Long-term effects of early postnatal nicotine exposure on cholinergic function in the mouse hippocampal CA1 region.

In rodent models of smoking during pregnancy, early postnatal nicotine exposure results in impaired hippocampus-dependent memory, but the underlying mechanism remains elusive. Given that hippocampal cholinergic systems modulate memory and rapid development of hippocampal cholinergic systems occurs during nicotine exposure, here we investigated its impacts on cholinergic function. Both nicotinic and muscarinic activation produce transient or long-lasting depression of excitatory synaptic transmission in the hippocampal CA1 region. We found that postnatal nicotine exposure impairs both the induction and nicotinic modulation of NMDAR-dependent long-term depression (LTD). Activation of muscarinic receptors decreases excitatory synaptic transmission and CA1 network activity in both wild-type and α2 knockout mice. These muscarinic effects are still observed in nicotine-exposed mice. M1 muscarinic receptor activity is required for mGluR-dependent LTD. Early postnatal nicotine exposure has no effect on mGluR-dependent LTD induction, suggesting that it has no effect on the function of m1 muscarinic receptors involved in this form of LTD. Our results demonstrate that early postnatal nicotine exposure has more pronounced effects on nicotinic function than muscarinic function in the hippocampal CA1 region. Thus, impaired hippocampus-dependent memory may arise from the developmental disruption of nicotinic cholinergic systems in the hippocampal CA1 region.

Effects of the putative lithium mimetic ebselen on pilocarpine-induced neural activity.

Lithium, commonly used to treat bipolar disorder, potentiates the ability of the muscarinic agonist pilocarpine to induce seizures in rodents. As this potentiation by lithium is reversed by the administration of myo-inositol, the potentiation may be mediated by inhibition of inositol monophosphatase (IMPase), a known target of lithium. Recently, we demonstrated that ebselen is a 'lithium mimetic' in regard to behaviours in both mice and man. Ebselen inhibits IMPase in vitro and lowers myo-inositol in vivo in the brains of mice and men, making ebselen the only known inhibitor of IMPase, other than lithium, that penetrates the blood-brain barrier. Our objective was to determine the effects of ebselen on sensitization to pilocarpine-induced seizures and neural activity. We administered ebselen at different doses and time intervals to mice, followed by injection of a sub-seizure dose of pilocarpine. We assessed seizure and neural activity by a subjective seizure rating scale, by monitoring tremors, and by induction of the immediate early gene c-fos. In contrast to lithium, ebselen did not potentiate the ability of pilocarpine to induce seizures. Unexpectedly, ebselen inhibited pilocarpine-induced tremor as well as pilocarpine-induced increases in c-fos mRNA levels. Both lithium and ebselen inhibit a common target, IMPase, but only lithium potentiates pilocarpine-induced seizures, consistent with their polypharmacology at diverse molecular targets. We conclude that ebselen does not potentiate pilocarpine-induced seizures and instead, reduces pilocarpine-mediated neural activation. This lack of potentiation of muscarinic sensitization may be one reason for the lack of side-effects observed with ebselen treatment clinically.

Carbachol dimers with primary carbamate groups as homobivalent modulators of muscarinic receptors.

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM1-5) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior. Classification, Molecular and cellular pharmacology.

Effects of Euterpe oleracea to Enhance Learning and Memory in a Conditioned Nicotinic and Muscarinic Receptor Response Paradigm by Modulation of Cholinergic Mechanisms in Rats.

Euterpe oleracea (EO) includes a large number of polyphenolic compounds such as phenolics, flavonoids, and anthocyanins that have antioxidant activities. E. oleracea was suggested to ease the oxidative stress and inflammation in brain cells. Our aim was to analyze the effects of E. oleracea on learning and memory. Seventy-two (250 ± 25 g) male Wistar albino rats were used for this study. The groups consisted of control, EO100 mg/kg, EO300 mg/kg, scopolamine 1.5 mg/kg, mecamylamine 7.5 mg/kg, combinations of scopolamine with EO100 mg/kg, EO300 mg/kg, and rivastigmine 1.5 mg/kg; and mecamylamine combined with EO100 mg/kg. Before the start of the study, E. oleracea doses were provided once a day for a period of 15 days and for a 6-day experimental period. Thirty minutes after intraperitoneal scopolamine and mecamylamine injections, gastrogavage was applied to each group. Ninety minutes after the drug treatments, locomotor activity and Morris water maze tests were performed. Rats were killed and each hippocampus was used for the quantification of acetylcholine (Ach). Statistical analyses were calculated using one-way and two-way analyses of variance (ANOVA), and a value of P < .05 was considered significant. In groups EO100 mg/kg and EO300 mg/kg the results did not show any significant changes on learning and memory compared with the control group. Mecamylamine and scopolamine enhanced the latency for the escape platform, and decreased the time spent in escape platform quadrant when the memory tests were applied in reference to the control value of P < .05. Scopolamine and mecamylamine combinations of EO100 mg/kg, EO300 mg/kg, and rivastigmine were proven to improve the memory. There was significant difference between the first and fifth days of the learning tests in all the groups, but no significant difference occurred between the groups. Ach levels in hippocampi supported all memory tests. We suggest that E. oleracea made no alterations on learning and memory, but still improved nicotinic and muscarinic receptor-mediated and impaired memory just as rivastigmine.

Anti-inflammatory and antioxidant effects of muscarinic acetylcholine receptor (mAChR) activation in the rat hippocampus.

Recently we found that acute treatment with Oxotremorine (Oxo), a non-selective mAChRs agonist, up-regulates heat shock proteins and activates their transcription factor heat shock factor 1 in the rat hippocampus. Here we aimed to investigate: a) if acute treatment with Oxo may regulate pro-inflammatory or anti-inflammatory cytokines and oxidative stress in the rat hippocampus; b) if chronic restraint stress (CRS) induces inflammatory or oxidative alterations in the hippocampus and whether such alterations may be affected by chronic treatment with Oxo. In the acute experiment, rats were injected with single dose of Oxo (0.4 mg/kg) and sacrificed at 24 h, 48 h and 72 h. In the CRS experiment, the rats were exposed for 21 days to the CRS and then were treated with Oxo (0.2 mg/kg) for further 10 days. The acute Oxo treatment showed an ability to significantly reduce reactive oxygen species (ROS), singlet oxygen (1O2), pro-inflammatory cytokines levels (IL-1ß and IL-6) and phosphorylated NF-κB-p65. Acute Oxo treatment also increased superoxide dismutase (SOD)-2 protein levels and stimulated SOD activity. No differences were detected in the anti-inflammatory cytokine levels, including IL-10 and TGF-ß1. In the group of rats exposed to the CRS were found increased hippocampal IL-1ß and IL-6 levels, together with a reduction of SOD activity level. These changes produced by CRS were counteracted by chronic Oxo treatment. In contrast, the upregulation of ROS and 1O2 levels in the CRS group was not counteracted by chronic Oxo treatment. The results revealed a hippocampal anti-inflammatory and antioxidant effect of Oxo treatment in both basal conditions and anti-inflammatory in the CRS rat model.

Ba-Wei-Die-Huang-Wan (Hachimi-jio-gan) can ameliorate ketamine-induced cystitis by modulating neuroreceptors, inflammatory mediators, and fibrogenesis in a rat model.

AIM: We investigated the effects of Ba-Wei-Die-Huang-Wan (BWDHW) on ketamine-induced cystitis (KIC) in a rat model. METHODS: Female Sprague-Dawley rats were distributed into three groups: control (saline), ketamine (25 mg/kg/day for 28 days), or ketamine (25 mg/kg/day for 28 days) plus BWDHW (90 mg/kg/day, started from day 14). Functional magnetic resonance imaging (fMRI), metabolic cage study, and cystometry were evaluated. Bladder histology was evaluated. Western blots of the bladder proteins were carried out. RESULTS: Compared with controls, ketamine-treated rats showed stronger fMRI intensity in the periaqueductal gray area and bladder overactivity in the bladder functional study, but the ketamine/BWDHW-treated rats did not. Furthermore, ketamine breached the uroplakin III membrane at the apical surface of the urothelium, enhanced substance P spread over the urothelium, induced suburothelial hemorrhage and monocyte/macrophage infiltration, and caused interstitial fibrosis deposition. By contrast, the BWDHW-treated rats exhibited less substance P spread, lower suburothelial monocyte/macrophage infiltration, and lower interstitial fibrosis deposition. The ketamine group showed significant overexpression of neuroreceptors in the bladder mucosa (the transient receptor potential vanilloid 1 and M2 - and M3 -muscarinic receptors) and detrusor (M2 - and M3 -muscarinic receptors); inflammatory mediators in the detrusor (interleukin-1ß [IL-1ß], IL-6, tumor necrosis factor-α, nuclear factor-κB, cyclooxygenase-2, and intercellular adhesion molecule-1); and fibrogenesis molecules in the detrusor (transforming growth factor-ß1, collagen I, collagen III, and fibronectin). However, no significant changes were noted between the ketamine/BWDHW and control groups. CONCLUSION: BWDHW could exert therapeutic effects by inhibiting the upregulation of neuroreceptors, modulating inflammatory mediators, suppressing fibrogenesis, and ameliorating bladder overactivity in rats with KIC.

Autonomic dysregulation at multiple sites is implicated in age-associated underactive bladder in female mice.

AIMS: To evaluate the functional and molecular alterations of contractile and relaxant machinery in the bladder and urethra that lead to the underactive bladder (UAB) in old female mice. METHODS: Female young (3-months) and old (18-months) C57BL/6 mice were used. Urodynamic was assessed in awake and anaesthetized mice. Electrical-field stimulation (EFS) and concentration-response curves to contractile and relaxing agents in isolated bladders and urethras were performed. Messenger RNA (mRNA) expressions of muscarinic, adrenergic, and transient receptor potential vanilloid-4 (TRPV4), and of the enzymes tyrosine hydroxylase and neuronal nitric oxide synthase (nNOS) were determined. Bladder cyclic adenosine monophosphate (cAMP) levels were measured. RESULTS: Cystometry in old mice showed incapacity to produce bladder emptying. On filter paper, old mice showed reduced urinary spots. Compared to the young group, bladder contractions induced by EFS and carbachol were lower in old mice. The ß3 -adrenoceptor agonist mirabegron promoted higher bladder relaxation and elevation of cAMP levels in old mice. In old mice urethras, the α1a -adrenoceptor agonist phenylephrine produced higher contractions, but no differences were found for the NO donor sodium nitroprusside-induced relaxations. In old mice, increased mRNA expressions of ß3 - and α1a -adrenoceptors in bladder and urethra were found, respectively, whereas the muscarinic M2 and M3 receptors and ß2 -adrenoceptors did not change between groups. Reduced mRNA expressions of tyrosine hydroxylase and nNOS were found in old mouse urethras. Additionally, TRPV4 expression was reduced in bladder urothelium from old mice. CONCLUSION: Age-associated mouse UAB is the result of autonomic dysfunction at multiple levels leading to the less sensitive and overrelaxed bladder, along with urethral hypercontractility.

Estrous cycle stage gates sex differences in prefrontal muscarinic control of fear memory formation.

The association of a sensory cue and an aversive footshock that are separated in time, as in trace fear conditioning, requires persistent activity in prelimbic cortex during the cue-shock interval. The activation of muscarinic acetylcholine receptors has been shown to facilitate persistent firing of cortical cells in response to brief stimulation, and muscarinic antagonists in the prefrontal cortex impair working memory. It is unknown, however, if the acquisition of associative trace fear conditioning is dependent on muscarinic signaling in the prefrontal cortex. Here, we delivered the muscarinic receptor antagonist scopolamine to the prelimbic cortex of rats prior to trace fear conditioning and tested their memories of the cue and training context the following day. The effect of scopolamine on working memory performance was also tested using a spatial delayed non-match to sample task. Male and female subjects were included to examine potential sex differences in the modulation of memory formation, as we have previously observed for pituitary adenylate cyclase-activating polypeptide signaling in the prefrontal cortex (Kirry et al., 2018). We found that pre-training administration of intra-prelimbic scopolamine impaired the formation of cued and contextual fear memories in males, but not females at a dose that impairs spatial working memory in both sexes. Fear memory formation in females was impaired by a higher dose of scopolamine and this impairment was gated by estrous cycle stage: scopolamine failed to impair memory in rats in the diestrus or proestrus stages of the estrous cycle. These findings add to the growing body of evidence that the prefrontal cortex is sexually dimorphic in learning and memory and additionally suggest that males and females differentially engage prefrontal neuromodulatory systems in support of learning.

Phencynonate S-isomer as a eutomer is a novel central anticholinergic drug for anti-motion sickness.

To compare and evaluate the differences of stereoselective activity, the binding affinity, metabolism, transport and molecular docking of phencynonate isomers to muscarinic acetylcholine receptor (mAChR) were investigated in this study. The rotation stimulation and locomotor experiments were used to evaluate anti-motion sickness effects. The competitive affinity with [3H]-QNB and molecular docking were used for studying the interactions between the two isomers and mAChR. The stereoselective mechanism of isomers was investigated by incubation with rat liver microsomes, a protein binding assay and membrane permeability assay across a Caco-2 cell monolayer using a chiral column HPLC method. The results indicated that S-isomer was more effective against motion sickness and had not anxiogenic action at therapeutic doses. S-isomer has the higher affinity and activity for mAChR in cerebral cortex and acted as a competitive mAChR antagonist. The stereoselective elimination of S-isomer was primarily affected by CYP1B1 and 17A1 enzymes, resulting in a higher metabolic stability and slower elimination. Phencynonate S isomer, as a eutomer and central anticholinergic chiral drug, is a novel anti-motion sickness drug with higher efficacy and lower central side effect. Our data assisted the development of a novel drug and eventual use of S-isomer in clinical practice.

An evaluation of the anti-inflammatory, antipyretic and analgesic effects of hydroethanol leaf extract of Albizia zygia in animal models.

CONTEXT: The leaves of Albizia zygia (DC.) J.F. Macbr. (Leguminosae-Mimosoideae) are used in Ghanaian traditional medicine for the treatment of pain, inflammatory disorders and fever (including malaria). OBJECTIVES: The present study evaluated the anti-inflammatory, antipyretic and analgesic effects of the hydroethanol leaf extract of Albizia zygia (AZE) in animal models. MATERIALS AND METHODS: The anti-inflammatory and antipyretic effects of AZE were examined in the carrageenan-induced foot oedema model and the baker's yeast-induced pyrexia test respectively. The analgesic effect and possible mechanisms of action were also assessed in the formalin test. RESULTS: AZE (30-300 mg/kg, p.o.), either preemptively or curatively, significantly inhibited carrageenan-induced foot edema in 7-day-old chicks (ED50 values; preemptive: 232.9 ± 53.33 mg/kg; curative: 539.2 ± 138.28 mg/kg). Similarly, the NSAID diclofenac (10-100 mg/kg, i.p.) significantly reduced the oedema in both preemptive (ED50: 21.16 ± 4.07 mg/kg) and curative (ED50: 44.28 ± 5.75 mg/kg) treatments. The extract (30-300 mg/kg, p.o.) as well as paracetamol (150 mg/kg, p.o.) also showed significant antipyretic activity in the baker's yeast-induced pyrexia test (ED50 of AZE: 282.5 ± 96.55 mg/kg). AZE and morphine (1-10 mg/kg, i.p.; positive control), exhibited significant analgesic activity in the formalin test. The analgesic effect was partly or wholly reversed by the systemic administration of naloxone, theophylline and atropine. CONCLUSION: The results suggest that AZE possesses anti-inflammatory, antipyretic and analgesic properties, which justifies its traditional use. Also, the results show the involvement of the opioidergic, adenosinergic and the muscarinic cholinergic pathways in the analgesic effects of AZE.

Non-neuronal cholinergic system contributes to corticosteroid resistance in chronic obstructive pulmonary disease patients.

BACKGROUND: Inhaled corticosteroid (ICS) with long-acting beta-2 agonists is a well-documented combination therapy for chronic obstructive pulmonary disease (COPD) based on its additive anti-inflammatory properties. By contrast, the recommendation of ICS in combination with long-acting muscarinic antagonist (LAMA) is not evidence-based. In this study, neutrophils obtained from COPD patients were used to compare the anti-inflammatory effects of aclidinium bromide (a long-acting muscarinic antagonist) with corticosteroids and their potential additive effect. METHODS: Human sputum and blood neutrophils were isolated from healthy individuals (n = 37), patients with stable COPD (n = 52) and those with exacerbated COPD (n = 16). The cells were incubated with corticosteroid fluticasone propionate (0.1 nM-1 µM), aclidinium bromide (0.1 nM-1 µM) or a combination thereof and stimulated with 1 µg of lipopolysaccharide/ml or 5 % cigarette smoke extract. Levels of the pro-inflammatory mediators interleukin-8, matrix metalloproteinase-9, CCL-5, granulocyte-macrophage colony-stimulating factor and interleukin-1ß were measured and the mechanisms of corticosteroid resistance evaluated at the end of the incubation. RESULTS: The non-neuronal cholinergic system was over-expressed in neutrophils from COPD patients, as evidenced by increases in the expression of muscarinic receptors (M2, M4 and M5), choline acetyltransferase and vesicular acetylcholine transporter. Aclidinium bromide demonstrated anti-inflammatory effects on neutrophils from COPD patients, reversing their resistance to corticosteroids. Additive effects of combined aclidinium bromide and fluticasone propionate in blocking M2 receptor levels, inhibiting phosphoinositide 3-kinase-δ and enhancing the glucocorticoid response element transcription factor were demonstrated and were accompanied by an increase in the corticosteroid-induced expression of anti-inflammatory-related genes. CONCLUSIONS: LAMAs potentiate the anti-inflammatory effects of corticosteroids in neutrophils from COPD patients in vitro, thus providing a scientific rationale for their use in combination with corticosteroids in the treatment of COPD.

The participation of the muscarinic receptors in the preoptic-anterior hypothalamic areas in the regulation of ovulation depends on the ovary.

BACKGROUND: Muscarinic receptors (mAChRs) of the preoptic and anterior hypothalamus areas (POA-AHA) regulate ovulation in an asymmetric manner during the estrous cycle. The aims of the present study were to analyze the effects of a temporal blockade of mAChRs on either side of the POA-AHA performed in diestrus-2 rats on ovulation, the levels of estradiol, follicle stimulating hormone (FSH) and luteinizing hormone (LH) and the mechanisms involved in changes in ovulation. METHODS: Cyclic rats on diestrus-2 day were anesthetized and randomly assigned to the following groups: 1) microinjection of 1 µl of saline or atropine solution (62.5 ng) in the left or right POA-AHA; 2) removal (unilateral ovariectomty, ULO) of the left (L-ULO) or right (R-ULO) ovary, and 3) rats microinjected with atropine into the left or right POA-AHA plus L-ULO or R-ULO. The ovulation rate and the number of ova shed were measured during the predicted estrus, as well as the levels of estradiol, FSH and LH during the predicted proestrus and the effects of injecting synthetic LH-releasing hormone (LHRH) or estradiol benzoate (EB). RESULTS: Atropine in the left POA-AHA decreased both the ovulation rate and estradiol and LH levels on the afternoon of proestrus, also LHRH or EB injection restored ovulation. L- or R-ULO resulted in a lower ovulation rate and smaller number of ova shed, and only injection of LHRH restored ovulation. EB injection at diestrus-2 restored ovulation in animals with L-ULO only. The levels of estradiol, FSH and LH in rats with L-ULO were higher than in animals with unilateral laparotomy. In the group microinjected with atropine in the left POA-AHA, ovulation was similar to that in ULO rats. In contrast, atropine in the right POA-AHA of ULO rats blocked ovulation, an action that was restored by either LHRH or EB injection. CONCLUSIONS: These results indicated that the removal of a single ovary at noon on diestrus-2 day perturbed the neuronal pathways regulating LH secretion, which was mediated by the muscarinic system connecting the right POA-AHA and the ovaries.

Protective effect of carbachol postconditioning on hypoxia/reoxygenation-induced injury in human gastric epithelial cells.

AIMS: We investigated the protective effects of carbachol postconditioning (CAR-P) on acute gastric mucosal injury induced by hypoxia/reoxygenation (H/R) and its possible mechanisms. MAIN METHODS: Cell viability was detected by methyl thiazolyl tetrazolium (MTT). The apoptotic cells were examined by Hoechst 33258 staining. Flow cytometric analysis, lactate dehydrogenate (LDH) release assay, immunocytochemistry, and western blotting were used to investigate the effects of CAR-P on acute gastric mucosal injury induced by H/R. The model of H/R was established by hypoxia induction(94% N2+1% O2+5% CO2 for 2 h) and reoxygenation (normoxic condition for 4 h, 8 h and 16 h). KEY FINDINGS: Our study observed the protective effect of carbachol postconditioning on H/R-induced injury in human gastric epithelial cell lines (hGES-1) cells, which is achieved by direct activation of vanilloid receptor subtype 1 (VR1) and production of calcitonin gene-related peptide (CGRP), and in the inhibition of cell apoptosis. In the study, we demonstrate that CAR-P has protective effects on the H/R-induced injury in hGES-1 cells, and these effects are associated with cholinergic muscarinic receptors (CMR), VR1, and extracellular signal-regulated kinase (ERK) signaling pathway. SIGNIFICANCE: Our findings might provide a new and improved understanding of CAR-P function and an effective treatment strategy for acute gastric mucosal injury induced by H/R.

BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation.

Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics, as well as changes in cytoplasmic Ins(1,4,5)P3 levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction.

The impact of prolapse mesh on vaginal smooth muscle structure and function.

OBJECTIVE: To evaluate the impact of prolapse meshes on vaginal smooth muscle structure (VaSM) and function, and to evaluate these outcomes in the context of the mechanical and textile properties of the mesh. DESIGN: Three months following the implantation of three polypropylene prolapse meshes with distinct textile and mechanical properties, mesh tissue explants were evaluated for smooth muscle contraction, innervation, receptor function, and innervation density. SETTING: Magee-Womens Research Institute at the University of Pittsburgh. POPULATION: Thirty-four parous rhesus macaques of similar age, parity, and pelvic organ prolapse quantification (POP-Q) scores. METHODS: Macaques were implanted with mesh via sacrocolpopexy. The impact of Gynemesh(™)  PS (Ethicon; n = 7), Restorelle(®) (Coloplast; n = 7), UltraPro(™) parallel and UltraPro(™) perpendicular (Ethicon; n = 6 and 7, respectively) were compared with sham-operated controls (n = 7). Outcomes were analysed by Kruskal-Wallis ANOVA, Mann-Whitney U-tests and multiple regression analysis (P < 0.05). MEAN OUTCOME MEASURES: Vaginal tissue explants were evaluated for the maximum contractile force generated following muscle, nerve, and receptor stimulation, and for peripheral nerve density. RESULTS: Muscle myofibre, nerve, and receptor-mediated contractions were negatively affected by mesh only in the grafted region (P < 0.001, P = 0.002, and P = 0.008, respectively), whereas cholinergic and adrenergic nerve densities were affected in the grafted (P = 0.090 and P = 0.008, respectively) and non-grafted (P = 0.009 and P = 0.005, respectively) regions. The impact varied by mesh property, as mesh stiffness was a significant predictor of the negative affect on muscle function and nerve density (P < 0.001 and P = 0.013, respectively), whereas mesh and weight was a predictor of receptor function (P < 0.001). CONCLUSIONS: Mesh has an overall negative impact on VaSM, and the effects are a function of mesh properties, most notably, mesh stiffness. TWEETABLE ABSTRACT: Prolapse mesh affects vaginal smooth muscle.

Functional and biochemical characteristics of urinary bladder muscarinic receptors in long-term alloxan diabetic rats.

OBJECTIVE: To re-examine the function of the urinary bladder in vivoas well as to determine the functional and biochemical characteristics of bladder muscarinic receptors in long-term alloxan-induced diabetes rats. METHODS: Two-month-old male Wistar rats were injected with alloxan and the animals showing blood glucose levels >300mg/dL together with age-paired untreated animals were kept for 11 months. Body weight, bladder weight, blood glucose, and urinary volume over a period of 24 hours were determined in both groups of animals. A voiding cystometry in conscious control and diabetic rats was performed to determine maximal micturition pressure, micturition contraction interval and duration as well as voided and post-voiding residual volume. In addition, concentration-response curves for bethanechol in isolated bladder strips, as well as [3H]-N methyl-scopolamine binding site characteristics in bladder homogenates were determined. RESULTS: Mean bladder weight was 162.5±21.2mg versus 290±37.9mg in control and treated animals, respectively (p<0.05). Micturition contraction amplitude (34.6±4.7mmHg versus 49.6±2.5mmHg), duration (14.5±1.7 seconds versus 23.33±4.6 seconds) and interval (87.5±17.02 seconds versus 281.11±20.24 seconds) were significantly greater in alloxan diabetic rats. Voided urine volume per micturition contraction was also significantly higher in diabetic animals. However the post-voiding residual volume was not statistically different. Bethanechol potency (EC50 3µM versus 5µM) and maximal effect (31.2±5.9g/g versus 36.1±6.8g/g) in isolated bladder strips as well as number (169±4fmol/mg versus 176±3fmol/mg protein) and affinity (0.69±0.1nM versus 0.57±0.1nM) of bladder muscarinic receptors were also not statistically different. CONCLUSION: Bladder function in vivo is altered in chronic alloxan-induced diabetes rats without changes in functional and biochemical characteristics of bladder muscarinic receptors.

Functional and biochemical characteristics of urinary bladder muscarinic receptors in long-term alloxan diabetic rats / Características funcionais e bioquímicas dos receptores muscarínicos da bexiga de ratos com diabetes crônico induzido por aloxana

Objective To re-examine the function of the urinary bladder in vivoas well as to determine the functional and biochemical characteristics of bladder muscarinic receptors in long-term alloxan-induced diabetes rats.Methods Two-month-old male Wistar rats were injected with alloxan and the animals showing blood glucose levels >300mg/dL together with age-paired untreated animals were kept for 11 months. Body weight, bladder weight, blood glucose, and urinary volume over a period of 24 hours were determined in both groups of animals. A voiding cystometry in conscious control and diabetic rats was performed to determine maximal micturition pressure, micturition contraction interval and duration as well as voided and post-voiding residual volume. In addition, concentration-response curves for bethanechol in isolated bladder strips, as well as [3H]-N methyl-scopolamine binding site characteristics in bladder homogenates were determined.Results Mean bladder weight was 162.5±21.2mg versus 290±37.9mg in control and treated animals, respectively (p<0.05). Micturition contraction amplitude (34.6±4.7mmHg versus 49.6±2.5mmHg), duration (14.5±1.7 seconds versus 23.33±4.6 seconds) and interval (87.5±17.02 seconds versus 281.11±20.24 seconds) were significantly greater in alloxan diabetic rats. Voided urine volume per micturition contraction was also significantly higher in diabetic animals. However the post-voiding residual volume was not statistically different. Bethanechol potency (EC50 3µM versus 5µM) and maximal effect (31.2±5.9g/g versus 36.1±6.8g/g) in isolated bladder strips as well as number (169±4fmol/mg versus 176±3fmol/mg protein) and affinity (0.69±0.1nM versus 0.57±0.1nM) of bladder muscarinic receptors were also not statistically different.Conclusion Bladder function in vivo is altered in chronic alloxan-induced diabetes rats without changes in functional and biochemical characteristics of bladder muscarinic receptors.

Objetivo Reestudar o funcionamento da bexiga in vivo e determinar as características funcionais e bioquímicas dos receptores muscarínicos vesicais de ratos com diabetes crônico induzido por aloxana.Métodos Ratos Wistar de dois meses de idade receberam injeção de aloxana, e os animais que apresentaram glicemia >300mg/dL foram mantidos por 11 meses junto de outros não tratados e pareados por idade. Nos dois grupos de animais, peso corpóreo, peso da bexiga, glicemia e volume urinário de 24 horas foram medidos. Em ambos os grupos, realizou-se a cistometria miccional em animais não anestesiados. Foram determinados os seguintes parâmetros: pressão máxima de micção, intervalo e contração de micção, bem como o volume de esvaziamento e o volume residual pós-miccional. Além disso, foram determinadas as curvas de concentração-resposta a betanecol em preparações isoladas de bexiga e também as características dos sítios de ligação da [3H]-N-metil-escopolamina em homogenatos de bexiga.Resultados O peso médio da bexiga foi de 162,5±21,2mg versus290±37,9mg nos animais controles e tratados, respectivamente (p<0,05). A amplitude de contração (34,6±4,7mmHg versus 49,6±2,5mmHg), a duração (14,5±1,7 segundos versus 23,33±4,6 segundos) e o intervalo (87,5±17,02 segundos versus 281,11±20,24 segundos) de micção foram significantemente maiores nos ratos tratados com aloxana. O volume de urina eliminada durante a contração miccional também foi maior nos animais diabéticos. Contudo, o volume residual pós-miccional não foi estatisticamente diferente. Não foram observadas diferenças na resposta ao betanecol (EC50 3µM versus 5µM) e no seu efeito máximo (31,2±5,9g/g versus 36,1±6,8g/g) em preparações isoladas de bexiga, bem como no número total (169±43fmol/mgversus 176±3fmol/mg) e na afinidade (0,69±0,1nMversus 0,57±0,1nM) dos receptores muscarínicos da bexiga.Conclusão O funcionamento da bexiga in vivo está alterado no diabetes crônico induzido por aloxana, porém sem alterações funcionais e bioquímicas nos receptores muscarínicos da bexiga.

Impairment of skin barrier function via cholinergic signal transduction in a dextran sulphate sodium-induced colitis mouse model.

Dry skin has been clinically associated with visceral diseases, including liver disease, as well as for our previously reported small intestinal injury mouse model, which have abnormalities in skin barrier function. To clarify this disease-induced skin disruption, we used a dextran sulphate sodium (DSS)-induced colitis mouse model. Following treatment with DSS, damage to the colon and skin was monitored using histological and protein analysis methods as well as the detection of inflammatory mediators in the plasma. Notably, transepidermal water loss was higher, and skin hydration was lower in DSS-treated mice compared to controls. Tumor necrosis factor-alpha (TNF-α), interleukin 6 and NO2-/NO3- levels were also upregulated in the plasma, and a decrease in body weight and colon length was observed in DSS-treated mice. However, when administered TNF-α antibody or an iNOS inhibitor, no change in skin condition was observed, indicating that another signalling mechanism is utilized. Interestingly, the number of tryptase-expressing mast cells, known for their role in immune function via cholinergic signal transduction, was elevated. To evaluate the function of cholinergic signalling in this context, atropine (a muscarinic cholinoceptor antagonist) or hexamethonium (a nicotinic cholinergic ganglion-blocking agent) was administered to DSS-treated mice. Our data indicate that muscarinic acetylcholine receptors (mAChRs) are the primary receptors functioning in colon-to-skin signal transduction, as DSS-induced skin disruption was suppressed by atropine. Thus, skin disruption is likely associated with DSS-induced colitis, and the activation of mast cells via mAChRs is critical to this association.

Muscarinic excitation of parvalbumin-positive interneurons contributes to the severity of pilocarpine-induced seizures.

OBJECTIVE: A common rodent model in epilepsy research employs the muscarinic acetylcholine receptor (mAChR) agonist pilocarpine, yet the mechanisms underlying the induction of pilocarpine-induced seizures (PISs) remain unclear. Global M1 mAChR (M1 R) knockout mice are resistant to PISs, implying that M1 R activation disrupts excitation/inhibition balance. Parvalbumin-positive (PV) inhibitory neurons express M1 Rs, participate in cholinergically induced oscillations, and can enter a state of depolarization block (DB) during epileptiform activity. Here, we test the hypothesis that pilocarpine activation of M1 Rs expressed on PV cells contributes to PISs. METHODS: CA1 PV cells in PV-CRE mice were visualized with a floxed YFP or hM3Dq-mCherry adeno-associated virus, or by crossing PV-CRE mice with the RosaYFP reporter line. To eliminate M1 Rs from PV cells, we generated PV-M1 knockout (KO) mice by crossing PV-CRE and floxed M1 mice. Action potential (AP) frequency was monitored during application of pilocarpine (200 µm). In behavioral experiments, locomotion and seizure symptoms were recorded in wild-type (WT) or PV-M1 KO mice during PISs. RESULTS: Pilocarpine significantly increased AP frequency in CA1 PV cells into the gamma range. In the continued presence of pilocarpine, a subset (5/7) of PV cells progressed to DB, which was mimicked by hM3Dq activation of Gq-receptor signaling. Pilocarpine-induced depolarization, AP firing at gamma frequency, and progression to DB were prevented in CA1 PV cells of PV-M1 KO mice. Finally, compared to WT mice, PV-M1 KO mice were associated with reduced severity of PISs. SIGNIFICANCE: Pilocarpine can directly depolarize PV+ cells via M1 R activation, but a subset of these cells progress to DB. Our electrophysiologic and behavioral results suggest that this mechanism is active during PISs, contributing to a collapse of PV-mediated γ-aminobutyric acid (GABA)ergic inhibition, dysregulation of excitation/inhibition balance, and increased susceptibility to PISs.

[Effect of anticholinergic drugs on cognitive impairment in the elderly]. / Efecto de los fármacos anticolinérgicos en el rendimiento cognitivo de las personas mayores.

The use of anticholinergic drugs is common in the elderly, even in people with cognitive impairment. A systematic search was conducted in PubMed (anticholinergic effects, anticholinergic and dementia) to define the effects of anticholinergic drugs in the elderly. We emphasized the search in patterns of use, the combined use with AChEIs, the measurement of the Serum Anticholinergic Activity, and the short-term and long-term cognitive effects. The conclusions are that the use of anticholinergic drugs is common in the elderly, even more so than the medical prescription of AChEIs in Alzheimer's disease. The use of anticholinergic drugs may result in cognitive impairment. In long-term use it may generate a worsening of cognitive functions. It can lead to a wrong diagnosis of mild cognitive impairment or dementia, and they can also initiate signs of dementia. Greater cognitive effects appear when there is a previous deficit, but cognitive effects from anticholinergic drugs disappear in severe dementia. The presence of ApoEɛ4 increases the vulnerability for cognitive impairment when these drugs are employed.