Nikki Watson

Muscarinic acetylcholine receptor subtypes in smooth muscle

Muscarinic acetylcholine M2 and M3 receptor subtypes are coexpressed in many types of smooth muscle including gastrointestinal smooth muscle, urinary bladder and vascular and airway tissue. Activation of M3 receptors, via the G protein Gq, results in increased polyphosphoinositide hydrolysis, release of Ca2+ ions from the sarcoplasmic reticulum and consequently causes contraction. Quantitation of the relative expression of M2 and M3 receptors has shown that the proportion of M2 receptors often predominates over the M3 receptor population by 4:1 or more. Although it is established that M2 receptors preferentially link, via a pertussis-toxin-sensitive G protein Gi, to inhibition of adenylate cyclase activity, relatively little is known concerning the physiological role of the M2 receptor population. In this review, Richard Eglen and colleagues discuss recent data concerning the possible role(s) of muscarinic receptor subtypes in smooth muscle and appraise the pharmacological methods for dissecting the function of muscarinic receptor subtypes in tissues co-expressing multiple receptors.

Therapeutic opportunities from muscarinic receptor research

Muscarinic acetylcholine receptor subtypes have been the subjects of research for at least a quarter of a century. Nonetheless, there are few selective muscarinic receptor ligands presently used as therapeutics. The extensive development of muscarinic M(1) receptor agonists for the treatment of cognitive dysfunction has culminated in a series of unsuccessful drug candidates, which reflects a lack of understanding of the disease and the role played by muscarinic cholinergic transmission. Paradoxically, the most successful antagonist approved for use in urinary incontinence is the nonselective muscarinic receptor antagonist tolterodine. This deficit in subtype-selective ligands could be circumvented by the development of transgenic mice, each lacking functional M(1), M(2), M(3), M(4) or M(5) receptors. In this article, the current status of muscarinic receptor research is critically assessed.

T cells in the central nervous system: messengers of destruction or purveyors of protection?

Although the destructive effects of an overactive adaptive immune system have been well established, especially in the context of autoimmune diseases, recently an understanding of the beneficial effects of the adaptive immunity in central nervous system (CNS) injuries has emerged. CD4+ T cells have been shown to benefit injured CNS tissue through various mechanisms; both traditional cytokine signalling and by modulating the phenotype of neural cells in the injury site. One of the major targets of the cytokine signalling in the CNS are myeloid cells, both resident microglia and monocytes, that infiltrate the tissue after injury and whose phenotype; protective or destructive, appears to be directly influenced by T cells. This cross‐talk between the adaptive and innate immune systems presents potential new targets that could provide tangible benefits in pathologies that currently have few treatment options.

Role of muscarinic M2 and M3 receptors in guinea-pig trachea : effects of receptor alkylation

Muscarinic M2 receptors account for more than half the muscarinic receptor population in smooth muscles of a number of species and yet it is the smaller M3 receptor population that mediates contraction of many of these tissues. The role of the majority of M2 receptors in the control of smooth muscle tone is unclear. In guinea-pig ileal smooth muscle, an indirect contractile role (re-contraction) for M2 receptors has been demonstrated in tissues subjected to M3 receptor alkylation and stimulation of adenylyl cyclase. The present studies have employed the technique of irreversible receptor alkylation in order to investigate the role of muscarinic M2 and M3 receptors in the control of guinea-pig tracheal smooth muscle tone. Experiments were performed to determine (i) whether an indirect contractile role for M2 receptors can be demonstrated in tracheal smooth muscle as described for ileum, and (ii) whether stimulation of M2 receptors can inhibit isoprenaline-induced relaxations of histamine pre-contracted trachea after selective M3 receptor alkylation. Our results suggest (i) that there is no evidence of M2 receptor-mediated re-contraction of tracheal smooth muscle after M3 receptor alkylation and stimulation of adenylyl cyclase, but (ii) that activation of M2 receptors, after M3 receptor alkylation, has a small inhibitory effect on relaxant responses to isoprenaline in guinea-pig tracheal smooth muscle. Therefore, it appears that the major role of postjunctional muscarinic M2 receptors in guinea-pig trachea remains to be determined.

Antagonism of β-adrenoceptor-mediated relaxations of human bronchial smooth muscle by carbachol

Activation of muscarinic M2 receptors has been suggested to account, in part, for the reduced relaxant potency of beta-adrenoceptor agonists in canine and guinea-pig tracheal smooth muscle pre-contracted with muscarinic agonists as compared to histamine. The aim of the present study was to determine whether the potency of isoprenaline is reduced in human bronchial ring preparations pre-contracted with carbachol as compared to histamine and whether activation of muscarinic M2 receptors contributes to this effect. Cumulative concentration-effect curves to isoprenaline were obtained in the absence and presence of muscarinic M2 receptor antagonism by methoctramine (0.3 microM) in bronchial ring preparations pre-contracted to equivalent isometric tensions with either histamine (10 microM) or carbachol (1 microM). The relaxant potency of isoprenaline was reduced in preparations pre-contracted with carbachol compared to histamine, but there was no significant effect of muscarinic M2 receptor antagonism on either the potency or maximal relaxation by isoprenaline. In conclusion, increased functional antagonism of beta-adrenoceptor-mediated relaxation by muscarinic agonists can be demonstrated in human bronchial smooth muscle, but muscarinic M2 receptors do not appear to contribute to this effect.

Characterization of the interaction of zamifenacin at muscarinic receptors in vitro

The interaction of zamifenacin ((3R)-(+)-diphenylmethoxy-1-(3,4)-methylenedioxyphenethyl)pi peridine) at muscarinic receptor subtypes was studied using radioligand binding and functional techniques, in vitro. In radioligand binding studies, zamifenacin acted as a competitive antagonist, with the following pKi values; rat cerebral cortex (M1) 7.90 +/- 0.08, myocardium (M2) 7.93 +/- 0.13, submaxillary gland (M3) 8.52 +/- 0.04 and rabbit lung (M4) 7.78 +/- 0.04. In functional studies zamifenacin acted as a surmountable antagonist, exhibiting the following apparent affinity values; canine saphenous vein (putative M1) 7.93 +/- 0.09, guinea-pig left atria (M2) 6.60 +/- 0.04, guinea-pig ileum (M3) 9.31 +/- 0.06, guinea-pig oesophageal muscularis mucosae (M3) 8.84 +/- 0.04, guinea-pig trachea (M3) 8.16 +/- 0.04, and guinea-pig urinary bladder (M3) 7.57 +/- 0.15. Therefore, zamifenacin is selective for muscarinic M3 receptors in guinea-pig ileum, oesophageal muscularis mucosae, trachea and bladder over muscarinic M2 receptors in atria. The degree of muscarinic M3/M2 receptor selectivity depends upon the muscarinic M3 receptor preparation studied.

Characterization of muscarinic receptor and β-adrenoceptor interactions in guinea-pig oesophageal muscularis mucosae

Smooth muscles of a number of species contain both muscarinic M2 and M3 receptors in differing proportions and while muscarinic M3 receptors mediate contraction, the role of muscarinic M2 receptors is unclear. Muscarinic M2 receptor-mediated inhibition of adenylyl cyclase activity has been demonstrated in smooth muscle and since beta-adrenoceptor relaxation of this tissue is mediated via stimulation of adenylyl cyclase, an interaction between muscarinic M2 receptors and beta-adrenoceptors in smooth muscle has been postulated. Such an interaction has been demonstrated in guinea-pig ileum and trachea using two different approaches. The present study investigates whether interactions between muscarinic M2 receptors and beta-adrenoceptors also occur in guinea-pig oesophageal muscularis mucosae. Using the technique of selective muscarinic M3 receptor alkylation, we were unable to demonstrate muscarinic M2 receptor-mediated re-contractions in oesophageal smooth muscle, as described previously in ileum. In addition, while increased functional antagonism of relaxant responses to isoprenaline could be demonstrated in tissues pre-contracted with oxotremorine M compared to histamine, muscarinic M2 receptor activation did not contribute to this effect, as described previously in trachea. These data suggest a lack of interaction between muscarinic M2 receptors and beta-adrenoceptors in guinea-pig oesophageal smooth muscle, but suggest an interaction between muscarinic M3 receptors and beta-adrenoceptors.

Breaking barriers through collaboration: the example of the Cell Migration Consortium.

Understanding complex integrated biological processes, such as cell migration, requires interdisciplinary approaches. The Cell Migration Consortium, funded by a Large-Scale Collaborative Project Award from the National Institute of General Medical Science, develops and disseminates new technologies, data, reagents, and shared information to a wide audience. The development and operation of this Consortium may provide useful insights for those who plan similarly large-scale, interdisciplinary approaches.

In Vitro Isolated Tissue Functional Muscarinic Receptor Assays

Muscarinic receptor (mAChRs) subtypes are viable targets for the design of novel agents for use in a number of central and peripheral disorders. In vitro isolated tissue functional assays for muscarinic receptor subtypes have played an invaluable role in basic research and drug discovery. The availability of biological assays for generation of quantitative estimates of affinity and potency of ligands allows evaluation of the contribution of a given mAChR to the functional end organ response and also enables drug discovery by facilitating the iterative process of screening and optimization of chemical leads. This unit describes isolated tissue functional assays for the quantification of ligand affinity and efficacy at the M1, M2, M3, M4, and M5 muscarinic receptor subtypes in tissues expressing the native receptor using organ bath techniques. Curr. Protoc. Pharmacol. 48:4.15.1‐4.15.29.

Functional roles of postjunctional muscarinic M2 receptors in airway smooth muscle

Acetylcholine, the main neurotransmitter of the parasympathetic nervous system, acts by binding to two major receptor types, the nicotinic and the muscarinic receptor classes. Muscarinic receptors are composed of five subtypes, M1—M5, encoded by intronless genes, with endogenously expressed correlates in several tissues, including the respiratory tract [1]. Given this diversity, it has been a challenge to define the physiological roles for each subtype. As discussed below, a paucity of selective ligands remains for use as defining pharmacological tools, but gene-targeting techniques, such as receptor antisense and transgenic animals, could assist in this respect. While the antisense techniques have not met with much success in the examination of smooth muscle function, it is fair to state that this has not been exten-sively studied. Alternatively, transgenic mice, lacking muscarinic M1, M2, M4 or M5 receptor genes, have now been constructed but, as yet, no studies have been reported relating to airway smooth muscle function.