Pancreas-specific CHRM3 activation causes pancreatitis in mice.

Hyperstimulation of the cholecystokinin 1 receptor (CCK1R), a G protein-coupled receptor (GPCR), in pancreatic acinar cells is commonly used to induce pancreatitis in rodents. Human pancreatic acinar cells lack CCK1R but express cholinergic receptor muscarinic 3 (M3R), another GPCR. To test whether M3R activation is involved in pancreatitis, a mutant M3R was conditionally expressed in pancreatic acinar cells in mice. This mutant receptor loses responsiveness to its native ligand, acetylcholine, but can be activated by an inert small molecule, clozapine-N-oxide (CNO). Intracellular calcium and amylase were elicited by CNO in pancreatic acinar cells isolated from mutant M3R mice but not WT mice. Similarly, acute pancreatitis (AP) could be induced by a single injection of CNO in the transgenic mice but not WT mice. Compared with the cerulein-induced AP, CNO caused more widespread acinar cell death and inflammation. Furthermore, chronic pancreatitis developed at 4 weeks after 3 episodes of CNO-induced AP. In contrast, in mice with 3 recurrent episodes of cerulein-included AP, pancreas histology was restored in 4 weeks. Furthermore, the M3R antagonist ameliorated the severity of cerulein-induced AP in WT mice. We conclude that M3R activation can cause the pathogenesis of pancreatitis. This model may provide an alternative approach for pancreatitis research.

Muscarinic receptors promote castration-resistant growth of prostate cancer through a FAK-YAP signaling axis.

Prostate cancer (PCa) innervation contributes to the progression of PCa. However, the precise impact of innervation on PCa cells is still poorly understood. By focusing on muscarinic receptors, which are activated by the nerve-derived neurotransmitter acetylcholine, we show that muscarinic receptors 1 and 3 (m1 and m3) are highly expressed in PCa clinical specimens compared with all other cancer types, and that amplification or gain of their corresponding encoding genes (CHRM1 and CHRM3, respectively) represent a worse prognostic factor for PCa progression free survival. Moreover, m1 and m3 gene gain or amplification is frequent in castration-resistant PCa (CRPC) compared with hormone-sensitive PCa (HSPC) specimens. This was reflected in HSPC-derived cells, which show aberrantly high expression of m1 and m3 under androgen deprivation mimicking castration and androgen receptor inhibition. We also show that pharmacological activation of m1 and m3 signaling is sufficient to induce the castration-resistant growth of PCa cells. Mechanistically, we found that m1 and m3 stimulation induces YAP activation through FAK, whose encoding gene, PTK2 is frequently amplified in CRPC cases. Pharmacological inhibition of FAK and knockdown of YAP abolished m1 and m3-induced castration-resistant growth of PCa cells. Our findings provide novel therapeutic opportunities for muscarinic-signal-driven CRPC progression by targeting the FAK-YAP signaling axis.

Cholinergic innervation of principal neurons in the cochlear nucleus of the Mongolian gerbil.

Principal neurons in the ventral cochlear nucleus (VCN) receive powerful ascending excitation and pass on the auditory information with exquisite temporal fidelity. Despite being dominated by ascending inputs, the VCN also receives descending cholinergic connections from olivocochlear neurons and from higher regions in the pontomesencephalic tegmentum. In Mongolian gerbils, acetylcholine acts as an excitatory and modulatory neurotransmitter on VCN neurons, but the anatomical structure of cholinergic innervation of gerbil VCN is not well described. We applied fluorescent immunohistochemical staining to elucidate the development and the cellular localization of presynaptic and postsynaptic components of the cholinergic system in the VCN of the Mongolian gerbil. We found that cholinergic fibers (stained with antibodies against the vesicular acetylcholine transporter) were present before hearing onset at P5, but innervation density increased in animals after P10. Early in development cholinergic fibers invaded the VCN from the medial side, spread along the perimeter and finally innervated all parts of the nucleus only after the onset of hearing. Cholinergic fibers ran in a rostro-caudal direction within the nucleus and formed en-passant swellings in the neuropil between principal neurons. Nicotinic and muscarinic receptors were expressed differentially in the VCN, with nicotinic receptors being mostly expressed in dendritic areas while muscarinic receptors were located predominantly in somatic membranes. These anatomical data support physiological indications that cholinergic innervation plays a role in modulating information processing in the cochlear nucleus.

Effect of muscarinic receptors agonist in the rat model of coronary heart disease: A potential therapeutic target in cardiovascular diseases.

Cardiac hypertrophy is a one of common type of CHD, responsible for cardiac mortality worldwide. The present study designed to investigate the effect of muscarinic receptors agonist in the rat model of cardiac hypertrophy. A total of 30 male adult Wistar rats having body weight 300-400 gram were equally distributed in two groups (Test group: Rats with Angiotensin II + M3 receptor agonist [acetylcholine]; Reference group: Rats with cardiac hypertrophy induced by Angiotensin II). Rat model of cardiac hypertrophy were induced by Angiotensin II. Effect of M3 receptor agonist on cardiac hypertrophy was evaluated by electrocardiography, hemodynamic and histological assessment. Also, expression of M3 receptor was analyzed using by real-time-PCR and Western blot analysis. Also, vital signs such as pulse rate, and blood pressure were measured. Echocardiographic related variable including ejection fraction were also assessed in both the groups. The results of this study showed acetylcholine attenuates the hypertrophic response triggered by Angiotensin II, by upregulation of M3 receptor. Upregulation of M3 receptor after administration of acetylcholine ameliorates hypertrophic responses induced by angiotensin II. Also acetylcholine treatment prevents Angiotensin II induced increase in level of ANP and ß-myosin, which are responsible for inducing cardiac hypertrophic responses. Moreover, acetylcholine ameliorates Angiotensin II induced cell enlargement by reducing the surface area of cells. Overall finding suggested that acetylcholine improves left ventricle hypertrophy and ejection fraction by activating M3 receptor in heart. The finding of this study gives the new vision to cardiovascular researchers to develop anti- hypertrophy therapy based on M3 receptor.

Cigarette smoke and non-neuronal cholinergic system in the airway epithelium of COPD patients.

Acetylcholine (ACh), synthesized by Choline Acetyl-Transferase (ChAT), exerts its physiological effects via mAChRM3 in epithelial cells. We hypothesized that cigarette smoke affects ChAT, ACh, and mAChRM3 expression in the airways from COPD patients promoting airway disease. ChAT, ACh, and mAChRM3 were assessed: "ex vivo" in the epithelium from central and distal airways of COPD patients, Healthy Smoker (S) and Healthy Subjects (C), and "in vitro" in bronchial epithelial cells stimulated with cigarette smoke extract (CSE). In central airways, mAChRM3, ChAT, and ACh immunoreactivity was significantly higher in the epithelium from S and COPD than in C subjects. mAChRM3, ChAT, and ACh score of immunoreactivity was high in the metaplastia area of COPD patients. mAChRM3/ChAT and ACh/ChAT co-localization of immunoreactivity was observed in the bronchial epithelium from COPD. In vitro, CSE stimulation significantly increased mAChRM3, ChAT, and ACh expression and mAChRM3/ChAT and ACh/ChAT co-localization in 16HBE and NHBE, and increased 16HBE proliferation. Cigarette smoke modifies the levels of mAChMR3, ChAT expression, and ACh production in bronchial epithelial cells from COPD patients. Non-neuronal components of cholinergic system may have a role in the mechanism of bronchial epithelial cell proliferation, promoting alteration of normal tissue, and of related pulmonary functions.

Regulation of molecular clock oscillations and phagocytic activity via muscarinic Ca2+ signaling in human retinal pigment epithelial cells.

Vertebrate eyes are known to contain circadian clocks, however, the intracellular mechanisms regulating the retinal clockwork remain largely unknown. To address this, we generated a cell line (hRPE-YC) from human retinal pigmental epithelium, which stably co-expressed reporters for molecular clock oscillations (Bmal1-luciferase) and intracellular Ca2+ concentrations (YC3.6). The hRPE-YC cells demonstrated circadian rhythms in Bmal1 transcription. Also, these cells represented circadian rhythms in Ca2+-spiking frequencies, which were canceled by dominant-negative Bmal1 transfections. The muscarinic agonist carbachol, but not photic stimulation, phase-shifted Bmal1 transcriptional rhythms with a type-1 phase response curve. This is consistent with significant M3 muscarinic receptor expression and little photo-sensor (Cry2 and Opn4) expression in these cells. Moreover, forskolin phase-shifted Bmal1 transcriptional rhythm with a type-0 phase response curve, in accordance with long-lasting CREB phosphorylation levels after forskolin exposure. Interestingly, the hRPE-YC cells demonstrated apparent circadian rhythms in phagocytic activities, which were abolished by carbachol or dominant-negative Bmal1 transfection. Because phagocytosis in RPE cells determines photoreceptor disc shedding, molecular clock oscillations and cytosolic Ca2+ signaling may be the driving forces for disc-shedding rhythms known in various vertebrates. In conclusion, the present study provides a cellular model to understand molecular and intracellular signaling mechanisms underlying human retinal circadian clocks.

Differential receptor dependencies: expression and significance of muscarinic M1 receptors in the biology of prostate cancer.

Recent reports on acetylcholine muscarinic receptor subtype 3 (CHRM3) have shown its growth-promoting role in prostate cancer. Additional studies report the proliferative effect of the cholinergic agonist carbachol on prostate cancer by its agonistic action on CHRM3. This study shows that the type 1 acetylcholine muscarinic receptor (CHRM1) contributes toward the proliferation and growth of prostate cancer. We used growth and cytotoxic assays, the prostate cancer microarray database and CHRM downstream pathways' homology of CHRM subtypes to uncover multiple signals leading to the growth of prostate cancer. Growth assays showed that pilocarpine stimulates the proliferation of prostate cancer. Moreover, it shows that carbachol exerts an additional agonistic action on nicotinic cholinergic receptor of prostate cancer cells that can be blocked by tubocurarine. With the use of selective CHRM1 antagonists such as pirenzepine and dicyclomine, a considerable inhibition of proliferation of prostate cancer cell lines was observed in dose ranging from 15-60 µg/ml of dicyclomine. The microarray database of prostate cancer shows a dominant expression of CHRM1 in prostate cancer compared with other cholinergic subtypes. The bioinformatics of prostate cancer and CHRM pathways show that the downstream signalling include PIP3-AKT-CaM-mediated growth in LNCaP and PC3 cells. Our study suggests that antagonism of CHRM1 may be a potential therapeutic target against prostate cancer.

Effect of umbilical cord blood stem cells transplantation on bladder dysfunction induced by cerebral ischemia in rats.

OBJECTIVE: To demonstrate the effect of human umbilical cord blood-derived CD34+ cells on bladder dysfunction induced by cerebral ischemia in rats. MATERIALS AND METHODS: Female rats were subjected to either 60 minute middle cerebral artery occlusion (MCAO) or a sham operation. Rats were divided into four groups: sham operation, MCAO without treatment, infusion with 1×106 CD34+ cells 30 minutes before MCAO, and infusion with 1×106 CD34+ cells 3 hours after MCAO. Bladder function was analyzed by cystometry at 1 day, 3 days, and 7 days after MCAO. Expressions of nerve growth factor (NGF), M2 and M3 muscarinic receptors were measured by immunohistochemistry and real time polymerase chain reaction. RESULTS: Cystometric results showed that, following MCAO, rats have a significant increase in peak voiding pressure and residual volume. Conversely, there is a significant decrease in voided volumes and intercontraction intervals. Cystometric variables after pre- and postischemic CD34+ treatment nearly returned to levels found in sham-operated rats. The expression of bladder NGF and M3 was decreased after MCAO, but significantly increased following preischemic CD34+ treatment. There was decreased expression of bladder M2 mRNA despite an increased level of M2 immunoreactivity at 3 days and 7 days after MCAO. Expression of bladder M2 immunoreactivity and mRNA nearly returned to sham group levels after preischemic CD34+ treatment. CONCLUSION: Bladder dysfunction in a rat model caused by MCAO may be restored to normal micturition by treatment with human umbilical cord blood-derived CD34+ cells and may be related to the expressions of NGF, M2, and M3 in the bladder.

Urinary Retention, Incontinence, and Dysregulation of Muscarinic Receptors in Male Mice Lacking Mras.

Here we show that male, but not female mice lacking expression of the GTPase M-Ras developed urinary retention with distention of the bladder that exacerbated with age but occurred in the absence of obvious anatomical outlet obstruction. There were changes in detrusor morphology in Mras-/- males: Smooth muscle tissue, which exhibited a compact organization in WT mice, appeared disorganized and became increasingly 'layered' with age in Mras-/- males, but was not fibrotic. Bladder tissue near the apex of bladders of Mras-/- males exhibited hypercontractility in response to the cholinergic agonist carbachol in in vitro, while responses in Mras-/- females were normal. In addition, spontaneous phasic contractions of detrusors from Mras-/- males were increased, and Mras-/- males exhibited urinary incontinence. We found that expression of the muscarinic M2 and M3 receptors that mediate the cholinergic contractile stimuli of the detrusor muscle was dysregulated in both Mras-/- males and females, although only males exhibited a urinary phenotype. Elevated expression of M2R in young males lacking M-Ras and failure to upregulate M3R with age resulted in significantly lower ratios of M3R/M2R expression that correlated with the bladder abnormalities. Our data suggests that M-Ras and M3R are functionally linked and that M-Ras is an important regulator of male bladder control in mice. Our observations also support the notion that bladder control is sexually dimorphic and is regulated through mechanisms that are largely independent of acetylcholine signaling in female mice.

Activation of M3 muscarinic receptor by acetylcholine promotes non-small cell lung cancer cell proliferation and invasion via EGFR/PI3K/AKT pathway.

Acetylcholine (ACh), which can be synthesized and secreted by cancer cells, has been reported to play an important role in tumor progression. ACh acts its role through activation of its receptors, muscarinic receptor (mAChR), and nicotinic receptor (nAChR). As a member of mAChR, M3 muscarinic receptor (M3R) is often highly expressed in many cancers. Activation of M3R by ACh participates in the proliferation, differentiation, transformation, and carcinogenesis of cancer. However, the effect of M3R activation on non-small cell lung cancer (NSCLC) remains unclear. Here, our study found that ACh dose-dependently promoted the proliferation, invasion, and migration of NSCLC cells. After silencing of M3R, the biological functions of ACh in NSCLC cells were greatly attenuated. Furthermore, ACh stimulation increased the production of IL-8 and time-dependently induced the activation of EGFR, PI3K, and AKT through M3R. In addition, ACh stimulated the activation of PI3K and AKT via EGFR activity, and blocking of PI3K/AKT pathway by special inhibitor LY294002 suppressed the ACh-mediated proliferation, invasion, and migration of NSCLC cells. Taken together, these findings indicate that activation of M3R by ACh enhances the proliferation, invasion, and migration of NSCLC cells. ACh-induced activation of EGFR/PI3K/AKT pathway and subsequent IL-8 upregulation may be one of the important mechanisms of M3R function. Thus, M3R could be a potential therapeutic target for the treatment of NSCLC.

Laser-capture microdissection for analysis of cell type-specific gene expression of muscarinic receptor subtypes in the rat bladder with cyclophosphamide-induced cystitis.

PURPOSE: This study examined whether the laser-capture microdissection (LCM) method can achieve separation of urothelial cells from detrusor cells or superficial urothelial cells from intermediate/basal urothelial cells, using α-smooth muscle actin (SMA) and cytokeratin 20 (CK20). In addition, we investigated the changes in expression of muscarinic receptors in laser-captured urothelial and detrusor cells in rats with chronic cystitis. METHODS: Female SD rats were injected with cyclophosphamide (75 mg/kg) intraperitoneally at day 1, 4, 7 and 10 to induce chronic cystitis. Saline was injected in the same protocol for controls. Bladder specimens were cut at 8 µm thickness, fixed in 70% ethanol and lightly stained by hematoxylin and eosin, and then superficial urothelium, intermediate/basal urothelium and detrusor muscles were laser-captured separately. Real-time PCR was performed to examine expressions of α-SMA, CK20, muscarinic 2 receptors (M2R) and muscarinic 3 receptors (M3R). RESULTS: The expression of α-SMA mRNA in detrusor muscle cells was 200 times higher than that in urothelial cells in controls. CK20 mRNA expression in apical urothelial cells was 55 times more than that in detrusor muscle and four times more than that in intermediate/basal urothelial cells. Expressions of M2R and M3R mRNA were increased in urothelial cells and decreased in detrusor muscles following chronic cystitis. CONCLUSIONS: The LCM could be useful for tissue collection of detrusor muscle and different layers of urothelial cells with minimal contamination of other cell types, and cell type-specific changes in molecular expression could accurately be analyzed. Increased expression of urothelial MR might enhance urothelial-afferent interactions to induce bladder overactivity/pain conditions associated with bladder inflammation.

Role of the M3 muscarinic acetylcholine receptor subtype in murine ophthalmic arteries after endothelial removal.

PURPOSE: We tested the hypothesis that the M3 muscarinic acetylcholine receptor subtype mediates cholinergic responses in murine ophthalmic arteries after endothelial removal. METHODS: Muscarinic receptor gene expression was determined in ophthalmic arteries with intact and with removed endothelium using real-time PCR. To examine the role of the M3 receptor in mediating vascular responses, ophthalmic arteries from M3 receptor-deficient mice (M3R(-/-)) and respective wild-type controls were studied in vitro. Functional studies were performed in nonpreconstricted arteries with either intact or removed endothelium using video microscopy. RESULTS: In endothelium-intact ophthalmic arteries, mRNA for all five muscarinic receptor subtypes was detected, but M3 receptor mRNA was most abundant. In endothelium-removed ophthalmic arteries, M1, M2, and M3 receptors displayed similar mRNA expression levels, which were higher than those for M4 and M5 receptors. In functional studies, acetylcholine evoked vasoconstriction in endothelium-removed arteries from wild-type mice that was virtually abolished after incubation with the muscarinic receptor blocker atropine, indicative of the involvement of muscarinic receptors. In concentration-response experiments, acetylcholine and carbachol concentration-dependently constricted endothelium-removed ophthalmic arteries from wild-type mice, but produced only negligible responses in arteries from M3R(-/-) mice. In contrast, acetylcholine concentration-dependently dilated ophthalmic arteries with intact endothelium from wild-type mice, but not from M3R(-/-) mice. Responses to the nitric oxide donor nitroprusside and to KCl did not differ between ophthalmic arteries from wild-type and M3R(-/-) mice, neither in endothelium-intact nor in endothelium-removed arteries. CONCLUSIONS: These findings provide evidence that in murine ophthalmic arteries the muscarinic M3 receptor subtype mediates cholinergic endothelium-dependent vasodilation and endothelium-independent vasoconstriction.

High expression of M3 muscarinic acetylcholine receptor is a novel biomarker of poor prognostic in patients with non-small cell lung cancer.

We assessed the expression of M3 receptor in non-small cell lung cancer (NSCLC) and determined its relationship with clinicopathological features and its impact on patient outcome. Specimens from 192 patients with NSCLC were investigated by immunohistochemistry for M3 receptor and Ki67 expression. Correlation between the expression of M3 receptor and Ki67 and various clinicopathological features of NSCLC patients was analyzed. We found that M3 receptor expression was gradually elevated from normal to metaplasia/dysplasia tissues to cancer tissues. Furthermore, there was a similar trend for Ki67 expression. Statistical analysis revealed that M3 receptor expression in tumor cells were correlated significantly with stage (P < 0.0001), histology type (P = 0.0003), Ki67 expression (P < 0.0001), tumor size (P < 0.0001), lymph node status (P < 0.0001), LVS invasion (P = 0.0002), and histology grade (P < 0.0001). Patients with M3 receptor high expression showed far lower disease-free survival (DFS) and overall survival (OS) rates than those with M3 receptor low expression. Multivariate Cox regression analysis demonstrated that high M3 receptor expression was an independent prognostic factor for both DFS and OS. High M3 receptor expression correlates with poor survival in NSCLC patients. M3 receptor expression may be related with tumor progression in NSCLC, indicating that M3 receptor may be a novel antineoplastic target in the future.

Botulinum toxin A detrusor injections reduce postsynaptic muscular M2, M3, P2X2, and P2X3 receptors in children and adolescents who have neurogenic detrusor overactivity: a single-blind study.

OBJECTIVE: To analyze the effect of OnabotulinumtoxinA detrusor injections on postsynaptic muscular receptors in children and adolescents with neurogenic detrusor overactivity. MATERIALS AND METHODS: A bladder augmentation became necessary in 10 children and adolescents (7 males, 3 females; median age, 12 years) who had neurogenic detrusor overactivity. Seven had previously received 1 to 8 (average 3.86) OnabotulinumtoxinA detrusor injections, but their detrusor pressure could not be maintained at tolerable levels because of low-compliance bladder. The last injection session had been completed an average of 3 months (range, 1.5-3.5 months) previously. Three patients had never received that therapy and were considered controls. On the bladder dome resections, a specific receptor analysis (muscarinic M2 and M3 and purinergic P2X1, P2X2, and P2X3) was performed with confocal immunofluorescence, and nerve fiber density was analyzed with light-microscopic 3,3'-diaminobenzidine-immunohistochemical staining. RESULTS: Receptor analysis showed a downregulation of all examined receptors after OnabotulinumtoxinA injections; the reductions in M2, M3, P2X2, and P2X3 receptors reached a significance level of P <.05 (Mann-Whitney test). The ratios of means (OnabotulinumtoxinA-to-control) were 0.26 for M2, 0.33 for M3, 0.35 for P2X1, 0.19 for P2X2, and 0.37 for P2X3. CONCLUSION: OnabotulinumtoxinA detrusor injections led to significant reductions in muscular M2, M3, P2X2, and P2X3 receptors. The reductions probably affect the generated force in the urinary bladder and could contribute to the clinically observed increase in residual urine.

Regulation of immediate-early gene transcription following activation of Gα(q)-coupled designer receptors.

G-protein coupled designer receptors that are specifically activated by designer drugs have been developed. Here, we have analyzed the regulation of gene transcription following activation of Gα(q)-coupled designer receptor (Rα(q)). Stimulation of human embryonic kidney (HEK) 293 cells expressing Rα(q) with clozapine-N-oxide (CNO), a pharmacologically inert compound, induced the expression of biologically active Egr-1, a zinc finger transcription factor. Expression of a dominant-negative mutant of the ternary complex factor (TCF) Elk-1, a key transcriptional regulator of serum response element (SRE)-driven gene transcription, prevented Egr-1 expression. Stimulation of Rα(q) with CNO increased the transcriptional activation potential of Elk-1 and enhanced transcription of an SRE regulated reporter gene. In addition, AP-1 transcriptional activity was significantly elevated. AP-1 activity was controlled by TCFs and c-Jun in cells expressing an activated Gα(q)-coupled designer receptor. CNO stimulation did not increase Egr-1 and AP-1 activity in neuroblastoma cells expressing endogenous M3 muscarinic acetylcholine receptors, indicating that CNO did not function as a ligand for these receptors. Rα(q) stimulation also increased the transcriptional activation potential of CREB and cAMP response controlled gene transcription. Pharmacological and genetic experiments revealed that the protein kinases Raf and ERK were essential to connect Rα(q) stimulation with enhanced Egr-1 and AP-1 controlled transcription. In contrast, MAP kinase phosphatase-1 functioned as a nuclear shut-off device of stimulus-transcription coupling. The fact that Rα(q) stimulation activates the transcription factors Egr-1, Elk-1, AP-1, and CREB indicates that regulation of gene transcription is an integral part of Gα(q)-coupled receptor signaling.

Neuroprotection of green tea catechins on surgical menopause-induced overactive bladder in a rat model.

OBJECTIVE: A rat model of ovariectomy-induced voiding dysfunction has been established, which mimicked the urge incontinence in postmenopausal women. Previous studies have identified strong anti-inflammatory/antioxidant properties of green tea and its associated polyphenols. The aim of this study was to evaluate whether the green tea extract, epigallocatechin gallate (EGCG), could prevent an ovariectomy-induced overactive bladder. METHODS: The study included 48 female Sprague-Dawley rats, which were divided into four groups. After bilateral ovariectomy during the following 6-month period, 12 rats received an intraperitoneal injection of saline, 24 rats received either a low-dose (1 µM kg(-1) d(-1)) or a high-dose (10 µM kg(-1) d(-1)) EGCG intraperitoneal injection. The sham group consisted of twelve rats that were not ovariectomized. In vivo isovolumetric cystometrograms were performed in all groups before the animals were euthanized. The immunofluorescence study used neurofilament stains to evaluate intramural nerve damage. Western immunoblots and real-time polymerase chain reaction were performed to determine M2 and M3 muscarinic cholinergic receptors (MChRs) at both protein and messenger RNA (mRNA) expressions. RESULTS: Long-term ovariectomy significantly increased non-voiding contractions, whereas treatment with EGCG significantly attenuated the frequency of non-voiding contractions. Ovariectomy significantly decreased the numbers of neurofilament and increased M2 and M3 MChR protein and mRNA expressions. Treatment with EGCG restored the amount of neurofilament staining and decreased M2 and M3 MChR protein and mRNA overexpressions. CONCLUSIONS: This study confirmed that ovary hormone deficiency induced overactive bladder dysfunction via intramural nerve damage and muscarinic receptor overexpression. EGCG prevented ovariectomy-induced bladder dysfunction through neuroprotective effects in a dose-dependent fashion.

Cholinergic and GABAergic receptor functional deficit in the hippocampus of insulin-induced hypoglycemic and streptozotocin-induced diabetic rats.

Neurotransmitter receptor functional regulation plays an important role in controlling the excitability and responsiveness of hippocampal neurons. Deregulation of its function is associated with seizure generation, motor deficits, and memory impairment. In the present study we investigated the changes in hippocampal cholinergic and GABA receptor binding and gene expression in insulin-induced hypoglycemic and streptozotocin-induced diabetic rats. Expression of cholinergic enzymes; acetylcholine esterase (AChE) and choline acetyltransferase (ChAT) upregulated and downregulated, respectively, in diabetic group, which was further exacerbated by hypoglycemia. Total muscarinic receptor, muscarinic M1, and GABA maximal binding (B(max)) significantly decreased in hypoglycemic and diabetic rats. In hypoglycemic group, the B(max) showed further decline compared with diabetes. Muscarinic M3 receptor B(max) and gene expression upregulated in hypoglycemic and diabetic group. Alpha7 nicotinic acetylcholine receptor (α7 nAChR) expression significantly downregulated in hypoglycemic and diabetic rats. Gene expression of glutamate decarboxylase (GAD), GABAAα1, and GABAB in hypoglycemic and diabetic rats downregulated, with more significant decrease in hypoglycemic group. Present findings show altered cholinergic, muscarinic, nicotinic receptor expression and thereby function. Decreased GABA receptor expression is associated with decline in GABAergic neurotransmission. Thus cholinergic receptor dysfunction and decreased GABAergic neuroprotective inhibitory function in the hippocampus of hypoglycemic and diabetic rats account for the increased vulnerability of hippocampus predisposing to neuronal damage, which is suggested to contribute to cognitive impairment and memory deficit reported in hypoglycemia and diabetes. Also, recurrent hypoglycemia in diabetes exacerbates the hippocampal dysfunction induced by diabetes, which has clinical significance in diabetes therapy.

Differential expression of muscarinic acetylcholine receptor subtypes in Jurkat cells and their signaling.

Muscarinic acetylcholine receptors expression and signaling in the human Jurkat T cell line were investigated. Semiquantitative real-time PCR and radioligand binding studies, using a wide set of antagonist compounds, showed the co-existence of M(3), M(4), and M(5) subtypes. Stimulation of these subpopulations caused a concentration and time- dependent activation of second messengers and ERK signaling pathways, with a major contribution of the M(3) subtype in a G(q/11)-mediated response. In addition, we found that T-cell stimulation leads to increased expression of M(3) and M(5) both at transcriptional and protein levels in a PLC/PKCθ dependent manner. Our data clarifies the functional role of AChR subtypes in Jurkat cells and pave the way to future studies on the potential cross-talk among these subpopulations and their regulation of T lymphocytes immune function.

Overproduction of human M3 muscarinic acetylcholine receptor: an approach toward structural studies.

Human M(3) muscarinic acetylcholine receptor (M3R), present in both the central and the peripheral nervous system, is involved in several neurodegenerative and autoimmune diseases. Recently, M3R overexpression has been suggested to play a role in certain forms of cancer, showing promise as a new potential pharmacological target. However, the lack of structural information hampered to develop a new potent selective and potent antagonist. We describe here different strategies for overexpressing functional M3R on the perspective of future biophysical studies. To achieve this goal, four tagged M3R genes were engineered and codon optimized. Different heterologous expression systems, including mammalian cells and viral transfection, were employed to overexpress M3R. Although codon optimization resulted in only twofold to threefold increase of M3R expression, we found that epitope tagging of the synthetic M3R, especially with hemagglutinin and Flag epitope tags, could improve M3R expression levels. On the other hand, viral transfection led to a yield of 27 pmol/mg protein that is the highest level reported so far for this receptor subtype in mammalian cells. Taking together several of the strategies used can help increasing M3R expression, not only to start purification efforts but also for secondary structural analysis trial and functional analyses.

The impact of simulated birth trauma and ovariectomy on the gene expression of detrusor muscarinic receptors in female rats.

INTRODUCTION AND HYPOTHESIS: This study aims to investigate the effects of simulated birth trauma and ovariectomy on detrusor muscarinic receptors (M2 and M3), urethral neuronal nitric oxide synthase (nNOS), and estrogen receptor beta (ER beta). METHODS: Forty primiparous rats were equally divided into five groups: group A--delivery, group B--delivery plus ovariectomy, group C--delivery plus balloon dilatation for 2 h, group D--delivery plus balloon dilatation for 4 h, and group E--delivery plus balloon dilatation for 2 h plus ovariectomy. The gene expression of M2, M3, nNOS, and ER beta were assessed by reverse transcription polymerase chain reaction. RESULTS: Significant decreases in mRNA expression of M2 receptors and nNOS (P < 0.05), and a significant increase in M3 mRNA expression (P < 0.05) were observed in groups D and E when compared with group A. CONCLUSIONS: Ovariectomy following birth trauma may synergistically impact the function of urinary tract, this being possibly related to the modification of the gene expression of muscarinic receptors.