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Muscarinic Receptor Subtypes (muscarinic + receptor_subtype)
Selected AbstractsDetection of muscarinic receptor subtypes in human urinary bladder mucosa: Age and gender-dependent modifications,,§NEUROUROLOGY AND URODYNAMICS, Issue 5 2008Nicola Arrighi Abstract Aims Muscarinic receptor subtypes expressed in the human urinary bladder mucosa were characterized, investigating whether there were gender-dependent differences and if aging could induce changes in their expression. Methods The study was carried out on 34 subjects, 22 men and 12 women, divided in four groups, based on gender and age. Gene expression was evaluated by quantitative RT-PCR. The Western blot was performed using the 4,12% NuPAGE Bis,Tris Gel System. Results The molecular expression of each subtype of the M1 receptor family was observed and it was not influenced either by gender or age. M2 receptor family transcripts revealed that both M2 and M4 were detected and that the M2 transcripts were modified by both gender and age. Indeed, M2 mRNA was lower in old rather than adult men (P,<,0.05), but higher in rather old than adult women (P,<,0.05). Further, adult men expressed more M2 mRNA than adult women (P,<,0.05), while the opposite was detected in old age (P,<,0.05). The Western blot followed by quantification confirmed that the mRNAs were translated into proteins, and that the M2 subtype showed similar modifications found at molecular level. Discussion The selective modification of M2 receptors observed at the urinary bladder mucosa levels indicates that this anatomical structure could play an active role in the pathophysiology of micturition and supports evidence suggesting an effect of antimuscarinic drugs at this level. Whether these results may influence the age-dependent development of micturition disorders remains to be determined. Neurourol. Urodynam. 27:421,428, 2008. © 2007 Wiley-Liss, Inc. [source] Muscarinic receptor subtypes in neuronal and non-neuronal cholinergic functionAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 3 2006R. M. Eglen Summary 1 Muscarinic M1,M5 receptors mediate the metabotropic actions of acetylcholine in the nervous system. A growing body of data indicate they also mediate autocrine functions of the molecule. The availability of novel and selective muscarinic agonists and antagonists, as well as in vivo gene disruption techniques, has clarified the roles of muscarinic receptors in mediating both functions of acetylcholine. 2 Selective M1 agonists or mixed M1 agonists/M2 antagonists may provide an approach to the treatment of cognitive disorders, while M3 antagonism, or mixed M2/M3 antagonists, are approved for the treatment of contractility disorders including overactive bladder and chronic obstructive pulmonary disease. Preclinical data suggest that selective agonism of the M4 receptor will provide novel anti-nociceptive agents, while therapeutics-based upon agonism or antagonism of the muscarinic M5 receptor have yet to be reported. 3 The autocrine functions of muscarinic receptors broadly fall into two areas , control of cell growth or proliferation and mediation of the release of chemical mediators from epithelial cells, ultimately causing muscle relaxation. The former particularly are involved in embryological development, oncogenesis, keratinocyte function and immune responsiveness. The latter regulate contractility of smooth muscle in the vasculature, airways and urinary bladder. 4 Most attention has focused on muscarinic M1 or M3 receptors which mediate lymphocyte immunoresponsiveness, cell migration and release of smooth muscle relaxant factors. Muscarinic M4 receptors are implicated in the regulation of keratinocyte adhesion and M2 receptors in stem cell proliferation and development. Little data are available concerning the M5 receptor, partly due to the difficulties in defining the subtype pharmacologically. 5 The autocrine functions of acetylcholine, like those in the nervous system, involve activation of several muscarinic receptor subtypes. Consequently, the role of these subtypes in autocrine, as well neuronal cholinergic systems, significantly expands their importance in physiology and pathophysiology. [source] Regulation of bladder muscarinic receptor subtypes by experimental pathologiesAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 3 2006M. R. Ruggieri Sr Summary 1 The M3 muscarinic receptor subtype is widely accepted as the receptor on smooth muscle cells that mediates cholinergic contraction of the normal urinary bladder and other smooth muscle tissues, however, we have found that the M2 receptor participates in contraction under certain abnormal conditions. The aim of this study was to determine the effects of various experimental pathologies on the muscarinic receptor subtype mediating urinary bladder contraction. 2 Experimental pathologies resulting in bladder hypertrophy (denervation and outlet obstruction) result in an up-regulation of bladder M2 receptors and a change in the receptor subtype mediating contraction from M3 towards M2. Preventing the denervation-induced bladder hypertrophy by urinary diversion prevents this shift in contractile phenotype indicating that hypertrophy is responsible as opposed to denervation per se. 3 The hypertrophy-induced increase in M2 receptor density and contractile response is accompanied by an increase in the tissue concentrations of mRNA coding for the M2 receptor subtype, however, M3 receptor protein density does not correlate with changes in M3 receptor tissue mRNA concentrations across different experimental pathologies. 4 This shift in contractile phenotype from M3 towards M2 subtype is also observed in aged male Sprague,Dawley rats but not females or either sex of the Fisher344 strain of rats. 5 Four repeated, sequential agonist concentration response curves also cause this shift in contractile phenotype in normal rat bladder strips in vitro, as evidenced by a decrease in the affinity of the M3 selective antagonist p -fluoro-hexahydro-sila-diphenidol (p -F-HHSiD). 6 A similar decrease in the contractile affinity of M3 selective antagonists (darifenacin and p -F-HHSiD) is also observed in bladder specimens from patients with neurogenic bladder as well as certain organ transplant donors. 7 It is concluded that although the M3 receptor subtype predominately mediates contraction under normal circumstances, the M2 receptor subtype can take over a contractile role when the M3 subtype becomes inactivated by, for example, repeated agonist exposures or bladder hypertrophy. This finding has substantial implications for the clinical treatment of abnormal bladder contractions. [source] Disparate cholinergic currents in rat principal trigeminal sensory nucleus neurons mediated by M1 and M2 receptors: a possible mechanism for selective gating of afferent sensory neurotransmissionEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2006Kristi A. Kohlmeier Abstract Neurons situated in the principal sensory trigeminal nucleus (PSTN) convey orofacial sensory inputs to thalamic relay regions and higher brain centres, and the excitability of these ascending tract cells is modulated across sleep/wakefulness states and during pain conditions. Moreover, acetylcholine release changes profoundly across sleep/wakefulness states and ascending sensory neurotransmission is altered by cholinergic agonists. An intriguing possibility is, therefore, that cholinergic mechanisms mediate such state-dependent modulation of PSTN tract neurons. We tested the hypotheses that cholinergic agonists can modulate PSTN cell excitability and that such effects are mediated by muscarinic receptor subtypes, using patch-clamp methods in rat and mouse. In all examined cells, carbachol elicited an electrophysiological response that was independent of action potential generation as it persisted in the presence of tetrodotoxin. Responses were of three types: depolarization, hyperpolarization or a biphasic response consisting of hyperpolarization followed by depolarization. In voltage-clamp mode, carbachol evoked corresponding inward, outward or biphasic currents. Moreover, immunostaining for the vesicle-associated choline transporter showed cholinergic innervation of the PSTN. Using muscarinic receptor antagonists, we found that carbachol-elicited PSTN neuron hyperpolarization was mediated by M2 receptors and depolarization, in large part, by M1 receptors. These data suggest that acetylcholine acting on M1 and M2 receptors may contribute to selective excitability enhancement or depression in individual, rostrally projecting sensory neurons. Such selective gating effects via cholinergic input may play a functional role in modulation of ascending sensory transmission, including across behavioral states typified by distinct cholinergic tone, e.g. sleep/wakefulness arousal levels or neuropathic pain conditions. [source] Role of M2, M3, and M4 muscarinic receptor subtypes in the spinal cholinergic control of nociception revealed using siRNA in ratsJOURNAL OF NEUROCHEMISTRY, Issue 4 2009You-Qing Cai Abstract Muscarinic acetylcholine receptors (mAChRs) are involved in the control of nociception in the spinal cord. The M2, M3, and M4 mAChR subtypes are present in the spinal dorsal horn. However, the role of the individual subtypes in the anti-nociceptive effect produced by mAChR agonists is uncertain. Here, we determined the contribution of M2, M3, and M4 subtypes to spinal muscarinic analgesia by using small-interference RNA (siRNA) targeting specific mAChR subtypes in rats. The neuronal uptake and distribution of a chitosan-siRNA conjugated fluorescent dye in the spinal cord and dorsal root ganglion were confirmed after intrathecal injection. The control and gene-specific siRNA-chitosan complexes were injected intrathecally for three consecutive days. Quantitative reverse-transcription polymerase chain reaction analysis showed that treatment with siRNA targeting M2, M3, or M4 subtype produced a large reduction in the corresponding mRNA levels in the dorsal root ganglion and dorsal spinal cord. Also, the protein levels of the mAChR subtypes in the spinal cord were significantly down-regulated by siRNA treatment, as determined by the immunoprecipitation and receptor-binding assay. Treatment with the M2 -siRNA caused a large reduction in the inhibitory effect of muscarine on the nociceptive withdrawal threshold. Furthermore, M4 knockdown at the spinal level significantly reduced the anti-nociceptive effect of muscarine. However, the anti-nociceptive effect of muscarine was not significantly changed by the M3 -specific siRNA. Our study suggests that chitosan nanoparticles can be used for efficient delivery of siRNA into the neuronal tissues in vivo. Our findings also provide important functional evidence that M2 and M4, but not M3, contribute to nociceptive regulation by mAChRs at the spinal level. [source] Muscarinic toxins: tools for the study of the pharmacological and functional properties of muscarinic receptorsJOURNAL OF NEUROCHEMISTRY, Issue 5 2009Denis Servent Abstract Muscarinic receptors mediate metabotropic actions of acetylcholine in the CNS and PNS and autocrine functions of acetylcholine in non-neuronal systems. Because of the lack of highly selective muscarinic ligands, the precise location, functional role, and roles in various diseases of the five muscarinic receptor subtypes remain unclear. Muscarinic toxins isolated from the venom of Dendroaspis snakes have a natural high affinity and selectivity, associated with roles as competitive antagonists, allosteric modulators, and potential agonists. These toxins may therefore be invaluable tools for studying muscarinic receptors. We review data on the structural and pharmacological characterization of the muscarinic toxins, focusing on recent structure,function studies on toxin,receptor interactions. We discuss the potential benefits of using these toxins for investigating muscarinic function in vivo. [source] Transmembrane signaling through phospholipase C-, in the developing human prefrontal cortexJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2006Iñigo Ruiz de Azúa Abstract To investigate changes in muscarinic receptor-stimulated phospholipase C-, (PLC-,) activity during brain development, we examined the functional coupling of each of the three major protein components of the phosphoinositide system (M1, M3, and M5 muscarinic receptor subtypes; Gq/11 proteins; PLC-,1,4 isoforms) in membrane preparations from post-mortem human prefrontal cerebral cortex collected at several stages of prenatal and postnatal development. In human prenatal brain membranes, PLC was found to be present and could be activated by calcium, but the ability of guanosine-5,-o-3 thiotriphosphate (GTP,S) or carbachol (in the presence of GTP,S) to modulate prenatal PLC-, was significantly weaker than that associated with postnatal PLC-,. Western blot analysis revealed that the levels of G,q/11 did not change significantly during development. In contrast, dramatically higher levels of expression of PLC-,1,4 isoforms and of M1, M3, and M5 muscarinic receptors were detected in the child vs. the fetal brain, a finding that might underlie the observed increased activity of PLC. Thus, inositol phosphate production may be more efficiently regulated by altering the amount of effectors (PLC-,1,4) and receptors (M1,3,5 subtypes) than by altering the level of G,q/11 subunits. These results demonstrate that different PLC isoforms are expressed in the prefrontal cortex of the developing human brain in an age-specific manner, suggesting specific roles not only in synaptic transmission but also in the differentiation and maturation of neurons in the developing brain. © 2006 Wiley-Liss, Inc. [source] Polymethylene tetraamine backbone as template for the development of biologically active polyaminesMEDICINAL RESEARCH REVIEWS, Issue 2 2003Carlo Melchiorre Abstract The concept that polyamines may represent a universal template in the receptor recognition process is embodied in the design of ligands for different biological targets. As a matter of fact, the insertion of different pharmacophores onto the polymethylene tetraamine backbone can tune both affinity and selectivity for any given receptor. The application of this approach provided a prospect of modifying benextramine (1) structure to achieve specific recognition of muscarinic receptors that led to the discovery of methoctramine (2), which is widely used as a pharmacological tool for muscarinic receptor characterization. In turn, appropriate structural modifications performed on the structure of methoctramine led to the discovery of new polyamines endowed with high affinity and selectivity for (a) muscarinic receptor subtypes, (b) Gi proteins, and (c) muscle-type nicotinic receptors. Thus, polyamines tripitramine (9) and spirotramine (33), among others, were designed, which were shown to be highly selective for muscarinic M2 and M1 receptors, respectively. Several polyamines have been discovered, which inhibit noncompetitively a closed state of the nicotinic receptor. These ligands, such as 66, resulted in important tools for elucidating the mode and site of interaction of polyamines with the ion channel. It was discovered that reducing the flexibility of the diaminohexane spacer of methoctramine led to polyamines, such as 70, which are endowed with a biological profile significantly different from that of the prototype. Most likely, tetraamine (70) is a potent activator of Gi proteins. Finally, the universal template approach formed the basis for modifying benextramine (1) structure to the design of ligands, which display affinity for acetylcholinesterase and muscarinic M2 receptors. Thus, these polyamines, such as caproctamine (78), could have potential in the investigation of Alzheimer disease. © 2002 Wiley Periodicals, Inc. Med Res Rev, 23, No. 2, 200,233, 2003 [source] Detection of muscarinic receptor subtypes in human urinary bladder mucosa: Age and gender-dependent modifications,,§NEUROUROLOGY AND URODYNAMICS, Issue 5 2008Nicola Arrighi Abstract Aims Muscarinic receptor subtypes expressed in the human urinary bladder mucosa were characterized, investigating whether there were gender-dependent differences and if aging could induce changes in their expression. Methods The study was carried out on 34 subjects, 22 men and 12 women, divided in four groups, based on gender and age. Gene expression was evaluated by quantitative RT-PCR. The Western blot was performed using the 4,12% NuPAGE Bis,Tris Gel System. Results The molecular expression of each subtype of the M1 receptor family was observed and it was not influenced either by gender or age. M2 receptor family transcripts revealed that both M2 and M4 were detected and that the M2 transcripts were modified by both gender and age. Indeed, M2 mRNA was lower in old rather than adult men (P,<,0.05), but higher in rather old than adult women (P,<,0.05). Further, adult men expressed more M2 mRNA than adult women (P,<,0.05), while the opposite was detected in old age (P,<,0.05). The Western blot followed by quantification confirmed that the mRNAs were translated into proteins, and that the M2 subtype showed similar modifications found at molecular level. Discussion The selective modification of M2 receptors observed at the urinary bladder mucosa levels indicates that this anatomical structure could play an active role in the pathophysiology of micturition and supports evidence suggesting an effect of antimuscarinic drugs at this level. Whether these results may influence the age-dependent development of micturition disorders remains to be determined. Neurourol. Urodynam. 27:421,428, 2008. © 2007 Wiley-Liss, Inc. [source] M2 mediated contractions of human bladder from organ donors is associated with an increase in urothelial muscarinic receptors,NEUROUROLOGY AND URODYNAMICS, Issue 1 2007Alan S. Braverman Abstract Aims Previous studies have shown increased density of M2 receptors in hypertrophied rat bladders that possess an M2 contractile phenotype. The aim of the current study is to determine whether human bladders with an M2 contractile phenotype also have a greater density of bladder M2 receptors. Materials and Methods Human bladders were obtained from 24 different organ transplant donors. Darifenacin and methoctramine affinity was determined by the rightward shift of cumulative carbachol concentration contractile response curves for each bladder. Radioligand binding and immunoprecipitation was used to quantify M2 and M3 subtypes in isolated detrusor muscle and urothelium. In addition, pig bladder muscle and urothelial receptors were quantified for comparison. Results In the human urothelium total, M2 and M3 muscarinic receptor density is significantly negatively correlated with the affinity of darifenacin for inhibition of contraction of the detrusor muscle. In the detrusor muscle there is no correlation between receptor density and darifenacin affinity for inhibition of contraction. Muscarinic receptor density is greater in the muscle than in the urothelium in human bladders whereas in the pig bladder the density is greater in the urothelium than in the muscle. Conclusions The greater density of urothelial muscarinic receptors in human bladders with lower darifenacin affinity, indicative of a greater contribution of M2 receptors to the contractile response, points towards a possible role of the urothelium in controlling M2 mediated contractile phenotype. In comparison between human and pig bladders, the distribution of muscarinic receptor subtypes in the muscle and urothelium are quite different. Neurourol. Urodynam. © 2006 Wiley-Liss, Inc. [source] Muscarinic receptor subtypes in neuronal and non-neuronal cholinergic functionAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 3 2006R. M. Eglen Summary 1 Muscarinic M1,M5 receptors mediate the metabotropic actions of acetylcholine in the nervous system. A growing body of data indicate they also mediate autocrine functions of the molecule. The availability of novel and selective muscarinic agonists and antagonists, as well as in vivo gene disruption techniques, has clarified the roles of muscarinic receptors in mediating both functions of acetylcholine. 2 Selective M1 agonists or mixed M1 agonists/M2 antagonists may provide an approach to the treatment of cognitive disorders, while M3 antagonism, or mixed M2/M3 antagonists, are approved for the treatment of contractility disorders including overactive bladder and chronic obstructive pulmonary disease. Preclinical data suggest that selective agonism of the M4 receptor will provide novel anti-nociceptive agents, while therapeutics-based upon agonism or antagonism of the muscarinic M5 receptor have yet to be reported. 3 The autocrine functions of muscarinic receptors broadly fall into two areas , control of cell growth or proliferation and mediation of the release of chemical mediators from epithelial cells, ultimately causing muscle relaxation. The former particularly are involved in embryological development, oncogenesis, keratinocyte function and immune responsiveness. The latter regulate contractility of smooth muscle in the vasculature, airways and urinary bladder. 4 Most attention has focused on muscarinic M1 or M3 receptors which mediate lymphocyte immunoresponsiveness, cell migration and release of smooth muscle relaxant factors. Muscarinic M4 receptors are implicated in the regulation of keratinocyte adhesion and M2 receptors in stem cell proliferation and development. Little data are available concerning the M5 receptor, partly due to the difficulties in defining the subtype pharmacologically. 5 The autocrine functions of acetylcholine, like those in the nervous system, involve activation of several muscarinic receptor subtypes. Consequently, the role of these subtypes in autocrine, as well neuronal cholinergic systems, significantly expands their importance in physiology and pathophysiology. [source] Regulation of bladder muscarinic receptor subtypes by experimental pathologiesAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 3 2006M. R. Ruggieri Sr Summary 1 The M3 muscarinic receptor subtype is widely accepted as the receptor on smooth muscle cells that mediates cholinergic contraction of the normal urinary bladder and other smooth muscle tissues, however, we have found that the M2 receptor participates in contraction under certain abnormal conditions. The aim of this study was to determine the effects of various experimental pathologies on the muscarinic receptor subtype mediating urinary bladder contraction. 2 Experimental pathologies resulting in bladder hypertrophy (denervation and outlet obstruction) result in an up-regulation of bladder M2 receptors and a change in the receptor subtype mediating contraction from M3 towards M2. Preventing the denervation-induced bladder hypertrophy by urinary diversion prevents this shift in contractile phenotype indicating that hypertrophy is responsible as opposed to denervation per se. 3 The hypertrophy-induced increase in M2 receptor density and contractile response is accompanied by an increase in the tissue concentrations of mRNA coding for the M2 receptor subtype, however, M3 receptor protein density does not correlate with changes in M3 receptor tissue mRNA concentrations across different experimental pathologies. 4 This shift in contractile phenotype from M3 towards M2 subtype is also observed in aged male Sprague,Dawley rats but not females or either sex of the Fisher344 strain of rats. 5 Four repeated, sequential agonist concentration response curves also cause this shift in contractile phenotype in normal rat bladder strips in vitro, as evidenced by a decrease in the affinity of the M3 selective antagonist p -fluoro-hexahydro-sila-diphenidol (p -F-HHSiD). 6 A similar decrease in the contractile affinity of M3 selective antagonists (darifenacin and p -F-HHSiD) is also observed in bladder specimens from patients with neurogenic bladder as well as certain organ transplant donors. 7 It is concluded that although the M3 receptor subtype predominately mediates contraction under normal circumstances, the M2 receptor subtype can take over a contractile role when the M3 subtype becomes inactivated by, for example, repeated agonist exposures or bladder hypertrophy. This finding has substantial implications for the clinical treatment of abnormal bladder contractions. [source] Pharmacological characterization of a novel investigational antimuscarinic drug, fesoterodine, in vitro and in vivoBJU INTERNATIONAL, Issue 8 2008Peter Ney OBJECTIVE To investigate the primary pharmacology of fesoterodine (a novel antimuscarinic drug developed for treating overactive bladder) and SPM 7605 (its active metabolite, considered to be the main pharmacologically active principle of fesoterodine in man) against human muscarinic receptor subtypes, and to investigate in vitro and in vivo functional activity of these agents on the rat bladder compared with existing standard agents. MATERIALS AND METHODS The displacement of radioligand binding by fesoterodine, SPM 7605 and standard agents in membrane preparations of Chinese hamster ovary (CHO) cells expressing the different human muscarinic receptors (M1,M5) was characterized. Agonistic and antagonistic activities were studied using different CHO cell lines stably expressing the human recombinant muscarinic receptor subtypes. The effects of fesoterodine and SPM 7605 on isolated bladder strips contracted by carbachol or electrical field stimulation (EFS) were investigated. In vivo the effects of fesoterodine and SPM 7605 on micturition variables were assessed using continuous cystometry in conscious female Sprague-Dawley rats, and compared to those of oxybutynin and atropine. RESULTS In vitro SPM 7605 potently inhibited radioligand binding at all five human muscarinic receptor subtypes with equal affinity across all five. Fesoterodine had a similar balanced selectivity profile but was less potent than SPM 7605. Both substances were competitive antagonists of cholinergic agonist-stimulated responses in human M1-M5 cell lines and had a similar potency and selectivity profile to the radioligand-binding studies. In rat bladder strips, fesoterodine and SPM 7605 caused a rightward shift of the concentration-response curve for carbachol with no depression of the maximum, and concentration-dependently reduced contractions induced by EFS. The potency of both drugs was similar to that of atropine and oxybutynin. In the presence of the esterase inhibitor neostigmine, the concentration-response curve of fesoterodine was shifted to the right, suggesting that part of the activity was caused by metabolism to SPM 7605 by tissue enzymes. In vivo, low doses (0.01 mg/kg) of fesoterodine and SPM 7605 reduced micturition pressure and increased intercontraction intervals and bladder capacity, but did not affect residual volume. CONCLUSIONS Fesoterodine and its active metabolite, SPM 7605, are nonsubtype selective, competitive antagonists of human muscarinic receptors, but SPM 7605 has greater potency than the parent compound. Pharmacodynamic studies in the rat bladder in vitro confirm the competitive muscarinic antagonist profile of these agents in a native tissue preparation, and in vivo studies in the rat showed effects on bladder function consistent with a muscarinic antagonist profile. [source] | |||||||||||||