Sialolithiasis Secondary to Xerostomia: A Complication of Chronic Antipsychotic and Anticholinergic Use

Class A G protein-coupled receptors (GPCRs) are able to form homodimers and/or oligomeric arrays. We recently proposed, based on bioluminescence resonance energy transfer studies with the M3 muscarinic receptor (M3R), a prototypic class A GPCR, that the M3R is able to form multiple, structurally distinct dimers that are probably transient in nature (McMillin, S. M., Heusel, M., Liu, T., Costanzi, S., and Wess, J. (2011) J. Biol. Chem. 286, 28584-28598). To provide more direct experimental support for this concept, we employed a disulfide cross-linking strategy to trap various M3R dimeric species present in a native lipid environment (transfected COS-7 cells). Disulfide cross-linking studies were carried out with many mutant M3Rs containing single cysteine (Cys) substitutions within two distinct cytoplasmic M3R regions, the C-terminal portion of the second intracellular loop (i2) and helix H8 (H8). The pattern of cross-links that we obtained, in combination with molecular modeling studies, was consistent with the existence of two structurally distinct M3R dimer interfaces, one involving i2/i2 contacts (TM4-TM5-i2 interface) and the other one characterized by H8-H8 interactions (TM1-TM2-H8 interface). Specific H8-H8 disulfide cross-links led to significant impairments in M3R-mediated G protein activation, suggesting that changes in the structural orientation or mobility of H8 are critical for efficient receptor-G protein coupling. Our findings provide novel structural and functional insights into the mechanisms involved in M3R dimerization (oligomerization). Because the M3R shows a high degree of sequence similarity with many other class A GPCRs, our findings should be of considerable general interest.

The release of acetylcholine from parasympathetic nerves and its interaction with muscarinic acetylcholine receptors (mAChRs) regulates many fundamental functions in the periphery (smooth muscle contraction, glandular secretion, modulation of cardiac output) and central nervous system (motor control

Pharmacological characterization of DA-8010, a novel muscarinic receptor antagonist selective for urinary bladder over salivary gland

BackgroundThe licencing of Cobenfy, a co-formulation of the muscarinic receptor agonist xanomeline and the peripherally active muscarinic receptor antagonist trospium, for the treatment of schizophrenia means the first drug not acting on the dopaminergic system of the CNS to treat the disorder will be available to clinicians. Notably, the discovery that drugs targeting the dopaminergic systems of the CNS could be used to treat schizophrenia preceded an understanding of how that system was involved in the molecular pathology of the disorder. By contrast, an understanding of how muscarinic receptors were affected by the molecular pathology of schizophrenia supported the rationale for the development of Cobenfy.Aims & ObjectivesTo explain how data from the study of muscarinic receptors in the human CNS support the notion that treating people with schizophrenia with muscarinic M1 and M4 receptor agonists would have beneficial therapeutic effects.MethodThis presentation will mainly focus on data from postmortem and neuroimaging studies of muscarinic receptors in the human CNS.ResultsData showing that levels of the muscarinic M1 and M4 receptors are lower in the cortex, caudate and hippocampus, but not that thalamus, from people with schizophrenia will be reviewed. Notably, in the cortex, available data suggests people with schizophrenia have lower levels of muscarinic M1 receptors and that a sub-group of people with the disorder have a marked loss of that receptor. However, it will also be shown that a loss of muscarinic M1 receptor positive pyramidal cells in the cortex is common across all people with schizophrenia. The loss of muscarinic M1 receptors on cortical pyramidal cells in people with schizophrenia would be predicted to affect glutamatergic neurotransmission with a feed down effect on GABAergic neurons. Finally, data will be presented that suggests that those in the sub-group with a marked loss of cortical muscarinic M1 receptors may have less severe cognitive deficits.Discussion & ConclusionsThe findings showing lower levels of muscarinic receptors in multiple CNS region supported the hypothesis that activating those receptors would be beneficial in treating the disorder. Moreover, given the role of muscarinic M1 and M4 receptors in sub-cortical functioning, activating those receptors would be predicted to down-regulate dopaminergic systems in the CNS and, by doing so, act on the severity of psychoses. Finally, activating cortical muscarinic M1 and M4 receptors would be predicted to impact on the severity of negative and cognitive symptoms. Data from clinical trials of Cobenfy provides support for all the proposed outcomes of activating muscarinic M1 and M4 receptors in people with schizophrenia. However, these early data need to be confirmed by using the drug in clinical practice. In addition, it would be of value to use neuroimaging techniques to confirm the presence of a sub-group of people with schizophrenia with markedly lower levels of muscarinic M1 receptors as data from postmortem CNS suggests they may be resistant to treatment with muscarinic M1 receptor agonists or positive allosteric modulators.

Recent studies suggest that the second extracellular loop (o2 loop) of bovine rhodopsin and other class I G protein-coupled receptors (GPCRs) targeted by biogenic amine ligands folds deeply into the transmembrane receptor core where the binding of cis-retinal and biogenic amine ligands is known to occur. In the past, the potential role of the o2 loop in agonist-dependent activation of biogenic amine GPCRs has not been studied systematically. To address this issue, we used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. Specifically, we subjected the o2 loop of the M3R to random mutagenesis and subsequently applied a novel yeast genetic screen to identity single amino acid substitutions that interfered with M3R function. This screen led to the recovery of about 20 mutant M3Rs containing single amino acid changes in the o2 loop that were inactive in yeast. In contrast, application of the same strategy to the extracellular N-terminal domain of the M3R did not yield any single point mutations that disrupted M3R function. Pharmacological characterization of many of the recovered mutant M3Rs in mammalian cells, complemented by site-directed mutagenesis studies, indicated that the presence of several o2 loop residues is important for efficient agonist-induced M3R activation. Besides the highly conserved Cys(220) residue, Gln(207), Gly(211), Arg(213), Gly(218), Ile(222), Phe(224), Leu(225), and Pro(228) were found to be of particular functional importance. In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are therefore consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. Given the high degree of structural homology found among all biogenic amine GPCRs, our findings should be of considerable general relevance.

The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M(3) muscarinic receptor, a prototypical G(q)-coupled receptor, as a model system. It is known that a tyrosine residue (Tyr(254)) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M(3) receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M(3) receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys(484)-Ser(493) at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M(3) receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing an in situ disulfide cross-linking strategy to examine agonist-dependent dynamic structural changes in a G protein-coupled receptor.

Benzodiazepine Use Among Patients With Schizophrenia in Taiwan: A Nationwide Population-Based Survey

Prior studies have suggested that heart expresses only the M2 isoform of the muscarinic receptor (Peralta, E.G., Ashkenazi, A., Winslow, J.W., Smith, D.H., Ramachandran, J., and Capon, D.J. (1987) EMBO J. 6, 3923-3929). Tietje and Nathanson (Tietje, K.M., and Nathanson, N. M. (1991) J. Biol. Chem. 266, 17382-17387) have recently demonstrated that the chick heart may be unique since it expresses both the M2 and M4 isoforms of the muscarinic receptor. In this study, in order to determine whether other isoforms of the muscarinic receptor were present in the chick heart, a chick M3 muscarinic receptor receptor was cloned, characterized, and its expression in chick tissues determined. Using a human M3 muscarinic receptor cDNA as a probe, a 2.4-kilobase pair cDNA was isolated from a chick brain cDNA library which contained an open reading frame coding for a 639 amino acid protein. This protein demonstrated an 87 and 86% homology to the human and rat M3 muscarinic receptor, respectively. Chinese hamster ovary (CHO-GRA) cells were stably transfected with the chick M3 muscarinic receptor and one clone (CHO-CM3) expressed the M3 receptor, as measured by the binding of quinuclidinly benzilate at 116 +/- 14 (+/- S.E., n = 3) fmol/mg protein with a Kd of 76 +/- 17 pM. This receptor demonstrated a rank order of potency for muscarinic antagonist binding characteristic for the M3 receptor: with high affinity binding for hexahydrosiladifenidol, Kd: 16 +/- 2 nM (+/- S.E., n = 3); intermediate affinity for pirenzepine, Kd: 383 +/- 47 nM, and low affinity for methoctramine, Kd: 533 +/- 185 nM (+/- S.E., n = 3). Carbamylcholine stimulation of CHO-CM3 cells resulted in a 1.6-fold increase in cyclic AMP accumulation and a 3.5-fold increase in a pertussis toxin-insensitive inositol phosphate release. These data demonstrate that the chick M3 muscarinic receptor has the properties characteristic of M3 receptors from other species. RNase protection studies demonstrated the presence of M3 muscarinic receptor mRNA in the brain, atria, and ventricle of chicks 17 days in ovo. Hence, the chick heart appears to have the unique capacity to express mRNAs coding not only for the M2 and M4 muscarinic receptors but also for the M3 muscarinic receptor.

Oral lichen planus (OLP) is a common, chronic immunological and inflammatory condition. Many of the OLP patients complain of xerostomia. The M3 muscarinic cholinergic receptors (MR3) are the main receptors in the salivary glands responsible for water secretion into the saliva. This study aimed to assess the level of M3 muscarinic cholinergic receptors in minor salivary glands of OLP patients. This case-control cross-sectional study evaluated 40 OLP patients and 22 controls. All participants completed two questionnaires (xerostomia and xerostomia inventory). Stimulated and unstimulated saliva samples were collected. The saliva flow rate was calculated by dividing the saliva volume (in milliliters) by time (in minutes). Six minor salivary glands were also surgically removed from the lower lip of patients and controls, and weighed using a digital scale with 10-4 g accuracy. They were then frozen at -80°C, and the level of M3 receptors of the glands was determined using the ELISA kit. The unstimulated and stimulated saliva flow rate was significantly lower in OLP patients. The xerostomia inventory score was significantly higher in the OLP group. The level of M3 muscarinic receptors in minor salivary glands of OLP patients was significantly higher than that in controls. It may be concluded that the reduction in saliva flow significantly increases the number of M3 receptors in an attempt to compensate for this shortage and prevent xerostomia (compensatory upregulation).

Sialorrhea is often treated with anticholinergic agents, but they can have undesirable side effects such as drowsiness, sedation, and constipation. Effective medication that acts selectively on the salivary glands is needed. We report the case of a patient with sialorrhea who was successfully treated by the combined use of pirenzepine and solifenacin (M1 and M3 muscarinic receptor antagonists, respectively). The patient was a 51-year-old man with mean unstimulated and stimulated salivary flow rates per 10 min of 6.1 mL and 41.7 mL, respectively (both were measured three times). 99mTcO4- salivary gland scintigraphy revealed characteristic spontaneous saliva secretion without stimulation. He was treated with Scopolia extract, escitalopram, solifenacin succinate, and the combined administration of solifenacin succinate and pirenzepine. A statistically significant decrease was observed from the pre-medication unstimulated and stimulated salivary flow rates only following the combined administration of solifenacin and pirenzepine. The major muscarinic receptor subtype expressed in the salivary glands is M3; however, M1 is also present. A study using knockout mice demonstrated that the presence of either M1 or M3 receptors was sufficient for salivation. Thus, the combined use of selective M1 and M3 antagonists could provide a good treatment option for sialorrhea.

Immunohistochemical Expression of Muscarinic Receptors in the Urothelium and Suburothelium of Neurogenic and Idiopathic Overactive Human Bladders, and Changes With Botulinum Neurotoxin Administration

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beta-Arrestins regulate the functioning of G protein-coupled receptors in a variety of cellular processes including receptor-mediated endocytosis and activation of signaling molecules such as ERK. A key event in these processes is the G protein-coupled receptor-mediated recruitment of beta-arrestins to the plasma membrane. However, despite extensive knowledge in this field, it is still disputable whether activation of signaling pathways via beta-arrestin recruitment entails paired activation of receptor dimers. To address this question, we investigated the ability of different muscarinic receptor dimers to recruit beta-arrestin-1 using both co-immunoprecipitation and fluorescence microscopy in COS-7 cells. Experimentally, we first made use of a mutated muscarinic M(3) receptor, which is deleted in most of the third intracellular loop (M(3)-short). Although still capable of activating phospholipase C, this receptor loses almost completely the ability to recruit beta-arrestin-1 following carbachol stimulation in COS-7 cells. Subsequently, M(3)-short was co-expressed with the M(3) receptor. Under these conditions, the M(3)/M(3)-short heterodimer could not recruit beta-arrestin-1 to the plasma membrane, even though the control M(3)/M(3) homodimer could. We next tested the ability of chimeric adrenergic muscarinic alpha(2)/M(3) and M(3)/alpha(2) heterodimeric receptors to co-immunoprecipitate with beta-arrestin-1 following stimulation with adrenergic and muscarinic agonists. beta-Arrestin-1 co-immunoprecipitation could be induced only when carbachol or clonidine were given together and not when the two agonists were supplied separately. Finally, we tested the reciprocal influence that each receptor may exert on the M(2)/M(3) heterodimer to recruit beta-arrestin-1. Remarkably, we observed that M(2)/M(3) heterodimers recruit significantly greater amounts of beta-arrestin-1 than their respective M(3)/M(3) or M(2)/M(2) homodimers. Altogether, these findings provide strong evidence in favor of the view that binding of beta-arrestin-1 to muscarinic M(3) receptors requires paired stimulation of two receptor components within the same receptor dimer.

BackgroundEvidence from postmortem CNS studies and a neuroimaging study suggest that, compared to controls, there are low levels of muscarinic receptors in a number of CNS regions from subjects with schizophrenia. Current data suggests the muscarinic M1 receptor is lower in the cortex of subjects with schizophrenia but other muscarinic receptors may be decreased in sub-cortical regions such as the striatum and hippocampus. In addition, it has been reported that ~25% of subjects with schizophrenia can be divided into a distinct sub-group because they have a marked decrease in cortical muscarinic M1 receptors (muscarinic receptor deficit schizophrenia (MRDS)). These findings have become of clinical significance because proof of principal data shows that treating subjects with schizophrenia with drugs that activate the muscarinic M1 receptor is effective in lessening the symptoms associated with the disorder.MethodsPublished and unpublished data will be reviewed to challenge the hypothesis that drugs that activate the muscarinic M1 receptor will be useful in treating schizophrenia.ResultsA proof of clinical trial has shown that treating subjects with treatment resistant schizophrenia with the muscarinic M1 and 4 receptor agonist, xanomeline, improves positive and negative symptoms as well as cognitive deficits. Moreover, it has more recently been reported that giving xanomeline on a transdermal patch with a peripheral muscarinic receptor antagonist can lessen the unwanted side effects of the drug to that of placebo. Relevant to these data is the finding that there is a sub-group of subjects with MRDS as the absence of cortical muscarinic M1 receptors in these receptors may make these subjects resistant to treatment with muscarinic M1 receptor agonists. However, novel studies using postmortem CNS from subjects with MRDS and non-MRDS has shown that whilst subjects with MRDS will likely be resistant to muscarinic M1 receptor orthosteric agonists (oxotremorine-M) they will, at least partially, respond to muscarinic M1 receptor allosteric agonists (AC-42) or positive allosteric modulators (BQCA).DiscussionMuscarinic receptor agonism appears to be a promising new treatment for schizophrenia. However, some subjects with MRDS may only respond to activation of the allosteric site on the muscarinic M1 receptor. Evidence from a neuroimaging study suggests subjects with MRDS can be identified whilst living. Hence, establishing the muscarinic receptor status of subjects involved in trials of muscarinic M1 receptor agonists may help in explaining varying levels of treatment responsiveness in subjects with schizophrenia. These conclusions, being directed by data from studies using postmortem CNS, reflect the need for drug discovery and delivery to be based on a growing understanding of the pathophysiology(ies) of schizophrenia.

Trends in Use of Antipsychotics and Mood Stabilizers Among Medicaid Beneficiaries With Bipolar Disorder, 2001-2004