Title: Contractions of the mouse prostate elicited by acetylcholine are mediated by M 3 muscarinic

Transcription

1 JPET Fast This Forward. article has not Published been copyedited on and September formatted. The 1, final 2011 version as may DOI: /jpet differ from this version. :Wd deeedd d Title: Contractions of the mouse prostate elicited by acetylcholine are mediated by M 3 muscarinic receptors Carl White, Jennifer Short, John Haynes, Minoru Matsui and Sabatino Ventura Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia (C.W., J.S., J.H., S.V.); Department of Clinical Research and General Medicine, Tokyo-Nishi Tokushukai Hospital, Tokyo, Japan (M.M) Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.

2 :Wd deeedd d Running title: M 3 muscarinic receptors in the mouse prostate Corresponding Author: Dr. Sabatino Ventura 381 Royal Parade, Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia Telephone: Fax: Sab.Ventura@monash.edu Pages: 29 Words in Abstract: 236 Words in Introduction: 575 Words in Discussion: 1473 Tables: 2 Figures: 8 References: 36 Non-standard abbreviations: 1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), analysis of variance (ANOVA), acetylcholine (Ach), benign prostatic hyperplasia (BPH), carbachol (Cch), cyclohexyl-hydroxy-phenyl- (3-piperidin-1-ylpropyl)silane (HHSiD), M 3 -muscarinic receptor (M3R), cyclohexyl-(4-fluorophenyl)- hydroxy-(3-piperidin-1-yl propyl)silane (p-f-hhsid), Section: Other

3 :Wd deeedd d Abstract Increased smooth muscle tone in the human prostate contributes to the symptoms associated with benign prostatic hyperplasia. In the mouse prostate gland, cholinergic innervation is responsible for a component of the nerve mediated contractile response. This study investigates the muscarinic receptor subtype responsible for the cholinergic contractile response in the mouse prostate gland. To characterise the muscarinic receptor subtype, mouse prostates taken from wild-type or M 3 muscarinic receptor knockout mice were mounted in organ baths. The isometric force that tissues developed in response to, electrical field stimulation or exogenously applied cholinergic agonists in the presence or absence of a range of muscarinic receptor antagonists, was evaluated. Carbachol elicited reproducible and concentration dependent contractions of the isolated mouse prostate, which were antagonised by the presence of muscarinic receptor antagonists. Calculation of antagonist affinities (pa 2 -values) indicated a rank order of antagonist potencies in the mouse prostate of: darifenacin (9.08) = atropine (9.07) = 4-DAMP (9.02) > HHSiD (7.85) > p-f-hhsid (7.39) > himbacine (7.19) > pirenzipine (6.88) > methoctramine (6.20). Furthermore, genetic deletion of the M 3 muscarinic receptor inhibited prostatic contractions to electrical field stimulation or exogenous administration of acetylcholine. In this study we identified that the cholinergic component of contraction in the mouse prostate is mediated by the M 3 muscarinic receptor subtype. Pharmacological antagonism of the M 3 muscarinic receptor may be a beneficial additional target for the treatment of benign prostatic hyperplasia in the human prostate gland.

4 :Wd deeedd d Introduction Muscarinic receptors are G-protein coupled receptors consisting of five subtypes denoted M 1-5, which are activated by the endogenous agonist acetylcholine. Muscarinic receptors can be divided into two groups depending on their G-protein coupling properties, M 1, M 3 and M 5 receptor subtypes couple to G q/11 proteins and signal through the inositol phosphate pathway, whereas the M 2 and M 4 receptor subtypes couple to G i/o proteins and inhibit adenylate cyclase activity. In the prostate gland, cholinergic innervation acting at muscarinic receptors in the glandular epithelium is thought to be primarily responsible for the secretion of prostatic fluids (Ventura et al., 2002). However in the mouse prostate gland, in addition to noradrenaline acting at α 1L -adrenoceptors (Gray and Ventura, 2006), we have shown a significant cholinergic component of the nerve mediated contractile response whereby acetylcholine acts at muscarinic receptors to mediate contraction (White et al., 2010). Currently, the muscarinic receptor subtype mediating the contractile response in the mouse prostate gland is unknown. In the canine prostate, M 2 muscarinic receptors are responsible for mediating contraction (Fernandez et al., 1998). In contrast, M 1 muscarinic receptors mediate the facilitation of contraction in the guinea pig prostate (Lau et al., 2000). In the rat prostate, the M 3 muscarinic receptor subtype has been reported to mediate contractile responses to carbachol (Lau and Pennefather, 1998). Moreover, immunohistochemical studies of the rat prostate have shown co-localisation of the M 3 muscarinic receptor subtype with the prostatic smooth muscle (Nadelhaft, 2003). However, a number of recent studies have shown that subtype specific muscarinic receptor antibodies are largely non-specific (Jositsch et al., 2009; Michel et al., 2009; Pradidarcheep et al., 2009). As such previous immunohistochemical studies of the prostate using muscarinic subtype specific antibodies should be viewed cautiously without additional functional evidence for the role of a particular muscarinic receptor subtype in the prostate. Both the M 1 and M 2 muscarinic receptor subtypes are observed in binding studies of whole human prostate homogenates (Anisuzzaman et al., 2008). However, Ruggieri et al. (1995) only observed the M 1 muscarinic subtype which was found to be localised immunohistochemically in the epithelium. In

5 :Wd deeedd d cultures of human prostatic smooth muscle cells only the M 2, M 3 and M 4 muscarinic receptor subtype mrna expression is observed (Obara et al., 2000), with the M 2 muscarinic receptor being most abundant. In a separate study of human prostatic smooth muscle cell cultures, M 2 muscarinic receptors were observed by radioligand binding experiments and mediated a decrease in camp accumulation (Yazawa et al., 1994). In the mouse prostate both the clinically selective M 3 muscarinic receptor antagonists darifenacin and solifenacin inhibited [ 3 H] NMS binding after oral administration indicating a population of muscarinic receptors (Oki et al., 2005; Yamada et al., 2006). With the availability of muscarinic receptor subtype knockout mice the M 1 and M 3, but not the M 2, M 4 or M 5 muscarinic receptor subtypes were shown to be present in mouse prostate by [ 3 H] NMS binding studies (Ito et al., 2009). However the location and function of either the M 1 or M 3 muscarinic receptor subtypes present in the mouse prostate was not elucidated. The muscarinic receptor subtype mediating contraction in the mouse prostate is of interest as an increase in prostatic smooth muscle tone contributes to the lower urinary tract symptoms associated with benign prostatic hyperplasia (BPH) in humans. The aim of this study was to pharmacologically characterise the muscarinic receptor subtype mediating the cholinergic contractile response in the mouse prostate gland and to confirm the muscarinic receptor subtype using muscarinic receptor knockout mice.

6 :Wd deeedd e Methods Animals Heterozygous (M3R +/-) breeding pairs of adult M 3 muscarinic receptor knockout mice were purchased from the Centre for Animal Resources and Development (Kumamoto University, Japan) and a colony was established at the Monash Animal Services facility (Clayton). Age matched littermates were used at 10 weeks for experimentation and were obtained by matings of M3R +/- breeding pairs maintained on a B6.129 background. The resulting litters of M 3 muscarinic receptor knockout (M3R -/-), M3R +/- and wild-type (M3R, +/+) mice were routinely genotyped by PCR using genomic DNA from tail samples obtained at weaning (21 days) as described previously (Matsui et al., 2000). For pharmacological characterisation experiments wild-type adult male mice ( 8 weeks) from a C57Bl/6 background were used. All mice were bred and housed at the Monash Animal Services facility (Clayton), exposed to a 12 hr light/dark photoperiod, and had free access to food and water. Mice were weighed before being sacrificed by cervical dislocation. Prior approval for animal experimentation was granted by the Monash University Standing Committee on Animal Ethics, ethics numbers VCPA 2009/14 and VCPA 2009/15, for the use of genetically modified and wild-type mice respectively. All studies conformed to the National Health and Medical Research Council, Australian code of practice for the care and use of animals for scientific purposes. Isolated organ bath studies The whole mouse ventral prostate was obtained by an incision along the midline of the lower abdomen which exposed the urogenital tract. To reveal the prostate; the penile muscles, excess fat and connective tissue were cut away. The prostate was then carefully dissected out and placed in a specimen jar containing Krebs-Henseleit solution (NaCl mm, NaHCO mm, glucose 11.7 mm, KCl 4.69 mm, KH 2 PO mm, MgSO mm, CaCl mm; ph 7.4). The prostate was mounted in a 10 ml water jacketed organ bath containing Krebs-Henseleit solution maintained at 37 C and bubbled with 95

7 :Wd deeedd e % O 2 / 5 % CO 2. One end of the tissue was attached to a perspex tissue holder and the other to a Grass FT03 force displacement transducer (Grass Instruments, Quincy, MA) for the measurement of isometric contractions. The force of contraction was recorded using a PowerLab 4/SP data acquisition system (ADInstruments Pty. Ltd., Castle Hill, NSW) and LabChart software version 5 (ADInstruments Pty. Ltd.). Tissues were maintained under a resting force of approximately 0.7 g. Prior to experimentation tissue preparations were equilibrated for a period of 1 hour, during which time prostates were stimulated with electrical pulses of 0.5 ms duration, 60 V at 0.01 Hz. Electrical stimulation occurred via two parallel platinum electrodes incorporated into the tissue holder, connected to a Grass S88 stimulator (Grass Instruments, Quincy, MA). Following experimentation, prostates were removed from the organ baths, blotted dry and weighed. Muscarinic receptor characterisation Following the equilibration period the prostates from wild-type C57Bl/6 mice were exposed to a priming dose of 1 mm carbamylcholine chloride (carbachol) to ensure reproducible responses. Once a maximum contractile response was observed the tissue was washed 4 times with the volume of the organ bath. After a 30 minute recovery period an initial discrete half-log concentration response curve to carbachol (10 nm 30 µm) was then constructed with 15 minute intervals between drug additions, whereby for each concentration the response was allowed to reach a maximum and plateau before the tissue was washed out with 4 times the volume of the organ bath. To assess confounding effects on potency by α 1 -adrenoceptors or cholinesterase activity, an appropriate time control curve to carbachol was constructed in the presence or absence of prazosin (0.3 µm) or physostigmine (10 µm). In subsequent experiments to prevent any confounding effects by α 1 -adrenoceptors on characterisation of the muscarinic receptor subtype, prazosin (0.3 µm) was added to the Krebs-Henseleit solution. To characterise the muscarinic subtype mediating contraction, following the initial concentration response curve, isolated prostates were exposed to a muscarinic receptor antagonist for one hour before a second

8 :Wd deeedd e concentration response curve to carbachol (10 nm 1 mm) was constructed in the same manner. The muscarinic receptor antagonist was replaced following each wash. Effect of M3 muscarinic receptor deletion on nerve mediated contraction Frequency response curves were constructed in prostates taken from M3R -/-, M3R +/- and M3R +/+ mice following the equilibration period. Prostates were exposed to electrical field stimulation at frequencies of 0.1, 0.2, 0.5, 1, 2, 5, 10 and 20 Hz (0.5 ms duration, 60 V) delivered at 10 min intervals in trains of pulses lasting 10 pulses or 10 s. An initial control frequency response curve was constructed to determine the contractile response of the tissue at each frequency. A second frequency response curve was then constructed after the prostate had been washed 3 times with the volume of the organ bath and exposed to atropine (1 µm) for a period of one hour. Where a third frequency response curve was constructed, the prostate was washed as described previously and exposed to atropine (1 µm) plus prazosin (0.3 µm). Following the final frequency response curve the prostate was washed again and allowed to rest for 1 hour before the Krebs-Henseleit solution was replaced with a high K + (80 mm) Krebs-Henseleit solution to measure the non specific contractile potential of the preparation. Effect of M3 muscarinic receptor deletion on acetylcholine mediated contraction Following the one hour equilibration period, an initial discrete concentration response curve to acetylcholine (10 nm - 1 mm), as described previously for carbachol, was constructed in prostates taken from M3R -/-, M3R +/- as well as M3R +/+ mice. After the initial concentration response curve, a second concentration response curve was constructed in the same manner; however the tissue was exposed to atropine (1 µm) for one hour prior to the first addition of acetylcholine and was replaced following each wash. In a separate experiment, using prostates taken from M3R -/- mice, the effect of mecamylamine (30 µm) on acetylcholine concentration response curves (control; 100 nm - 1 mm, in the presence of mecamylamine; 100 nm - 10 mm) was observed.

9 :Wd deeedd e Data analysis The peak force (g) elicited by electrical field stimulation or the exogenous application of muscarinic receptor agonists was measured at each frequency or concentration, in the presence and absence of antagonists. Mean frequency and concentration response curves were constructed using GraphPad Prism version 5.00 for windows (GraphPad Software, San Diego, CA). The mean curves were formed from the average of data from n experiments, where n is equal to the number of mice used. Results are expressed as mean ± S.E.M.. For characterisation studies, the contractile responses to carbachol were expressed as a percentage of the maximum response of the initial concentration response curve. The nonlinear regression function of GraphPad Prism version 5.00 was then used to fit an unconstrained sigmoidal concentration-response curve to the data. For each antagonist, concentration ratios (CR) were determined by dividing the EC 50 value of carbchol in the presence of the antagonist by the EC 50 value in the absence of the antagonist. Following which, Arunlakshana - Schild plots were constructed whereby log (CR-1) was plotted versus log (antagonist concentration) (Arunlakshana and Schild, 1959). When the slope of the linear regression did not differ from unity, the slope was constrained to 1 and the pa 2 value was calculated. Correlation and linear regression analysis was used to determine the muscarinic receptor subtype mediating contraction of the mouse prostate. pa 2 values of the antagonists at the muscarinic receptors in the mouse prostate were compared against the mean published antagonist pk D or pk i values at each of the five muscarinic receptor subtypes (Table 1). Ionic strength of assay buffer is known to influence the binding affinity of muscarinic receptor antagonists (Loury et al., 1999). As such pk D or pk i values were selected from Loury et al. (1999) and the International Union of Basic and Clinical Pharmacology (IUPHAR) GPCR database only from studies using buffers of comparable ionic strength to the Krebs-Henseleit solution used in this study. Where different antagonist affinities were given for the same subtype, the mean value was used for the correlation analysis. Pearson correlation coefficients (r) and associated p-values, as well as the slope of linear regression, were calculated using GraphPad Prism version 5.00.

10 :Wd deeedd dd In mean frequency and concentration response curves, differences between the initial and subsequent drug exposed curve were analysed by GraphPad Prism version 5.00 using a two way repeated measure analysis of variance (ANOVA), followed by a Bonferroni post-test where required. The p-values stated were used to evaluate the statistical significance of any difference between the two curves and represent the probability of the drug treatment causing a change. p < 0.05 was considered significant. All receptor nomenclature conforms to the IUPHAR nomenclature guidelines. Reagents used (+)-himbacine, acetylcholine chloride, atropine sulphate, carbamoylcholine chloride (carbachol), eserine hemisulfate salt (physostigmine), mecamylamine hydrochloride, methoctramine hemi-hydrate and prazosin hydrochloride were obtained from Sigma-Aldrich (St. Louis, MO). 1,1-Dimethyl-4- diphenylacetoxypiperidinium iodide (4-DAMP), cyclohexyl-hydroxy-phenyl-(3-piperidin-1- ylpropyl)silane (HHSiD), cyclohexyl-(4-fluorophenyl)-hydroxy-(3-piperidin-1-yl propyl)silane (p-f- HHSiD) were obtained from Research Biochemical International (Natick, MA). Darifenacin hydrobromide was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and pirenzipine dihydrochloride from Boehringer Ingelheim (Artamon, NSW). All drugs were dissolved and diluted in distilled water except HHSiD, which was dissolved in 10 % dimethyl sulfoxide, all further dilutions were made in distilled water.

11 :Wd deeedd dd Results Pharmacological characterisation In the isolated wild-type mouse prostate administration of exogenously applied carbachol (10 nm 1 mm) produced reproducible concentration dependant contractile responses with a log (EC 50 ) of 5.99 ± 0.05 (n=18, p=0.95). The contractile response to carbachol was unaffected by physostigmine (10 µm; Fig. 1a, p=0.20), however prazosin (0.3 µm; Fig. 1b, p<0.05) caused a three fold rightward shift in the concentration response curve to carbachol. The muscarinic receptor antagonists used were: 4-DAMP (3, 10, 30 nm), atropine (6, 20, 60 nm), p-f-hhsid (0.3, 1, 3 µm), HHSiD (0.1, 0.3, 1 µm), darifenacin (Fig. 2; 10, 30, 300 nm), himbacine (0.1, 0.3, 1 µm), methoctramine (1, 3, 10 µm), and pirenzipine (0.6, 1, 3 µm). All caused concentration dependent parallel rightward shifts in the concentration response curve to carbachol. The slopes and pa 2 values calculated by Arunlakshana Schild analysis are shown in Table 2. None of the antagonists produced a slope that was significantly different from unity (Fig. 3; p > 0.05). The rank order of antagonist affinities (pa 2 -values) at the muscarinic receptor mediating contraction in the isolated mouse prostate (Table 2) was: Darifenacin (9.08) = atropine (9.07) = 4-DAMP (9.02) > HHSiD (7.85) > p-f- HHSiD (7.39) > himbacine (7.19) > pirenzipine (6.88) > methoctramine (6.20). Correlation and linear regression analysis revealed significant correlation and linear regression for the M 3 (Fig. 4c; r = 0.98, p < 0.001, slope = 1.05 ± 0.08) and M 5 (Fig. 4e; r = 0.88, p < 0.01, slope = 1.02 ± 0.22) muscarinic receptor subtypes. Whereas, weaker linear regressions and correlations were observed for the M 1 (Fig. 4a; r = 0.77, p < 0.05, slope = 0.64 ± 0.21), M 2 (Fig. 4b; r = 0.51, slope = 0.44 ± 0.30) and M 4 subtypes (Fig. 4d; r = 0.78, p = 0.05, slope = 0.66 ± 0.22).

12 :Wd deeedd dd Genetic confirmation Deletion of the M 3 muscarinic receptor resulted in a reduction in mouse weight (Fig. 5a; p<0.001) however no effect on prostate weight was observed (Fig. 5b). Furthermore, the contractile response to high K + (80 mm) Krebs-Henseleit was reduced in prostates taken from both M3R +/- and M3R -/- mice (Fig. 5c; p<0.01). In prostates taken from M3R +/+, M3R +/- and M3R -/- mice, electrical field stimulation ( Hz) elicited frequency-dependent contractions. Contractile responses were reduced in prostates taken from M3R -/- mice but not M3R +/- mice when compared to prostates taken from M3R +/+ mice (Fig. 6). A maximum inhibition of contraction of 76 % for M3R -/- mice compared to M3R +/+ mice was observed at 2 Hz. Furthermore, atropine (1 µm) attenuated electrical field stimulation mediated contraction in prostates taken from both M3R +/+ (Fig.7a; p<0.001) and M3R +/- mice (Fig. 7b; p<0.001) with a maximum inhibition of contraction observed at 5 Hz of 76 % and 70% respectively. However, atropine (Fig. 7c; p=0.54, 1 µm) had no effect on the magnitude of contraction in prostates taken from M3R -/- mice, whereas prazosin (Fig. 7c; p<0.001, 0.3 µm) abolished the residual non-cholinergic contraction. The administration of the endogenous agonist acetylcholine (10 nm 100 µm) elicited concentration dependent contractile responses (Fig. 8a) that were inhibited by atropine (1 µm) in prostates taken from M3R +/+ and M3R +/- mice. A slightly decreased potency for acetylcholine was observed in prostates taken from M3R +/- mice (Fig. 8a; -log(ec 50 ) = 6.06, 95% confidence intervals ) when compared to M3R +/+ mice (Fig. 8a; -log(ec 50 ) = 6.46, 95% confidence intervals ). The maximum contractile response was unchanged (Fig. 8a; p=0.83). In prostates taken from M3R -/- mice acetylcholine elicited a small contractile response at concentrations greater than 10 µm (Fig. 8a). This response was unaffected by the addition of atropine (Fig. 8b; p=0.47, 1 µm), but was abolished by mecamylamine (Fig. 8c; p<0.001, 30 µm).

13 :Wd deeedd dd Discussion We have previously identified that in the mouse prostate gland acetylcholine acting at muscarinic receptors mediates a non-adrenergic contractile response; however the muscarinic receptor subtype mediating this response was not elucidated. Furthermore, it has been reported that both the M 1 and M 3 muscarinic receptor subtypes are present in the mouse prostate (Ito et al., 2009) however; their locations and functions are unknown. This study used pharmacological and genetic techniques to examine the muscarinic receptor subtype responsible for the functional contractile response. The contractile response to carbachol was unaffected by cholinesterase activity and was therefore seen as more suitable than acetylcholine for the use in the pharmacological characterisation study as it eliminated this confounding factor. However, the response to carbachol was inhibited by the α 1 - adrenoceptor antagonist prazosin suggesting that carbachol facilitates the adrenergic response. This is potentially due to the release of noradrenaline mediated by pre-junctional muscarinic receptors on adrenergic nerves, or nicotinic receptor activation of post-ganglionic adrenergic nerves. In the guinea-pig prostate, cholinergic agonists facilitate nerve mediated contraction hypothesised to be mediated by noradrenaline release by pre-juctional muscarinic receptors (Lau et al., 2000). In contrast, in both the human (Hedlund et al., 1985) and canine (Arver and Sjostrand, 1982) prostate, muscarinic receptors produce a slight reduction in noradrenaline release. Similarly, sympathetic neurotransmitter release is inhibited in the mouse atria, urinary bladder and vas deferens by muscarinic receptors (Trendelenburg et al., 2005). In the present study, atropine had no detectable effect on the contractile response to high concentrations of acetylcholine in prostates taken from M3R -/- mice, whereas the non-competitive nicotinic antagonist mecamylamine abolished the contractile response. This suggests that post-ganglionic nicotinic receptors rather than pre-junctional muscarinic receptors mediate the release of neuronal noradrenaline.

14 :Wd deeedd dd Correlation plots show that the antagonist affinities obtained in the pharmacological characterisation studies correlate most noticeably with the binding affinities of the antagonists at the M 3 muscarinic receptor subtype. Progressively poorer correlations were observed for the M 5, M 4, M 1 and M 2 muscarinic receptor subtypes. The slope of the regression analysis was also used to determine the muscarinic receptor subtype, with a slope of the line closest to unity (x = y) considered to be the best correlation. The best regression analysis was observed for the M 3 and M 5 muscarinic receptor subtypes. Taken together these results are indicative of the M 3 muscarinic receptor subtype mediating contraction in the mouse prostate gland. These results are consistent with findings in the rat prostate, where the M 3 muscarinic receptor subtype mediates contraction (Lau and Pennefather, 1998) and is localised in the stromal tissue (Nadelhaft, 2003). However, this is in contrast to findings in the human (Yazawa et al., 1994) and canine (Fernandez et al., 1998) prostates, where M 2 muscarinic receptors are thought to mediate contraction and in the guinea-pig prostate where M 1 muscarinic receptors facilitate contraction (Lau et al., 2000). It should be noted that a good correlation, with poor linear regression, was also observed for the M 1 subtype. Given a population of M 1 receptors have previously been reported in the mouse prostate (Ito et al., 2009) this is intriguing. At the M 1 muscarinic receptor the binding affinities for all the antagonists used, except for darifenacin and pirenzpine, are close to the binding affinities for the M 3 muscarinic receptor subtype (Birdsall et al.). Therefore darifenacin and pirenzipine impart the greatest strength when differentiating between M 1 and M 3 muscarinic receptors. The binding affinity for darifenacin (pa 2 = 9.08) exclusively suggests the M 3 subtype over the M 1. For pirenzipine however, the binding affinity (pa 2 = 6.88) falls within the range for the M 3 subtype and somewhat within the range of the M 1 subtype. The Schild slope (Table 2) for pirenzipine is steeper than what is expected of a truly competitive antagonist, albeit not significantly. The lack of statistical significance in this difference may be the result of a broad S.E.M. (± 0.38) confounding the analysis and can be explained by the concentration range of antagonist used (0.6 3 µm) being insufficiently large (Kenakin, 2009). When the binding affinity is calculated as a

15 :Wd deeedd dd pk B -value, instead of pa 2 value using the equation described by Furchogott (1972), the binding affinity obtained is 5.89, which is well within the range of the M 3 but not the M 1 muscarinic receptor subtype. Similarly, good linear regression analysis was observed for both the M 3 and M 5 subtypes. This is unsurprising given the dearth of muscarinic receptor antagonists capable of distinguishing between M 3 and M 5 muscarinic receptors. Given Ito et al. (2009) failed to find a significant population of M 5 receptors in the mouse prostate gland, in addition to the results presented here using M 3 muscarinic receptor knockout mice, it appears unlikely that M 5 muscarinic receptors mediate contraction M 3 muscarinic receptor knockout (M3R -/-) mice were used to confirm the muscarinic receptor subtype mediating the cholinergic contractile response in the mouse prostate gland. However a significant difference in the response to 80 mm K + Krebs-Henseleit solution between genotypes was also observed. This result is interesting and unexplained however; it may suggest that endogenously released acetylcholine acting at M 3 muscarinic receptors contributes to the contractile response mediated by 80 mm K + Krebs-Henseleit solution. Equally, it may suggest that the deletion of the M 3 muscarinic receptor affects the contractile machinery in the mouse prostate gland. Nevertheless, to allow fair comparison between genotypes, results to electrical field stimulation were plotted as a percentage of the contractile response to 80 mm K + Krebs-Henseleit solution. In prostates taken from M3R -/- mice, we observed that the nerve mediated contractile response was inhibited. Furthermore, atropine was without effect in M3R -/- mice. These observations suggest that the M 3 muscarinic receptor subtype is solely responsible for the cholinergic component of nerve mediated contraction and that compensatory changes by up-regulation of other muscarinic receptor subtypes do not occur in prostates taken from M3R -/- mice. This does not exclude the possibility of up regulation of noncholinergic mechanisms such as purinergic mechanisms, as observed in bladders of M3R -/- mice (Igawa et al., 2004). However, this appears unlikely in the prostate as the residual contractile response to electrical field stimulation in prostates taken from M3R -/- mice is abolished by the α 1 -adrenoceptor

16 :Wd deeedd de antagonist prazosin. Furthermore this confirms that the nerve mediated contractile response in the mouse prostate is mediated by both muscarinic receptors and α 1L -adrenoceptors (White et al., 2010). The decrease in the potency of acetylcholine observed in prostates taken from M 3 muscarinic receptor heterozygous mice may be a product of fewer M 3 muscarinic receptors being available, while the response to acetylcholine in prostates taken from M3R +/+ mice was comparable with our previous results (White et al., 2010). The absence of a contractile response to acetylcholine, except at high concentration (as discussed previously) in M3R -/- mice further confirms the M 3 muscarinic receptor as the subtype mediating contraction in the mouse prostate gland. The weight of the prostate was unaffected by the deletion of the M 3 muscarinic receptor, indicating the M 3 muscarinic receptor and/or gene does not play a role in growth of the mouse prostate gland. In primary human cell cultures and prostate cancer cell lines (Witte et al., 2008), as well as in the rat prostate (McVary et al., 1998), growth can be regulated by cholinergic agonists or parasympathetic innervation respectively. While the results of the present study exclude a role for M 3 muscarinic receptors in regulating growth, it can not exclude the possibility of additional muscarinic receptor subtypes regulating mouse prostate growth. It should be noted that the reduction in mouse weight at this age in M3R -/- mice, caused by an impairment of saliva production (Matsui et al., 2000) or appetite (Yamada et al., 2001), may have influenced this result. Unlike the mouse prostate, the cholinergic component of nerve mediated contraction in the human prostate is small compared to the adrenergic response (Caine et al., 1975; Hedlund et al., 1985; Gup et al., 1989; Kester et al., 2003). However, a recent study has observed an adreno-muscarinic synergy in contraction of the human prostate (Roosen et al., 2009). Although the M 3 muscarinic receptor subtype has not been demonstrated in the human prostate, recent reviews of clinical trials using muscarinic receptor antagonists (which included the M 3 muscarinic receptor antagonists solifenacin and darifenacin) for the treatment of lower urinary tract symptoms, usually in combination with an adrenoceptor

17 :Wd deeedd de antagonist, show varying improvement in storage symptoms without worsening of voiding symptoms or significant development of acute urinary retention (Athanasopoulos, 2010; Chapple, 2010). Although these drugs may act by inhibition of detrusor instability in the bladder, prostatic smooth muscle tone can not be ruled out as an additional site of action for the treatment of lower urinary tract symptoms by muscarinic receptor antagonists. In conclusion, the results of the present study have shown that the M 3 muscarinic receptor subtype is solely responsible for the nerve mediated cholinergic component of contraction in the mouse prostate gland.

18 :Wd deeedd de Acknowledgements: Authorship Contributions: Participated in research design: White, Ventura and Short Conducted experiments: White Contributed new reagents or analytic tools: Matsui Performed data analysis: White Wrote or contributed to the writing of the manuscript: White, Ventura, Short and Haynes

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22 :Wd deeedd dd Lau WAK and Pennefather JN (1998) Muscarinic receptor subtypes in the rat prostate gland. Eur J Pharmacol 343: Lau WAK, Pennefather JN and Mitchelson FJ (2000) Cholinergic facilitation of neurotransmission to the smooth muscle of the guinea-pig prostate gland. Br J Pharmacol 130: Loury DN, Hegde SS, Bonhaus DW and Eglen RM (1999) Ionic strength of assay buffers influences antagonist binding affinity estimates at muscarinic M1-M5 cholinoceptors. Life Sci 64:557. Matsui M, Motomura D, Karasawa H, Fujikawa T, Jiang J, Komiya Y, Takahashi S and Taketo MM (2000) Multiple functional defects in peripheral autonomic organs in mice lacking muscarinic acetylcholine receptor gene for the M3 subtype. Proc Natl Acad Sci U S A 97: McVary KT, McKenna KE and Lee C (1998) Prostate innervation. The Prostate Supplement 8:2-13. Michel MC, Wieland T and Tsujimoto G (2009) How reliable are G-protein-coupled receptor antibodies? Naunyn Schmiedebergs Arch Pharmacol 379: Nadelhaft I (2003) Cholinergic axons in the rat prostate and neurons in the pelvic ganglion. Brain Res 989:52-57.

23 :Wd deeedd dd Obara K, Arai K, Miyajima N, Hatano A, Tomita Y and Takahashi K (2000) Expression of m2 muscarinic acetylcholine receptor mrna in primary culture of human prostate stromal cells. Urol Res 28: Oki T, Sato S, Miyata K and Yamada S (2005) Muscarinic receptor binding, plasma concentration and inhibition of salivation after oral administration of a novel antimuscarinic agent, solifenacin succinate in mice. Br J Pharmacol 145: Pradidarcheep W, Stallen J, Labruyere WT, Dabhoiwala NF, Michel MC and Lamers WH (2009) Lack of specificity of commercially available antisera against muscarinergic and adrenergic receptors. Naunyn Schmiedebergs Arch Pharmacol 379: Roosen A, Blake-James BT, Wood D and Fry CH (2009) Clinical and experimental aspects of Adrenomuscarinic synergy in the bladder base and prostate. Neurourol Urodyn 28: Ruggieri MR, Colton MD, Wang P, Wang J, Smyth RJ, Pontari MA and Luthin GR (1995) Human prostate muscarinic receptor subtypes. J Pharmacol Exp Ther 274:

24 :Wd deeedd dd Trendelenburg A-U, Meyer A, Wess J and Starke K (2005) Distinct mixtures of muscarinic receptor subtypes mediate inhibition of noradrenaline release in different mouse peripheral tissues, as studied with receptor knockout mice. Br J Pharmacol 145: Ventura S, Pennefather JN and Mitchelson F (2002) Cholinergic innervation and function in the prostate gland. Pharmacol Ther 94: White CW, Short JL, Haynes JM, Evans RJ and Ventura S (2010) The residual nonadrenergic contractile response to nerve stimulation of the mouse prostate is mediated by acetylcholine but not ATP in a comparison with the mouse vas deferens. J Pharmacol Exp Ther 335: Witte LPW, Chapple CR, de la Rosette J and Michel MC (2008) Cholinergic innervation and muscarinic receptors in the human prostate. Eur Urol 54: Yamada M, Miyakawa T, Duttaroy A, Yamanaka A, Moriguchi T, Makita R, Ogawa M, Chou CJ, Xia B, Crawley JN, Felder CC, Deng C-X and Wess J (2001) Mice lacking the M3 muscarinic acetylcholine receptor are hypophagic and lean. Nature 410: Yamada S, Maruyama S, Takagi Y, Uchida S and Oki T (2006) In vivo demonstration of M3 muscarinic receptor subtype selectivity of darifenacin in mice. Life Sci 80:

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26 :Wd deeedd de Footnotes This work was supported by the ANZ Trustees [09/3164] (to S.V.) and the National Health & Medical Research Council (Australia) [334136] (to S.V.). Part of this work has previously been presented in abstract form. White C, Short J, Ventura S. Characterisation of the muscarinic receptor subtype mediating contraction in the mouse prostate gland. Basic Clin Pharmacol Toxicol 107 Suppl th World Congress on Basic and Clinical Pharmacology Copenhagen, Denmark, July 2010, poster presentation

27 :Wd deeedd de Legends for figures Figure 1 Mean log concentration response curves to carbachol in the isolated mouse prostate expressed as a percentage of the maximum response in the initial curve, before and after the administration of: (a) physostigmine (10 µm) or (b) prazosin (0.3 µm). Points represent mean force ± S.E.M., n=6. p values determined by a two-way repeated-measures of ANOVA represent the probability of a significant interaction between concentration and treatment. Histogram on right axis represent the mean ± S.E.M., n=6 maximum contractile response in the absence (open bar), or presence of: (a) physostigmine (10 µm) or (b) prazosin (0.3 µm) (closed bar). Figure 2 Representative graph showing the contractile response of the isolated mouse prostate to carbachol in the absence and presence of increasing concentrations of the muscarinic receptor antagonist darifenacin (10 nm, 30 nm and 100 nm). Responses expressed as a percentage of the mean ± S.E.M., n=6 maximum contractile response of carbachol in the absence of antagonist. Histogram represents the mean ± S.E.M., n=6 maximum contractile response in the absence (open bar), as well as presence of darifenacin 10 nm (grey bar), 30 nm (black bar) and 100 nm (crosshatched grey bar). Figure 3 Schild analysis of the muscarinic receptor antagonists atropine, pirenzepine, 4-DAMP, himbacine, methoctramine, HHSiD, p-f-hhsid and darifenacin. Each point represents the mean ± S.E.M., n=6. Slopes of the linear regression and pa 2 values are shown in Table 2.

28 :Wd deeedd de Figure 4 Correlation of the pa 2 values observed for the seven muscarinic receptor antagonists (x-axis) with the published (Table 1, y-axis) muscarinic receptor antagonist pk i values at the M 1 (a), M 2 (b), M 3 (c), M 4 (d) and M 5 (e) muscarinic receptor subtypes. Solid lines represent the linear regression analyses, whereas broken lines represent a line with a slope of 1. Inserts indicate the slope ± S.E.M of linear regression as well as the Pearsons coefficient (r), calculated by correlation analysis. Figure 5 Comparison of mean mouse weights (a), isolated prostate weights (b) and contractile responses to 80 mm K + Krebs-Henseleit solution (c) from wild-type (M3R +/+, open bars), M 3 muscarinic receptor heterozygous mice (M3R +/-, grey bars) and M 3 muscarinic receptor knockout mice (M3R -/-, black bars). Bars represent mean weight/contractile force ± S.E.M., n=6. **, p<0.01 ***, p<0.001 versus control calculated by an unpaired t-test. Figure 6 Comparison of mean contractile responses to electrical field stimulation (0.5 ms, 60 V, Hz, 10 s pulses) in prostates taken from wild-type mice (M3R +/+, open bars), M 3 muscarinic receptor heterozygous mice (M3R +/-, grey bars) and M 3 muscarinic receptor knockout mice (M3R -/-, black bars). Bars represent mean force ± S.E.M., n=6. ANOVA p-values determined by a two-way repeatedmeasures of ANOVA and represent the probability of the M 3 muscarinic receptor deletion causing a significant change in response. Figure 7 Mean contractile responses to electrical field stimulation (0.5ms, 60V, 1-20 Hz, 10 s pulses) in prostates taken from: (a) wild-type mice (M3R +/+), (b) M 3 muscarinic receptor heterozygous mice (M3R +/-) and (c) M 3 muscarinic receptor knockout mice (M3R -/-) in the absence (open bars) or presence of (a,b,c)

29 :Wd deeedd de atropine (1 µm) (closed bars) as well as (c) atropine (1 µm) plus prazosin (0.3 µm) (grey bar). Bars represent mean force ± S.E.M., n=6. ANOVA p-values determined by a two-way repeated-measures of ANOVA represent the probability of the drug treatment causing a significant change. Figure 8 (a) Comparison of mean contractile response to acetylcholine in prostates taken from wild-type (M3R +/+, closed circles), M 3 muscarinic receptor heterozygous mice (M3R +/-, closed squares) and M3 muscarinic receptor knockout mice (M3R -/-, closed triangles). (b,c) Mean log concentration response curves to acetylcholine in prostates taken from M 3 muscarinic receptor knockout mice (M3R -/-), before and after the administration of (b) atropine (1 µm) or (c) mecamylamine (30 µm). Points represent mean force ± S.E.M., n=4-6. ANOVA p-values determined by a two-way repeated-measures of ANOVA and represent the probability of the M 3 muscarinic receptor deletion, or test drug, causing a significant change in response.

30 :Wd deeedd dd Tables Table 1: pk D or pk i values reported for muscarinic receptor antagonists at the five muscarinic receptor subtypes Antagonist M1 M2 M3 M4 M5 Atropine r 9.0 r 9.3 r 9.7 r 9.4 r Darifenacin h 7.0 h 8.8 h 7.7 h 8.0 h 4-DAMP r 8.2 r 9.2 r 9.1 r 8.9 r Himbacine h 7.9 h 6.9 h 7.8 h 6.1 h Methoctramine * h (6.85) 7.3* h * h (6.2) 7.0* h 6.3* h Pirenzepine h h (6.2) 6.6 h 7.3 h 6.6 h p-f-hhsid h 6.4 h 7.6 h 7.3 h 6.3 h HHSiD h h (6.75) 7.7 h 7.7 h 6.8 h Values shown are selected pk D (indicated by *) or pk i values from, 1, (Birdsall et al.) IUPHAR database, Acetylcholine receptors (muscarinic) and 2, (Loury et al., 1999). h, r indicates values from studies using tissues containing muscarinic receptors taken from humans or rats respectively. Where a range is given the mean value in parentheses was used for the correlation analysis. Only estimates obtained from studies that employed a physiological buffer are included in this table.

31 :Wd deeedd dd Table 2: pa 2 values and slope factors (slope ± SEM) calculated from characterisation studies in the mouse prostate gland by the range of muscarinic antagonists used to carbachol Antagonist Slope ±S.E.M. pa 2 ±S.E.M Atropine 0.92± ±0.02 Darifenacin 1.02± ± DAMP 1.01± ±0.11 Himbacine 0.98± ±0.02 Methoctramine 0.95± ±0.06 Pirenzepine 1.25± ±0.09 p-f-hhsid 1.16± ±0.05 HHSiD 0.91± ±0.07

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