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 .

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.

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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; , ACh; long acting , LAMA; chronic obstructive pulmonary disease, COPD;

Chinese hamster ovary, CHO.

Section assignment: Cellular and Molecular

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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 )

and as inverse agonists were seen for some compounds, namely N-methyl 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.

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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 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 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.

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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), chloride, , oxotremorine sesquifumarate, Downloaded from sulphate, 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, , 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, et al., 2009). CHO-hM3 cells were grown in Ham’s F12 medium

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JPET#163188 supplemented with 10% fetal calf serum in the presence of the selection agent G418

(400 µg/ml). Cells were maintained at 37°C in humidified air containing 5% CO2.

Transient transfection for reportergene

One day in advance, two million CHO-hM3 cells were plated in a 6 well dish in Ham´s

F12 medium containing 10% FCS. The following day, cells, typically 50 to 60 %

confluent, were transiently transfected with the AP-1 luciferase reportergene (PathDetect Downloaded from

AP-1 Cis-Reporter Plasmid, Stratagene, La Jolla, CA) using the transfection reagent

FuGENE 6 (Roche Diagnostics, Mannheim, Germany) optimized at 2 µg of DNA / 6 µl jpet.aspetjournals.org of FuGENE 6 per well, according to the manufacturer’s protocol.

Detection of functional antagonism with the AP-1 luciferase assay at ASPET Journals on September 27, 2021

30 hours after transfection of the AP-1 luciferase cDNA, CHO-hM3 cells were resuspended in DMEM-F12 medium without phenol red containing 2% FCS and transferred to 384 white well plates (Perkin Elmer, Waltham, MA), where they were stimulated with a range of carbachol concentrations (from 10-9 to 10-2 M), in the presence or absence of at least 7 different concentrations of antagonists. After overnight stimulation (typically 20h) at 37°C, luciferase activity was quantified with the LucLite

Reporter Gene Assay System (Perkin Elmer, Waltham, MA) according to the manufacturer’s protocol. Briefly, 30 μl substrate solution were added in each well and the plate was further incubated for 30 min in the dark. Afterwards, light emission was quantified using VICTOR² (Perkin Elmer, Waltham, MA) measuring 1 second per well.

Schild plots were created by linear regression in GraphPad Prism 5.02 plotting the dose ratios against the concentration of antagonist to determine pA2 values for antagonists.

7 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.

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Detection of constitutive activity and inverse agonism with the AP-1 luciferase assay

30 hours after transfection of the AP-1 luciferase cDNA, CHO-hM3 cells were resuspended in DMEM-F12 medium without phenol red containing 2% FCS and transferred to 96 white well plates, where they were incubated with a range of antagonists’ concentrations (from 10-12 to 10-6 M), in the absence of any added agonist. Downloaded from To rule out the presence of acetylcholine released by the cells, some experiments were performed in the presence of 10 units/ml of Acetylcholinesterase from Electrophorus electricus (electric eel) Type V-S (Sigma, St. Louis, MO). After overnight incubation jpet.aspetjournals.org

(typically 20h) at 37*C, luciferase activity was quantified with the LucLite kit as described above. Data were analyzed with GraphPad Prism 5.02 by non-linear regression using at ASPET Journals on September 27, 2021 equation: “log(inhibitor) vs. response”. The best fit between a variable Hill coefficient and a Hill coefficient fixed to unity was determined using an F test.

Receptor Up-Regulation Studies.

CHO-hM3 cells, approximately 70-80% confluent in 24 well plates, were incubated with the indicated concentrations of antagonists for 30 hours. At the end of this period, cells were subjected to an acidic wash to remove surface-bound ligands. Briefly, acidic washes with Hams´ F12 (with pH set at 2) were allowed to proceed for 30 min, with medium being exchanged every 4 min. Control binding experiments were conducted and showed that this procedure fully removed surface-bound ligands while leaving the receptor surface intact (see figure 5A in the Results section). Afterwards, cells were rinsed twice with ice-cold binding buffer (HBSS, 10 mM HEPES and 0.05% BSA) to set the pH back to neutral, then receptor expression on the cell surface was monitored by 8 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 binding of [³H]-tiotropium at saturating concentrations (approximately 2 nM). After 1 h incubation, free radioligand was removed by rapid washing of wells with 3 x 1 ml of ice- cold buffer. Cell monolayers were dissolved in 250 µl of 0.1 M NaOH, and radioactivity was determined by liquid scintillation counting in a Tricarb beta counter device (Perkin

Elmer, Waltham, MA).

Chemical stability studies Downloaded from

To analyze their degradation kinetics, compounds were dissolved in 0.1 mol/l KH2PO4 buffer pH 7.4 at a concentration of ca. 0.35 mg/ml. The solutions were filled in 2 ml jpet.aspetjournals.org HPLC glass vials and were placed in a thermostated autosampler which was held at

37 °C. HPLC-diagrams were collected every 2 hours for a total of 20 hours. The RP-

HPLC (Agilent HP 1100 with diode array detector, equipped with Prontosil C18 ace-EPS at ASPET Journals on September 27, 2021 column, 125 mm length, 4.6 mm inner diameter) setup consisted in a gradient between eluent A (10 mmol/l ammonium acetate sol. pH 7.0) and eluent B (acetonitrile) with a flow rate of 1ml/min and quantitative detection at 240 nm. The obtained diagrams were analysed using a first order degradation approach.

Data analysis

All experiments were analysed by either linear or non-regression analysis with the equations mentioned under the different assay methodology using Prism version 5.02

(GraphPad Software, San Diego, CA). Individual estimates (either pA2, pIC50 or pEC50 values) were obtained from each experiment and then averaged to provide mean data (±

S.E.M). The statistical significance of differences between data were determined using either Student's t test, or one-way analysis of variance with Dunnett's post test for

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JPET#163188 multiple comparisons, as indicated in detail in the different tables’ legends. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

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JPET#163188

Results

Selection of an appropriate reporter gene for monitoring hM3 receptor activation

The hM3-R couples to Gq proteins, resulting in activation of phospholipase C (PLC)

(Wess, 1993). Previously, functional antagonism of a series of muscarinic antagonists was characterized in our laboratory by measuring changes in the intracellular concentrations of inositol phosphates (Casarosa, et al., 2009). Although having several Downloaded from advantages, such as the ease of performance, sensitivity and reproducibility, this assay has a major limitation, namely the fact that its stimulation time cannot be prolonged for jpet.aspetjournals.org more than 1-2 hours. This aspect is of particular importance when considering ligands with slower kinetics of dissociation from the hM3 receptor, the so-called long acting

muscarinic antagonists (LAMAs), e.g. tiotropium (dissociaton t1/2 27h), aclidinium (t1/2 at ASPET Journals on September 27, 2021

11h) and glycopyrrolate (t1/2 6h). LAMAs are unable to reach pseudo-equilibrium within this time frame, resulting in deviation of the Schild plot from unity (Casarosa, et al.,

2009).

To overcome this problem, we intended to set up a reporter-gene assay with longer incubation times, using the same CHO-hM3 cell line previously used for the InsP assay, to allow for direct comparison of results. Given the Gq coupling of the hM3 receptor, efforts concentrated on the firefly-luciferase reporter construct under the control of the

AP-1 promoter, which typically depends on PKC activation (Angel and Karin, 1991). As expected, the agonist carbachol activated the AP-1-luciferase in a concentration dependent manner (fig 1 and 2). As a control, empty CHO cells were transfected with

AP-1: expectedly, carbachol was unable to induce AP-1 driven luciferase in the absence of the hM3 receptor (data not shown).

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Subsequently, different parameters, such as cell number and amount of DNA transfected, were optimized to increase assay sensitivity and reproducibility (see the

Materials and Methods section for detailed information). Most importantly, different incubation times were tested to ensure reaching of pseudo-equilibrium in the presence of LAMAs. Fig 1 shows results obtained with CHO-hM3 cells transfected with the AP-1 reporter and incubated with carbachol in the presence of tiotropium for different time

periods, namely 5, 8 and 18 hours. Increasing the assay incubation to 18 hours Downloaded from generated a more robust signal with an improved assay window (the signal to background ratio is above 8, compared to less than 2 obtained with shorter incubations); jpet.aspetjournals.org also, 18 hours was the only time point investigated that delivered a correct determination of a pA2 value through Schild analysis, with a slope of 1.06 (figure 1D). As a consequence, incubation was allowed to proceed for 20 h in all subsequent luciferase at ASPET Journals on September 27, 2021 experiments.

Under these conditions, the effects of the different agonists carbachol, muscarine, oxotremorine and pilocarpine were tested (figure 2). Each agonist stimulated a concentration-dependent increase in luciferase, with the following pEC50 values: carbachol, 6.5 ± 0.1; muscarine, 6.7 ± 0.1; oxotremorine, 6.9 ± 0.1 and pilocarpine, 6.0 ±

0.1; n ≥3). The reporter assay also allowed for a sensitive assessment of agonist intrinsic activity: compared to carbachol and muscarine, which behaved as full agonists, oxotremorine and pilocarpine exhibited intrinsic activities of 81 ± 3% and 52 ± 2%, respectively (n = 3).

Characterization of antagonistic behaviour at the hM3-R with the AP-1 driven reporter gene

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To measure the functional antagonism of different anticholinergics, the shift in the carbachol response curve induced in the presence of different antagonist concentrations were plotted according to Schild analysis (e.g. in Fig. 3 shown for atropine and tiotropium) and pA2 values were determined (table 1). All anticholinergics tested showed a competitive and surmountable antagonistic behaviour (Fig. 3), as indicated by the parallel right shift of the agonist curves in the presence of increasing antagonist

concentrations without any significant change in the maximal responses. For all tested Downloaded from antagonists, including LAMAs, the slope of Schild regression obtained with the reporter gene assay was not significantly different from unity over a wide range of antagonists’ jpet.aspetjournals.org concentrations. This represents an advantage to previous results obtained with the inositol phosphate (InsP) assay (Casarosa, et al., 2009), where fitting the dose ratios over a large range of LAMAs concentrations resulted in linear regressions with a slope at ASPET Journals on September 27, 2021 significantly higher than 1 (slopes were 1.29 ± 0.05; 1.27 ± 0.07 and 1.27 ± 0.05 for tiotropium, aclidinium, and glycopyrrolate, respectively; see Fig. 3b for Schild analysis of tiotropium). These results indicate that, in contrast to the insP accumulation, the reporter gene assay is ideal for testing antagonists with slower kinetics of receptor dissociation, since its longer incubation time allows the LAMAs to reach pseudo-equilibrium and therefore a correct determination of pA2 values.

The pA2 values obtained for each muscarinic antagonist against carbachol in the InsP assay and the AP-1 reporter gene are in close agreement with each other (table 1), as expected for competitive (i.e. orthosteric, surmountable and reversible) antagonists inhibiting a common pathway (i.e. Gq coupling). The only exception is represented by aclidinium, which shows a significantly weaker inhibitory potency in the AP-1 luciferase reporter gene assay (pA2 = 9.2) than in the InsP assay (pA2 = 10.0). Given the direct

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JPET#163188 correlation between decrease in antagonistic potency and duration of the assay, instability of aclidinium in aqueous solution was investigated as a potential cause.

Indeed, kinetic studies addressing the stability of aclidinium showed that its half-life is

3.1 hours in buffered solution (pH 7.4) at 37°C (figure 4). As a control, chemical degradation of other two muscarinic antagonists which also possess the ester function, namely ipratropium and NMS, was investigated as well. As shown in figure 4, the

degradation half lives are 257 hours for NMS and ca. 2300 hours for ipratropium. These Downloaded from data, in agreement with the functional read-outs, indicate that chemical degradation is not a necessary consequence of the presence of an ester function, but rather a specific jpet.aspetjournals.org characteristic of aclidinium.

at ASPET Journals on September 27, 2021

Detection of hM3-R constitutive activity

Changes induced by muscarinic antagonists on the basal levels of luciferase expressed in CHO-hM3 cells were investigated as well. In the absence of any added agonist, all tested antagonists decreased AP-1 driven luciferase activity below basal values, suggesting the presence of constitutive activity of the hM3 receptor in this CHO-hM3 system (figure 5). To confirm the M3-mediated constitutive signalling and rule out potential contamination with endogenous acetylcholine, experiments were performed in the presence of acetylcholinesterase from Electrophorus electricus (electric eel) Type V-

S. The addition to the cell culture media of as much as 10 units/ml, which are sufficient to hydrolyze 10 μmoles of acetylcholine per minute, did not attenuate the basal hM3- mediated signalling nor the observed negative intrinsic activity displayed by muscarinic antagonists in this assay (data not shown). As an additional control, empty CHO cells

14 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 transfected with AP-1 luciferase were incubated with NMS: the muscarinic antagonist was unable to affect the basal luciferase activity in the absence of the hM3 receptor

(data not shown).

Compared to the agonist-induced response, approximately 30% of the total hM3 response corresponds to basal, agonist independent, signalling (figure 5A). All the antagonists tested behaved as full inverse agonists concentration-dependently

decreasing the M3-R basal activity to the same minimum (figure 5B and C). Downloaded from

The half-maximal inhibitory concentrations of the different anticholinergics necessary to block the hM3-R constitutive signalling are reported in table 1. When comparing their jpet.aspetjournals.org potencies in inhibiting the agonist-induced response (pA2 values against carbachol) and the constitutive signalling of the hM3 receptor (pIC50) in the same luciferase assay, the tested anticholinergics revealed different behaviours: whereas most (i.e. atropine, at ASPET Journals on September 27, 2021 pirenzepine, ipratropium, glycopyrrolate, DAMP) were equi-potent as antagonists and inverse agonists, others (i.e. NMS and tiotropium) showed a significantly higher propensity to inhibit the agonist response than the constitutive activity of the receptor

(3.16- and 8.3-fold difference for NMS and tiotropium, respectively). Aclidinium was the only compound to show the opposite trend, i.e. higher potency as inverse agonist; however, due to the compound inherent instability, large variability was present in the different assays (see e.g. figure 5B) and therefore a large error was associated with the measurements, hampering the reaching of statistical significance.

Taken together, these results suggest that the constitutively active M3-R* and the agonist stabilized M3-AR*, although activating the same pathway, represent distinct conformational states of the receptor, which are differently recognized by some but not all antagonists.

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Effects of Chronic Exposure to Inverse Agonists on Receptor Expression Levels.

Next we assessed whether co-incubation with ligands shown to suppress hM3-R constitutive activity could affect receptor expression levels. In these experiments, CHO cells were incubated for 30 hours with the different antimuscarinics before measuring

M3-R expression at the cell surface with a binding assay using [3H]-tiotropium as marker.

Given the slow dissociation kinetics of some of the muscarinic antagonists, the so called Downloaded from

LAMAs (Casarosa, et al., 2009), care was taken to develop a washing procedure which would fully remove these compounds bound to the M3-R and allow [3H]-tiotropium jpet.aspetjournals.org occupation of the entire cell-surface receptor population. For this control experiment, cells were incubated for 1 hour at 4°C (to prevent any change in receptor expression) with the LAMAs (here data shown for tiotropium, figure 6A), then several buffers set at at ASPET Journals on September 27, 2021 different pH were used to extensively wash the cells. As can be seen in figure 6A, complete ligand removal could be obtained by washing with buffers at low pH.

Consequently, following the 30 hours incubation at 37°C in the presence of the different concentrations of muscarinic antagonists, cells underwent a 40 min washing procedure, followed by binding with saturating concentrations of [3H]-tiotropium, as described in the

Materials and Methods section in detail.

All tested compounds caused concentration-dependent increases in receptor expression levels (Fig. 6B and C and table 2), with EC50 values in good agreement with their potency as inverse agonists (cfr. table 1 and 2). However, the changes in receptor expression differed significantly in the presence of the different ligands: whereas most antagonists (i.e. atropine, ipratropium, glycopyrrolate, pirenzepine and aclidinium) caused a significant upregulation above 40% of basal hM3-R expression levels, a

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JPET#163188 second group of antagonists, namely tiotropium, 4-DAMP and NMS, induced less than

20% upregulaton.

As a control, a second cell line was used (CHO-hß2AR), where the human ß2 adrenoceptor is stably transfected as part of the pkCREH mammalian expression vector

(i.e. the same vector used for the hM3-R). CHO-hß2 cells were incubated for 30 hours with different muscarinic antagonists (namely ipratropium, tiotropium, atropine and NMS,

3 100 nM), then ß2 receptor expression was monitored by radioligand binding (using [ H]- Downloaded from

CGP12,177). No changes in the total hß2-receptor expression were seen upon exposure to the different muscarinic antagonists (data not shown), suggesting that jpet.aspetjournals.org ligand-induced upregulation of the hM3-R takes place through binding and stabilization of the receptor expressed at the cell membrane, and not through regulation of mRNA transcription. at ASPET Journals on September 27, 2021

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Discussion

Detection of different hM3-R active conformations

Here we describe the set-up of a read-out system, namely an AP-1-driven reporter-gene, which allows the detection of both agonist-induced as well as constitutive activity for the hM3 receptor (hM3-R). Interestingly, pharmacological characterization of a series of anticholinergics indicates disparate pharmacological behaviour upon receptor interaction. Downloaded from

Although all the antagonists studied here can be classified as full inverse agonists based on their ability to decrease agonist-independent activation of the AP-1 transcription jpet.aspetjournals.org factor, we found interesting differences in the pharmacology of these compounds when comparing their respective potencies as neutral antagonists (i.e. their pA2 values against

carbachol) and as inverse agonists (table 1), respectively. Our interpretation of these at ASPET Journals on September 27, 2021 data implies the presence of different active hM3-R conformations, either stabilized by agonists such as carbachol and muscarine (M3-AR*) or by the agonist-free active conformation itself (M3-R*). Importantly, antagonists tested in these assays are orthosteric to each other and to the agonist carbachol (Casarosa, et al., 2009), ruling out the probe-dependency seen with allosteric antagonists as a potential explanation for these pharmacological discrepancies.

The present finding that M3-R* and M3-AR* represent different conformations, which are differently recognized by some antagonists, is in general agreement with the emerging concept of multiple active states for a given receptor. Observations supporting this novel concept have been reported for many GPCRs (Kenakin, 2003). However, most studies have focused on inversion of agonists’ potencies or efficacies when looking at different signalling pathways mediated by the same receptor, supporting the idea that agonists

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JPET#163188 select among distinct receptor conformations to activate different downstream effectors, as seen e.g. with GPCRs showing pleiotropism in G protein coupling (Kenakin, 2003).

Instead, our current results, i.e. the pharmacological differences observed with some antagonists in blocking agonist-induced and constitutive activation of the AP-1 transcription factor, suggest that for the hM3-R multiple active conformations exist for the regulation of the same signalling pathway, namely Gq coupling. These findings are in

agreement with biophysical studies obtained using ß2-adrenoceptors fluorescently Downloaded from labelled at Cys265, a region that is sensitive to receptor conformational changes

(Ghanouni, et al., 2001). Fluorescence lifetime spectroscopy showed that different ß- jpet.aspetjournals.org agonists produced different arrays of receptor conformations, consistent with ligand- selective active states. Instead of a single receptor active state for G-protein activation, there is an ensemble of microconformations (Kenakin, 2002) that are all capable of at ASPET Journals on September 27, 2021 producing the same pharmacological effect (in this case, Gq-protein activation) but having different overall tertiary conformations.

Detection of constitutive activity also allowed for pharmacological differentiation among the tested anticholinergics: although for most compounds pA2 values and inverse agonist potencies were in close agreement, NMS and tiotropium showed a significantly lower potency as inverse agonists (without affecting their negative intrinsic activity).

These data suggest that the different anticholinergics stabilize different conformations, and possibly that they achieve their inverse agonistic effect via distinct mechanisms of action. Classically, inverse agonists are envisioned as ligands that stabilize the inactive

R conformation (Leff, 1995). One alternative mode of inverse agonist action has been proposed (Strange, 2002): this does not involve a redistribution of receptor states but one in which the inverse agonist switches the active conformation of the receptor to an

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JPET#163188 inactive state that still retains the ability to bind and sequester G proteins but is unable to activate them. A species of receptor that is inactive but still binds G protein is one of the key features of the cubic ternary complex model (Weiss, et al., 1996) which distinguishes it from the extended ternary complex model.

Differences in antagonists’ mediated hM3-R upregulation

It is generally assumed that inverse agonists might cause receptor upregulation: their

inhibition of the spontaneous formation of receptor active states and consequent Downloaded from phosphorylation and internalization, coupled with normal receptor synthesis and expression, would lead to an increased surface density of receptor (Milligan and Bond, jpet.aspetjournals.org 1997). However, most studies analyzing the effects of inverse agonists on receptor upregulation were performed with constitutively active (CAM) receptor mutants (Dowling, et al., 2006;Zeng, et al., 2003). Unfortunately, interpreting results obtained with CAM at ASPET Journals on September 27, 2021 receptors is not always straight-forward: the creation of a CAM can diminish stabilizing constraints within the receptor, leading to an inherently unstable receptor that is more susceptible to destabilization and/or proteolytic degradation (Milligan and Bond, 1997).

Consequently, the expression level of the mutant is increased by any ligand, either agonist or antagonist, regardless of its efficacy, as shown e.g. with a constitutively active chimeric M3-R (Zeng, et al., 2003). Here instead, we tested the ability of the different anticholinergics to upregulate the WT hM3-R, ruling out potential artefacts. Again, different profiles emerged: although most compounds induced a similar level of receptor upregulation (40 to 50% versus untreated) with a good correlation between their potency as inverse agonists and EC50 values for receptor upregulation, a second set of compounds, namely NMS, tiotropium and DAMP, caused a significantly reduced hM3-R upregulation (less than 20%). Differences in upregulation do not relate to differences in

20 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 ligands’ negative intrinsic activity, as all tested anthicholinergics behaved as full inverse agonists. However, a correlation between induction of upregulation and differential propensity to behave as inverse agonists seems plausible, as two of the three compounds which are causing a less pronounced M3-R upregulation, namely tiotropium and NMS, show a significant difference in their potencies as neutral antagonists (pA2 values) and as inverse agonists (table 1). The different ability to induce receptor

upregulation supports a model where the anticholinergics stabilize distinct M3-R Downloaded from conformations, all equally effective in blocking G-protein signalling (though possibly through different mechanisms), but differently able to affect receptor internalization and jpet.aspetjournals.org its steady-state expression at the cell membrane. This is in agreement with a model where internalization and G-protein activation are mediated by distinct receptor conformations, as suggested by the ability of antagonists or inverse agonists to elicit at ASPET Journals on September 27, 2021 internalization (Azzi, et al., 2003).

Physiological relevance of hM3-R constitutive activity

Presently, the physiological importance of constitutive activity for GPCRs is unclear.

Accumulating evidence for its potential role comes from studies showing constitutive signalling in cells endogenously expressing the receptor of interest (de Ligt, et al., 2000).

However, evidence of GPCR constitutive activity in vivo is still scarce, with few excellent studies detailing inverse agonism at GPCRs expressed in vivo in native systems

(e.g.(De, et al., 2004;Morisset, et al., 2000)). Additionally, somatic receptor mutations leading to constitutively active receptors are a causal factor in certain diseases, e.g. retinitis pigmentosa, congenital night blindness and familial hyperthyroidism (de Ligt, et al., 2000). The extent to which GPCRs are constitutively active is dependent on the level of receptor and G protein expression levels: generally, in efficiently coupled tissues with

21 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 large receptor reserves, this may be an important factor in the pharmacological control of tissue function (Kenakin, 2004).

The physiological role of hM3-R constitutive signaling is currently unknown. There is however evidence that an increased cholinergic tone may be an important feature of chronic pulmonary diseases such as COPD (Barnes, 2004b;Barnes, 2004a). This increased cholinergic tone might, at least in part, result from an increase in the

constitutive signalling of hM3 receptors, as suggested by recent studies indicating an Downloaded from upregulation of M3-R in COPD patients, compared to healthy individuals (Profita, et al.,

2005;Profita, et al., 2009). Additionally, increasing the expression levels of effector jpet.aspetjournals.org proteins also influences the basal signaling of GPCRs: for example, artificial overexpression of Gαq proteins greatly enhances the constitutive activity of hM3-R in a cellular model (Burstein, et al., 1995). To this end, it has been shown that at ASPET Journals on September 27, 2021 proinflammatory stimuli known to play a role in the pathogenesis of COPD, such as

TNF-α and cigarette smoke, increase the expression of Gαq and Rho proteins (Chiba, et al., 2005;Hotta, et al., 1999), which are known signalling partners for the hM3-R. If indeed constitutive activity were operative in this pathology, inverse agonists would be uniquely valuable in the retardation of disease progression. An important appendix to the use of inverse agonists in the clinical practice relates to their potential to induce receptor upregulation and tolerance upon chronic treatment. Indeed, studies have indicated the upregulation of muscarinic receptors as a consequence of chronic atropine administration in animals (Chevalier, et al., 1991;Wall, et al., 1992); in particular, chronic exposure to atropine resulted in an upregulation of M3-R that was associated with enhanced airway smooth muscle contraction in rabbits (Witt-Enderby, et al., 1995),

22 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 suggesting that the phenomenon might be of relevance with chronic treatment in the clinical use.

Negative efficacy is a molecular property of anticholinergics which might be desirable in clinical use for COPD, but attention should be paid to their potential for receptor upregulation, leading to tolerance and possibly a rebound effect upon antagonist withdrawal. Importantly, our results indicate that it is possible to dissociate inverse

agonistic properties from potential to induce upregulation, as it has been seen for Downloaded from example with tiotropium. In agreement with these results, recent data from the UPLIFT

(Understanding Potential Long-term Impacts on Function with Tiotropium) study jpet.aspetjournals.org involving almost 6000 COPD patients indicated no loss of tiotropium bronchodilatory effects throughout the trial, which lasted 4 years, ruling out tolerance to this drug in the clinical setting (Tashkin, et al., 2008). at ASPET Journals on September 27, 2021

23 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

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BK (2001) Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta 2 adrenergic receptor. J Biol Chem 276:24433-24436.

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Zeng FY, McLean AJ, Milligan G, Lerner M, Chalmers DT and Behan DP (2003) Ligand specific up-regulation of a Renilla reniformis luciferase-tagged, structurally unstable jpet.aspetjournals.org muscarinic M3 chimeric G protein-coupled receptor. Mol Pharmacol 64:1474-1484.

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Legends for Figures

Fig. 1. Effect of different stimulation times on the hM3-R-mediated induction of

AP-1 luciferase. A, B, C: CHO-hM3 cells were transiently transfected with the AP-1 luciferase reporter gene, as described in the Material & Methods section. 30 hours after transfection, cells were stimulated with a range of carbachol concentrations, in the absence or presence of 8 different concentrations of tiotropium, as indicated in the legend of inset B. Incubation was allowed to proceed for 5 (inset A), 8 (inset B) or 18 Downloaded from hours (inset C), before cell lysis and luciferase quantification. RLU, relative light units. D:

Schild regression of tiotropium is shown for the data obtained with 18 hours incubation. jpet.aspetjournals.org

A slope of 1.07 of the linear regression over a large range of tiotropium concentrations ensures that equilibrium conditions were reached within this time frame.

at ASPET Journals on September 27, 2021

Fig. 2. Agonist-stimulated increases in AP-1-driven luciferase in CHO-hM3 cells.

CHO-hM3 cells transiently transfected with the AP-1 luciferase reporter gene were stimulated with the indicated concentrations of carbachol (filled squares), muscarine

(filled circles), oxotremorine (empty circles), or pilocarpine (empty squares) for 20 hours.

Data are presented as percentage of maximal hM3-R response in the presence of an agonist. The mean ± S.E.M. from at least three separate experiments is shown.

Fig. 3. The AP-1 luciferase reporter gene allows for correct determination of pA2 values of muscarinic antagonists in CHO-hM3 cells. A, C: atropine (A) and tiotropium (C) induce a concentration-dependent rightward shift in the carbachol curve, measured as AP-1 driven induction of luciferase activity in CHO-hM3 cells. B, D: Schild regression of the data obtained with atropine (B) and tiotropium (D) in the AP-1 reporter

29 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 gene (filled circles, slopes: 1.00 ± 0.03 and 1.05 ± 0.02 for atropine and tiotropium, respectively), compared to the data previously obtained with the inositol phosphate assay (empty circles) are shown (Casarosa, et al., 2009). Whereas for atropine both regressions have a slope of unity, Schild analysis of the data obtained with the inositol phosphates assay over a large range of tiotropium concentrations results in a slope of

1.29 ± 0.05 (shown as dashed regression line). As a consequence, Schild analysis of

the LAMAs in the inositol phosphate assay was previously performed with the Downloaded from concentrations in the range 10-8M to 10-5M (tiotropium slope: 0.99 ± 0.03, shown as filled regression line) (Casarosa, et al., 2009). Data are the mean of at least three jpet.aspetjournals.org independent experiments (± S.E.M.).

Fig. 4. Chemical instability of aclidinium. Degradation kinetics of aclidinium (open at ASPET Journals on September 27, 2021 squares), NMS (open triangles) and ipratropium (open diamonds) were monitored in a phosphate buffer at 37 °C, as described in the materials and methods section. The obtained diagrams were analysed using a first order degradation approach and the corresponding half lives (t1/2) were extrapolated from the degradation curves according to the equation t1/2 = 0.693 / k.

Fig. 5. The AP-1 reporter gene allows for detection of constitutitve activity of the hM3-R and inverse agonism of the anticholinergics. A: CHO-hM3 cells transiently transfected with the AP-1 luciferase reporter gene were stimulated with the indicated concentrations of carbachol (filled circles) or atropine (empty circles) for 20 hours. Data are presented as mean ± S.D. from one representative out of 3 independent experiments performed in triplicate. B, C: CHO-hM3 cells transiently transfected with the

30 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

AP-1 luciferase reporter gene were incubated with the indicated concentrations of the different anticholinergics in the absence of any added agonist for 20 hours. The mean ±

S.E.M. from three separate experiments is shown.

Fig. 6. Effects of chronic treatment with anticholinergics on hM3-R upregulation.

A: Effect of pH and temperature of the wash buffer on antagonist removal. In this control

experiment, CHO-hM3 cells were preincubated 1 hour with tiotropium 100 nM, then Downloaded from extensively washed for 40 minutes with buffers set at different pH (pH7, 4 and 2) and temperature (4°C, shown as filled bars versus 37°C, empty bars). Afterwards, total hM3- jpet.aspetjournals.org R quantification at the cell surface was monitored in a radioligand binding assay using

[3H]-tiotropium (2 nM). Data are presented as percentage of total hM3-R expression, defined for each treatment group as the amount of binding obtained with cells which at ASPET Journals on September 27, 2021 were not pretreated with tiotropium but underwent the same washing procedures. The specific binding obtained with [3H]-tiotropium in CHO-hM3 cells which were not pretreated with tiotropium (control cells) (mean DPM ± SEM): 5232±161; 4192 ± 91;

3236 ± 151 at 4°C and 5071 ± 45; 4540 ± 72; 3224 ± 23 at 37°C after washing with buffers set at pH7, 4 and 2, respectively. B, C: CHO-hM3 cells were incubated in the presence of increasing anticholinergics’ concentrations for 30h. Following an extensive washing procedure with buffer set at pH2, which ensures fully removal of ligands (as shown in inset A), [3H]-tiotropium was added to establish a Bmax value. In all the cases, data are shown as percentages, where 100% is the hM3-R expression in basal conditions (i.e. in the absence of a preincubation with anticholinergics) and 0% is the unspecific binding, as determined in the presence of atropine 10 µM. The mean ± S.E.M. for at least three experiments performed in triplicate is shown.

31 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.

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Table 1. Comparison of functional properties of the anticholinergics at the hM3-R in different assays. pA2 values of the different antagonists were determined in CHO- hM3 cells with the inositol phosphate accumulation (InsP) and the AP-1-driven luciferase reportergene assays. pA2 values were experimentally obtained by extrapolation of agonistic (carbachol) concentration response shifts, according to Schild analysis, as shown in Figure 3. Values shown are the average of at least 3 independent experiments

± SEM, with each point determined in triplicate. Schild slopes were not significantly Downloaded from different from unity. The third column of the table reports the inverse agonistic potencies

(pIC50 ± SEM) of the different anticholinergics, determined as the concentration that jpet.aspetjournals.org blocks 50% of the hM3-R constitutive activity, as measured in the AP-1 driven luciferase assay in the absence of any added agonist.

InsP3 Reporter-gene Reporter-gene at ASPET Journals on September 27, 2021 pA2 pA2 pIC50 Atropine 9.21 ± 0.05 9.01 ± 0.08 9.14 ± 0.10

NMS 9.58 ± 0.09 9.83 ± 0.04 9.35 ± 0.05##

Pirenzepine 6.67 ± 0.09* 7.09 ± 0.06 6.80 ± 0.10

Ipratropium 9.21 ± 0.07 9.25 ± 0.03 9.10 ± 0.07

Tiotropium 10.68 ± 0.10 10.52 ± 0.09 9.61 ± 0.06##

Aclidinium 10.04 ± 0.07** 9.23 ± 0.10 9.72 ± 0.24

Glycopyrrolate 9.63 ± 0.07 9.83 ± 0.07 9.61 ± 0.10

4-DAMP N.D. 9.23 ± 0.10 9.28 ± 0.06

N.D.: not determined

*/**: Significant differences between pA2 values obtained with the reporte rgene assay and the InsP3 assay (P < 0.05; Student’s t test). ## : Significant differences between pA2 and pIC50 values obtained with the reporter gene assay (P < 0.05; Student’s t test). 32 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.

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Table 2. Differential ability of anticholinergics to cause hM3-R upregulation. The maximal hM3-R upregulation caused by the different anticholinergics and their respective half maximal effective concentration, following 30 hours incubation with CHO- hM3 cells, are reported. The average of at least three independent experiments performed in triplicate is reported. The data concerning receptor upregulation were Downloaded from analyzed by using one-way ANOVA followed by Dunnet's multiple-comparison test.

Statistical significance is denoted compared with the atropine group (*: p < 0.05). jpet.aspetjournals.org

% hM3

pEC50 upregulation at ASPET Journals on September 27, 2021

Atropine 54 ± 7% 9.10 ± 0.08

NMS 19 ± 4%* 9.03 ± 0.10

Pirenzepine 39 ± 2% 6.14 ± 0.06

Ipratropium 41 ± 2% 9.02 ± 0.07

Tiotropium 18 ± 5%* 10.27 ± 0.10

Aclidinium 40 ± 11% 9.51 ± 0.38

Glycopyrrolate 43 ± 1% 9.39 ± 0.08

4-DAMP 18 ± 2%* 9.79 ± 0.08

*: p < 0.05

33 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. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 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. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 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. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 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. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 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. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 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. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021