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Pharmacovigilance aims at a better understanding of the molecular events triggered by medications to prevent adverse effects, which despite significant advances in our analytical repertoire plague the use of drugs until today. In this study, we find that clinically prescribed and commercially available pirenzepine may not be the correct compound. Pirenzepine can undergo an unexpected scaffold rearrangement from the pharmaceutical active ingredient (API) to a previously uncharacterized benzimidazole. The rearrangement occurs under highly acidic conditions, which were believed to favour the dihydrochloride formation of pirenzepine. The rearranged products of pirenzepine and the structurally related telenzepine have significantly decreased affinity for the muscarinic acetylcholine receptor, the pharmacological target of these compounds. Fortunately, in situ rearrangement after oral application is no safety issue, as we show that reaction kinetics in gastric acid prevent rearrangement. The research community should consider appropriate measures to perform reliable receiving inspections in the commercial supply of well described and frequently used chemicals, in particular if experiments yield unexpected results., (© 2021. The Author(s).)

Divided attention may be more important than ever to comprehend, given ubiquitous distractors in modern living. In humans, concern has been expressed about the negative impact of distraction in education, the home, and the workplace. While acetylcholine supports divided attention, in part via muscarinic receptors, little is known about the specific muscarinic subtypes that may contribute. We designed a novel, high-response rate test of auditory sustained attention, in which rats complete variable-ratio runs on one of two levers, rather than emitting a single response. By doing this, we can present a secondary visual distractor task during some trials, for which a correct nosepoke response is reinforced with a more palatable food pellet. The nonspecific muscarinic antagonist scopolamine impaired performance, and slowed and reduced lever press activity. We then explored antagonists that preferentially block the M1 and M4 subtypes, because these receptors are potential therapeutic targets for cognitive enhancers. Telenzepine, an M1-preferring antagonist, impaired divided attention performance, but not performance of the attention task without distraction. Telenzepine also had fewer nonspecific effects than scopolamine. In contrast, the M4-preferring antagonist tropicamide had no effects. Analysis of overall behavior also indicated that accuracy in the main attention task decreased as a function of engagement with the distractor task. These results implicate the M1 receptor in divided attention., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: This study aimed to investigate the combined effects of dose, age, sex, body weight, and smoking on plasma concentrations of olanzapine (OLA) and N-desmethyl olanzapine (DMO) in Chinese inpatients with schizophrenia., Methods: A retrospective study including 185 inpatients was conducted. The steady-state plasma concentrations of OLA (COLA) and DMO (CDMO) were measured using high-performance liquid chromatography-tandem mass spectrometry. The combined effects of dose, age, sex, body weight, and smoking on COLA and CDMO were evaluated., Findings: Multiple linear regression analyses revealed that dose, age, body weight, and smoking had significant effects on COLA and CDMO in inpatients with schizophrenia treated with OLA. The dose was the most important determinant of COLA and CDMO and was positively correlated with both. Furthermore, smokers exhibited a significantly lower COLA and COLA + DMO, whereas higher body weight led to the reduction of COLA, CDMO, and COLA + DMO. Advanced age was associated with lower CDMO., Implications: These results suggest that dose, age, body weight, and smoking have a significant influence on the plasma concentration of OLA and its metabolite DMO. Clinicians should consider the combined effects when prescribing OLA to patients with schizophrenia., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Hair analysis is a useful tool for establishing long-term drug intake. Segmental analysis, in particular, where the hair is cut into defined segments, can potentially provide a calendar of patients' drug intake as drugs are incorporated into the growing hair through the bloodstream with an average growth rate of 1 cm per month. Forensic investigations of hair require knowledge of typical concentrations of common pharmaceuticals in hair, which are rarely reported. The aim of this study was to provide values for olanzapine and N-desmethyl-olanzapine concentrations in postmortem hair from chronic olanzapine consumers to contribute to the establishment of a reference interval for this drug. We analyzed postmortem head hair samples from 37 suspected mentally ill patients, who were part of the SURVIVE population, a Danish national autopsy-based study. Each sample was cut into 1 cm segments, and up to six segments, corresponding to up to six months of hair growth prior to death, were analyzed depending on the hair length. The hair extracts were analyzed by liquid chromatography tandem mass spectrometry. Olanzapine and N-desmethyl-olanzapine were added to a published and validated method. The 37 patients were 12 females and 25 males aged 25-81 years. Their hair colors varied from blond to black, with the majority brown, thus no trend could be discerned from the hair colors. Drugs other than olanzapine were found in all cases except one, and illicit drugs were found in the hair samples of 38 % of the cases. We report olanzapine concentrations ranging from 0.005-20.9 ng/mg (median 0.128 ng/mg) and N-desmethyl-olanzapine concentrations from 0.027 to 0.187 ng/mg (median 0.068 ng/mg) for all 141 analyzed segments. Metabolite-to-drug ratios ranged from 0.010 to 3.31 (median 0.590). Dose calculations based on prescription pick-up demonstrated no correlation with the concentrations in hair, but olanzapine concentrations in the proximal hair segment correlated significantly with olanzapine concentrations in postmortem blood. Olanzapine concentrations decreased considerably from the proximal to distal segments, emphasizing the importance of reporting the length of the measured hair when reporting drug concentrations in hair. This study can contribute to the establishment of a reference interval for olanzapine and N-desmethyl-olanzapine concentrations in hair by reporting concentrations in hair from chronic consumers., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study analyzed the effect of inflammation on acetylcholine (ACh)-induced muscarinic receptors (MR)2 and MR3 conducted contractility of the porcine uterus. On Day 3 of the estrous cycle, either E.coli suspension (E.coli group) or saline (SAL group) was injected into uterine horns or laparotomy was performed (CON group). Eight days later, infected gilts developed severe acute endometritis. Compared to the period before ACh treatment, ACh (10 -5  M) increased the tension in myometrium (MYO) and endometrium/myometrium (ENDO/MYO) of the CON group (P < 0.01) and in ENDO/MYO of the SAL group (P < 0.01), the amplitude in strips of the CON (P < 0.05) and SAL (MYO: P < 0.05, ENDO/MYO: P < 0.001) groups and the frequency in strips of the CON (MYO: P < 0.01, ENDO/MYO: P < 0.001) and SAL (P < 0.01) groups. In the E.coli group, ACh (10 -5  M) reduced the amplitude in MYO (P < 0.05) and ENDO/MYO (P < 0.001), increased the frequency in MYO (P < 0.01) and ENDO/MYO (P < 0.001) and did not change (P > 0.05) the tension. ACh (10 -5  M) in ENDO/MYO of the E.coli group, reduced the tension compared to the CON group (P < 0.05) and the amplitude compared to other groups (P < 0.001), while increased the frequency in relation to the SAL group (P < 0.05). MR2 antagonist (AF-DX 44 116) and ACh (10 -5  M) reduced (by 16.92%, P < 0.01) the tension in MYO of the CON group and increased (P < 0.01) it in the E.coli group compared to the period before antagonist and ACh addition. In MYO of the SAL group, the tension was increased (P < 0.01) in response to MR3 antagonist (4-DAMP) and ACh (10 -7 , 10 -6  M). In the E.coli group, these substances did not change (P > 0.05) the tension, but it was lower (P < 0.001) in MYO (ACh: 10 -7  M) and ENDO/MYO (ACh: 10 -5  M) than in the SAL group. MR2 or MR3 antagonists and ACh (10 -5  M) increased (P < 0.05-0.001) the amplitude in strips of the CON and SAL groups and reduced it in the E.coli group (P < 0.001) compared to the period before antagonists and ACh use. This parameter in the E.coli group was lower (P < 0.001) after using MR2 or MR3 antagonists and ACh (10 -6 , 10 -5  M) than in other groups. Both antagonists and ACh (10 -5  M) reduced the frequency in the CON, SAL (P < 0.05) and E.coli (MR2 antagonist: P < 0.01, MR3 antagonist: P < 0.05) groups compared to period before antagonists and ACh addition. Data show that ACh reduces the contractility of the inflamed porcine uterus by MR2 and MR3, which suggests that pharmacological modulation of these receptors can be used to raise the contractility of an inflamed uterus., (Copyright © 2019. Published by Elsevier Inc.)

Aim: Food intake regulated by a complex of physiologic mechanisms in the nervous system. Muscarinergic system has an important role in the central regulation of appetite in mammals, but there is no information for Muscarinic receptors in avian. The purpose of this study was to examine the effects of intracerebroventricular injection of carbachol (cholinergic agonist), Telenzepine (M 1 receptor antagonist), AF-DX116 (M 2 receptor antagonist), 4-DAMP (M 3 receptor antagonist), and PD102807 (M 4 receptor antagonist) on feeding behavior in 3-h food-deprived (FD 3 ) neonatal broiler chicken. Materials and Methods: In experiment 1, chicken intracerebroventricular injected with carbachol (125, 250, and 500 nmol). In experiment 2, birds intracerebroventricular injected with telenzepine (125, 250, and 500 nmol). In experiments 3-5, birds intracerebroventricular injected with AF-DX 116 (125, 250, and 500 nmol), 4-DAMP (125, 250, and 500 nmol), and PD102807 (125, 250, and 500 nmol), respectively. In experiment 6, broilers intracerebroventricular injected with carbacol (500 nmol), co-injection of telenzepine (125 nmol)+carbacol (500 nmol), and 4-DAMP (125 nmol)+carbacol (500 nmol). In experiment 7, injection procedure was carbacol (500 nmol), co-injection of AF-DX116 (125 nmol)+carbacol (500 nmol), and PD102807 (125 nmol)+carbacol (500 nmol). Then, food intake measured until 120 min after injection. Results: According to the data, carbachol (250 and 500 nmol) significantly decreased food intake in comparison with control group ( P  < 0.05). Intracerebroventricular injection of telenzepine (250 and 500 nmol) and 4-DAMP (250 and 500 nmol) significantly increased food intake ( P  < 0.05). In addition, carbacol-induced hypophagia was significantly attenuated by co-injection of telenzepine + carbacol ( P  < 0.05). Also, co-injection of 4-DAMP + carbacol decreased the effect of carbacol on food intake ( P  < 0.05). However, AF-DX116 and PD102807 had no effect on hypophagia induced by carbacol ( P  > 0.05). Conclusion: These results suggest, hypophagic effect of muscarinergic system is mediated via M 1 and M 3 receptors in neonatal chicken.

Background: To compare pharmacologic effects of pirenzepine and AF-DX116, a selective competitive antagonist for M1 and M2 subtype muscarinic cholinergic receptors (mAChRs), respectively, with atropine, a non-selective competitive antagonist for mAChRs, on Lipopolysaccharide (LPS)., Methods: Male C57BL/6 mice were used to establish models of LPS-induced experimental endotoxemia. Mice were intraperitoneally injected 10 min prior to LPS injection with control (saline), atropine, pirenzepine and AF-DX116, respectively. Overall survival time was estimated using Kaplan-Meier plots. Inflammatory cytokine tumor necrosis factor-α (TNF-α) was monitored at various intervals after LPS injection and individual reagent administration. Pathological alternations in lungs and liver were analyzed., Results: Pirenzepine and atropine pretreatment improved survival rate of LPS-induced septic shock; in contrast, AF-DX116 accelerated death from sepsis. Moreover, TNF-α plasma level was decreased in response to pirenzepine or atropine, whereas increased in response to AF-DX116. Pirenzepine and atropine relieved whereas AF-DX116 accelerated LPS-induced pulmonary and hepatic injury. Pirenzepine reduced proportion of M1 subtype of macrophages, while AF-DX116 promoted polarization of macrophages to M1 subtype. Pirenzepine pretreatment reduced while AF-DX116 enhanced expression of SOCS3 at mRNA level., Conclusions: The administration of pirenzepine and atropine may have beneficial effects on septic shock., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Clinical use of the antipsychotic drug olanzapine (OLA) is associated with metabolic side effects to variable degrees. N-desmethyl-olanzapine (DMO) is one major metabolite of OLA, but its potential involvement in the metabolic responses remains unclear. Here we examined whether DMO can directly impact the metabolic, endocrinal and inflammatory parameters under conditions of metabolic disturbance. DMO administration (2 mg/kg, i.g.) to high-fat diet induced obesity mice for 4 weeks induced a remarkable loss of body weight and fat mass. DMO improved insulin resistance and energy expenditure in mice, but had no significant effects on dyslipidemia or hepatic steatosis. Moreover, DMO induced morphological changes in the white adipose tissue, accompanied by reduced interleukin-1β (IL-1β) production and increased UCP1 expression. These findings demonstrate that DMO is devoid of the metabolic side effects commonly observed for OLA during obesity, which suggests that the N-desmethyl metabolism may function to regulate the metabolic responses to OLA., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Human muscarinic receptor M 2 is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M 2 receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M 2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M 2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M 2 receptors compared to M 3 receptors leads to subtype selectivity of AF-DX 384.

Early non-motor symptoms such as mood disorders and cognitive deficits are increasingly recognised in Parkinson's disease (PD). They may precede the characteristic motor symptomatology caused by dopamine (DA) neuronal loss in the substantia nigra pars compacta (SNc). It is well known that striatal cholinergic interneurons (ChIs) are emerging as key regulators of PD motor symptom, however, their involvement in the cognitive and affective alterations occurring in the premotor phase of PD is poorly understood. We used optogenetic photoinhibition of striatal ChIs in mice with mild nigrostriatal 6-hydroxydopamine (6-OHDA) lesions and assessed their role in anxiety-like behaviour in the elevated plus maze, social memory recognition of a congener and visuospatial object recognition. In transgenic mice specifically expressing halorhodopsin (eNpHR) in cholinergic neurons, striatal ChIs photoinhibition reduced the anxiety-like behaviour and reversed social and spatial short-term memory impairment induced by moderate DA depletion (e.g., 50% loss of tyrosine hydroxylase TH-positive neurons in the SNc). Systemic injection of telenzepine (0.3 mg/kg), a preferential M1 muscarinic cholinergic receptors antagonist, improved anxiety-like behaviour, social memory recognition but not spatial memory deficits. Our results suggest that dysfunction of the striatal cholinergic system may play a role in the short-term cognitive and emotional deficits of partially DA-depleted mice. Blocking cholinergic activity with M1 muscarinic receptor antagonists may represent a possible therapeutic target, although not exclusive, to modulate these early non-motor deficits., (© 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Protein kinase C (PKC) is a family of serine/threonine kinases related to several phenomena as cell proliferation, differentiation and survival. Our previous data demonstrated that treatment of axotomized neonatal rat retinal cell cultures for 48 h with phorbol 12-myristate 13-acetate (PMA), a PKC activator, increases retinal ganglion cells (RGCs) survival. Moreover, this treatment decreases M1 receptors (M1R) and modulates BDNF levels. The aim of this work was to assess the possible involvement of neurotrophins BDNF and NGF in the modulation of M1R levels induced by PKC activation, and its involvement on RGCs survival. Our results show that PMA (50 ng/mL) treatment, via PKC delta activation, modulates NGF, BDNF and M1R levels. BDNF and NGF mediate the decrease of M1R levels induced by PMA treatment. M1R activation is essential to PMA neuroprotective effect on RGCs as telenzepine (M1R selective antagonist) abolished it. Based on our results we suggest that PKC delta activation modulates neurotrophins levels by a signaling pathway that involves M1R activation and ultimately leading to an increase in RGCs survival in vitro., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The mitogen-activated protein kinase (MAPK), especially its extracellular signal-regulated kinase (ERK) subfamily, is a group of kinases enriched in the mammalian brain. While ERK is central to cell signaling and neural activities, the regulation of ERK by transmitters is poorly understood. In this study, the role of acetylcholine in the regulation of ERK was investigated in adult rat striatum in vivo. We focused on muscarinic M1 and M4 receptors, two principal muscarinic acetylcholine (mACh) receptor subtypes in the striatum. A systemic injection of the M1-preferring antagonist telenzepine did not alter ERK phosphorylation in the two subdivisions of the striatum, the caudate putamen and nucleus accumbens. Similarly, telenzepine did not affect ERK phosphorylation in the medial prefrontal cortex (mPFC), hippocampus, and cerebellum. Moreover, telenzepine had no effect on the ERK phosphorylation induced by dopamine stimulation with the psychostimulant amphetamine. In contrast to telenzepine, the M4-preferring antagonist tropicamide consistently increased ERK phosphorylation in the striatum and mPFC. This increase was rapid and transient. Tropicamide and amphetamine when coadministered at subthreshold doses induced a significant increase in ERK phosphorylation. These results demonstrate that mACh receptors exert a subtype-specific modulation of ERK in striatal and mPFC neurons. While the M1 receptor antagonist has no effect on ERK phosphorylation, M4 receptors inhibit constitutive and dopamine-stimulated ERK phosphorylation in these dopamine-innervated brain regions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aims: Rho/Rho-kinase (ROCK) signaling has extensively been shown to take part in mammalian smooth muscle contractions in response to diverse agents yet its role in the contraction of amphibian smooth muscle has not been investigated. Therefore, we aimed to explore any role of this pathway in the contractions of frog stomach smooth., Main Methods: The strips were prepared and suspended in organ baths filled with Ringer solution. Changes in the circular strips of the frog stomach muscle length were recorded isotonically with a force transducer in organ baths., Key Findings: Carbachol (CCh) exerted both phasic and tonic contractions. In contrast, atropin abolished all types of contractions by CCh. The phasic contractions were suppressed by a Ca 2+ channel blocker, nifedipine but not by the ROCK inhibitor, Y-27632. However, the tonic contractions were markedly attenuated by Y-27632. Selective M 1 receptor blocker, pirenzepin, selective M 3 receptor blocker and DAMP had no effects on CCh-elicited contractions. On the other hand, selective M 2 receptor blocker, AF-DX suppressed all types of contractile activity by CCh., Significance: These data suggest that M 2 receptor activation could mainly mediate CCh-induced phasic and tonic contractions, and ROCK seems to be involved in the CCh-induced tonic but not phasic contractions of the frog stomach smooth muscle., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The effect of sarin on the binding parameters (KD & Bmax) of M2 muscarinic acetylcholine receptor (mAChR) was studied 24h and 1 week post exposure. Male & female Sprague-Daweley rats were poisoned with 1XLD50 sarin (80μg/kg, im) followed by treatment of trimedoxime bromide and atropine (7.5:5mg/kg, im) 1min later. Brains were removed and analyzed for M2 mAChR binding, using [ 3 H]AFDX384, an M2 selective antagonist. A significant increase in KD of M2 mAChR was found in the cortex 24h post poisoning, displaying elevation from 4.65±1.16 to 8.45±1.06nM and 5.24±0.93 to 9.29±1.56nM in male and female rats, respectively. A rise in KD was also noted 1 week following exposure from 5.04±1.20 to 11.75±2.78 and from 5.37±1.02 to 11.66±1.73nM, presenting an added increase of 51 and 40% (compared to 24h) in males and females, respectively. Analysis of M2 receptor density (Bmax) revealed a significant reduction of 68% in males and insignificant reduction of 22% in females, 24h after sarin exposure which was followed by 37% recovery in males and 100% recovery in females, 1 week later. These results indicate that sarin induces a long-term decreased affinity in M2 mAChR (elevated KDs) and a transient effect on the number of this receptor subtype (Bmax). We hypothesize that the reduced affinity of the M2 receptors (negative auto-regulatory receptors) may cause long-term brain deficits by impairing the normal regulation release of ACh into the synaptic cleft., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Metabolic disturbance is a common side effect of olanzapine (OLZ); however, the relationships between plasma OLZ concentration (C OLZ ) and metabolic disturbance remain unclear. Our previous study revealed that C OLZ ≧22.77ng/mL was a positive predictor of therapeutic efficacy in patients with schizophrenia. This study aimed to investigate the roles of OLZ or N-desmethyl-olanzapine (DMO) in metabolic outcomes among OLZ-treated patients with schizophrenia. The metabolic syndrome (MS) was diagnosed based on the modified the National Cholesterol Education Program Adult Treatment Panel III criteria for Asians. HPLC-ECD analytical system was applied to determine the C OLZ and DMO concentration (C DMO ). The absolute drug levels and concentration-to-dose ratios (C/D ratios) were tested for their correlations to metabolic parameters. Total 151 fasting blood samples from patients with schizophrenia were collected. DMO C/D ratio negatively correlated with weight, body mass index, waist circumference, and C-peptide level. The receiver operator characteristic analysis determined a threshold C DMO >5.63ng/mL and DMO C/D ratio>0.35ng/mL/mg were negative predictors of MS. The C OLZ /C DMO ratio>6.03 was identified as positive predictor of MS. Combined with previous study result, we proposed that the optimal OLZ treatment should maintain C OLZ /C DMO ratio between 3 and 6 to maximize the clinical efficacy and minimize the metabolic side effects. Our findings suggested that therapeutic drug monitoring on OLZ and DMO is a valuable tool to monitor metabolic side effects in OLZ-treated patients with schizophrenia., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Parkinson's disease (PD) is a devastating disorder, affecting approximately 2% of people aged 60 and above. It is marked by progressive neurodegeneration that has long been known to impact dopaminergic cells and circuits, but more recently the acetylcholine system has also been implicated in the complex aetiology and symptomatology of the disease. While broad changes in cholinergic markers have been described, insight into the contribution of specific acetylcholine receptors is less clear. To address this important unknown, in this study we performed [ 3 H] pirenzepine, [ 3 H] 4DAMP, and [ 3 H] AF-DX 384 in situ radioligand binding on postmortem tissues from Brodmann's area 6, 9, 46, and the caudate putamen, from PD and matched controls to detect muscarinic M1, M3, and M1/2/4 receptors, respectively. We found no difference in [ 3 H] pirenzepine binding between PD and controls across all regions assessed. [ 3 H] 4DAMP binding was found to be higher in PD CPu and BA9 than in controls. [ 3 H] AF-DX 384 was higher in BA9 of PD compared with controls. In sum, we show selective increase in M3 receptors in cortical and subcortical regions, as well as increased M2/M4 in cortical area BA9, which together support a role for cholinergic dysfunction in PD.

Understanding how episodic memories are formed and retrieved is necessary if we are to treat disorders in which they malfunction. Muscarinic acetylcholine receptors (mAChR) in the hippocampus and cortex underlie memory formation, but there is conflicting evidence regarding their role in memory retrieval. Additionally, there is no consensus on which mAChR subtypes are critical for memory processing. Using pharmacological and genetic approaches, we found that (1) encoding and retrieval of contextual memory requires mAChR in the dorsal hippocampus (DH) and retrosplenial cortex (RSC), (2) memory formation requires hippocampal M 3 and cooperative activity of RSC M 1 and M 3, and (3) memory retrieval is more impaired by inactivation of multiple M 1 -M 4 mAChR in DH or RSC than inactivation of individual receptor subtypes. Contrary to the view that acetylcholine supports learning but is detrimental to memory retrieval, we found that coactivation of multiple mAChR is required for retrieval of both recently and remotely acquired context memories. Manipulations with higher receptor specificity were generally less potent than manipulations targeting multiple receptor subtypes, suggesting that mAChR act in synergy to regulate memory processes. These findings provide unique insight into the development of therapies for amnestic symptoms, suggesting that broadly acting, rather than receptor-specific, mAchR agonists and positive allosteric modulators may be the most effective therapeutic approach., (© 2016 Leaderbrand et al.; Published by Cold Spring Harbor Laboratory Press.)

Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands are unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by using spatial intensity distribution analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules·μm(-2) human muscarinic M1 receptor identified a ∼75:25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked up-regulation of the receptor, simple mass action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population, but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Increasingly, data are implicating muscarinic receptors in the aetiology and treatment of mood disorders. This led us to measure levels of different muscarinic receptor-related parameters in the cortex from people with mood disorders and the CNS of rats treated with mood stabilisers and antidepressant drugs., Methods: We measured [(3)H]AF-DX 384 binding in BA 46 and BA 24 from subjects with bipolar disorders (n = 14), major depressive disorders (n = 19), as well as age- and sex-matched controls (n = 19) and the CNS of rats treated with fluoxetine or imipramine. In addition, we used Western blots to measure levels of CHRM2 protein and oxotremorine-M stimulated [(35)S]GTPγS binding as a measure of CHRM 2 / 4 signaling., Results: Compared with controls, [(3)H]AF-DX 384 binding was lower in BA 24 and BA 46 in bipolar disorders and major depressive disorders, while CHRM2 protein and oxotremorine-M stimulated [(35)S]GTPγS binding was only lower in BA 24. Compared with vehicle, treatment with mood stabilisers, antidepressant drugs for 10 days, or imipramine for 28 days resulted in higher levels of in [(3)H]AF-DX 384 binding select regions of rat CNS., Conclusions: Our data suggest that levels of CHRM2 are lower in BA 24 from subjects with mood disorders, and it is possible that signalling by that receptor is also less in this cortical region. Our data also suggest increasing levels of CHRM2 may be involved in the mechanisms of action of mood stabilisers and tricyclic antidepressants., (© The Author 2015. Published by Oxford University Press on behalf of CINP.)

Neurons in the vestibular nuclei have a vital function in balance maintenance, gaze stabilization, and posture. Although muscarinic acetylcholine receptors (mAChRs) are expressed and involved in regulating vestibular function, it remains unclear how individual mAChR subtypes regulate vestibular neuronal activity. In this study, we determined which specific subtypes of mAChRs control synaptic input and excitability of medial vestibular nucleus (MVN) neurons that project to the cerebellum. Cerebellum-projecting MVN neurons were labeled by a fluorescent retrograde tracer and then identified in rat brainstem slices. Quantitative PCR analysis suggested that M2 and M3 were the possible major mAChR subtypes expressed in the MVN. The mAChR agonist oxotremorine-M significantly reduced the amplitude of glutamatergic excitatory post-synaptic currents evoked by stimulation of vestibular primary afferents, and this effect was abolished by the M2-preferring antagonist AF-DX 116. However, oxotremorine-M had no effect on GABA-mediated spontaneous inhibitory post-synaptic currents of labeled MVN neurons. Furthermore, oxotremorine-M significantly increased the firing activity of labeled MVN neurons, and this effect was blocked by the M3-preferring antagonist J104129 in most neurons tested. In addition, AF-DX 116 reduced the onset latency and prolonged the excitatory effect of oxotremorine-M on the firing activity of labeled MVN neurons. Our findings suggest that M3 is the predominant post-synaptic mAChR involved in muscarinic excitation of cerebellum-projecting MVN neurons. Pre-synaptic M2 mAChR regulates excitatory glutamatergic input from vestibular primary afferents, which in turn influences the excitability of cerebellum-projecting MVN neurons. This new information has important therapeutic implications for treating vestibular disorders with mAChR subtype-selective agents. Medial vestibular nucleus (MVN) neurons projecting to the cerebellum are involved in balance control. We found that activation of pre-synaptic M2 muscarinic receptors inhibit glutamatergic input from vestibular primary afferents, whereas stimulation of post-synaptic M3 muscarinic receptors increases the firing activity of cerebellum-projecting MVN neurons. This new information advances our understanding of the cholinergic mechanism regulating the vestibular system., (© 2016 International Society for Neurochemistry.)

Background: This therapeutic drug monitoring (TDM) study aimed to determine the role of olanzapine (OLZ) and N-desmethyl-OLZ (DMO) levels in the therapeutic efficacy of OLZ in patients with schizophrenia., Method: Plasma concentrations of OLZ (COLZ) and DMO (CDMO) in schizophrenic patients 12 hours post-dose were assessed. The correlations of COLZ and CDMO with the various scores of the Positive and Negative Syndrome Scale (PANSS) were evaluated. A receiver operating characteristic curve (ROC) was utilized to identify the threshold COLZ and COLZ/CDMO ratio for maintenance of satisfactory efficacy., Results: A total of 151 samples from patients with schizophrenia were analyzed for individual COLZ and CDMO levels. The mean COLZ and CDMO levels were 37.0 ± 25.6 and 6.9 ± 4.7 ng/mL, respectively, and COLZ was ~50% higher in female or nonsmokers (p<0.01). In all patients, the daily dose of OLZ was positively correlated with COLZ and CDMO. Linear relationships between COLZ and OLZ dose were observed in both nonsmokers and smokers (rs = 0.306, 0.426, p<0.01), although CDMO was only correlated with OLZ dose in smokers (rs = 0.485, p<0.01) and not nonsmokers. In all patients, COLZ was marginally negatively correlated with the total PANSS score. The total PANSS score was significantly negatively correlated with the COLZ/CDMO ratio (p<0.005), except in smokers. The ROC analysis identified a COLZ/CDMO ratio ≥2.99 or COLZ ≥22.77 ng/mL as a predictor of maintenance of an at least mildly ill status (PANSS score ≤58) of schizophrenia in all patients., Conclusions: A significantly negative correlation between the steady-state COLZ/CDMO ratio and total PANSS score was observed in Taiwanese schizophrenic patients. TDM of both OLZ and DMO levels could assist clinical practice when individualizing OLZ dosage adjustments for patients with schizophrenia.

Rationale: The acetylcholinesterase inhibitors donepezil, galantamine, and rivastigmine are used for the treatment of Alzheimer's disease. We previously demonstrated that donepezil and galantamine differentially affect isolation rearing-induced prepulse inhibition (PPI) deficits and that this might be due to differential effects on brain muscarinic acetylcholine (mACh) receptor function in mice., Objectives: We examined the effects of rivastigmine on isolation rearing-induced PPI deficits, brain ACh levels, and mACh receptor function in mice., Methods: Acoustic startle responses were measured in a startle chamber. Microdialysis was performed, and the levels of dopamine and ACh in the prefrontal cortex were measured., Results: Rivastigmine (0.3 mg/kg) improved PPI deficits, and this improvement was antagonized by the mACh receptor antagonist telenzepine but not by the nicotinic ACh receptor antagonist mecamylamine. Rivastigmine increased extracellular ACh levels by approximately 2-3-fold, less than the increase produced by galantamine. Rivastigmine enhanced the effect of the mACh receptor agonist N-desmethylclozapine on prefrontal dopamine release, a marker of mACh receptor function, and this increase was blocked by telenzepine. In contrast, galantamine did not affect N-desmethylclozapine-induced dopamine release. Furthermore, rivastigmine did not affect cortical dopamine release induced by the serotonin1A receptor agonist osemozotan, suggesting that the effect of rivastigmine has specificity for mACh receptors., Conclusions: Taken together with our previous finding that marked increases in ACh levels are required for the PPI deficit improvement induced by galantamine, our present results suggest that rivastigmine improves isolation rearing-induced PPI deficits by increasing ACh levels and by concomitantly enhancing mACh receptor function.

Background/objectives: Having separated a sub-group of people with schizophrenia based on a marked loss of cortical [(3)H]pirenzepine binding (MRDS); we wished to determine if MRDS had lower levels of [(3)H]pirenzepine and other muscarinic receptor antagonist binding to the striatum and if this was due to loss of pre- or post-synaptic neurons or glia measured using surrogate markers (25 kilodalton synaptosomal-associated protein (SNAP 25), postsynaptic density protein 95 (PSD 95), glial fibrillary acidic protein (GFAP) 41/43) of cell number., Methods: [(3)H]pirenzepine, [(3)H]AF-DX 384 and [(3)H]4-DAMP binding to the striatum from 37 subjects with schizophrenia (19 MRDS) and 20 controls as well as SNAP 25, PSD 95 and GFAP 41/43 in crude particulate membrane were measured., Results: [(3)H]pirenzepine and [(3)H]AF-DX 384 binding to the striatum were significantly lower in schizophrenia due to lower binding of both radioligands in the striatum from MRDS. Levels of PSD 95 were higher in schizophrenia, predominantly due to higher levels in MRDS., Conclusions: Our data suggest muscarinic M1 ([(3)H]pirenzepine) and M2 and/or M4 receptors ([(3)H]AF-DX 384) are lower in the striatum from MRDS which could mediate inappropriate adaption to internal and external cues which, in turn, would affect motivation, cognition and motor control. Increased levels of PSD 95 could indicate increased post-synaptic boutons or changes in NMDA receptor-mediated signalling in MRDS., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A simple, sensitive, and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of olanzapine (OLZ) and its metabolite N-desmethylolanzapine (DMO) in human plasma for therapeutic drug monitoring. Sample preparation was performed by one-step protein precipitation with methanol. The analytes were chromatographed on a reversed-phase YMC-ODS-AQ C18 Column (2.0 × 100 mm,3 µm) by a gradient program at a flow rate of 0.30 mL/min. Quantification was performed on a triple quadrupole tandem mass spectrometer via electrospray ionization in positive ion mode. The method was validated for selectivity, linearity, accuracy, precision, matrix effect, recovery and stability. The calibration curve was linear over the concentration range 0.2-120 ng/mL for OLZ and 0.5-50 ng/mL for DMO. Intra- and interday precisions for OLZ and DMO were <11.29%, and the accuracy ranged from 95.23 to 113.16%. The developed method was subsequently applied to therapeutic drug monitoring for psychiatric patients receiving therapy of OLZ tablets. The method seems to be suitable for therapeutic drug monitoring of patients undergoing therapy with OLZ and might contribute to prediction of the risk of adverse reactions., (Copyright © 2014 John Wiley & Sons, Ltd.)

The hippocampal formation receives strong cholinergic input from the septal/diagonal band complex. Although the functional effects of cholinergic activation have been extensively studied in pyramidal neurons within the hippocampus and entorhinal cortex, less is known about the role of cholinergic receptors on dentate gyrus neurons. Using intracellular recordings from rat dentate hilar neurons, we find that activation of m1-type muscarinic receptors selectively increases the excitability of glutamatergic mossy cells but not of hilar interneurons. Following brief stimuli, cholinergic modulation reveals a latent afterdepolarization response in mossy cells that can extend the duration of stimulus-evoked depolarization by >100 msec. Depolarizing stimuli also could trigger persistent firing in mossy cells exposed to carbachol or an m1 receptor agonist. Evoked IPSPs attenuated the ADP response in mossy cells. The functional effect of IPSPs was amplified during ADP responses triggered in the presence of cholinergic receptor agonists but not during slowly decaying simulated ADPs, suggesting that modulation of ADP responses by IPSPs arises from destabilization of the intrinsic currents underlying the ADP. Evoked IPSPs also could halt persistent firing triggered by depolarizing stimuli. These results show that through intrinsic properties modulated by muscarinic receptors, mossy cells can prolong depolarizing responses to excitatory input and extend the time window where multiple synaptic inputs can summate. By actively regulating the intrinsic response to synaptic input, inhibitory synaptic input can dynamically control the integration window that enables detection of coincident inputs and shape the spatial pattern of hilar cell activity.

Myopia is a huge public health problem worldwide, reaching the highest incidence in Asia. Identification of susceptible genes is crucial for understanding the biological basis of myopia. In this paper, we have identified and characterized a functional myopia-associated gene using a specific mouse-knockout model. Mice lacking the muscarinic cholinergic receptor gene (M2; also known as Chrm2) were less susceptible to lens-induced myopia compared with wild-type mice, which showed significantly increased axial length and vitreous chamber depth when undergoing experimental induction of myopia. The key findings of this present study are that the sclera of M2 mutant mice has higher expression of collagen type I and lower expression of collagen type V than do wild-type mice and mice that are mutant for other muscarinic subtypes, and, therefore, M2 mutant mice were resistant to the development of experimental myopia. Pharmacological blockade of M2 muscarinic receptor proteins retarded myopia progression in the mouse. These results suggest for the first time a role of M2 in growth-related changes in extracellular matrix genes during myopia development in a mammalian model. M2 receptor antagonists might thus provide a targeted therapeutic approach to the management of this refractive error.

While acetylcholine (ACh) and muscarinic receptors in the bladder are mainly known for their role in the regulation of smooth muscle contractility, in other tissues they are involved in tissue remodelling and promote cell growth and proliferation. In the present study we have used primary cultures of human detrusor smooth muscle cells (HDSMCs), in order to investigate the role of muscarinic receptors in HDSMC proliferation. Samples were obtained as discarded tissue from men >65 years undergoing radical cystectomy for bladder cancer and cut in pieces that were either immediately frozen or placed in culture medium for the cell culture establishment. HDSMCs were isolated from samples, propagated and maintained in culture. [(3)H]-QNB radioligand binding on biopsies revealed the presence of muscarinic receptors, with a Kd of 0.10±0.02nM and a Bmax of 72.8±0.1fmol/mg protein. The relative expression of muscarinic receptor subtypes, based on Q-RT-PCR, was similar in biopsies and HDSMC with a rank order of M2≥M3>M1>M4>M5. The cholinergic agonist carbachol (CCh, 1-100μM) concentration-dependently increased [(3)H]-thymidine incorporation (up to 46±4%). This was concentration-dependently inhibited by the general muscarinic receptor antagonist atropine and by subtype-preferring antagonists with an order of potency of darifenacin >4-DAMP>AF-DX 116. The CCh-induced cell proliferation was blocked by selective PI-3 kinase and ERK activation inhibitors, strongly suggesting that these intracellular pathways mediate, at least in part, the muscarinic receptor-mediated cell proliferation. This work shows that M2 and M3 receptors can mediate not only HDSM contraction but also proliferation; they may also contribute bladder remodelling including detrusor hypertrophy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bitopic binding properties apply to a variety of muscarinic compounds that span and simultaneously bind to both the orthosteric and allosteric receptor sites. We provide evidence that fluorescent pirenzepine derivatives, with the M1 antagonist fused to the boron-dipyrromethene [Bodipy (558/568)] fluorophore via spacers of varying lengths, exhibit orthosteric/allosteric binding properties at muscarinic M1 receptors. This behavior was inferred from a combination of functional, radioligand, and fluorescence resonance energy transfer binding experiments performed under equilibrium and kinetic conditions on enhanced green fluorescent protein-fused M1 receptors. Although displaying a common orthosteric component, the fluorescent compounds inherit bitopic properties from a linker-guided positioning of their Bodipy moiety within the M1 allosteric vestibule. Depending on linker length, the fluorophore is allowed to reach neighboring allosteric domains, overlapping or not with the classic gallamine site, but distinct from the allosteric indolocarbazole "WIN" site. Site-directed mutagenesis, as well as molecular modeling and ligand docking studies based on recently solved muscarinic receptor structures, further support the definition of two groups of Bodipy-pirenzepine derivatives exhibiting distinct allosteric binding poses. Thus, the linker may dictate pharmacological outcomes for bitopic molecules that are hardly predictable from the properties of individual orthosteric and allosteric building blocks. Our findings also demonstrate that the fusion of a fluorophore to an orthosteric ligand is not neutral, as it may confer, unless carefully controlled, unexpected properties to the resultant fluorescent tracer. Altogether, this study illustrates the importance of a "multifacet" experimental approach to unravel and validate bitopic ligand binding mechanisms.

Our knowledge about the mechanisms of human motor cortex facilitation induced by repetitive transcranial magnetic stimulation (rTMS) is still incomplete. Here we used pharmacological conditioning with carbamazepine, dextrometorphan, lorazepam, and placebo to elucidate the type of plasticity underlying this facilitation, and to probe if mechanisms reminiscent of long-term potentiation are involved. Over the primary motor cortex of 10 healthy subjects, we applied biphasic rTMS pulses of effective posterior current direction in the brain. We used six blocks of 200 pulses at 5-Hz frequency and 90% active motor threshold intensity and controlled for corticospinal excitability changes using motor-evoked potential (MEP) amplitudes and latencies elicited by suprathreshold pulses before, in between, and after rTMS. Target muscle was the dominant abductor digiti minimi muscle; we coregistered the dominant extensor carpi radialis muscle. We found a lasting facilitation induced by this type of rTMS. The GABAergic medication lorazepam and to a lesser extent the ion channel blocker carbamazepine reduced the MEP facilitation after biphasic effective posteriorly oriented rTMS, whereas the N-methyl-d-aspartate receptor-antagonist dextrometorphan had no effect. Our main conclusion is that the mechanism of the facilitation induced by biphasic effective posterior rTMS is more likely posttetanic potentiation than long-term potentiation. Additional findings were prolonged MEP latency under carbamazepine, consistent with sodium channel blockade, and larger MEP amplitudes from extensor carpi radialis under lorazepam, suggesting GABAergic involvement in the center-surround balance of excitability.

Background: Olanzapine (OLZ) is one of the most prescribed atypical antipsychotic drugs but its use is associated with unfavorable metabolic abnormalities. N-desmethyl-olanzapine (DMO), one of the OLZ metabolites by CYP1A2, has been reported to have a normalizing action on metabolic abnormalities, but this remains unclear. Our aim was to explore the correlation between the concentrations of OLZ or DMO with various metabolic parameters in schizophrenic patients., Methods: The chromatographic analysis was carried out with a solvent delivery system coupled to a Coulochem III coulometric detector to determine OLZ and DMO simultaneously in OLZ-treated patients. The correlation between the concentration of OLZ or DMO and the metabolic parameters was analyzed by the Spearman rank order correlation method (r s)., Principal Findings: The established analytical method met proper standards for accuracy and reliability and the lower limitation of quantification for each injection of DMO or OLZ was 0.02 ng. The method was successfully used for the analysis of samples from nonsmoking patients (n = 48) treated with OLZ in the dosage range of 5-20 mg per day. There was no correlation between OLZ concentrations and tested metabolic parameters. DMO concentrations were negatively correlated with glucose (r s = -0.45) and DMO concentrations normalized by doses were also negatively correlated with insulin levels (r s = -0.39); however, there was a marginally positive correlation between DMO and homocysteine levels (r s = +0.38)., Conclusions: The observed negative correlations between levels of DMO and glucose or insulin suggest a metabolic normalization role for DMO regardless of its positive correlation with a known cardiovascular risk factor, homocysteine. Additional studies of the mechanisms underlying DMO's metabolic effects are warranted.

Large airway bronchoconstriction acts mainly through cholinergic pathways via muscarinic M3 receptors with some contribution from M2 receptors. By contrast, the mechanisms of small airway contraction are largely unknown. This study used precision cut lung slices to examine the role of muscarinic M2 and M3 receptors in the contractile response of rat and human small airways. In rat small airways, the M3 preferential antagonist, 4-DAMP, inhibited carbachol-mediated contraction (1×10(-6) M) more than that of the M2 selective antagonist, AF-DX116 (pIC50 values: 8.85±0.18 and 6.31±0.19, n=6-8 respectively). Tiotropium, inhibited the contractile response to carbachol with (pIC50: 9.86±0.07, n=6), but could not distinguish between M2 and M3 mediated effects. Similar experiments using human small airways with tiotropium and AF-DX116, gave a pIC50 of 10.35±0.05 and a pKB of 6.37±0.13, n=5 respectively. Therefore, M3 receptors play a key role in muscarinic contraction of small airways in both rats and humans but the effect of M2 receptors cannot be excluded. To investigate the role of M2 receptors, carbachol-induced contraction of small airways was performed in the presence and absence of a β2-agonist in order to elevate intracellular cAMP levels prior to contraction. Isoproterenol-induced relaxation was significantly increased by AF-DX116 (P<0.001) in rat small airways and by AF-DX116 (P<0.01), gallamine (P<0.05) and pertussis toxin (P<0.05) in human small airways. Taken together, these data suggest that cholinergic antagonism of muscarinic receptors in human and rat small airways inhibits airway contraction via direct inhibition of contraction through M3 receptors, and by M2 receptor mediated inhibition of relaxation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Organophosphates are a class of highly toxic chemicals that includes many pesticides and chemical weapons. Exposure to organophosphates, either through accidents or acts of terrorism, poses a significant risk to human health and safety. Existing antidotes, in use for over 50 years, have modest efficacy and undesirable toxicities. Therefore, discovering new organophosphate antidotes is a high priority. Early life stage zebrafish exposed to organophosphates exhibit several phenotypes that parallel the human response to organophosphates, including behavioral deficits, paralysis, and eventual death. Here, we have developed a high-throughput zebrafish screen in a 96-well plate format to find new antidotes that counteract organophosphate-induced lethality. In a pilot screen of 1200 known drugs, we identified 16 compounds that suppress organophosphate toxicity in zebrafish. Several in vitro assays coupled with liquid chromatography/tandem mass spectrometry-based metabolite profiling enabled determination of mechanisms of action for several of the antidotes, including reversible acetylcholinesterase inhibition, cholinergic receptor antagonism, and inhibition of bioactivation. Therefore, the in vivo screen is capable of discovering organophosphate antidotes that intervene in distinct pathways. These findings suggest that zebrafish screens might be a broadly applicable approach for discovering compounds that counteract the toxic effects of accidental or malicious poisonous exposures.

In this study, the ability to modulate rheological and degradation properties of temperature-responsive gelling systems composed of aqueous blends of poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide) (PCLA-PEG-PCLA) triblock copolymers (i.e. uncapped) and their fully capped derivatives was investigated. Uncapped and capped PCLA-PEG-PCLA triblock copolymers, abbreviated as degree of modification 0 and 2 (DM0 and DM2, respectively), were composed of identical PCLA and PEG blocks but different end groups: namely hydroxyl and hexanoyl end groups. DM0 was synthesized by ring opening polymerization of l-lactide and ε-caprolactone in toluene using PEG as initiator and tin(II) 2-ethylhexanoate as the catalyst. A portion of DM0 was subsequently reacted with an excess of hexanoyl chloride in solution to yield DM2. The cloud point and phase behaviour of DM0 and DM2 in buffer as well as that of their blends were determined by light scattering in a diluted state and by vial tilting and rheological measurements in a concentrated state. Degradation/dissolution properties of temperature-responsive gelling systems were studied in vitro at pH 7.4 and 37°C. The cloud points of DM0/DM2 blends were ratio-dependent and could be tailored from 15 to 40°C for blends containing 15 to 100wt.% DM0. Vial tilting and rheological experiments showed that, with solid contents between 20 and 30wt.%, DM0/DM2 blends (15/85 to 25/75w/w) had a sol-to-gel transition temperature at 10-20°C, whereas blends with less than 15wt.% DM0 formed gels below 4°C and the ones with more than 25wt.% DM0 did not show a sol-to-gel transition up to 50°C. Complete degradation of temperature-responsive gelling systems took ∼100days, independent of the DM0 fraction and the initial solid content. Analysis of residual gels in time by GPC and (1)H-NMR showed no chemical polymer degradation, but indicated gel degradation by dissolution. Preferential dissolution of lactoyl-rich polymers induced enrichment of the residual gels in caproyl-rich polymers. To the best of our knowledge, degradation of temperature-responsive gelling systems by dissolution has not been reported or hypothesized as being the consequence of acylation of polymers. In conclusion, blending of PCLA-PEG-PCLA triblock polymers composed of identical backbones but different end groups provides for a straightforward preparation of temperature-responsive gelling systems with well-characterized rheological properties and potential in drug delivery. Furthermore, acylation of triblock copolymers may allow for the design of bioerodible systems with control over degradation by polymer dissolution., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Acetylcholine has been implicated in higher cortical functions such as learning, memory and cognition, yet the cellular effects of muscarinic acetylcholine receptor (mAChR) activation are poorly understood in the human cortex. Here we investigated the effect of the mAChR agonist carbachol (CCh) and various mAChR antagonists in human cortical slices (from tissue removed during neurosurgical treatment of epilepsy) by intracellular and extracellular recordings. CCh increased neuronal firing, which was antagonised by atropine (non-selective mAChR antagonist) and pirenzepine (M(1)/M(4) mAChRs antagonist) when applied before or after CCh application. AF-DX 116 (M(2)/M(4) mAChRs antagonist) had no effect on CCh-induced increase of firing. CCh also reduced evoked excitatory postsynaptic potentials (EPSP), and the CCh-induced depression of EPSP was fully reversed by atropine. Pirenzepine reversed the depression of CCh on EPSP, but failed to prevent the depression when applied before CCh. AF-DX 116 prevented the CCh-induced depression of evoked EPSP when applied before CCh. CCh also depressed GABAergic transmission and this effect was antagonised by AF-DX 116. Xanomeline (M(1)/M(4) mAChR agonist) increased neuronal firing and decreased EPSP, but had no effect on GABAergic transmission. Reduction (with linopirdine) and enhancement (with retigabine) of the M-current (mediated by K(V)7 channels), increased and decreased neuronal firing, respectively, but had marginal effects on the evoked EPSP. Our results indicate that three pharmacologically distinct mAChRs modulate neuronal firing, glutamatergic and GABAergic transmissions in the human epileptogenic neocortex. The data are discussed towards possible implications of altered mAChR signalling in hyperexcitability and cognitive functions in the human neocortex., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Acetylcholinesterase (AChE) rapidly hydrolyzes acetylcholine. At the neuromuscular junction, AChE is mainly anchored in the extracellular matrix by the collagen Q, whereas in the brain, AChE is tethered by the proline-rich membrane anchor (PRiMA). The AChE-deficient mice, in which AChE has been deleted from all tissues, have severe handicaps. Surprisingly, PRiMA KO mice in which AChE is mostly eliminated from the brain show very few deficits. We now report that most of the changes observed in the brain of AChE-deficient mice, and in particular the high levels of ambient extracellular acetylcholine and the massive decrease of muscarinic receptors, are also observed in the brain of PRiMA KO. However, the two groups of mutants differ in their responses to AChE inhibitors. Since PRiMA-KO mice and AChE-deficient mice have similar low AChE concentrations in the brain but differ in the AChE content of the peripheral nervous system, these results suggest that peripheral nervous system AChE is a major target of AChE inhibitors, and that its absence in AChE- deficient mice is the main cause of the slow development and vulnerability of these mice. At the level of the brain, the adaptation to the absence of AChE is nearly complete., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

The atrioventricular node (AVN) is a vital component of the pacemaker-conduction system of the heart, co-ordinating conduction of electrical excitation from cardiac atria to ventricles and acting as a secondary pacemaker. The electrical behaviour of the AVN is modulated by vagal activity via activation of muscarinic potassium current, IKACh. However, it is not yet known if this response exhibits 'fade' or desensitization in the AVN, as established for the heart's primary pacemaker--the sinoatrial node. In this study, acute activation of IKACh in rabbit single AVN cells was investigated using whole-cell patch clamp at 37 °C. 0.1-1 μM acetylcholine (ACh) rapidly activated a robust IKACh in AVN myocytes during a descending voltage-ramp protocol. This response was inhibited by tertiapin-Q (TQ; 300 nM) and by the M2 muscarinic ACh receptor antagonist AFDX-116 (1 μM). During sustained ACh exposure the elicited IKACh exhibited bi-exponential fade (τf of 2.0 s and τs 76.9 s at -120 mV; 1 μM ACh). 10 nM ET-1 elicited a current similar to IKACh, which faded with a mono-exponential time-course (τ of 52.6 s at -120 mV). When ET-1 was applied following ACh, the ET-1 activated response was greatly attenuated, demonstrating that ACh could desensitize the response to ET-1. For neither ACh nor ET-1 was the rate of current fade dependent upon the initial response magnitude, which is inconsistent with K+ flux mediated changes in electrochemical driving force as the underlying mechanism. Collectively, these findings demonstrate that TQ sensitive inwardly rectifying K+ current in cardiac AVN cells, elicited by M2 muscarinic receptor or ET-1 receptor activation, exhibits fade due to rapid desensitization., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: We investigated the role of muscarinic receptor subtypes in calcium sensitization and their contribution to rho-kinase (ROK) and protein kinase C (PKC) pathways in carbachol (CCh)-induced contraction of human detrusor smooth muscle (DSM)., Materials and Methods: α-toxin-permeabilized human DSM strips were prepared and mounted horizontally to record isometric force. The roles of M(2) and M(3) muscarinic receptors in Ca(2+) sensitization were studied using selective antagonists of M(2) (AF-DX116) and M(3) (4-DAMP) receptor subtypes. The effects of a selective inhibitor of ROK, Y-27632, and a selective inhibitor of PKC, bisindolylmaleimide I (GF-109203X), were also studied on contraction induced by 10 μM CCh with 100 μM guanosine triphosphate at a fixed 1 μM [Ca(2+)](i) after preincubation with 1 μM AF-DX116 or 1 μM 4-DAMP., Results: Carbachol-induced Ca(2+) sensitization was predominantly inhibited by 4-DAMP compared with AF-DX116. Four-DAMP equivalently inhibited the relaxation effect of 5 μM GF-109203X as well as that of 5 μM Y-27632 on CCh-induced Ca(2+) sensitization. AF-DX116 reduced the relaxation effect of Y-27632 to a greater degree than GF-109203X., Conclusion: The results of the present study have demonstrated the predominant role of M(3) receptor subtype in Ca(2+) sensitization and the relative contribution to ROK and PKC pathways. Our study also shows that the ROK pathway is dominant compared with the PKC pathway after M(2) receptor activation, which in turn is inferior, but not negligible, in producing Ca(2+) sensitization., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Introduction: The purpose of the study is to understand whether the cholinergic stimulation is important, not only in inducing contraction of the detrusor muscle, but also in modulating the proliferation of smooth muscle cells. These results could help to better understand the role of antimuscarinic drugs, which are currently used for the treatment of many urological diseases., Patients and Methods: Primary cultures were prepared from biopsies of human detrusor muscle of subjects >65 years. From the cell culture set-up for each patient, mRNA was extracted and both the gene expression and the influence of increasing passages on the expression of muscarinic receptor subtypes were evaluated by semi-quantitative and quantitative PCR (RT-PCR and Q-RT-PCR). The rate of cell proliferation induced by cholinergic drugs was assessed by the evaluation of the [3H]-thymidine incorporation., Results: The gene expression analysis demonstrated that the range of expression of muscarinic subtypes in human detrusor smooth muscle cells (HDSMCs) is M2 > M3 > M1 > M4 >> M5. The exposure to the cholinergic agonist carbachol induced a concentration-dependent increase in cell proliferation rate. The pharmacological characterization indicated that this effect was mainly mediated by the receptor subtypes M3 and M2., Discussion: The cholinergic stimulation led to an increase in HDSMC proliferation, suggesting that this phenomenon might be involved in the pathogenic mechanism through which the cervico-urethral obstruction causes a detrusor hypertrophy, followed by a loss of function. These results could then provide an indication of the use of antimuscarinic drugs in the treatment of lower urinary tract disorders.

Behavioral flexibility, the ability to modify responses due to changing task demands, is detrimentally affected by aging with a shift towards increased cognitive rigidity. The neurobiological basis of this cognitive deficit is not clear although striatal cholinergic neurotransmission has been implicated. To investigate the possible association between striatal acetylcholine signaling with age-related changes in behavioral flexibility, young, middle-aged, and aged F344 X Brown Norway F1 rats were assessed using an attentional set-shifting task that includes two tests of behavioral flexibility: reversal learning and an extra-dimensional shift. Rats were also assessed in the Morris water maze to compare potential fronto-striatal-dependent deficits with hippocampal-dependent deficits. Behaviorally characterized rats were then assessed for acetylcholine muscarinic signaling within the striatum using oxotremorine-M-stimulated [(35)S]GTPγS binding and [(3)H]AFDX-384 receptor binding autoradiography. The results showed that by old age, cognitive deficits were pronounced across cognitive domains, suggesting deterioration of both hippocampal and fronto-striatal regions. A significant decline in oxotremorine-M-stimulated [(35)S]GTPγS binding was limited to the dorsomedial striatum of aged rats when compared to young and middle-aged rats. There was no effect of age on striatal [(3)H]AFDX-384 receptor binding. These results suggest that a decrease in M2/M4 muscarinic receptor coupling is involved in the age-associated decline in behavioral flexibility., (Copyright © 2011 Elsevier B.V. All rights reserved.)

It has been previously shown that cross-tolerance to the discriminative stimulus properties of clozapine can be demonstrated with the drug discrimination paradigm. This study examined the ability of N-desmethylclozapine and N-desmethylolanzapine (metabolites of the atypical antipsychotic drugs clozapine and olanzapine, respectively) to induce cross-tolerance to the discriminative stimulus effects of clozapine. After C57BL/6 mice were trained to reliably discriminate 2.5 mg/kg clozapine from vehicle, a clozapine generalization curve was generated. Next, training was suspended and the mice received a maintenance dosing regimen in which they were injected twice daily with 10 mg/kg N-desmethylclozapine for 10 days. Then a second clozapine generalization curve was generated. This was followed by a 10-day washout period during which the mice did not receive drug injections or discrimination training. Finally, a third clozapine generalization curve was generated. These same procedures were followed for N-desmethylolanzapine (10 mg/kg twice daily during maintenance dosing). Both N-desmethylclozapine and N-desmethylolanzapine produced significant rightward shifts in the clozapine generalization curve indicating cross-tolerance between N-desmethylclozapine and clozapine and between N-desmethylolanzapine and clozapine. After a washout period with no training or drug administration this cross-tolerance effect was lost for both metabolites. This cross-tolerance drug discrimination procedure demonstrated in-vivo similarities between these two metabolites and clozapine and suggests that common underlying pharmacological mechanisms were responsible for the cross-tolerance that was observed. These findings also demonstrated that this procedure may be useful for identifying drugs with therapeutic efficacy similar to the atypical antipsychotic clozapine under repeated dosing conditions.

The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges.

Aim: To investigate the potential interaction between olanzapine, a CYP1A2 substrate, and ethinylestradiol-containing contraceptives (ECC)., Methods: The study was carried out at a routine therapeutic drug monitoring service. To identify patients who were co-administered ECC or other contraceptives, a questionnaire was sent to the physician who ordered serum monitoring of olanzapine for women aged 18-40 years during an 18 month period. The physicians were asked to provide information about contraceptive use and smoking habits. When questionnaires were returned by the physicians, the respective serum concentration data were included in the analysis. Patients were stratified into users of ECC, progestogen-based contraceptives (PBC) or no contraceptives. Dose-adjusted serum concentrations of olanzapine and the metabolite N-desmethyl olanzapine were compared between the subgroups., Results: A total of 149 patients were included in the study (10 ECC users and 10 PBC users). In users of ECC, we found no differences in serum concentrations of olanzapine, but significantly lower concentrations of the CYP1A2-mediated metabolite N-desmethyl olanzapine compared with users of PBC (P = 0.019) and non-contraceptive users (P = 0.012)., Conclusion: The present study confirms that ECC exhibit CYP1A2-inhibitory properties in terms of significantly lower exposure of N-desmethyl olanzapine. However, the inhibition does not provide clinically relevant changes in serum concentrations of olanzapine., (© 2011 The Authors. British Journal of Clinical Pharmacology © 2011 The British Pharmacological Society.)

Background and Purpose: We have previously reported that galantamine, a weak acetylcholinesterase inhibitor, improves prepulse inhibition (PPI) deficits in mice reared in social isolation. ACh receptors are involved in the underlying mechanism of PPI, but whether rearing in social isolation causes dysfunction of the cholinergic system is unknown. In this study, we examined the involvement of muscarinic receptors in the improvement of PPI deficits induced by galantamine, and whether the cholinergic system is altered in mice reared in isolation., Experimental Approach: Three-week-old male ddY mice were housed in isolated cages for 6 weeks before the initiation of experiments to create PPI deficits. Cholinergic functions were determined by measuring the behavioural and neurochemical responses to nicotinic and muscarinic receptor agonists., Key Results: The improvement by galantamine of social isolation-induced PPI deficits was blocked by scopolamine, a non-selective muscarinic antagonist, and telenzepine, a preferential M₁ receptor antagonist. Activation of M₁ receptors improved social isolation-induced PPI deficits. Social isolation did not affect choline acetyltransferase and acetylcholinesterase activities in the prefrontal cortex and hippocampus, but it reduced the locomotor-suppressive response to muscarinic agonist oxotremorine, but not to nicotine. The isolation also attenuated the M₁ receptor agonist N-desmethylclozapine-induced increase in prefrontal dopamine release., Conclusions and Implications: Galantamine improves PPI deficits of mice reared in social isolation via activation of M₁ receptors. Social isolation reduces the muscarinic, especially M₁, receptor function and this is involved in PPI deficits., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Gastrin-releasing peptide (GRP) has been implicated in the itch-scratch cycle. We investigated if this gut-brain-skin peptide plays a role in the compulsive, hindleg scratching of the neck of mice by 5'-guanidinonaltrindole (GNTI), the kappa opioid receptor antagonist, and in the antipruritic activity of nalfurafine, the kappa opioid agonist. Previously, we showed that GNTI (0.03-1mg/kg, s.c.) elicits dose-related scratching and that nalfurafine (0.001-0.02mg/kg, s.c.) inhibits this behavior in mice. Utilizing immunohistochemistry, GRP positive nerve fibers were detected in mouse skin and superficial layer of the dorsal horn of the spinal cord as well as GRP positive cells in the dorsal root ganglion. Pretreating mice with either a pseudopeptide GRP receptor antagonist, RC-3095 (10-30mg/kg, s.c. at -15min), or a peptide GRP receptor antagonist, [d-Phe(6)]bombesin(6-13) methyl ester (2-100nmol, i.t. at -10min), did not suppress GNTI-induced scratching. However, pretreating mice with either antagonist inhibited scratching precipitated by the GRP receptor agonist, GRP(18-27) (2nmol, i.t.). Pretreating mice with a muscarinic M(1) receptor agonist, McN-A-343 (1.5-15μg/5μl, i.t. at -10min) antagonized GNTI-induced scratching. Norbinaltorphimine (20mg/kg, i.p. at -18 to -20h), a kappa opioid antagonist, countered the antiscratch activity of nalfurafine. We conclude that (a) the GRP receptor system does not mediate GNTI-induced scratching and (b) the kappa opioid system is involved, at least in part, in the scratch suppressing activity of nalfurafine., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The study used the paw withdrawal test to investigate the role of the cholinergic system on the modulation of inflammatory pain induced by carrageenan (Cg) at the peripheral level, through activation of muscarinic and nicotinic receptors. Intraplantar administration of the specific M(1) receptor antagonist telenzepine (TEL; 6, 12 and 24 μg/paw) caused a dose-dependent reduction in the nociceptive threshold induced by Cg (125 μg/paw). This effect was not observed with increasing doses (4, 10 and 40 μg) of other specific receptor antagonists: M(2) (dimethindene), M(3) (4-DAMP) and M(4) (tropicamide). The nicotinic antagonist mecamylamine (MEC; 25, 50 and 100 μg/paw) also caused a dose-dependent reduction in the nociceptive threshold induced by Cg (125 μg). To exclude a non-local effect, Cg (125 μg) was injected into both hind paws, while TEL (12 μg) and MEC (50 μg) were administered only in the right paw. At these doses, the muscarinic antagonists increased inflammatory pain only in the treated right paw, suggesting a peripheral effect. In the presence of prostaglandin E(2) (1 μg/paw), TEL (12 μg) and MEC (50 μg) did not reduce the nociceptive threshold, suggesting that this hyperalgesic agent does not induce the release of endogenous acetylcholine. These data suggest that muscarinic M(1) and nicotinic receptors participate in the modulation of endogenous cholinergic inflammatory pain at the peripheral level., (Copyright © 2011 S. Karger AG, Basel.)

A validated, accurate and sensitive LC-MS/MS method for determination of olanzapine and its metabolite N-desmethylolanzapine has been developed. The analytes were quantified by tandem mass spectrometry operating in positive electrospray ionization mode with multiple reaction monitoring. Olanzapine and desmethylolanzapine were extracted from serum or cerebral spinal fluid samples, 200 microl, with tert-butyl methyl ether using olanzapine-D3 as internal standard. Calibrations for olanzapine and desmethylolanzapine were linear within the selected range of 0.2-30 ng/ml (6-96 nM) in cerebral spinal fluid and for olanzapine in plasma, in the range of 5-100 ng/ml (16-320 nM). The method was successfully used for the analysis of samples from patients treated with olanzapine in the dose range of 2.5-25mg/day., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Objectives: To clarify the basic mechanism involved in the pathophysiology of cystitis by characterizing the urodynamic parameters, pharmacologically relevant (muscarinic and purinergic) receptors, and the in vivo release of adenosine triphosphate (ATP) in the bladder of hydrochloric acid (HCl)-treated rats., Methods: The muscarinic and purinergic receptors in rat tissue were measured by radioreceptor assays using (N-methyl-³H) scopolamine methyl chloride ([³H]NMS) and αβ-methylene-ATP (2,8-³H) tetrasodium salt ([³H]αβ-MeATP), respectively. The urodynamic parameters and ATP levels were measured using a cystometric method and the luciferin-luciferase assay, respectively., Results: In the HCl-treated rats, the micturition interval and micturition volume were significantly (48% and 55%, respectively, P <.05) decreased and the number of micturitions was significantly (3.2-fold, P <.05) increased compared with those of the control rats. The maximal number of binding sites for [³H]NMS and [³H]αβ-MeATP was significantly (55% and 72%, respectively, P <.001) decreased in the bladder of HCl-treated rats, suggesting downregulation of both muscarinic and purinergic receptors. In the HCl-treated rats, the inhibition constant, K(i), values for oxybutynin, solifenacin, and darifenacin were significantly (1.3-1.4-fold, P <.05) increased, but those for tolterodine and AF-DX116 were unchanged. Similarly, the inhibition constant for A-317491, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium, and MRS2273 was significantly (5.5, 11, and 7.6-fold, respectively, P <.001) increased. Furthermore, the in vivo release of ATP was significantly (P <.05) enhanced in the HCl-treated rat bladder., Conclusions: Both muscarinic and purinergic mechanisms might be, at least in part, associated with the urinary dysfunction due to cystitis., (Copyright © 2010 Elsevier Inc. All rights reserved.)