M1 Acetylcholine Receptors Research Articles

Cyclic Stretch Promotes Proliferation and Contraction of Human Bladder Smooth Muscle Cells by Cajal-Mediated c-kit Expression in Interstitial Cells.

BackgroundThe present study was performed to assess the effect of mechanical stretch on the proliferation and contractile function of hBSMCs.Material/MethodshBSMCs and ICCs were seeded at 8×104 cells/well in 6-well silicone elastomer-bottomed culture plates coated with type I collagen, and grown to 80% confluence in DMEM/10% FBS and a 5% CO2 humidified atmosphere at 37°C. Cells of hBSMCs and hBSMCs/ICCs of co-culture were then subjected to continuous cycles of stretch-relaxation using a computer-driven, stretch-inducing device. The treated concentration of imatinib was 10 μM. Mechanisms underlying observed hBSMCs contraction were examined using Western blotting and RT-PCR. The 0.1 μM carbachol was separately added to the experimental groups, and 300 s was recorded by laser scanning confocal microscope.ResultsWe found that mechanical stretch increased contraction and proliferation of hBSMCs. Calcium ion activity increased significantly after mechanical stretch. The number of hBSMCs was significantly increased after the combination mechanical stretch with ICCs treatment. After combination mechanical stretch with hBSMCs/ICCs treatment, the mRNA and protein level of M2, M3, and c-kit were significantly increased. After combination of mechanical stretch with no imatinib treatment, the proliferation of hBSMCs was higher than others, and the mRNA and protein level of M2 and M3 were significantly increased.ConclusionsWe revealed that ICCs could promote hBSMC proliferation and contraction, and cyclic stretch could promote acetylcholine receptor M2 and M3 caused by c-kit in the ICCs, which promoted the contraction of hBSMCs.

Development of Enhanced Conformational Sampling Methods for GPCRs

G protein‐coupled receptors (GPCRs) are known to possess multiple active conformational states in nature, however, studying these conformations is extremely challenging as majority of the functionally important conformations have high energy compared to the lowest energy conformation. We are developing a method called Enhanced Conformational Markov‐state Sampling in Membrane BiLayer Environment (EnCoMSeMBLE) that would enhance our search of the conformational landscape of GPCRs and that can be applied to a‐helical transmembrane proteins in general. It enables a level of conformational sampling not achievable by classical or accelerated molecular dynamics (MD) simulations or Markov‐State Models (MSM). This method combines brute force conformational sampling of helix‐helix interactions in the membrane with MD simulations and Markov‐State modeling to identify functionally important conformations. This method is being tested by application to the Glucagon Like peptide‐1 receptor (GLP1‐R), a class B GPCR, and the muscarinic acetylcholine M2 receptor, a class A GPCR, both of which have been crystallized in inactive as well as active conformations. The comparison of the activation landscapes of class A and class B GPCRs is beginning to provide key similarities and differences in activation across these very distinct GPCRs. This detailed understanding of the GPCR activation, enabled by our method, complements major structural biology efforts underway targeting GPCRs, where it is very challenging to map out the structural landscape and activation pathways of GPCRs.Support or Funding InformationBUILD PODER is supported through a grant from the National Institutes of General Medical Sciences (NIGMS), grants # # 8TL4GM11897This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ligand biological activity predicted by cleaning positive and negative chemical correlations

Predicting ligand biological activity is a key challenge in drug discovery. Ligand-based statistical approaches are often hampered by noise due to undersampling: The number of molecules known to be active or inactive is vastly less than the number of possible chemical features that might determine binding. We derive a statistical framework inspired by random matrix theory and combine the framework with high-quality negative data to discover important chemical differences between active and inactive molecules by disentangling undersampling noise. Our model outperforms standard benchmarks when tested against a set of challenging retrospective tests. We prospectively apply our model to the human muscarinic acetylcholine receptor M1, finding four experimentally confirmed agonists that are chemically dissimilar to all known ligands. The hit rate of our model is significantly higher than the state of the art. Our model can be interpreted and visualized to offer chemical insights about the molecular motifs that are synergistic or antagonistic to M1 agonism, which we have prospectively experimentally verified.

Comparison of cerebral and cutaneous microvascular dysfunction with the development of type 1 diabetes.

Rationale: Diabetes can lead to cerebral and cutaneous vascular dysfunction. However, it is still unclear how vascular function changes with the development of diabetes and what differences exist between cerebral and cutaneous vascular dysfunction. Thus, it is very important to monitor changes in cerebral and cutaneous vascular function responses in vivo and study their differences during diabetes development.Methods: With the assistance of newly developed skull and skin optical clearing techniques, we monitored the responses of sodium nitroprusside (SNP)- and acetyl choline (ACh)-induced cerebral and cutaneous vascular blood flow and blood oxygen in diabetic mice in vivo during the development of type 1 diabetes (T1D) by combining laser speckle contrast imaging with hyperspectral imaging. We then compared the differences between cerebral and cutaneous vascular responses and explored the reasons for abnormal changes induced in response to different vascular beds.Results: In the early stage of diabetes (T1D-1 week), there were abnormal changes in the cerebral vascular blood flow and blood oxygen responses to SNP and ACh as well as cutaneous vascular blood oxygen. The cutaneous vascular blood flow response also became abnormal from T1D-3 weeks. Additionally, the T1D-induced abnormal blood flow response was associated with changes in vascular myosin light chain phosphorylation and muscarinic acetylcholine receptor M3 levels, and the aberrant blood oxygen response was related to an increase in glycated hemoglobin levels.Conclusion: These results suggest that the abnormal cutaneous vascular blood oxygen response occurred earlier than the blood flow response and therefore has the potential to serve as a good assessment indicator for revealing cerebrovascular dysfunction in the early stage of diabetes.

Effect of green flickering light on myopia development and expression of M1 muscarinic acetylcholine receptor in guinea pigs.

To investigate the effects of green flickering light on refractive development and expression of muscarinic acetylcholine receptor (mAChR) M1 in the eyes of guinea pigs. Thirty guinea pigs (15-20 days old) were randomly divided into three groups (n=10/group). Animals in group I were raised in a completely closed carton with green flickering light illumination. Those in group II were kept in the open top closed carton under normal natural light. Guinea pigs were raised in a sight-widen cage under normal natural light in group III. The refractive status and axial length were measured before and after 8 weeks' illumination. Moreover, total RNA extracted from retinal, choroidal, and scleral tissues were determined by real-time reverse transcription polymerase chain reaction (RT-PCR). The expressions of the receptor M1 were also explored in the retina, choroid, and sclera using immunohistochemistry. There was a remarkable reduction in refractive error and increase in axial length after 8-weeks' green flickering light stimulation (P<0.001). The expression of M1 receptor mRNA in sclera and retina in myopia group were remarkably lower than that in group II and III (P<0.01). Significant reduced expression of M1 receptor stimulated by green flickering light in retina and sclera tissues were also observed (P<0.05). However, there was no M1 receptor expression in choroid in 3 groups. Myopia can be induced by 8 weeks' green flickering light exposure in the animal model. M1 receptor may be involved causally or protectively in myopia development.

Of molecules, memories and migration: M1 acetylcholine receptors facilitate spatial memory formation and recall during migratory navigation.

Many animals use complex cognitive processes, including the formation and recall of memories, for successful navigation. However, the developmental and neurological processes underlying these cognitive aspects of navigation are poorly understood. To address the importance of the formation and recollection of memories during navigation, we pharmacologically manipulated turtles (Chrysemys picta) that navigate long distances using precise, complex paths learned during a juvenile critical period. We treated freely navigating turtles both within and outside of their critical learning period with a specific M1 acetylcholine receptor antagonist, a drug known to disrupt spatial cognition. Experienced adult turtles lost all navigational ability under the influence of the drug, while naive juveniles navigated successfully. We retested these same juveniles the following year (after they had passed their critical period). The juveniles that initially navigated successfully under the influence of the antagonist (but were unable to form spatial memories) were unable to do so subsequently. However, the control animals (who had the opportunity to form memories previously) exhibited typical navigational precision. These results suggest that the formation of spatial memories for navigation occur during a critical period, and successful navigation after the critical period is dependent upon the recall of such memories.

Effects of a Pasty Bone Cement Containing Brain-Derived Neurotrophic Factor-Functionalized Mesoporous Bioactive Glass Particles on Metaphyseal Healing in a New Murine Osteoporotic Fracture Model.

The development of new and better implant materials adapted to osteoporotic bone is still urgently required. Therefore, osteoporotic muscarinic acetylcholine receptor M3 (M3 mAChR) knockout (KO) and corresponding wild type (WT) mice underwent osteotomy in the distal femoral metaphysis. Fracture gaps were filled with a pasty α-tricalcium phosphate (α-TCP)-based hydroxyapatite (HA)-forming bone cement containing mesoporous bioactive CaP-SiO2 glass particles (cement/MBG composite) with or without Brain-Derived Neurotrophic Factor (BDNF) and healing was analyzed after 35 days. Histologically, bone formation was significantly increased in WT mice that received the BDNF-functionalized cement/MBG composite compared to control WT mice without BDNF. Cement/MBG composite without BDNF increased bone formation in M3 mAChR KO mice compared to equally treated WT mice. Mass spectrometric imaging showed that the BDNF-functionalized cement/MBG composite implanted in M3 mAChR KO mice was infiltrated by newly formed tissue. Leukocyte numbers were significantly lower in M3 mAChR KO mice treated with BDNF-functionalized cement/MBG composite compared to controls without BDNF. C-reactive protein (CRP) concentrations were significantly lower in M3 mAChR KO mice that received the cement/MBG composite without BDNF when compared to WT mice treated the same. Whereas alkaline phosphatase (ALP) concentrations in callus were significantly increased in M3 mAChR KO mice, ALP activity was significantly higher in WT mice. Due to a stronger effect of BDNF in non osteoporotic mice, higher BDNF concentrations might be needed for osteoporotic fracture healing. Nevertheless, the BDNF-functionalized cement/MBG composite promoted fracture healing in non osteoporotic bone.

Variation of nicotinic subtype α7 and muscarinic subtype M3 acetylcholine receptor expression in three main types of leukemia.

Cholinergic receptors, such as α7-nicotinic acetylcholine receptor (α7-nAChR) and M3-muscarinic acetylcholine receptor (M3-mAChR), have been demonstrated to serve a significant role in the proliferation, differentiation and apoptosis of leukemic cells. However, the expression of these receptors in samples from patients with leukemia remains unclear. The present study aimed to determine the expression of M3-mAChR and α7-nAChR in the bone marrow or peripheral blood of 51 patients with leukemia, including acute myeloid leukemia (AML; n=33), acute lymphoblastic leukemia (ALL; n=13), and chronic myeloid leukemia (CML; n=5). Peripheral blood mononuclear cells (PBMCs) were also isolated from healthy subjects (n=5) for comparison. Western blot analysis was performed to determine the protein expression profiles, and a pattern of decreased α7-nAChR levels in patients with leukemia was observed. Among the leukemia types, the lowest expression of α7-nAChR and M3-mAChR were identified in patients with T-cell ALL/lymphoma (T-ALL). CML exhibited the highest level of M3-mAChR, which was significantly different from APL and AML-M4, yet not from healthy subjects (P<0.05). Therefore, different expression profiles of α7-nACR and M3-mAChR were detected amongst the leukemia types. Collectively, the present study supports the potential role of cholinergic signaling in mediating leukemogenesis. However, further studies in larger cohorts are required to validate these findings.

The Anti-Inflammatory Effects of Fermented Herbal Roots of Asparagus cochinchinensis in an Ovalbumin-Induced Asthma Model.

Introduction: Roots of Asparagus cochinchinensis, which have pharmacologically active ingredients, have received great attention because they show good therapeutic effects for various inflammatory diseases without specific toxicity. This study investigated the anti-asthmatic effects of a butanol extract of Asparagus cochinchinensis roots that had been fermented with Weissella cibaria (BAW) and its possible underlying cholinergic regulation. Methods: Alterations of the anti-asthmatic markers and the molecular response factors were measured in an ovalbumin (OVA)-induced asthma model after treatment with BAW. Results: Treatment with BAW decreased the intracellular reactive oxygen species (ROS) production in lipopolysaccharides (LPS) activated RAW264.7 cells. The results of the animal experiments revealed lower infiltration of inflammatory cells and bronchial thickness, and a significant reduction in the number of macrophages and eosinophils, concentration of OVA-specific IgE, and expression of Th2 cytokines in the OVA + BAW treated group. In addition, a significant recovery of goblet cell hyperplasia, MMP-9 expression, and the VEGF signaling pathway was observed upon airway remodeling in the OVA + BAW treated group. Furthermore, these responses of BAW were linked to recovery of acetylcholine esterase (AChE) activity and muscarinic acetylcholine receptor (mAChR) M3 downstream signaling pathway in epithelial cells, smooth muscle cells, and afferent sensory nerves of OVA + BAW-treated mice. Conclusion: Overall, these findings are the first to provide evidence that the therapeutic effects of BAW can prevent airway inflammation and remodeling through the recovery of cholinergic regulation in structural cells and inflammatory cells of the chronic asthma model.

Correction: The combination of Cassia obtusifolia L. and Foeniculum vulgare M. exhibits a laxative effect on loperamide-induced constipation of rats.

[This corrects the article DOI: 10.1371/journal.pone.0195624.].

Most calbindin-immunoreactive neurons, but few calretinin-immunoreactive neurons, express the m1 acetylcholine receptor in the middle temporal visual area of the macaque monkey.

IntroductionRelease of the neuromodulator acetylcholine into cortical circuits supports cognition, although its precise role and mechanisms of action are not well understood. Little is known about functional differences in cholinergic modulatory effects across cortical model systems, but anatomical evidence suggests that such differences likely exist because, for example, the expression of cholinergic receptors differs profoundly both within and between species.MethodsIn the primary visual cortex (V1) of macaque monkeys, cholinergic receptors are strongly expressed by inhibitory interneurons. Using dual‐immunofluorescence confocal microscopy, we examine m1 muscarinic acetylcholine receptor expression by two subclasses of inhibitory interneurons—identified by their expression of the calcium‐binding proteins calbindin and calretinin—in the middle temporal extrastriate area (MT) of the macaque.Results and ConclusionsWe find that the majority of calbindin‐immunoreactive neurons (55%) and only few calretinin‐immunoreactive neurons (10%) express the m1 acetylcholine receptor. These results differ from the pattern observed in V1 of the same species, lending further support to the notion that cholinergic modulation in the cortex is tuned such that different cortical compartments will respond to acetylcholine release in different ways.

Effects of Cortisol-Induced Acetylcholine Accumulation on Tissue Damage and Regeneration in Steatotic Livers in the Context of Partial Hepatectomy Under Vascular Occlusion

Introduction Steatosis is a known risk factor in hepatic surgery and it is associated with postoperative morbidity and mortality. Acetylcholine (ACh) is the major neurotransmitter of vagus nerve and it is released as a response to stressful events like trauma or infection. Stress can also activate the HPA axis that drives the production of cortisol. Both cortisol and ACh have been proved beneficial in experimental models of sepsis, liver transplantation or myocardial ischemia, among other pathologies. In this study, we will study the relationship between cortisol and ACh and their effects on regeneration and injury in seatotic livers submitted to partial hepatectomy (PH) under vascular occlusion, a common technique used in hepatic surgery to avoid bleeding. Materials & Methods We determined hepatic levels of ACh and its receptors in obese Zucker rats that underwent partial hepatectomy under vascular occlusion. The ACTH-cortisol pathway was pharmacologically modulated and the effects on hepatic injury and regeneration, as well as its underlying mechanisms of action, were evaluated. Results There is a significant ACh accumulation driven by the administration of cortisol in steatotic livers submitted to PH under vascular occlusion, that consequently leads to an overexpression of a7 nicotinic and M3 muscarinic ACh receptors. However, administration of ACh prior to surgery increases injury and impairs regeneration via M3 mACh receptor. Interestingly, M3 mAChR antagonist, but not a7 nAChR, alone or in combination with cortisol, did confer eprotection against liver damage caused by the surgical procedure and enhanced regeneration. Moreover, administration of ACh reduced PI3K/Akt hepatic expression and consequently reduced levels of markers of oxidative stress, neutrophil accumulation and edema. In consonance with the results presented above, treatment with an M3 mAChR antagonist, but not a7 nAChR, alone or in combination with cortisol, reduced the hepatic levels of these harmful markers as a result of an overexpression of PI3K and pAKT in these steatotic livers. Thus, this indicates a lesser role of a7 nAChR. Conclusions The reported benefits of cortisol in PH cannot be extrapolated in situations of PH under vascular occlusion in steatotic livers. Cortisol is responsible of the accumulation of acetylcholine in these livers submitted to partial hepatectomy with vascular occlusion. ACh exerts its action through the M3 ACh receptor, as it exacerbates liver damage and oxidative stress and impairs regeneration. Strategies aimed to block cortisol activity in order to block ACh accumulation and/or blockade of M3 mAChR are probably worthwhile to protect steatotic livers and reduce the incidence of postoperative complications following PH under vascular occlusion.

Mnemonic and behavioral effects of biperiden, an M1-selective antagonist, in the rat.

There is a persistent pressing need for valid animal models of cognitive and mnemonic disruptions (such as seen in Alzheimer's disease and other dementias) usable for preclinical research. We have set out to test the validity of administration of biperiden, an M1-acetylcholine receptor antagonist with central selectivity, as a potential tool for generating a fast screening model of cognitive impairment, in outbred Wistar rats. We used several variants of the Morris water maze task: (1) reversal learning, to assess cognitive flexibility, with probe trials testing memory retention; (2) delayed matching to position (DMP), to evaluate working memory; and (3) "counter-balanced acquisition," to test for possible anomalies in acquisition learning. We also included a visible platform paradigm to reveal possible sensorimotor and motivational deficits. A significant effect of biperiden on memory acquisition and retention was found in the counter-balanced acquisition and probe trials of the counter-balanced acquisition and reversal tasks. Strikingly, a less pronounced deficit was observed in the DMP. No effects were revealed in the reversal learning task. Based on our results, we do not recommend biperiden as a reliable tool for modeling cognitive impairment.

Ligand-Triggered Structural Changes in the M2 Muscarinic Acetylcholine Receptor.

The muscarinic M2 acetylcholine receptor, one of the few G-protein coupled receptors that has not only been crystallized in both active and inactive conformations but also in the presence of a positive allosteric modulator, is an interesting system to study the molecular mechanisms of GPCR activation and ligand allosterism. Here, we have employed molecular dynamics (MD) simulations (adding to 14 μs in total) to study conformational changes triggered by the inverse agonist R-(-)-3-quinuclidinyl-benzilate (QNB) in the structure of the active M2 receptor (PBD ID 4MQS ) after replacement of the agonist iperoxo by the inverse agonist QNB. This permitted us to identify the sequence of events in the deactivation mechanism of the M2 acetylcholine receptor, which results first in the rearrangement of the transmission switch, the subsequent opening of the extracellular portion of the receptor and finally, the closure of the intracellular part. We also evaluate the effect of the positive allosteric modulator LY2119620 when bound simultaneously with the orthosteric agonist iperoxo and find that it restricts the conformation of Trp4227.35 in a position that modulates the orientation of the Tyr4267.39 at the orthosteric-binding pocket.

In Vitro Pharmacological Characterization and In Vivo Validation of LSN3172176 a Novel M1 Selective Muscarinic Receptor Agonist Tracer Molecule for Positron Emission Tomography

In the search for improved symptomatic treatment options for neurodegenerative and neuropsychiatric diseases, muscarinic acetylcholine M1 receptors (M1 mAChRs) have received significant attention. Drug development efforts have identified a number of novel ligands, some of which have advanced to the clinic. However, a significant issue for progressing these therapeutics is the lack of robust, translatable, and validated biomarkers. One valuable approach to assessing target engagement is to use positron emission tomography (PET) tracers. In this study we describe the pharmacological characterization of a selective M1 agonist amenable for in vivo tracer studies. We used a novel direct binding assay to identify nonradiolabeled ligands, including LSN3172176, with the favorable characteristics required for a PET tracer. In vitro functional and radioligand binding experiments revealed that LSN3172176 was a potent partial agonist (EC50 2.4-7.0 nM, Emax 43%-73%), displaying binding selectivity for M1 mAChRs (Kd = 1.5 nM) that was conserved across species (native tissue Kd = 1.02, 2.66, 8, and 1.03 at mouse, rat, monkey, and human, respectively). Overall selectivity of LSN3172176 appeared to be a product of potency and stabilization of the high-affinity state of the M1 receptor, relative to other mAChR subtypes (M1 > M2, M4, M5 > M3). In vivo, use of wild-type and mAChR knockout mice further supported the M1-preferring selectivity profile of LSN3172176 for the M1 receptor (78% reduction in cortical occupancy in M1 KO mice). These findings support the development of LSN3172176 as a potential PET tracer for assessment of M1 mAChR target engagement in the clinic and to further elucidate the function of M1 mAChRs in health and disease.

The combination of Cassia obtusifolia L. and Foeniculum vulgare M. exhibits a laxative effect on loperamide-induced constipation of rats.

Chronic constipation is a functional gastrointestinal disease that is detrimental to the quality of patient life. Cassia obtusifolia L. (CO) and Foeniculum vulgare M. (FV) are commonly used as medicinal foods in many countries. We aimed to examine the laxative effect and their underlying mechanism of CO and FV mixture on loperamide (lop)-induced constipated rats. To determine the laxative effects of these compounds, Sprague-Dawley rats were divided into six groups: the control, lop-induced constipated (2mg/kg), and three doses (100, 300, and 500mg/kg) of CO and FV mixture-, and Bisacodyl (bis, 3.3mg/kg)-treated groups. The mixture of CO and FV and bis were orally administered once a day for 4 weeks. For induction of constipation, the lop were treated with a dose of 2 mg/kg twice a day on the 3rd week after treatments of CO and FV extracts and bis. The results were revealed that the CO and FV mixture has the laxative effects more than those in CO and FV-alone treatments on constipated rats by determining the stool parameters, including stool number and weight. Indeed, stool parameters, such as, stool number, weight, and water contents and colonic peristalsis from the intestinal transit length and ratio were dramatically improved by CO and FV mixture treatment. Histological study also revealed that CO and FV mixture enhanced the thicknesses of mucosa and muscular layers of the colon in constipated rats. For their underlying mechanism, the mRNAs and proteins expression of muscarinic acetylcholine receptors (mAchR) M2 and M3 and their downstream signaling were preserved by CO and FV mixture treatment in constipated rats. Therefore, this study suggests that treatment with CO and FV mixture has beneficial effects against constipation. We further suggest that CO and FV mixture may be utilized as an alternative therapeutic strategy for constipation.

MP09-14 ESTABLISHMENT OF IMMORTALIZED HUMAN BLADDER SMOOTH MUSCLE CELLS

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2018MP09-14 ESTABLISHMENT OF IMMORTALIZED HUMAN BLADDER SMOOTH MUSCLE CELLS Masaki Yoshida, Naohiro Hashimoto, Hisae Nishii, and Masanori Nomiya Masaki YoshidaMasaki Yoshida More articles by this author , Naohiro HashimotoNaohiro Hashimoto More articles by this author , Hisae NishiiHisae Nishii More articles by this author , and Masanori NomiyaMasanori Nomiya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.340AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Smooth muscle cell (SMC) cultures are an excellent tool for the study of cellular events and molecular mechanisms by which the phenotypic plasticity is controlled. Although many SMC systems have been previously analyzed, human bladder cell biology has not been fully evaluated. Therefore we attempted to establish the immortalized human bladder smooth muscle cells. METHODS In immortalization of human smooth muscle cells, primary cultured human bladder smooth muscle cells derived from a 68 year old man were transduced with recombinant lentiviruses and retroviruses encoding human cyclin D1, human mutant CDK4, and human telomerase. Immunoblotting analysis were performed using primary antibodies included mouse monoclonal antibodies for calponin, α-smooth muscle actin (α-SMA), ?-smooth muscle actin (γ-SMA) and rabbit monoclonal antibodies for muscarinic acetylcholine receptor M2 and M3 and rabbit polyclonal antibodies for smooth muscle myosin heavy chain 11 (MYH11). In addition, immunofluorescence analyses for connexin 43 were also performed. Live imaging of intracellular calcium evaluated with Calcium Kit-Fluo 4 after stimulation with high potassium medium and carbachol. Smooth muscle contraction were also evaluated live imaging of F-actin using SiR-A. RESULTS We have established an immortalized human bladder SMC line designated as hBS11. hBS11 cells exhibited continuous cell proliferation for more than 50 populations under optimized culture conditions. The cell cycle interval was 18.0±3.2h. A chromosome analysis of the cells revealed a normal 46XY diploid karyotype. hBS11 cells expressed smooth muscle differentiation marker proteins, including MYH11, α-SMA, γ-SMA, and calponin. The connexin 43 signals were also detected as spotty signals on the surface of hBS11 cells. M2 and M3 receptors expressed in differentiated hBS11 cells. Both the cholinergic receptor agonist calbachol and a high concentration of extracellular potassium increased intracellular calcium in differentiated hBS11 cells. Differentiated hBS11 cells stimulated by calcium ionophore (A23187) exhibited contractility. CONCLUSIONS The present study demonstrates that the hBS11 cell maintains plasticity of the differentiation that a smooth muscle cell originally has. Immortalized hBS11 cell provides useful analytical system for elucidation of the pathophysiological mechanism of lower urinary tract dysfunction and the development of the new treatment options, in addition to the elucidation of molecular differentiation mechanism, reproduction mechanism, and the function control mechanism of the smooth muscle cell. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e111-e112 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Masaki Yoshida More articles by this author Naohiro Hashimoto More articles by this author Hisae Nishii More articles by this author Masanori Nomiya More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Molecular Biological Mechanisms of Yuan Zhi Powder in the Treatment of Alzheimer’s Disease: an Analysis Based on Network Pharmacology

Objective To predict the main active ingredients, potential targets and molecular mechanisms of Yuan Zhi powder in treatment of dementia by using network pharmacology. Methods A database of chemical constituents of Yuan Zhi powder was constructed by using databases and literatures. Potential targets were predicted by reverse molecular docking, and then a component-target network was constructed. The target network of Alzheimer’s disease (AD) was mapped and analyzed to obtain the “active ingredient-AD target” network. The key targets were screened through network analysis. Finally, the rationality of the prediction was analyzed by comparing with the target reported in the literatures. Results There were 180 chemical constituents acting on the AD target, and the targets included three key targets (cyclooxygenase-2, muscarinic acetylcholine receptor M1, and muscarinic acetylcholine receptor M2) and an important target (acetylcholine esterase). Alzheimer’s disease may be treated by regulating the activity of acetylcholine receptors and the binding to β-amyloid protein, and its biological process may be concentrated in the acetylcholine receptor signal pathway, negative regulation of synaptic transmission and so on. Conclusion The fact that Yuan Zhi powder can treat AD is consistent with the characteristics of multi-components-multi-targets-multiple pathways of traditional Chinese medicine. The important targets obtained from network analysis have a large proportion in literature research, so network analysis have some rationality.

Neuronal Growth and Behavioral Alterations in Mice Deficient for the Psychiatric Disease-Associated Negr1 Gene.

Neuronal growth regulator 1 (NEGR1), a member of the immunoglobulin superfamily cell adhesion molecule subgroup IgLON, has been implicated in neuronal growth and connectivity. In addition, genetic variants in or near the NEGR1 locus have been associated with obesity and more recently with learning difficulties, intellectual disability and psychiatric disorders. However, experimental evidence is lacking to support a possible link between NEGR1, neuronal growth and behavioral abnormalities. Initial expression analysis of NEGR1 mRNA in C57Bl/6 wildtype (WT) mice by in situ hybridization demonstrated marked expression in the entorhinal cortex (EC) and dentate granule cells. In co-cultures of cortical neurons and NSC-34 cells overexpressing NEGR1, neurite growth of cortical neurons was enhanced and distal axons occupied an increased area of cells overexpressing NEGR1. Conversely, in organotypic slice co-cultures, Negr1-knockout (KO) hippocampus was less permissive for axons grown from EC of β-actin-enhanced green fluorescent protein (EGFP) mice compared to WT hippocampus. Neuroanatomical analysis revealed abnormalities of EC axons in the hippocampal dentate gyrus (DG) of Negr1-KO mice including increased numbers of axonal projections to the hilus. Neurotransmitter receptor ligand binding densities, a proxy of functional neurotransmitter receptor abundance, did not show differences in the DG of Negr1-KO mice but altered ligand binding densities to NMDA receptor and muscarinic acetylcholine receptors M1 and M2 were found in CA1 and CA3. Activity behavior, anxiety-like behavior and sensorimotor gating were not different between genotypes. However, Negr1-KO mice exhibited impaired social behavior compared to WT littermates. Moreover, Negr1-KO mice showed reversal learning deficits in the Morris water maze and increased susceptibility to pentylenetetrazol (PTZ)-induced seizures. Thus, our results from neuronal growth assays, neuroanatomical analyses and behavioral assessments provide first evidence that deficiency of the psychiatric disease-associated Negr1 gene may affect neuronal growth and behavior. These findings might be relevant to further evaluate the role of NEGR1 in cognitive and psychiatric disorders.

Autoantibodies against muscarinic acetylcholine receptor M3 in Sjogren's syndrome and corresponding mouse models.

Muscarinic acetylcholine receptor M3 (M3R) is a GPCR on exocrine gland cells involved in fluid secretion. In the last two decades, evidence has been accumulated arguing for a role of autoantibodies (aab) against M3R in the development of Sjögren's syndrome (SS). In this review, we provide an updated overview on this issue and critically discuss the relation between autoimmunity to M3R and SS pathogenesis. Clinical data as well as findings from experimental disease were summarized in categories addressing the presence of aab against M3R in SS patients, the function of anti-M3R aab, the association of aab against M3R with SS-related phenotypes, in vivo pathogenicity of transferred aab against M3R in mice, and mouse models induced via immunization with M3R. Based on these comprehensive data, we propose a hypothetic model for the role of aab against M3R in the pathogenesis of SS.