dose administration of this M4 PAM. Together, these studies are the first to demonstrate that cognitive enhancing and antipsychotic-like activity are not subject to the development of tolerance following repeated dosing with a selective M4 PAM in mice and further suggest that activation of M4 mAChRs may modulate both acquisition and consolidation of memory functions.

Role of muscarinic receptors in the contraction of jejunal smooth muscle in the horse: An in vitro study

Nonselective antimuscarinic drugs are clinically useful in several pathologic conditions of horses, but, blocking all muscarinic receptor (MR) subtypes, may cause several side effects. The availability of selective antimuscarinic drugs could improve therapeutic efficacy and safety.We aimed to enlighten the role of different MR subtypes by evaluating the effects of nonselective, and selective M1, M2 and M3 MR antagonists on the contractions of horse jejunum.Segments of circular muscle of equine jejunum, were put into organ baths, connected to isotonic transducers, and the effects on ACh concentration-response curves, and on electrical field stimulation (EFS)-evoked contractions of intestinal preparations, induced by nonselective or selective MR antagonists, compared to pre-drug level, were studied. Atropine (nonselective MR antagonist), pirenzepine (selective M1 antagonist), and p-FHHSiD (selective M3 antagonist) competitively antagonized ACh (pA2=9.78±0.21; 7.14±0.25 and 7.56±0.17, respectively). Methoctramine (selective M2 antagonist) antagonized ACh in a concentration-unrelated fashion; however, it competitively antagonized carbachol, a nonselective muscarinic agonist (pA2=6.42±0.23). Atropine dose-dependently reduced EFS-evoked contractions, reaching a maximal effect of −45.64±6.54%; the simultaneous block of neurokinin receptors, almost completely abolished the atropine-insensitive contractions. p-FHHSiD dose-dependently reduced EFS-induced contractions, while pirenzepine caused a minor decrease. Methoctramine, ineffective up to 10−7M, enhanced the contractions at 10−6M; the block of neurokinin receptors abolished the increase of contraction. Cholinergic contractions of horse jejunum are mainly mediated by M3 receptors; M2 selective antagonists seem to scarcely affect cholinergic, and to enhance neurokininergic contractions of equine jejunum, thus their use entails a lower risk of causing intestinal hypomotility, compared to nonselective drugs.

Abstract 3339: Muscarinic acetylcholine receptor subtype 3 regulates gastric stem cell expansion and gastric cancer progression by controlling YAP activation

Abstract Within the gastrointestine, nerves help to regulate both normal and neoplastic stem cell dynamics. Several previous studies suggested that cholinergic nerve signaling plays an important role in gastrointestinal cancer development, but the exact underlying mechanism has not been clarified. In this study, we examined the role of muscarinic acetylcholine receptor subtype 3 (M3R) in gastric homeostasis and cancer development by using mouse models and human cancer cell lines. In situ hybridization revealed M3R expression in gastric stem cell zone, and its expression was markedly upregulated in gastric cancer cells. We knocked out M3R in Lgr5+ gastric stem cells in Lgr5-CreERT; M3Rflox/flox mice, and found that deletion of M3R inhibited clonal expansion of Lgr5+ cells in regenerative states. In a gastric tumor model of Mist1-CreERT; Apcflox/flox mice, knockout of M3R dramatically suppressed tumor growth. RNA sequencing analysis of these tumors revealed that several important pathways were significantly inhibited in M3R knockout samples, including YAP/TAZ pathway. We established M3R-expressing gastric cancer cell lines, and western blotting, luciferase assay, and RT-PCR analysis confirmed that acetylcholine (ACh) agonist activates YAP pathway through M3R. YAP is upregulated in approximately half of gastric cancer patients, and its expression is significantly associated with disease stage and histological form. This M3R-YAP axis activates the gastric stem cell niche and offers a compelling target for tumor treatment and prevention. Note: This abstract was not presented at the meeting. Citation Format: Yoku Hayakawa, Mitsuru Konishi, Kosuke Sakitani, Kazuhiko Koike, Timothy Wang. Muscarinic acetylcholine receptor subtype 3 regulates gastric stem cell expansion and gastric cancer progression by controlling YAP activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3339. doi:10.1158/1538-7445.AM2017-3339

AN APPROACH TO DISCOVER NOVEL MUSCARINIC M1 RECEPTOR-POSITIVE ALLOSTERIC MODULATORS WITH POTENT COGNITIVE IMPROVEMENT AND MINIMIZED GASTROINTESTINAL DYSFUNCTION

Activation of the muscarinic M1 receptor (M1R) is a promising approach to improve cognitive impairment in Alzheimer's disease (AD). However, we found that an M1 selective positive allosteric modulator (PAM) induced severe diarrhea through M1R activation, by using wild-type and M1R knock-out (KO) mice. In this study, we established a drug screening strategy to discover novel M1 PAMs with potent cognitive improvement and minimized gastrointestinal dysfunction. PAM parameters of M1 PAMs were assessed by using in vitro binding and functional analysis in human M1R expressing Chinese hamster ovary (CHO-K1) cells. Effects of M1 PAMs on contraction of mouse ileum were assessed by using in vitro Magnus assay. Y-maze task was used to evaluate effects of M1 PAMs on cognitive function in scopolamine-treated mice. Seven M1 PAMs with ≥ 100-fold selectivity over other muscarinic receptor subtypes were used. Their PAM parameters such as PAM activity (the inflection point-value), cooperativity value (α-value), and efficacy value (β-value) were assessed by the in vitro assay. Evaluation of these M1 PAMs in the Magnus assay revealed that there was a strong and positive correlation between % ileum contraction at 1 μM and their cooperativity value (log α), a PAM parameter associated with the effect of allosteric modulation on the affinity of the orthosteric site. Next, we characterized in vivo profiles of two M1 PAMs: compound A (log α = 1.18) and compound B (log α = 3.30). Compound A at 30 mg/kg significantly improved scopolamine-induced cognitive deficits in Y-maze without prominent signs of diarrhea in mice. In contrast, compound B at 10 mg/kg showed both significant amelioration of scopolamine-induced cognitive deficits in Y-maze and severe diarrhea. Fine adjustment of the α-value could be a key to discover M1 PAMs with potent cognitive improvement and lower risk of diarrhea. The current study provides the original screening strategy to identify safer M1 PAMs for the treatment of cognitive deficits related to AD. Based on this strategy, we have identified TAK-071. TAK-071 is currently in clinical development (ClinicalTrials.gov Identifier: NCT02769065).

IN VITRO CHARACTERIZATION OF TAK-071: A NOVEL MUSCARINIC M1 RECEPTOR-POSITIVE ALLOSTERIC MODULATOR WITH LOW COOPERATIVITY

Muscarinic M1 receptor (M1R) is a promising target for CNS disorders with cholinergic deficits such as Alzheimer's disease. We previously reported that the cooperativity (α-value) was a key to lower the risk of diarrhea by M1R positive allosteric modulators (M1PAMs). In this study, we characterized in vitro profiles of TAK-071, a novel M1R selective PAM with a low α-value and compared with that of T-662, an M1PAM with a high α-value. PAM parameters were assessed by using in vitro binding and functional analysis in Chinese hamster ovary cells stably expressing human M1R. Evaluation of mouse ileum contraction was conducted under the conditions to assess spontaneous activity or electric field stimulation (EFS)-induced activity by using the in vitro Magnus method. Electrophysiological experiments were performed by using current clamp recordings in slices of mouse medial prefrontal cortex. TAK-071 had an inflection point (IP)-value of 2.7 nM, and showed an α-value of 199 for human M1R with more than 3700-fold selectivity over other muscarinic receptor subtypes. T-662 also had potent M1PAM activity with an IP-value of 2.1 nM, and showed an α-value of 1786. In the in vitro Magnus method, T-662, but not TAK-071, strengthened spontaneous ileum contraction in a concentration-dependent manner, and augmented EFS-induced ileum contraction. Tonic activation of M1Rs is known to produce neuronal excitability through three actions; depolarizing the resting membrane potential, suppressing the after hyperpolarization that follows the spike, and revealing the afterdepolarization (ADP) that can initiate repetitive firing. Among then, T-662 induced all three actions, while TAK-071 selectively induced ADP in layer 5 pyramidal neurons. We searched M1PAMs with low α-value and discovered TAK-071. TAK-071 induced ADP in layer 5 pyramidal neurons, while it did not cause mouse ileum contraction in vitro. Thus, TAK-071 may have a promising therapeutic potential for CNS disorders without causing diarrhea. TAK-071 is currently in clinical development (ClinicalTrials.gov, Identifier: NCT02769065).

Long Term Effects of Neonatal Hypoglycemia on Muscarinic Receptor Function in the Cerebellum

Neonatal stress conditions like hypoglycemia cause brain damage by affecting various signaling pathways thereby causing long term effects on brain functions. A proper understanding of the signaling pathways affected by this stress will help to devise better neonatal care. The focus of the current study was to evaluate the effect of neonatal hypoglycemic insult on cerebellar metabotropic cholinergic receptor function in one month old rats. The receptor analysis of cholinergic muscarinic receptors were done by radioreceptor assays and gene expression was analysed using Real Time PCR. Neonatal hypoglycemia significantly reduced (p&lt;0.001) the cerebellar muscarinic receptor density with a down regulation (p&lt;0.001) of muscarinic M3 receptor subtype gene expression in one month old rats. Both muscarinic M1 and M2 receptor subtype expression were not significantly altered. The catabolic enzyme in acetyl choline metabolism- acetylcholine esterase – showed a significant (p&lt;0.001) up regulation with no siginificant change in the anabolic enzyme – choline acetyl transferase, signifying a change in the turnover ratio. Targeting these pathways at different levels can be exploited to devise better treatment for neonatal stress management and also for diseases with impaired insulin secretion such as diabetes.

Antinociceptive Effect of the Essential Oil from Croton conduplicatus Kunth (Euphorbiaceae).

Medicinal plants have been widely used in the treatment of chronic pain. In this study, we describe the antinociceptive effect of the essential oil from Croton conduplicatus (the EO 25, 50, and 100 mg/kg, i.p.), a medicinal plant native to Brazil. Antinociceptive activity was investigated by measuring the nociception induced by acetic acid, formalin, hot plate and carrageenan. A docking study was performed with the major constituents of the EO (E-caryophyllene, caryophyllene oxide, and camphor). The EO reduced nociceptive behavior at all doses tested in the acetic acid-induced nociception test (p < 0.05). The same was observed in both phases (neurogenic and inflammatory) of the formalin test. When the hot-plate test was conducted, the EO (50 mg/kg) extended the latency time after 60 min of treatment. The EO also reduced leukocyte migration at all doses, suggesting that its antinociceptive effect involves both central and peripheral mechanisms. Pretreatment with glibenclamide and atropine reversed the antinociceptive effect of the EO on the formalin test, suggesting the involvement of KATP channels and muscarinic receptors. The docking study revealed a satisfactory interaction profile between the major components of the EO and the different muscarinic receptor subtypes (M2, M3, and M4). These results corroborate the medicinal use of C. conduplicatus in folk medicine.

Contribution of muscarinic receptors to in vitro and in vivo effects of Ruscus extract

The objectives of this study were to evaluate, in vitro and in vivo, the contribution of muscarinic receptors to the effects of Ruscus extract.Ruscus extract was tested in competition binding experiments at recombinant human muscarinic receptors, heterologous expressed in Chinese Hamster Ovary (CHO) cells and in cellular assays measuring Ca2+ liberation and activator protein-1 (AP-1) reporter gene activation. The impact of muscarinic blockade on prolonged treatment outcome was evaluated using the hamster cheek pouch (HCP) microcirculation examining macromolecular permeability increase induced by histamine or ischemia/reperfusion (I/R), mean arteriolar and venular diameters, functional capillary density and I/R-induced leukocyte rolling and sticking.Ruscus extract exhibited affinities for muscarinic receptor subtypes at a range of 50–100μg/ml and behaved as partial agonist at human recombinant M1 and M3 receptors for Ca2+ liberation, confirmed in an AP-1 reporter gene assay. In the HCP model, topical application of atropine completely or partially blocked Ruscus extract-induced reductions of histamine- and I/R-induced increases of macromolecular permeability and leukocyte-endothelium interaction.Our results showed that Ruscus extract in vitro binds and activates different subtypes of muscarinic receptors and in vivo its anti-inflammatory effects are, at least partially, mediated via muscarinic receptors.

PREPARATION AND CHARACTERIZATION OF TOLTERODINE TARTRATE PRONIOSOMES

Objective: Proniosomes are dry free flowing, granular products that are water soluble carrier particles, coated with surfactants and can be hydrated to form niosomal dispersion immediately before use in hot aqueous media. Tolterodine, is medication used to treat frequent urination, urinary incontinence, or urinary urgency. It acts on M2 and M3 subtypes of muscarinic receptors. Methods: Tolterodine tartrate proniosome formulations were prepared by coacervation phase separation method by using different surfactants in different ratios. The prepared proniosomal formulations were evaluated for vesicle size, rate of spontaneity, encapsulation efficiency, drug content, In vitro release study and stability studies. Results: The size range was found to be 15.28±0.33 to 16.43±0.22 µm. Viscosity of all formulations lies in the range of 7244-9314 cp. Maximum release was shown by formulations of batch PG4 (87.45 %), and minimum for formulations of batch PG4 (50%), after 12 h. Optimized formulations PG4 shows stability at different temperatures. Conclusion: Tolterodine tartrate proniosome formulations prepared by using coacervation phase separation method are capable of releasing drug for the extended period of time. Peer Review History: Received 2 April 2017; Revised 5 May; Accepted 10 May, Available online 15 May 2017 Academic Editor: Dr. DANIYAN Oluwatoyin Michael, Obafemi Awolowo University, ILE-IFE, Nigeria, toyinpharm@gmail.com UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency. Received file: Reviewer's Comments: Average Peer review marks at initial stage: 3.0/10 Average Peer review marks at publication stage: 7.0/10 Reviewer(s) detail: Dr. Mohammed Abdel-Wahab Sayed Abourehab, Umm Al-Qura University; Makkah Al-Mukarramah, Saudi Arabia, mohawahab2002@yahoo.com Dr. Sally A. El-Zahaby, Pharos University in Alexandria, Egypt, sally.elzahaby@yahoo.com Similar Articles: INVESTIGATION OF PRONIOSOMES GEL AS A PROMISING CARRIER FOR TRANSDERMAL DELIVERY OF GLIMEPIRIDE FORMULATION AND EVALUATION OF ETODOLAC NIOSOMES BY MODIFIED ETHER INJECTION TECHNIQUE

Effects of M1 and M4 activation on excitatory synaptic transmission in CA1.

Hippocampal networks are particularly susceptible to dysfunction in many neurodegenerative diseases and neuropsychiatric disorders including Alzheimer's disease, Lewy body dementia, and schizophrenia. CA1, a major output region of the hippocampus, receives glutamatergic input from both hippocampal CA3 and entorhinal cortex, via the Schaffer collateral (SC) and temporoammonic (TA) pathways, respectively. SC and TA inputs to CA1 are thought to be differentially involved in the retrieval of previously stored memories versus the encoding of novel information, and switching between these two crucial hippocampal functions is thought to critically depend on acetylcholine (ACh) acting at muscarinic receptors. In this study, we aimed to determine the roles of specific subtypes of muscarinic receptors in mediating the neuromodulatory effects of ACh on glutamatergic synaptic transmission in the SC and TA pathways of CA1. Using selective pharmacological activation of M1 or M4 receptors along with extracellular and intracellular electrophysiology recordings from adult rat hippocampal slices, we demonstrate that activation of M1 receptors increases spontaneous spike rates of neuronal ensembles in CA1 and increases the intrinsic excitability of pyramidal neurons and interneurons. Selective activation of M4 receptors inhibits glutamate release in the SC pathway, while leaving synaptic transmission in the TA pathway comparatively intact. These results suggest specific mechanisms by which M1 and M4 activation may normalize CA1 circuit activity following disruptions of signaling that accompany neurodegenerative dementias or neuropsychiatric disorders. These findings are of particular interest in light of clinical findings that xanomeline, an M1/M4 preferring agonist, was able to improve cognitive and behavioral symptoms in patients with Alzheimer's disease or schizophrenia.

Synergistic Action of Presynaptic Muscarinic Acetylcholine Receptors and Adenosine Receptors in Developmental Axonal Competition at the Neuromuscular Junction

The development of the nervous system involves the initial overproduction of synapses, which promotes connectivity. Hebbian competition between axons with different activities leads to the loss of roughly half of the overproduced elements and this refines connectivity. We used quantitative immunohistochemistry to investigate, in the postnatal day 7 (P7) to P9 neuromuscular junctions, the involvement of muscarinic receptors (muscarinic acetylcholine autoreceptors and the M₁, M₂, and M₄ subtypes) and adenosine receptors (A₁ and A_2A subtypes) in the control of axonal elimination after the mouse levator auris longus muscle had been exposed to selective antagonists in vivo. In a previous study we analyzed the role of each of the individual receptors. Here we investigate the additive or occlusive effects of their inhibitors and thus the existence of synergistic activity between the receptors. The main results show that the A_2A, M₁, M₄, and A₁ receptors (in this order of ability) delayed axonal elimination at P7. M₄ produces some occlusion of the M₁ pathway and some addition to the A₁ pathway, which suggests that they cooperate. M₂ receptors may modulate (by allowing a permissive action) the other receptors, mainly M₄ and A₁. The continued action of these receptors (now including M₂ but not M₄) finally promotes axonal loss at P9. All 4 receptors (M₂, M₁, A₁, and A_2A, in this order of ability) are necessary. The M₄ receptor (which in itself does not affect axon loss) seems to modulate the other receptors. We found a synergistic action between the M₁, A₁, and A_2A receptors, which show an additive effect, whereas the potent M₂ effect is largely independent of the other receptors (though can be modulated by M₄). At P9, there is a full mutual dependence between the A₁ and A_2A receptors in regulating axon loss. In summary, postnatal axonal elimination is a regulated multireceptor mechanism that involves the cooperation of several muscarinic and adenosine receptor subtypes.

Acetylcholinesterase Inhibitors and Drugs Acting on Muscarinic Receptors- Potential Crosstalk of Cholinergic Mechanisms During Pharmacological Treatment.

BackgroundPharmaceuticals with targets in the cholinergic transmission have been used for decades and are still fundamental treatments in many diseases and conditions today. Both the transmission and the effects of the somatomotoric and the parasympathetic nervous systems may be targeted by such treatments. Irrespective of the knowledge that the effects of neuronal signalling in the nervous systems may include a number of different receptor subtypes of both the nicotinic and the muscarinic receptors, this complexity is generally overlooked when assessing the mechanisms of action of pharmaceuticals.MethodsWe have search of bibliographic databases for peer-reviewed research literature focused on the cholinergic system. Also, we have taken advantage of our expertise in this field to deduce the conclusions of this study.ResultsPresently, the life cycle of acetylcholine, muscarinic receptors and their effects are reviewed in the major organ systems of the body. Neuronal and non-neuronal sources of acetylcholine are elucidated. Examples of pharmaceuticals, in particular cholinesterase inhibitors, affecting these systems are discussed. The review focuses on salivary glands, the respiratory tract and the lower urinary tract, since the complexity of the interplay of different muscarinic receptor subtypes is of significance for physiological, pharmacological and toxicological effects in these organs.ConclusionMost pharmaceuticals targeting muscarinic receptors are employed at such large doses that no selectivity can be expected. However, some differences in the adverse effect profile of muscarinic antagonists may still be explained by the variation of expression of muscarinic receptor subtypes in different organs. However, a complex pattern of interactions between muscarinic receptor subtypes occurs and needs to be considered when searching for selective pharmaceuticals. In the development of new entities for the treatment of for instance pesticide intoxication, the muscarinic receptor selectivity needs to be considered. Reactivators generally have a muscarinic M2 receptor acting profile. Such a blockade may engrave the situation since it may enlarge the effect of the muscarinic M3 receptor effect. This may explain why respiratory arrest is the major cause for deaths by esterase blocking.

Muscarinic cholinoreceptors (M1-, M2-, M3- and M4-type) modulate the acetylcholine secretion in the frog neuromuscular junction

Muscarinic cholinoreceptors regulate the neurosecretion process in vertebrate neuromuscular junctions. The diversity of muscarinic effects on acetylcholine (ACh) secretion may be attributed to the different muscarinic subtypes involved in this process. In the present study, the location of five muscarinic receptor subtypes (M1, M2, M3, M4 and M5) on the motor nerve terminals of frog cutaneous pectoris muscle was shown using specific polyclonal antibodies. The modulatory roles of these receptors were investigated via assessment of the effects of muscarine and specific muscarinic antagonists on the quantal content of endplate currents (EPCs) and the time course of secretion, which was estimated from the distribution of “real” synaptic delays of EPCs recorded in a low Ca2+/high Mg2+ solution.The agonist muscarine decreased the EPC quantal content and synchronized the release process. The depressing action of muscarine on the EPC quantal content was abolished only by pretreatment of the preparation with the M3 blockers 4-DAMP (1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide) and J 104129 fumarate ((αR)-α-Cyclopentyl-α-hydroxy-N-[1-(4-methyl-3-pentenyl)-4-piperidinyl]benzeneacetamide fumarate). Moreover, antagonists of the M1, M2, M3 and M4 receptors per se diminished the intensity of secretion, which suggests a putative up-regulation of the release by endogenous ACh.