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.

Inter- and intra-specific differences in muscarinic acetylcholine receptor expression in the neural pathways for vocal learning in songbirds.

Acetylcholine receptors (AChRs) abound in the central nervous system of vertebrates. Muscarinic AChRs (mAChRs), a functional subclass of AChRs, mediate neuronal responses via intracellular signal transduction. They also play roles in sensorimotor coordination and motor skill learning by enhancing cortical plasticity. Learned birdsong is a complex motor skill acquired through sensorimotor coordination during a critical period. However, the functions of AChRs in the neural circuits for vocal learning and production remain largely unexplored. Here, we report the unique expression of mAChRs subunits (chrm2-5) in the song nuclei of zebra finches. The expression of excitatory subunits (chrm3 and chrm5) was downregulated in the song nuclei compared with the surrounding brain regions. In contrast, the expression of inhibitory mAChRs (chrm2 and chrm4) was upregulated in the premotor song nucleus HVC relative to the surrounding nidopallium. Chrm4 showed developmentally different expression in HVC during the critical period. Compared with chrm4, individual differences in chrm2 expression emerged in HVC early in the critical period. These individual differences in chrm2 expression persisted despite testosterone administration or auditory deprivation, which altered the timing of song stabilization. Instead, the variability in chrm2 expression in HVC correlated with parental genetics. In addition, chrm2 expression in HVC exhibited species differences and individual variability among songbird species. These results suggest that mAChRs play an underappreciated role in the development of species and individual differences in song patterns by modulating the excitability of HVC neurons, providing a potential insight into the gating of auditory responses in HVC neurons.

Muscarinic type-1 receptors contribute to IK,ACh in human atrial cardiomyocytes and are upregulated in patients with chronic atrial fibrillation.

BACKGROUND: Basal and acetylcholine-gated inward-rectifier K+-currents (IK1 and IK,ACh, respectively) are altered in atrial fibrillation (AF). Gi-protein-coupled muscarinic (M) receptors type-2 are considered the predominant receptors activating IK,ACh. Although a role for Gq-coupled non-M2-receptor subtypes has been suggested, the precise regulation of IK,ACh by multiple M-receptor subtypes in the human atrium is unknown. Here, we investigated M1-receptor-mediated IK,ACh regulation and its remodeling in chronic AF (cAF). METHODS AND RESULTS: M1-receptor mRNA and protein abundance were increased in atrial cardiomyocyte fractions and atrial homogenates from cAF patients, whereas M2-receptor levels were unchanged. The regulation of IK,ACh by M1-receptors was investigated in right-atrial cardiomyocytes using two applications of the M-receptor agonist carbachol (CCh, 2µM), with pharmacological interventions during the second application. CCh application produced a rapid current increase (Peak-IK,ACh), which declined to a quasi-steady-state level (Qss-IK,ACh). In sinus rhythm (Ctl) the selective M1-receptor antagonists pirenzepine (10nM) and muscarinic toxin-7 (MT-7, 10nM) significantly inhibited CCh-activated Peak-IK,ACh, whereas in cAF they significantly reduced both Peak- and Qss-IK,ACh, with no effects on basal inward-rectifier currents in either group. Conversely, the selective M1-receptor agonist McN-A-343 (100µM) induced a current similar to the CCh-activated current in Ctl atrial cardiomyocytes pretreated with pertussis toxin to inhibit M2-receptor-mediated Gi-protein signaling, which was abolished by MT-7. Computational modeling indicated that M1- and M2-receptors redundantly activate IK,ACh to abbreviate APD, albeit with predominant effects of M2-receptors. CONCLUSION: Our data suggest that Gq-coupled M1-receptors also regulate human atrial IK,ACh and that their relative contribution to IK,ACh activation is increased in cAF patients. We provide novel insights about the role of non-M2-receptors in human atrial cardiomyocytes, which may have important implications for understanding AF pathophysiology.

An improved, high-efficiency assay for assessing serum anticholinergic activity using cultured cells stably expressing M1 receptors.

Assessments of total anticholinergic activity (SAA) in serum are of considerable interest for its potential involvement in cognitive impairment associated with polydrug states in the elderly and other populations. Such estimations have been based on the displacement of radioligand binding in rat brain tissues. The validity of such measurements has been questioned, as a potentially distorting effect of large serum proteins was identified. We sought to develop a modified assay that would be more efficient and free of this potential confound. Cultured CHO cells stably expressing M1 receptors M1WT3 were used. Binding of 3H-radioligands was conducted in 96-well plates and tested in serum containing known amounts of anticholinergic medications. Effects of endogenous serum proteins were assessed by pre-assay filtration and also by deproteinization with perchloric acid (PCA). Binding of [3H]quinuclidinyl benzilate ([3H]QNB) or [3H]N-methyl-scopolamine ([3H]NMS) to M1WT3 cells proved reliable and equally sensitive to varying concentrations of anticholinergic agents. In agreement with previous findings (Cox, Kwatra, Shetty, & Kwatra, 2009), filtration of proteins heavier than 50kDa essentially reduced SAA values to zero. In contrast, PCA preserved more than 70% of the binding seen untreated cell membranes. Cell-based assays also showed significant signal increases compared to the conventional rat brain-based protocol. Further advantages of the cell-based protocol described here include increased sensitivity and reliability, smaller amounts of radioligand needed, and higher throughput. PCA pretreatment eliminates potential artifacts attributable to serum proteins. This step, together with improvements in efficiency, should contribute significantly to the usefulness of the assay.

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.

Urtica dioica leaves modulates muscarinic cholinergic system in the hippocampus of streptozotocin-induced diabetic mice.

Diabetes mellitus is a chronic metabolic disorder and has been associated with cognitive dysfunction. In our earlier study, chronic Urtica dioica (UD) treatment significantly ameliorated diabetes induced associative and spatial memory deficit in mice. The present study was designed to explore the effect of UD leaves extract on muscarinic cholinergic system, which has long been known to be involved in cognition. Streptozotocin (STZ) (50 mg/kg, i.p., consecutively for 5 days) was used to induce diabetes followed by treatment with UD extract (50 mg/kg, oral) or rosiglitazone (5 mg/kg, oral) for 8 weeks. STZ-induced diabetic mice showed significant reduction in hippocampal muscarinic acetylcholine receptor-1 and choline acetyltransferase expressions. Chronic diabetes significantly up-regulated the protein expression of acetylcholinesterase associated with oxidative stress in hippocampus. Besides, STZ-induced diabetic mice showed hypolocomotion with up-regulation of muscarinic acetylcholine receptor-4 expression in striatum. Chronic UD treatment significantly attenuated the cholinergic dysfunction and oxidative stress in the hippocampus of diabetic mice. UD had no effect on locomotor activity and muscarinic acetylcholine receptor-4 expression in striatum. In conclusion, UD leaves extract has potential to reverse diabetes mediated alteration in muscarinic cholinergic system in hippocampus and thereby improve memory functions.

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.

Expression of cholinergic, insulin, vitamin D receptors and GLUT 3 in the brainstem of streptozotocin induced diabetic rats: effect of treatment with vitamin D3.

Complications arising from diabetes mellitus include cognitive deficits, neurophysiological and structural changes in the brain. The current study investigated the expression of cholinergic, insulin, Vitamin D receptor and GLUT 3 in the brainstem of streptozotocin-induced diabetic rats. Radioreceptor binding assays and gene expression were done in the brainstem of male Wistar rats. Our results showed that B(max) of total muscarinic, muscarinic M3 receptors was increased and muscarinic M1 receptor was decreased in diabetic rats compared to control. A significant increase in gene expression of muscarinic M3, α7 nicotinic acetylcholine, insulin, Vitamin D3 receptors, acetylcholine esterase, choline acetyl transferase and GLUT 3 were observed in the brainstem of diabetic rats. Immunohistochemistry studies of muscarinic M1, M3 and α7 nicotinic acetylcholine receptors confirmed the gene expression at protein level. Vitamin D3 and insulin treatment reversed diabetes-induced alterations to near control. This study provides an evidence that diabetes can alter the expression of cholinergic, insulin, Vitamin D receptors and GLUT 3 in brainstem. We found that Vitamin D3 treatment could modulate the Vitamin D receptors and plays a pivotal role in maintaining the glucose transport and expressional level of cholinergic receptors in the brainstem of diabetic rats. Thus, our results suggest a therapeutic role of Vitamin D3 in managing neurological disorders associated with diabetes.

The snake venom peptide Bj-PRO-7a is a M1 muscarinic acetylcholine receptor agonist.

Proline-rich peptides from Bothrops jararaca venom (Bj-PRO) were characterized based on the capability to inhibit the somatic angiotensin-converting enzyme. The pharmacological action of these peptides resulted in the development of Captopril, one of the best examples of a target-driven drug discovery for treatment of hypertension. However, biochemical and biological properties of Bj-PROs were not completely elucidated yet, and many recent studies have suggested that their activity relies on angiotensin-converting enzyme-independent mechanisms. Here, we show that Bj-PRO-7a (

Expression of muscarinic M1 and M2 receptors in the anterior cingulate cortex associated with neuropathic pain.

The anterior cingulate cortex (ACC) and muscarinic receptors modulate pain. This study investigates changes in the expression of muscarinic-1 and -2 receptors (M1R, M2R) in rats' ACC (cg1-rostral- and cg2-caudal) using a model of neuropathic pain by denervation, measured as autotomy score (AS) for 8 days. Changes were analysed with painful stimuli and with scopolamine into the ACC prior to this scheme. We used reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence to determine M1R and M2R's mRNA and protein levels, respectively. Animals were divided in low, medium and high AS groups. Cg1 showed decreased mRNA levels for both M1R and M2R in the low AS group, as opposed to an increased expression in the medium and high AS groups. Both receptors correlated positively with AS in these groups. In the scopolamine-treated animals there was an increase in mRNA levels for both receptors in cg1, whereas in cg2, mRNA levels of M1R decreased in all the AS and scopolamine groups. The increased M2R mRNA in cg2 correlated with AS in the low, medium and high AS groups whereas all the scopolamine groups showed an increase. Immunoreactivity of the M2R in cg1 decreased in the medium AS group in comparison to controls but scopolamine treatment produced an increase in the medium scopolamine AS group compared to the medium AS group. The M1R in cg1 and both receptors in cg2 showed no immunoreactivity changes. These results highlight the role of the M2R in cg1 related to the degree of autotomy.

Contrasting effects of allosteric and orthosteric agonists on m1 muscarinic acetylcholine receptor internalization and down-regulation.

A new class of subtype-selective muscarinic acetylcholine (mACh) receptor agonist that activates the receptor through interaction at a site distinct from the orthosteric acetylcholine binding site has been reported recently. Here, we have compared the effects of orthosteric (oxotremorine-M, arecoline, pilocarpine) and allosteric [4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine (AC-42); 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1)] agonists on M(1) mACh receptor internalization and down-regulation, as well as functional coupling in a Chinese hamster ovary (CHO) cell line. In contrast to full and partial orthosteric agonists, which cause significant receptor internalization and down-regulation, prolonged exposure to AC-42 did not significantly alter either cell-surface or total cellular M(1) mACh receptor expression. 77-LH-28-1, an AC-42 homolog, did cause some receptor internalization, but not down-regulation. The presence of atropine completely prevented the orthosteric agonist-induced adaptive changes in receptor populations; however, in contrast, the copresence of atropine and AC-42 significantly increased both cell-surface receptor and total M(1) mACh receptor expression. Maximal phosphoinositide hydrolysis responses to the partial agonist arecoline were similar in CHO-M(1) cells pretreated for 24 h with either AC-42 or vehicle; in contrast, these responses were markedly reduced when cells were pretreated with oxotremorine-M or pilocarpine. These data indicate that, whereas AC-42 binding to the M(1) mACh receptor can initiate signal transduction, the AC-42-liganded receptor is resistant to the usual mechanisms regulating receptor internalization and down-regulation. In addition, our data suggest unusual interactions between allosteric agonists and orthosteric antagonists to regulate cell-surface and total cellular receptor expression.

The muscarinic M1 receptor activates Nrf2 through a signaling cascade that involves protein kinase C and inhibition of GSK-3beta: connecting neurotransmission with neuroprotection.

In this study, we provide evidence that the muscarinic M1 receptor targets NF-E2-related factor-2 (Nrf2), a transcription factor that regulates the expression of genes containing antioxidant response elements (AREs) in their promoters and that collectively constitute the phase II antioxidant response. In hippocampal primary and cerebellar granule neuron cultures expressing endogenous M1 receptor, carbachol increased the levels of a prototypical phase II antioxidant enzyme, heme oxygenase-1. Moreover, in a heterologous system, based on lentiviral expression of M1 receptor in PC12 pheochromocytoma cells, we found that M1 increased total and nuclear Nrf2 protein levels and heme oxygenase-1 messenger RNA and protein levels. Luciferase reporter constructs for AREs and the use of two inhibitors of protein kinase C (PKC), chelerythrine and 2-aminoethyl diphenylborinate, or transfection with relevant expression vectors allowed us to identify Galphaq, phospholipase C-beta and the classical PKC-gamma isoenzyme, as responsible for the regulation of Nrf2. A PKC-insensitive Nrf2S40A single-point mutant partially channeled M1 signaling to AREs, therefore suggesting the participation of additional intermediates. Inhibition of glycogen synthase kinase-3beta (GSK-3beta) augmented M1-dependent activation of AREs while a PKC-insensitive mutant of GSK-3beta (GSK-3beta-Delta9) blocked this effect and prevented M1-induced accumulation of Nrf2 in the nucleus. Our results demonstrate a previously unidentified role of the Galphaq/phospholipase C-beta/PKC/GSK-3beta axis in regulation of Nrf2 by M1. Such role provides additional conceptual support for the use of cholinemimetics in the treatment of pathologies that, like Alzheimer's disease, require a reinforcement of the cell antioxidant capacity.

Protein kinase C regulates the expression of M1 receptors and BDNF in rat retinal cells.

Protein kinase C (PKC) plays a key role in cellular events including proliferation, survival and differentiation. Our previous study showed the effect of phorbol 12-myristate 13-acetate (PMA), a PKC activator, inducing a decrease in retinal cells proliferation. This effect was mediated by muscarinic type 1 receptors (M1) activation and brain derived neurotrophic factor (BDNF) treatment also induced a decrease in cell proliferation. Based on these results we analyzed the expression of either M1 receptors or BDNF following PMA treatment of retinal cell cultures. Our data demonstrated that PMA induced a decrease in both protein expressions after 48 h in culture. However, after 45 min, PMA induced a transient increase in BDNF expression and a decrease in M1 receptors expression. Analyzing the expression of M1 receptors and BDNF during the postnatal development in vivo, we observed a decrease in both proteins. Taken together our results suggest the involvement of PKC in the control of M1 expression in retinal cells.

Chronic treatment with simvastatin upregulates muscarinic M1/4 receptor binding in the rat brain.

Statins are increasingly being used for the treatment of a variety of conditions beyond their original indication for cholesterol lowering. We previously reported that simvastatin affected the dopaminergic system in the rat brain. This study aims to investigate regional changes of muscarinic M1/4 receptors in the rat brain after 4-week administration of simvastatin (1 or 10 mg/kg/day). M1/4 receptor distribution and alterations in the post-mortem rat brain were detected by [(3)H]pirenzepine binding autoradiography. Simvastatin (1 mg/kg/day) increased [(3)H]pirenzepine binding, predominantly in the prefrontal cortex (171%, P<0.001), primary motor cortex (153%, P=0.001), cingulate cortex (109%, P<0.001), hippocampus (138%, P<0.001), caudate putamen (122%, P=0.002) and nucleus accumbens (170%, P<0.001) compared with controls; while lower but still significant increases of [(3)H]pirenzepine binding were observed in the examined regions following simvastatin (10 mg/kg/day) treatment. Our results also provide strong evidence that chronic simvastatin administration, especially at a low dosage, up-regulates M1/4 receptor binding, which is likely to be independent of its muscarinic agonist-like effect. Alterations in [(3)H]pirenzepine binding in the examined brain areas may represent the specific regions that mediate the clinical effects of simvastatin treatment on cognition and memory via the muscarinic cholinergic system. These findings contribute to a better understanding of the critical roles of simvastatin in treating neurodegenerative disorders, via muscarinic receptors.

Decreased muscarinic M1 receptor gene expression in the hypothalamus, brainstem, and pancreatic islets of streptozotocin-induced diabetic rats.

The brain neurotransmitters' receptor activity and hormonal pathways control many physiological functions in the body. Acetylcholine (ACh), a major neurotransmitter from autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The objective of the present study was to investigate the changes in the total muscarinic and muscarinic M1 receptor ([(3)H]quinuclidinyl benzilate; QNB) binding and gene expression in the hypothalamus, brainstem, and pancreatic islets of streptozotocin (STZ)-induced diabetic and insulin-treated diabetic rats. In the hypothalamus and brainstem, total muscarinic receptor numbers were increased in diabetic rats with increase in affinity. Hypothalamic and brainstem muscarinic M1 receptors number were decreased in STZ diabetic rats with increase in affinity. In the pancreatic islets, muscarinic M1 receptors of diabetic rats were decreased, with a decrease in affinity. In all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR data also showed a decrease in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. Thus our results suggest that insulin modulates binding parameters and gene expression of total and muscarinic M1 receptors.

Effects of antipsychotic medication on muscarinic M1 receptor mRNA expression in the rat brain.

Alterations in muscarinic M1 receptor protein and mRNA expression have been revealed in post-mortem brains of schizophrenia patients. Most patients had been treated with antipsychotics, so medication effects cannot be excluded as a possible explanation for these results. With in situ hybridization, this study investigated M1 receptor mRNA expression in rats treated with the typical antipsychotic haloperidol (0.3 mg/kg/day) and the atypical antipsychotics olanzapine (1.5 mg/kg/day) and aripiprazole (2.25 mg/kg/day) for 1 or 12 weeks. Compared with the control group, haloperidol significantly increased (approximately 13-21%, P < 0.05) M1 mRNA expression in the CA1, CA2, and CA3 regions of the hippocampus after both 1 and 12 weeks of treatment, and it also increased (approximately 17%, P < 0.01) M1 mRNA expression in the substantia nigra compacta after 1 week of treatment. Olanzapine significantly increased (14-22%, P < 0.05) M1 mRNA expression in the hippocampus (CA1, CA2, and CA3) and substantia nigra compacta after 12 weeks of treatment, but not after 1 week. Aripiprazole significantly increased (17%, P < 0.01) M1 mRNA expression in the hippocampus (CA1) after both 1 and 12 week treatments and increased (12%, P < 0.05) M1 mRNA expression in the nucleus accumbens after 1 week of treatment. Despite their different affinities for muscarinic M1 receptors, all three antipsychotic medications induced a similar trend of change in M1 mRNA expression in selected brain regions. These data suggest that the decreased M1 receptor protein and mRNA expression observed in schizophrenia patients is unlikely to be a consequence of drug treatments and implicates muscarinic M1 receptors in the pharmacotherapy of the disease.

Infection with the cestode Hymenolepis diminuta induces changes in acetylcholine metabolism and muscarinic receptor mRNA expression in the rat jejunum.

Total and neuron-specific uptake of [3H] choline into smooth muscle/myenteric plexus (SM/MP) preparations from the jejunum of rats infected with five Hymenolepis diminuta for 30 days compared to uninfected rats was significantly increased, as was choline acetyltransferase activity and acetylcholine biosynthesis. Although acetylcholinesterase and total cholinesterase activity levels in SM/MP preparations from infected rats were not significantly different from uninfected animals, pseudocholinesterase activity was significantly elevated in infected rats. Infection resulted in a significant elevation in the relative expression of muscarinic 2 (M2) receptor mRNA in jejunum compared to uninfected rats. Conversely, in rats infected with 50 worms for 30 days, the relative expression of muscarinic 1 (M1) receptor mRNA in the jejunum was significantly depressed, while the expression of M2 receptor mRNA was not significantly different from that in five worm infections. The relative expression of muscarinic 3 receptor mRNA was unaffected by infection. The present study shows that infection of rats with low numbers of an enteric cestode leads to a significant modulation of the cholinergic components of the myenteric plexus and M2 receptor mRNA, and that large number of worms result in suppression in the relative expression of M1 receptor mRNA.

High level stable expression of pharmacologically active human M1-M5 muscarinic receptor subtypes in mammalian cells.

cDNAs encoding for five mAChR subtypes (M1-M5) were cloned under different promoters in various eukaryotic vectors and each subtype was expressed in different mammalian cell lines. CHO-K1 cell line was the best for generating stable cell lines expressing muscarinic receptors. Immunofluorescence and flow cytometry revealed that expression of M1-M5 was primarily localized on the cell membrane. Western blotting and radio-ligand binding studies revealed that expression of each receptor was stable at higher passages.

Cholinergically stimulated gastric acid secretion is mediated by M(3) and M(5) but not M(1) muscarinic acetylcholine receptors in mice.

Muscarinic acetylcholine receptors play an important role in the regulation of gastric acid secretion stimulated by acetylcholine; nonetheless, the precise role of each receptor subtype (M(1)-M(5)) remains unclear. This study examined the involvement of M(1), M(3), and M(5) receptors in cholinergic regulation of acid secretion using muscarinic receptor knockout (KO) mice. Gastric acid secretion was measured in both mice subjected to acute gastric fistula production under urethane anesthesia and conscious mice that had previously undergone pylorus ligation. M(3) KO mice exhibited impaired gastric acid secretion in response to carbachol. Unexpectedly, M(1) KO mice exhibited normal intragastric pH, serum gastrin and mucosal histamine levels, and gastric acid secretion stimulated by carbachol, histamine, and gastrin. Pirenzepine, known as an M(1)-receptor antagonist, inhibited carbachol-stimulated gastric acid secretion in a dose-dependent manner in M(1) KO mice as well as in wild-type (WT) mice, suggesting that the inhibitory effect of pirenzepine on gastric acid secretion is independent of M(1)-receptor antagonism. Notably, M(5) KO mice exhibited both significantly lower carbachol-stimulated gastric acid secretion and histamine-secretory responses to carbachol compared with WT mice. RT-PCR analysis revealed M(5)-mRNA expression in the stomach, but not in either the fundic or antral mucosa. Consequently, cholinergic stimulation of gastric acid secretion is clearly mediated by M(3) (on parietal cells) and M(5) receptors (conceivably in the submucosal plexus), but not M(1) receptors.