JPET Fast Forward. Published on December 24, 2009 as DOI: 10.1124/jpet.109.163188 JPET FastThis Forward. article has not Published been copyedited on andDecember formatted. The 24, final 2009 version as mayDOI:10.1124/jpet.109.163188 differ from this version. JPET#163188 Title Page The constitutive activity of the human muscarinic M3 receptor Downloaded from unmasks differences in the pharmacology of anticholinergics. Paola Casarosa, Tobias Kiechle, Peter Sieger, Michael Pieper and Florian Gantner. jpet.aspetjournals.org Dept. of Pulmonary Diseases Research (P.C., T.K., M.P. and F.G.) and Drug Discovery Support (P.S.), Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, at ASPET Journals on September 27, 2021 Germany 1 Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on December 24, 2009 as DOI: 10.1124/jpet.109.163188 This article has not been copyedited and formatted. The final version may differ from this version. JPET#163188 Running Title Page Running Title: Evidence for Differential Inverse Agonism at the hM3 Receptor Corresponding author: Paola Casarosa, Ph.D. Contact Information: Dept. of Pulmonary Diseases Research, BI Pharma GmbH & Co. KG, Birkendorferstrasse 65, Biberach an der Riss, Germany E-mail: [email protected] Phone: +49-7351-5493196 Downloaded from Fax: +49-7351-8393196 Number of text pages: 33 jpet.aspetjournals.org Number of tables: 2 Number of figures: 6 Number of references: 29 at ASPET Journals on September 27, 2021 Number of words in the abstract: 250 Number of words in the introduction: 466 Number of words in the discussion: 1496 Non-standard abbreviations: human muscarinic receptor, hM-R; acetylcholine, ACh; long acting muscarinic antagonist, LAMA; chronic obstructive pulmonary disease, COPD; Chinese hamster ovary, CHO. Section assignment: Cellular and Molecular 2 JPET Fast Forward. Published on December 24, 2009 as DOI: 10.1124/jpet.109.163188 This article has not been copyedited and formatted. The final version may differ from this version. JPET#163188 Abstract An AP-1 driven luciferase reporter-assay was developed to monitor the activation of the human muscarinic M3 receptor (hM3-R) and evaluate functional potencies of different anticholinergics in CHO cells. This assay proved to be superior to previously used functional assays (i.e. inositol phosphate accumulation (Casarosa, et al., 2009)), thanks to the longer incubation times which allow reaching of pseudo-equilibrium also for ligands with slower dissociation kinetics, the so-called LAMAs (Long-Acting Muscarinic Downloaded from Antagonists). Interestingly, within this system the hM3-R efficiently signalled in an agonist-independent manner. All the antagonists tested were able to inhibit the hM3-R jpet.aspetjournals.org constitutive activity in a concentration-dependent fashion, behaving as full inverse agonists. Curiously, significant differences in potency as antagonists (against carbachol) and as inverse agonists were seen for some compounds, namely N-methyl scopolamine at ASPET Journals on September 27, 2021 and tiotropium. Given the potential for inverse agonists to cause receptor upregulation, the effect of chronic exposure to anticholinergics on the expression levels of hM3-R was also tested. Again, significant differences were seen, with some ligands (e.g. tiotropium) producing less than half of the receptor up-regulation caused by other anticholinergics. This study shows that anticholinergics can exhibit differential behaviours, which are dependent on the pathway investigated, and therefore provides evidence that the molecular mechanism of inverse agonism is likely to be more complex than the stabilization of a single inactive receptor conformation. Also, differences in the potential of anticholinergics to induce hM3-R upregulation might have clinical relevance, since many are on the market or in clinical trials as chronic treatment for e.g. chronic obstructive pulmonary disease. 3 JPET Fast Forward. Published on December 24, 2009 as DOI: 10.1124/jpet.109.163188 This article has not been copyedited and formatted. The final version may differ from this version. JPET#163188 Introduction Substantial experimental evidence now exists to show that G protein-coupled receptors (GPCRs) can productively couple to G proteins in the absence of agonist to produce a measurable downstream response (Casarosa, et al., 2009;Kenakin, 2004), a phenomenon which is termed constitutive activity. To accommodate these empirical Downloaded from observations, the “extended ternary complex” model (Samama, et al., 1993) and the more thermodynamically complete “cubic ternary complex model” (Weiss, et al., 1996a) were developed. Central to these models is the concept that receptors exist in an jpet.aspetjournals.org equilibrium between inactive (R) and active (R*) receptor conformations. The enhanced ability to detect constitutive activity led to the discovery of a unique subclass of ligands, at ASPET Journals on September 27, 2021 which exert their actions by actively reducing basal receptor activity. This novel pharmacological property, termed “inverse agonism,” has been modeled by assuming that inverse agonists preferentially bind and stabilize the inactive R state of the receptor (Leff, 1995). According to the simplest interpretation of the two-state receptor model, the constitutively active R* conformation ought to be identical with the agonist induced AR*, because there is only one single active conformational state. However, there is no a priori reason that this has to be the case. By analogy to ionic channels and enzymes, it is likely that a receptor may possess multiple, distinct active conformations, as supported by increasing evidence (Kenakin, 2003). Here, we report novel findings on the human muscarinic acetylcholine receptor subtype 3 (hM3-R), which belongs to the seven transmembrane-containing superfamily 4 JPET Fast Forward. Published on December 24, 2009 as DOI: 10.1124/jpet.109.163188 This article has not been copyedited and formatted. The final version may differ from this version. JPET#163188 of G protein-coupled receptors (GPCRs) and stimulates intracellular inositol trisphosphate production by a Gq/11-mediated mechanism (Wess, 1993). In airway diseases such as chronic obstructive pulmonary disease (COPD) and asthma, the hM3- R, which is highly expressed on airway smooth muscle cells, is an important pharmacological target: its activation in response to acetylcholine (ACh), which is released from parasympathetic nerve endings, causes bronchoconstricton. Indeed, muscarinic antagonists such as ipratropium or tiotropium are effective bronchodilators Downloaded from which have a particular value in the treatment of COPD, because they block the effects of an increased vagal cholinergic tone (Barnes, 2004). jpet.aspetjournals.org In the present study, we examined the inhibitory properties of several muscarinic antagonists in a system where both constitutive activity and agonist-induced responses at the human M3 receptor could be observed. Our results support the idea that, despite at ASPET Journals on September 27, 2021 inducing the same intracellular signalling cascade (here Gq coupling), the constitutively active M3-R* and the agonist-stabilized M3-AR* may represent distinct conformational states of the receptor, which are differently recognized by some antagonists. These findings support a model of G protein-coupled receptor activation in which the assumption of two states (active and inactive) is expanded to include two or more active conformations. Also, we found evidence that the different anticholinergics have distinct pharmacological behaviours. Therefore, the assumption that inverse agonists operate by stabilizing a common inactive conformation of the receptor may be too simplistic. 5 JPET Fast Forward. Published on December 24, 2009 as DOI: 10.1124/jpet.109.163188 This article has not been copyedited and formatted. The final version may differ from this version. JPET#163188 Methods Chemicals and reagents [N-methyl-3H]scopolamine methyl chloride ([3H]-NMS specific activity 82 Ci / mmol) was obtained from Perkin Elmer (Waltham, MA). MgCl2, carbamoylcholine chloride (carbachol), muscarine chloride, pilocarpine, oxotremorine sesquifumarate, atropine Downloaded from sulphate, pirenzepine dihydrochloride, N-methyl scopolamine bromide, 4- Diphenylacetoxy-N-methylpiperidine (4-DAMP), EDTA, NaCl and HEPES were obtained jpet.aspetjournals.org from Sigma (St. Louis, MO). Acetylcholinesterase from Electrophorus electricus (electric eel) Type V-S, lyophilized powder, ≥1,000 units/mg protein was obtained from Sigma (St. Louis, MO) and reconstituted in distilled water at 1 mg/ml in the presence of 0.1% BSA. at ASPET Journals on September 27, 2021 Ipratropium bromide, tiotropium bromide, aclidinium bromide and glycopyrrolate bromide were synthesized in the chemical laboratories of Boehringer Ingelheim, Biberach an der Riss, Germany. The tritiation of tiotropium was carried out by RC Tritec AG (Teufen, Switzerland). [3H]-tiotropium was purified by HPLC on a XBridge (Waters GmbH, Eschborn, Germany) C-8 column resulting in a radiochemical purity ≥98%, and a specific activity of 65 Ci/mmol. All cell culture reagents were purchased from GIBCO (Invitrogen, Carlsbad, CA). Cell culture techniques Chinese hamster ovary (CHO) cells stably transfected with the cDNA encoding the human M3 muscarinic acetylcholine receptor (hM3-R) were previously described (Casarosa,