Expression Of M3 Receptors Research Articles

Progressive changes in detrusor function and micturition patterns with chroinc bladder ischemia

PurposeLower urinary tract symptoms (LUTS) are bothersome constellation of voiding symptoms in men and women as they age. Multiple factors and comorbidities are attributed to this problem but underlying mechanisms of nonobstructive nonneurogenic detrusor overactivity, detrusor underactivity and LUTS remain largely unknown. Our goal was to characterize detrusor function and voiding patterns in relation to muscarinic receptors expression, nerve fiber density, and neural ultrastructure in chronic bladder ischemia.Materials and MethodsIliac artery atherosclerosis and bladder ischemia were produced in male Sprague-Dawley rats. At 8 and 16 weeks after ischemia, micturition patterns and cystometrograms were recorded in conscious rats then bladder blood flow and nonvoiding spontaneous contractions were measured under general anesthesia. Bladder tissues were processed for Western blotting, immunostaining, and transmission electron microscopy.ResultsBladder responses to ischemic insult depended on the duration of ischemia. Micturition patterns and cystometric changes at 8-week ischemia suggested detrusor overactivity, while voiding behavior and cystometrograms at 16-week ischemia implied abnormal detrusor function resembling underactivity. Upregulation of muscarinic M2 receptor was found after 8- and 16 weeks of ischemia. Downregulation of M3 and upregulation of M1 were detected at 16-week ischemia. Neural structural damage and marked neurodegeneration were found after 8 and 16 weeks of ischemia, respectively.ConclusionsProlonged ischemia may be a mediating variable in progression of overactive bladder to dysfunctional patterns similar to detrusor underactivity. The mechanism appears to involve differential expression of M1, M2, and M3 receptors, neural structural injury, and progressive loss of nerve fibers.

Enteric Dysfunctions in Experimental Parkinson's Disease: Alterations of Excitatory Cholinergic Neurotransmission Regulating Colonic Motility in Rats.

Parkinson's disease is frequently associated with gastrointestinal symptoms, mostly represented by constipation and defecatory dysfunctions. This study examined the impact of central dopaminergic denervation, induced by injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle, on distal colonic excitatory cholinergic neuromotor activity in rats. Animals were euthanized 4 and 8 weeks after 6-OHDA injection. In vivo colonic transit was evaluated by radiologic assay. Electrically induced and carbachol-induced cholinergic contractions were recorded in vitro from longitudinal and circular muscle colonic preparations, whereas acetylcholine levels were assayed in the incubation media. Choline acetyltransferase (ChAT), HuC/D (pan-neuronal marker), muscarinic M2 and M3 receptors were assessed by immunohistochemistry or western blot assay. As compared with control rats, at week 4, 6-OHDA-treated animals displayed the following changes: decreased in vivo colonic transit rate, impaired electrically evoked neurogenic cholinergic contractions, enhanced carbachol-induced contractions, decreased basal and electrically stimulated acetylcholine release from colonic tissues, decreased ChAT immunopositivity in the neuromuscular layer, unchanged density of HuC/D immunoreactive myenteric neurons, and increased expression of colonic muscarinic M2 and M3 receptors. The majority of such alterations were also detected at week 8 post 6-OHDA injection. These findings indicate that central nigrostriatal dopaminergic denervation is associated with an impaired excitatory neurotransmission characterized by a loss of myenteric neuronal ChAT positivity and decrease in acetylcholine release, resulting in a dysregulated smooth muscle motor activity, which likely contributes to the concomitant decrease in colonic transit rate.

The activation of M2 muscarinic receptor inhibits cell growth and survival in human glioblastoma cancer stem cells

The involvement of muscarinic receptors in cancer has been reported. Recently we have demonstrated that the activation of M2 muscarinic receptors, through arecaidine propargyl ester, arrests cell proliferation and induces apoptosis in primary and established glioblastoma cell lines. Considering the inability of conventional drugs to completely counteract the growth of glioblastoma cancer stem cells (GSCs), we have investigated the effect produced by arecaidine on GSC growth and survival. The expression of M2 receptors has been analyzed in GSC cell lines derived from human biopsies. Based on the M2 receptor expression levels, we have selected two gliolastoma cell lines (GB7 and GB8). In both cell lines the treatment with arecaidine decreased GCS cell growth. GB7 cells exhibited a time- and dose-dependent decrease of cell proliferation. Moreover arecaidine caused a reduced cell survival in particular in GB8 cell line. These effects appear to be mediated by M2 receptor activation as suggested by pharmacological experiments performed in the presence of M1 and M3 preferring antagonists (pirenzepine and 4-DAMP respectively) and M2/M4 antagonist methoctramine. M2 receptor silencing by siRNA has further confirmed that the inhibition of cell growth arecaidine-induced was mediated by the M2 receptor activation. These results suggest that the M2 receptors may represent a new interesting therapeutic tool to counteract glioblastoma cancer stem cell growth and survival.

Pharmacologic and Molecular Characterization of Underactive Bladder Induced by Lumbar Canal Stenosis

To investigate the voiding function in a rat model of lumbar canal stenosis (LCS) using pharmacologic and molecular approaches. Sixty-one female Sprague-Dawley rats were broadly split into a sham and an LCS group. A hole was surgically drilled in the L5-L6 epidural space and filled with a rectangular piece of silicone rubber. Metabolic cage study at week 2 and continuous cystometry (CMG) under urethane anesthesia at weeks 2 and 4 were performed. During CMG, prostaglandin E2 or sulprostone, an prostaglandin E receptor 1 and prostaglandin E receptor 3 agonist was administered locally and intravenously, respectively, and the bladder was then harvested for histology and Western blot. Compared with sham, the LCS group showed dribbling urination and progressive increase in bladder size. CMG under urethane anesthesia in the LCS group was marked by overflow incontinence and acontractile bladder. Administration of intravesical prostaglandin E2 (200 μM) or intravenous sulprostone (0.1 mg/kg) in the sham group induced bladder overactivity, but decreased the compliance and failed to restore the bladder emptying function in the LCS group. The LCS group showed edematous changes and muscle thinning at week 2, which were partially restored by week 4. Histologic changes were accompanied by downregulation of agrin protein (64.0%) at week 2 and upregulation of M2 receptor (65.4%) at week 4. Expression of M3, protein gene product 9.5, and nerve growth factor did not differ between groups. LCS-induced underactive bladder is associated with altered expression of agrin and M2 receptor. The underactive bladder model is clinically relevant, and the findings indicate potential molecular targets for new therapies.

A long-acting β2-adrenergic agonist increases the expression of muscarine cholinergic subtype‑3 receptors by activating the β2-adrenoceptor cyclic adenosine monophosphate signaling pathway in airway smooth muscle cells.

The persistent administration of β2-adrenergic (β2AR) agonists has been demonstrated to increase the risk of severe asthma, partly due to the induction of tolerance to bronchoprotection via undefined mechanisms. The present study investigated the potential effect of the long-acting β2-adrenergic agonist, formoterol, on the expression of muscarinic M3 receptor (M3R) in rat airway smooth muscle cells (ASMCs). Primary rat ASMCs were isolated and characterized following immunostaining with anti-α-smooth muscle actin antibodies. The protein expression levels of M3R and phospholipase C-β1 (PLCβ1) were characterized by western blot analysis and the production of inositol 1,4,5-trisphosphate (IP3) was determined using an enzyme-linked immunosorbent assay. Formoterol increased the protein expression of M3R in rat ASMCs in a time- and dose-dependent manner, which was significantly inhibited by the β2AR antagonist, ICI118,551 and the cyclic adenosine monophosphate (cAMP) inhibitor, SQ22,536. The increased protein expression of M3R was positively correlated with increased production of PLCβ1 and IP3. Furthermore, treatment with the glucocorticoid, budesonide, and the PLC inhibitor, U73,122, significantly suppressed the formoterol-induced upregulated protein expression levels of M3R and PLCβ1 and production of IP3. The present study demonstrated that formoterol mediated the upregulation of M3R in the rat ASMCs by activating the β2AR-cAMP signaling pathway, resulting in increased expression levels of PLCβ1 and IP3, which are key to inducing bronchoprotection tolerance. Administration of glucocorticoids or a PLC antagonist prevented formoterol-induced bronchoprotection tolerance by suppressing the protein expression of M3R.

Changes of muscarinic receptors and connexin-43 expression as a mechanism of overactive bladder in ovariectomized rats.

After menopause, the bladder is known to become overactive. To investigate the mechanisms involved in these changes, we examined the muscarinic receptors M2, M3 and gap junction protein connexin-43 in an ovariectomized rat bladder. Twenty 10-week-old female SD rats were used. Ten rats were ovariectomized, (Ovx group) and 10 rats received a sham operation (Con group). Four weeks after the operation, urodynamic tests were performed to verify overactive bladder, and the animals were killed. The body, bladder and uterus weights were measured. The bladder specimens were prepared for immunohistochemical staining for muscarinic receptors M2, M3 and connexin-43. Western blotting was also used for the same protein measurement (M2, M3 and connexin-43). A t test with a p value of 0.05 was considered significant, and SPSS 12.0 for Windows was used to analyze the data. The mean body weight of the Ovx group (315.8 ± 18.1 g) was heavier than the Con group (270.0 ± 23.6 g) (p = 0.009). The mean uterus weight of the Ovx group (260.4 ± 186.8 g) was lighter than the Con group, (600.6 ± 175.9 g) (p = 0.028) and the mean bladder weight of the Ovx group (80.2 ± 15.9 g) was lighter than the Con group (97.4 ± 10.6 g) (p = 0.041). The mean bladder contraction of the Ovx group (5.5 ± 2.3/10 min) was more frequent than that of the Con group (3.2 ± 2.8) (p < 0.05). The expressions of M2 and M3 were not different between the Ovx and the Con group, but the expression of connexin-43 in the Ovx group was more intense than in the Con group in immunohistochemical staining. These findings were also confirmed by Western blotting results. Ovariectomized rats showed frequent bladder contraction and increased connexin-43 expression without changes in M2 and M3 receptor expression. These results imply that ovariectomy-induced overactive bladder may be due to an altered gap junction protein function rather than muscarinic receptor modification.

Long-Term Caloric Restriction in Rats may Prevent Age Related Impairment of In Vitro Bladder Function

Bladder function is often impaired with aging. In other organs caloric restriction has had a prophylactic effect on the biological changes associated with aging. We tested the hypothesis that long-term caloric restriction can prevent age related impaired bladder function in the rat. Fisher 344 male rats were divided into 3 groups, including 16 young rats at age 6 months with free access to normal food, 15 old rats at age 25 to 28 months with free access to normal food and 16 old rats at age 25 to 28 months fed with normal food 3 days per week since age 6 weeks (caloric restriction). We performed frequency volume measurements, in vitro organ bath functional studies using full-thickness longitudinal detrusor strips, evaluation of muscarinic and purinergic receptor mRNA expression, and histological examination with Masson trichrome staining of bladder tissue. Frequency volume changes did not significantly differ among the 3 groups. The old group fed normal food showed weaker contractile responses to carbachol and electrical field stimulation (especially in the cholinergic component), lower M3 receptor mRNA expression and higher collagen deposition compared to the young group. These age related bladder changes were milder in the old group with caloric restriction than in the old group fed normal food. This study suggests that in the rat long-term caloric restriction has a preventive effect against age related functional and morphological bladder changes. These changes include impaired detrusor contractility that may be related to decreased expression of M3 receptors and to bladder wall fibrosis.

Chronic obstructive sleep apnea causes atrial remodeling in canines: mechanisms and implications.

Obstructive sleep apnea (OSA) is closely related to atrial fibrillation (AF). However, the roles and mechanisms of chronic OSA in atrial remodeling are still unclear. Canine model of chronic OSA was simulated by stopping the ventilator and closing the airway for 4 h per day and lasting for 12 weeks. AF inducibility and duration was increased while atrial effective refractory period (AERP) was shortened after chronic apnea. Meanwhile, upregulation of proteins encoding inward rectifier K(+) current (IK1), delayed rectifier K(+) current (IKr and IKs), acetylcholine activated K(+) current (IKACh), transient outward K(+) current (Ito) and ultra-rapid delayed rectifier potassium current (IKur) as well as downregulation of protein encoding L-type Ca(2+) current (ICa,L) were found after chronic OSA. Besides abnormal electrical activity, chronic OSA induced apoptosis and interstitial fibrosis of atrial myocytes, which was partly mediated by caspase 9, phosphorylation of extracellular-regulated kinase 1/2, and α-smooth muscle actin. In addition, atrial sympathetic and parasympathetic hyperinnervation were found manifesting by enhanced growth-associated protein 43, tyrosine hydroxylase and elevated choline acetyltransferase. Moreover, protein expression of β1, β2, and M2 receptor were markedly increased by chronic OSA. In summary, we firstly demonstrated in canine model that chronic OSA could shorten AERP and lead to altered expression of important channel proteins, moreover, induce atrial structure remodeling by increased atrial apoptosis, fibrosis, and autonomic remodeling, eventually promoting the development of a substrate of AF. Our findings suggested that reversing atrial remodeling might be a potential therapeutic strategy for OSA-induced AF.

Effect of hyperglycaemia on muscarinic M3 receptor expression and secretory sensitivity to cholinergic receptor activation in islets.

Islets are innervated by parasympathetic nerves which release acetylcholine (ACh) to amplify glucose-induced insulin secretion, primarily via muscarinic M3 receptors (M3R). Here we investigate the consequence of chronic hyperglycaemia on islet M3R expression and secretory sensitivity of mouse islets to cholinergic receptor activation. The impact of hyperglycaemia was studied in (i) islets isolated from ob/ob mice, (ii) alginate-encapsulated mouse islets transplanted intraperitoneally into streptozotocin-induced diabetic mice and (iii) mouse and human islets maintained in vitro at 5.5 or 16 mmol/l glucose. Blood glucose levels were assessed by a commercial glucose meter, insulin content by RIA and M3R expression by qPCR and immunohistochemistry. M3R mRNA expression was reduced in both ob/ob islets and islets maintained at 16 mmol/l glucose for 3 days (68 and 50% control, respectively). In all three models of hyperglycaemia the secretory sensitivity to the cholinergic receptor agonist, carbachol, was reduced by 60-70% compared to control islets. Treatment for 72 h with the irreversible PKC activator, PMA, or the PKC inhibitor, Gö6983, did not alter islet M3R mRNA expression nor did incubation with the PI3K-inhibitor, LY294002, although enhancement of glucose-induced insulin secretion by LY294002 was reduced in islets maintained at 16 mmol/l glucose, as was mRNA expression of the PI3K regulatory subunit, p85α. Cholinergic regulation of insulin release is impaired in three experimental islet models of hyperglycaemia consistent with reduced expression of M3 receptors. Our data suggest that the receptor downregulation is a PKC- and PI3K-independent consequence of the hyperglycaemic environment, and they imply that M3 receptors could be potential targets in the treatment of type 2 diabetes.

Muscarinic Control of MIN6 Pancreatic β Cells Is Enhanced by Impaired Amino Acid Signaling

We have shown recently that the class C G protein-coupled receptor T1R1/T1R3 taste receptor complex is an early amino acid sensor in MIN6 pancreatic β cells. Amino acids are unable to activate ERK1/2 in β cells in which T1R3 has been depleted. The muscarinic receptor agonist carbachol activated ERK1/2 better in T1R3-depleted cells than in control cells. Ligands that activate certain G protein-coupled receptors in pancreatic β cells potentiate glucose-stimulated insulin secretion. Among these is the M3 muscarinic acetylcholine receptor, the major muscarinic receptor in β cells. We found that expression of M3 receptors increased in T1R3-depleted MIN6 cells and that calcium responses were altered. To determine whether these changes were related to impaired amino acid signaling, we compared responses in cells exposed to reduced amino acid concentrations. M3 receptor expression was increased, and some, but not all, changes in calcium signaling were mimicked. These findings suggest that M3 acetylcholine receptors are increased in β cells as a mechanism to compensate for amino acid deficiency.

Differential effects of lipopolysaccharide and cigarette smoke exposure on genes associated with contractile agonist responsiveness in mouse lungs (660.2)

Introduction: Inflammation is a key driver of many lung diseases, including asthma and chronic obstructive pulmonary disease. Exposure to lipopolysaccharide (LPS) and cigarette smoke (CS) are two mouse models that induce inflammation, however studies assessing changes in expression of contractile receptors in these models are limited.Methods: Female Balb/C mice were exposed to inhaled LPS once a day for 4 days or PBS. Male Balb/C mice were exposed to 9 cigarettes 3 times a day for 4 days and sham mice were exposed to air. On day 5, mice were euthanized, bronchoalveolar lavage fluid (BALF) was taken and lungs were harvested for PCR.Results: Inflammation was confirmed in both LPS and CS models by an increase in total cell number in the BALF. In both groups, there were no changes in gene expression levels of serotonin 2A, bradykinin, or muscarinic M3 receptors. There were no changes in G‐protein expression or downstream inositol trisphosphate receptor expression. In the LPS‐treated mice, there was an upregulation of muscarinic M2 receptors and ryanodine isoform 2 receptors (RyR2). In contrast, these receptors were down regulated in CS mice.Discussion: Despite both LPS and CS inducing inflammation, these stimuli have opposite effects on expression of M2 and RyR2 receptors. The influence of these changes on responsiveness to muscarinic and other contractile agonists remain to be determined.Grant Funding Source: Supported by NHMRC

MP1-15 FUNCTIONAL AND MOLECULAR CHARACTERISTIC OF UNDERACTIVE BLADDER INDUCED BY LUMBAR CANAL STENOSIS IN RATS

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Basic Research I1 Apr 2014MP1-15 FUNCTIONAL AND MOLECULAR CHARACTERISTIC OF UNDERACTIVE BLADDER INDUCED BY LUMBAR CANAL STENOSIS IN RATS Yao-Chi Chuang, Hung-Jen Wang, Naoki Yoshimura, and Michael Chancellor Yao-Chi ChuangYao-Chi Chuang More articles by this author , Hung-Jen WangHung-Jen Wang More articles by this author , Naoki YoshimuraNaoki Yoshimura More articles by this author , and Michael ChancellorMichael Chancellor More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.113AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES It has been recognized that lumbar canal stenosis (LCS) compromised neurological function is a common problem in elderly patients and may cause underactive bladder (UAB). However, the mechanism and molecular change underlying the UAB is not clear. METHODS One small hole was drilled at the fifth lumbar vertebral arch (sham), and a rectangular piece of silicone rubber was inserted into the L5¡VL6 epidural space (LCS) of female Sprague-Dawley rats. LCS rats underwent bladder expression twice a day. Before and after surgery, a metabolic cage study was performed. Continuous cystometry (CMG) was performed at day 14, or 28 under urethane anesthesia. The bladder was then harvested for histology, expression of M2 and M3 receptors, NGF, and agrin by western blotting. Some animals received intravesical PGE2 instillation. RESULTS LCS induced acute urinary retention and progressive increase of bladder weight. The LCS rats showed a significant decrease in voided volume and a significant increase in postvoid residual urine compared with Sham rats. CMG showed that the LCS bladder was no contraction, high compliance and overflow incontinence. Intravesical PGE2 induced decreased bladder compliance, but no recovery of bladder contraction in the LCS rats. In contrast, there was a significant decrease in intercontraction interval (ICI) and increased in contraction amplitude in response to PGE2 in sham rats. LCS rats showed no obvious difference in detrusor morphology (Fig 1), M2, M3 receptor, NGF, and agrin compared with the sham rats. CONCLUSIONS LCS induced UAB model preserved bladder morphology and functional receptor. It might be a useful model for the mechanism and treatment of neurogenic UAB. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e7 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Yao-Chi Chuang More articles by this author Hung-Jen Wang More articles by this author Naoki Yoshimura More articles by this author Michael Chancellor More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP1-16 STRIKING DOSE- AND TIME-DEPENDENCE OF RADIATION CYSTITIS IN THE RAT: WEEKLY CYSTOMETRIC EVALUATION UP TO NINE WEEKS AFTER PELVIC IRRADIATION

INTRODUCTION AND OBJECTIVES: It has been recognized that lumbar canal stenosis (LCS) compromised neurological function is a common problem in elderly patients and may cause underactive bladder (UAB). However, the mechanism and molecular change underlying the UAB is not clear. METHODS: One small hole was drilled at the fifth lumbar vertebral arch (sham), and a rectangular piece of silicone rubber was inserted into the L5iVL6 epidural space (LCS) of female Sprague-Dawley rats. LCS rats underwent bladder expression twice a day. Before and after surgery, a metabolic cage study was performed. Continuous cystometry (CMG) was performed at day 14, or 28 under urethane anesthesia. The bladder was then harvested for histology, expression of M2 and M3 receptors, NGF, and agrin by western blotting. Some animals received intravesical PGE2 instillation. RESULTS: LCS induced acute urinary retention and progressive increase of bladder weight. The LCS rats showed a significant decrease in voided volume and a significant increase in postvoid residual urine compared with Sham rats. CMG showed that the LCS bladder was no contraction, high compliance and overflow incontinence. Intravesical PGE2 induced decreased bladder compliance, but no recovery of bladder contraction in the LCS rats. In contrast, there was a significant decrease in intercontraction interval (ICI) and increased in contraction amplitude in response to PGE2 in sham rats. LCS rats showed no obvious difference in detrusor morphology (Fig 1), M2, M3 receptor, NGF, and agrin compared with the sham rats. CONCLUSIONS: LCS induced UAB model preserved bladder morphology and functional receptor. It might be a useful model for the mechanism and treatment of neurogenic UAB.

Expression Imbalance of Cholinergic M&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt; and M&amp;lt;sub&amp;gt;3&amp;lt;/sub&amp;gt; Receptors Contributes to the Motility Reduction of the Small Intestine in Spleen Qi Deficiency

Objective: To study roles of cholinergic M2 and M3 receptors in the motility reduction of small intestine (SI) in spleen qi deficiency. Methods: 16 male SD rats were randomly divided in the control group and spleen qi deficiency group (model group)—8 rats each group; spleen qi deficiency model of the improper diet and overfatigue was established; the SI propelling rate (SIPR) was used to evaluate the SI motility; ELISA was used to measure concentrations of acetylcholine (ACh), cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in the SI tissue; immohistochemistry was employed to detect expressions of cholinergic M2 and M3 receptors. Results: Compared with those in the control group, SIPR was reduced; expression of M2 receptors was increased; and expression of M3 receptors and concentrations of cAMP and PKA were decreased, significantly, in the model group. Conclusions: Expression imbalance of cholinergic M2 and M3 receptors might contribute to the motility reduction of the SI in spleen qi deficiency.

A Decreased Expression and Functionality of Muscarinic Cholinergic Receptor in Acute Chagas Myocarditis

Background: Chagas disease is still a public health problem because of the remaining high prevalence, the expansion of the disease into developed countries and the re-emergences by oral transmission outbreaks. Chagas cardiomyopathy evolves as a consequence of an autonomic un-balance where the parasympathetic tone is undermined. Objective: To determine the functionality and expression of muscarinic cholinergic receptors in acute Chagas disease. Methodology: 62 male, 3-week-old Sprague Dawley rats were assayed; 32 were infected with Trypanosoma cruzi trypo-mastigotes and 30 were healthy controls. Electrocardiographic studies were conducted in the absence or presence of direct muscarinic (oxotremorine and McN-A-343) or indirect agonists (phenylephrine) or antagonist (pirenzepine). Muscarinic M1 and M2 receptor expression was determined by radioligand [3H]-QNB binding assay and immunoblot. Results: Chagasic acute myocarditis was sustained by electrocardiographic signs and histopathological findings. Bradycardia induced by oxotremorine was significantly higher in healthy rats (HR) and the differences were enhanced by CsCl. In the absence of the agonist, CsCl induced a greater bradycardia in chagasic rats (ChR). In HR McN-A-343 induced tachycardia, however it induces bradycardia in the presence of a acetylcholinesterase inhibitor (neostigmine); no effects were observed in ChR. Pirenzepine induced a higher tachycardia in HR. Phenylephrine in the presence of pirenzepine induced a similar bradycardia in both groups, but recovery was faster in ChR. Muscarinic M1 and M2 receptor density was higher in HR. Conclusion: Muscarinic receptor expression and functionality are decreased in the acute Chagas disease that could impact the evolution and prognosis of the disease.

Effects of diabetic hyperlipidemia on bladder function and its possible mechanisms in a male rabbit model

To evaluate the effects of chronic hyperlipidemia on bladder function and its possible mechanisms and examine the functional and histological changes of bladder in a diabetic hyperlipidemic model. A total of 30 male New Zealand white rabbits (2.5 to 3.0 kg) were numbered sequentially and randomly divided into 2 groups (hyperlipidemic group and control group). Then urodynamic test and detrusor strip contraction study were performed to assess bladder function.Histological studies of bladder and internal iliac arteries were performed with hematoxylin and eosin staining. The ultrastructure of bladder detrusor tissue was examined by transmission electron microscopy. Also the bladder tissue M2, 3-muscarinic receptor protein levels were examined by immunohistochemistry. After 12 weeks, the urodynamic test of the test group showed significantly increased non-voiding contractions (10/14 vs 1/13, P < 0.05) and decreased compliance ((2.16 ± 0.17) vs (4.18 ± 0.21) ml/cm H2O, P < 0.01) versus the control group. The functional experiments showed that carbachol-induced contractions significantly decreased in hyperlipidemic rabbits than those in controls. As to the relaxant actions of selective muscarinic receptor M3 antagonist solifenacin, the bladder strips of hyperlipidemic rabbits were significantly more sensitive than those of the controls.Histological examination showed thickened intima of internal iliac arteries, urothelial disruption and mild fibrosis in the hyperlipidemic rabbits. Transmission electron microscopy of hyperlipidemic bladder muscle tissues showed swollen mitochondrial damage. As to the immunohistochemical results, compared with the control group, the hyperlipidemic group showed significantly increased M3 receptor expression both in bladder urothelium (146 ± 14 vs 108 ± 9, P < 0.01) and smooth muscle layer (131 ± 17 vs 116 ± 15, P < 0.05) . And the expression was more obvious in urothelium layer. Involuntary detrusor overactivity with decreased bladder compliance and decreased contractions of detrusor are present in hyperlipidemic rabbits. And chronic bladder ischemia and increased M3 receptor expression may contribute to bladder dysfunction.

Changes of salivary functions in experimental periodontitis model rats

ObjectiveThis study was designed to investigate the mechanism of salivary dysfunction in an experimental periodontitis rat model and to examine the improvements in salivary secretion following treatment of the experimental periodontitis. MethodsIn the experimental periodontitis rat model, which included a unilateral ligature for 4 weeks around the second upper molar, several salivary functions were investigated. Changes in the salivary function were evaluated 4 weeks after removal of the ligature in some rats. ResultsThe periodontitis model showed significant reductions in the weight of the bilateral major salivary glands and pilocarpine-induced salivary secretion. The model also showed an increase in the number of apoptotic cells in bilateral salivary glands. According to Ca2+ imaging and Western blotting, there were no differences in the muscarine-induced intracellular Ca2+ mobilization in acinar cells or in the M3 receptor and AQP5 expression levels in the salivary glands between the sham and the periodontitis model. Following removal of the ligature, differences in the weights of salivary glands and pilocarpine-induced salivary secretion between the sham and the periodontitis model animals were not found. ConclusionThese results suggest that experimental periodontitis leads to hyposalivation and that relief from it improves salivary function. It is likely that lower levels of salivary secretion are caused by the decrease of functional acinar cells in salivary glands in the experimental periodontitis model, and the bilateral gland effects in the unilateral periodontitis model are caused by systemic rather than by local effects.

Muscarinic receptor subtype mRNA expression in the human prostate: association with age, pathological diagnosis, prostate size, or potentially interfering medications?

As the prostate abundantly expresses muscarinic receptors and antagonists for such receptors are increasingly used in the treatment of men with voiding function and large prostates, we have explored an association of the mRNA expression of human M1, M2, M3, M4, and M5 receptors in human prostate with patient age, prostate size, prostate-specific antigen level, pathological diagnosis, and concomitant medication. mRNA was isolated from prostate chips of 110 consecutive patients undergoing transurethral resection of the prostate for the treatment of benign prostatic hyperplasia or prostate cancer. Expression of each of the five muscarinic receptor subtype transcripts was assessed by real-time PCR and association with patient age, prostate size, prostate-specific antigen level, pathological diagnosis, and concomitant medication were explored. M1 and M4 receptors were the most and least prevalently expressed subtypes in the human prostate, respectively. M1 receptor mRNA expression was weakly but significantly associated with prostate size (r = 0.2494, p = 0.0451), but mRNA expression of none of the five subtypes was significantly associated with age, prostate-specific antigen level, pathological diagnosis (benign prostatic hyperplasia vs. prostate cancer), or concomitant medication (5α-reductase inhibitors, α1- or β-adrenoceptor antagonists). We conclude that human prostate muscarinic receptor subtype transcripts apparently undergo only a very limited regulation by a variety of physiological, pathophysiological, or treatment factors. In light of the growing use of muscarinic receptor antagonists in men with voiding dysfunction including those with large prostates, the functional role of the weak association between M1 receptor mRNA expression and prostate size merits further investigation.

Botulinum toxin A inhibits salivary secretion of rabbit submandibular gland

Botulinum toxin A (BTXA) has been used in several clinical trials to treat excessive glandular secretion; however, the precise mechanism of its action on the secretory function of salivary gland has not been fully elucidated. In this study, we aimed to investigate the effect of BTXA on secretion of submandibular gland in rabbits and to identify its mechanism of action on the secretory function of salivary gland. At 12 weeks after injection with 5 units of BTXA, we found a significant decrease in the saliva flow from submandibular glands, while the salivary amylase concentration increased. Morphological analysis revealed reduction in the size of acinar cells with intracellular accumulation of secretory granules that coalesced to form a large ovoid structure. Expression of M3-muscarinic acetylcholine receptor (M3 receptor) and aquaporin-5 (AQP5) mRNA decreased after BTXA treatment, and distribution of AQP5 in the apical membrane was reduced at 1, 2 and 4 weeks after BTXA injection. Furthermore, BTXA injection was found to induce apoptosis of acini. These results indicate that BTXA decreases the fluid secretion of submandibular glands and increases the concentration of amylase in saliva. Decreased expression of M3 receptor and AQP5, inhibition of AQP5 translocation, and cell apoptosis might involve in BTXA-reduced fluid secretion of submandibular glands.

Inhibitory effect of sanguinarine on PKC-CPI-17 pathway mediating by muscarinic receptors in dispersed intestinal smooth muscle cells

This study investigated the inhibitory effects of sanguinarine (SA) on PKC-CPI-17 pathway in rat intestinal smooth muscle cells (ISMC). Previous studies indicate that the inhibitory effects of SA on ISMC contraction are possibly mediated by the Ca2+ influx. ISMC was treated with 1μM SA for 24h remarkably inhibited the mRNA expression of m2 and m3 receptors. ISMC treated with 1 or 3μM SA for 30min significantly decreased the mRNA expression of PKC-δ, PKC-ε, PKC-η, and CPI-17. 1μM SA could markedly inhibit carbachol (CCh)-mediated increase PKC-δ, PKC-η, and CPI-17 mRNA but had no effect in PKC-ε.Treatment of ISMC with SA (1μM, 30min) caused a decrease in protein expression of PKC-δ. However, the expression of CPI-17 was significantly inhibited in a time-dependent manner. These results demonstrate that the inhibitory effect of SA is coupled with alteration of PKC-mediated signal transduction and intracellular Ca2+ concentration.