Pulmonary, gastrointestinal and urogenital pharmacology

Rho kinase activation mediates adrenergic and cholinergic smoothmuscle contractile responses in the mouse prostate gland

Carl W. White, Jennifer L. Short, Sabatino Ventura n

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville Victoria, 3052, Australia

a r t i c l e i n f o

Article history:Received 22 March 2013Received in revised form27 August 2013Accepted 4 September 2013Available online 17 September 2013

Keywords:NoradrenalineAcetylcholineAdrenoceptor signallingMuscarinic receptor signallingBenign prostatic hyperplasia

a b s t r a c t

With age an increase in prostatic smooth muscle tone mediated by α1L-adrenoceptors contributes to thelower urinary tract symptoms associated with benign prostatic hyperplasia. Current treatments forbenign prostatic hyperplasia include α1-adrenoceptor antagonists which inhibit smooth muscle contrac-tion. However, muscarinic receptors also mediate prostatic smooth muscle contraction and targeting aconvergent signalling pathway may be a more effective treatment strategy. This study determinedsignalling pathways involved in contraction by measuring isometric force developed by prostates fromwild type, α1A-adrenoceptor and M3-muscarinic receptor knockout mice mounted in organ baths inresponse to, electrical field stimulation or exogenously applied agonists, in the presence or absence ofsignalling pathway inhibitors. Fluorescence immunohistochemistry was also used to confirm functionalobservations. Contractile responses mediated by carbachol were reduced by inhibitors of phospholipaseC (U73122; 3–10 mM), L-type Ca2þ channels (nifedipine; 1 mM), Rho kinase (Y-27632; 10–30 mM), but notprotein kinase C (GF109203 X;10 mM). Nifedipine (1 mM), Y-27632 (10 mM), and GF109203 X (10 mM)inhibited nerve mediated contractile responses. Y-27632 (10–30 mM) inhibited noradrenaline mediatedcontractions. RhoA and ROCK2 were found to be immunolocalised with prostatic smooth-muscle.Contractions mediated by M3-muscarinic receptors in the mouse prostate involve the prototypicalphospholipase C signalling pathway, as well as L-type Ca2þ channels. Adrenergic and cholinergiccomponents of smooth muscle contraction in the mouse prostate both involve the activation of the Rho-kinase pathway, which may be a suitable common pathway for more effective treatments of symptomsassociated with benign prostatic hyperplasia.

& 2013 Elsevier B.V. All rights reserved.

1. Introduction

Nerve mediated contraction of mouse prostatic smooth muscleinvolves both adrenergic and cholinergic mechanisms with nora-drenaline acting at α1L-adrenoceptors (an α1A-adrenoceptor phe-notype found in the lower urinary tract) (Gray et al., 2008; Grayand Ventura, 2006; Muramatsu et al., 2008; Nishimune et al.,2012) and acetylcholine acting at M3 muscarinic receptors (Whiteet al., 2010, 2011). In the human, an increase in prostatic smoothmuscle tone mediated by noradrenaline acting at α1L-adrenocep-tors contributes to the lower urinary tract symptoms associatedwith benign prostatic hyperplasia. In addition, muscarinic recep-tors also mediate a contractile response in the human prostate(Caine et al., 1975; Gup et al., 1989; Hedlund et al., 1985; Kesteret al., 2003; Roosen et al., 2009).

Prostatic smooth muscle contraction is mediated via activationof Ca2þ-dependent and Rho kinase-dependent Ca2þ sensitisationsignalling pathways (Christ and Andersson, 2007). Typically, fol-lowing activation, G-protein coupled receptors involved in smoothmuscle contraction cause an increase in phospholipase C (PLC)activity resulting in the increased production of the secondmessengers inositol trisphosphate (IP3) and diacylglycerol (DAG).IP3 causes an increase in cytosolic Ca2þ by inducing its releasefrom intracellular stores, whereas DAG activates the protein kinaseC (PKC) signalling pathway (Haynes et al., 2002; Preston andHaynes, 2003) to mediate an influx of extracellular Ca2þ throughL-type Ca2þ channels (Drescher et al., 1994; Eckert et al., 1995;Guh et al., 1995; Haynes and Hill, 1997; Marshall et al., 1999).Binding of cytosolic Ca2þ to calmodulin causes a conformationalchange in calmodulin thus enabling the Ca2þ/calmodulin complexto activate myosin light chain kinase (MLCK), which in turnphosphorylates myosin light chain (MLC). Phosphorylated MLCinteracts with actin to give rise to contraction. Following contrac-tion, myosin light chain phosphatase (MLCP) dephosphorylates theMLC filaments causing relaxation of the contracted smooth muscle(see Fig. 1). In a variety of smooth muscles, including those in the

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European Journal of Pharmacology

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.ejphar.2013.09.012

Abbreviations: DAG, diacylglycerol; IP3, inositol trisphosphate; MLC, myosin lightchain; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase;ROCK2, Rho-kinase 2; PLC, phospholipase C; PKC, protein kinase C.

n Corresponding author. Tel.: þ61 3 9903 9566.E-mail address: Sab.Ventura@monash.edu (S. Ventura).

European Journal of Pharmacology 721 (2013) 313–321

lower urinary tract (Christ and Andersson, 2007), Ca2þ sensitisa-tion via regulation of myosin light chain phosphatase (MLCP) alsocontributes to smooth muscle contraction. Therefore, RhoA/Rhokinase and PKC, both of which inhibit MLCP, promote prostaticsmooth muscle contraction (Christ and Andersson, 2007).

There are conflicting reports as to the relative importance ofthe role of PLC activation which results in IP3-mediated Ca2þ

release from intracellular stores (Eckert et al., 1995; Marshall et al.,1999; Preston and Haynes, 2003). Additionally, the RhoA/Rho-kinase Ca2þ sensitisation pathway is also involved in α1-adren-ceptor mediated contractile responses in the human and ratprostates (Rees et al., 2003; Saito et al., 2011; Takahashi et al.,2007). Furthermore, recent studies have shown that activation ofc-jun N-terminal kinase (Strittmatter et al., 2012) as well asphosphorylation of caldesmon (Walther et al., 2012) are involvedin smooth muscle contraction of the prostate following α1-adre-noceptor stimulation. In contrast, little is known about the signal-ling pathways involved in muscarinic receptor contraction of theprostate. Both an increase in cytosolic Ca2þ (Witte et al., 2008) andinhibition of cAMP (Yazawa et al., 1994) accumulation havepreviously been reported. Furthermore, RhoA/Rho-kinase Ca2þ

sensitisation is involved in the muscarinic receptor mediatedcontractile response of the rat prostate (Saito et al., 2011). Theprototypical signalling pathway for M3 muscarinic receptors is viaactivation of PLC (Caulfield and Birdsall, 1998) but multiplesignalling pathways may be involved in smooth muscle contrac-tion as demonstrated in the bladder where contractions mediatedby M3 muscarinic receptors are elicited by L-type Ca2þ channelswith an apparent lack of PLC involvement (Frazier et al., 2007;Schneider et al., 2004a, 2004b).

It is known that the most effective treatments of benignprostatic hyperplasia are α1-adrenoceptor antagonists (Mianoet al., 2008). However, there are multiple mediators of prostaticcontraction. Furthermore, α1-adrenoceptor antagonists in vitrocommonly only inhibit a proportion of the nerve mediatedcontractile response (for review see Ventura et al., 2011). There-fore, identifying and targeting convergent signalling pathways formultiple mediators of contraction would be beneficial for a moreeffective treatment of benign prostatic hyperplasia. To that end,one such possible convergent pathway identified in the prostateis Rho-kinase mediated Ca2þ sensitisation and as such inhibitorsof this pathway have been suggested as possible treatments of

benign prostatic hyperplasia (Andersson, 2007; Ventura et al.,2011). However, the role of the RhoA/Rho-kinase pathway in themouse prostate is unknown. This study uses pharmacological toolsto investigate the RhoA/Rho-kinase and other signal transductionpathways responsible for the M3 muscarinic receptor mediatedcontractile response in the mouse prostate. With the endpoint ofmeasuring prostate contractility, this study aimed to identifyconvergent signalling pathways for noradrenaline and acetylcho-line. Such an investigation may be beneficial for the understandingof the increase in prostatic tone in hyperplastic human prostates.

2. Materials and methods

2.1. Animals

Male wild-type C57Bl/6 mice were obtained at Z8 weeks ofage from the Monash Animal Research Platform (MARP) RodentBreeding & Research Facility (Clayton) and transported to theMARP Small Animal Facility (Parkville) before experimentation.Breeding pairs of α1A-adrenoceptor and M3 muscarinic receptorknockout mice were obtained from The Jackson Laboratory (BarHarbor, USA) and the Centre for Animal Resources and Develop-ment (Kumamoto University, Kumamoto, Japan), respectively.Colonies of knockout mice were maintained on a C57Bl/6 back-ground at the MARP Small Animal Facility (Parkville) by hetero-zygous breeding pairs. Offspring were routinely genotyped by PCRusing genomic DNA from tail samples obtained at weaning (21days) as described previously (Matsui et al., 2000; Rokosh andSimpson, 2002). All mice were exposed to a 12 h light/darkphotoperiod, and had free access to food and water. Prior approvalfor animal breeding and experimentation was granted by theMonash University Standing Committee on Animal Ethics, ethicsnumber BCSV 2009.03 for breeding and VCPA 2009/14 and VCPA2009/15, for the use of genetically modified and wild-type micerespectively. All studies conformed to the National Health andMedical Research Council, Australian code of practice for the careand use of animals for scientific purposes.

2.2. Tissue collection

Mice for experimentation Z8 weeks were killed by cervicaldislocation and the whole mouse ventral prostate was carefullydissected out and placed in a specimen jar containing Krebs–Henseleit solution (NaCl 118.1 mM, NaHCO3 25.0 mM, glucose11.7 mM, KCl 4.69 mM, KH2PO4 1.2 mM, MgSO4 0.5 mM, CaCl22.5 mM; pH 7.4) for isolated organ bath studies or phosphatebuffered saline (PBS; NaCl 136.9 mM, KCl 2.7 mM, KH2PO4 1.5 mM,Na2HPO4 8.1 mM) for immunohistochemical studies.

2.3. Isolated organ bath studies

Isolated prostates were mounted in 10 ml water jacketed organbaths containing Krebs–Henseleit solution maintained at 37 1Cand bubbled with 95% O2/5% CO2. One end of the tissue wasattached to a perspex tissue holder and the other to a Grass FT03force displacement transducer (Grass Instruments, Quincy, MA) forthe measurement of isometric contractions. The force of contrac-tion was recorded using a PowerLab 4/SP data acquisition system(ADInstruments Pty. Ltd., Castle Hill, NSW) and LabChart softwareversion 5 (ADInstruments Pty. Ltd.). Tissues were maintainedunder a resting force of approximately 0.7 g. Prior to experimenta-tion tissue preparations were equilibrated for a period of 1 h,during which time prostates were stimulated with electricalpulses of 0.5 ms duration, 60 V at 0.01 Hz. Electrical stimulationoccurred via two parallel platinum electrodes incorporated into

Fig. 1. A simplified schematic diagram of probable signalling pathways in prostaticsmooth muscle contraction. GPCR: G protein-coupled receptor; PIP2: phosphati-dylinositol bisphosphate; PLC: phospholipase C; IP3: Inositol 1,4,5-triphosphate;DAG: diacylglycerol; PLC: phospholipase C; PKC: protein kinase C; MLC: myosinlight chain; MLC-P: myosin light chain phosphate; SR: sarcoplasmic reticulum;Ca2þ: calcium; CPI-17: a 17 kDa PKC-potentiated phosphatase inhibitor.

C.W. White et al. / European Journal of Pharmacology 721 (2013) 313–321314

the tissue holder, connected to a Grass S88 stimulator (GrassInstruments, Quincy, MA).

2.3.1. Electrical field stimulationFrequency response curves were constructed in tissues follow-

ing the equilibration period. Prostates were exposed to electricalfield stimulation at frequencies of 1, 2, 5, 10 and 20 Hz (0.5 msduration, 60 V) delivered at 10 min intervals in trains of pulseslasting 10 s. Using tetrodotoxin, we have previously establishedthat electrical field stimulation induced contractions of the mouseprostate using these parameters are caused by the activation ofintramural nerves (Gray and Ventura, 2005; White et al., 2010). Aninitial control frequency response curve was constructed todetermine the contractile response of the tissue at each frequency.A second frequency response curve was then constructed after thetissue had been washed three times with the volume of the organbath and exposed to a test drug for a period of 1 h. In preliminaryexperiments, appropriate time control curves in the absence oftest drugs were constructed in parallel tissues to determine theviability of experimental preparations.

2.3.2. Agonist mediated contractionFollowing the equilibration period the tissue was washed and

the tension readjusted. A priming dose of 1 mM of the appropriateagonist was then added to the organ bath and allowed to elicit amaximal response before the tissue was washed. We have deter-mined through numerous previous experiments that this primingdose stabilises isolated prostate tissue and yields reproducibility ofseveral subsequent concentration-response curves. After a 30 minrecovery period an initial discrete half-log concentration responsecurve to carbachol (10 nM to 30 mM) or noradrenaline (10 nM to100 mM) was then constructed with 15 min intervals between drugadditions, whereby for each concentration the response wasallowed to reach a maximum and plateau before the tissue waswashed out with 4 times the volume of the organ bath. Followingthe initial concentration response curve, isolated prostates wereexposed to an inhibitor for 1 h before a second concentrationresponse curve to carbachol (10 nM to 1 mM) or noradrenaline(10 nM to 300 mM) was constructed in the same manner. Theantagonist was replaced following each wash. To prevent anyconfounding effects by off target receptors as we have describedpreviously (Gray and Ventura, 2006; White et al., 2010, 2011), theKrebs–Henseleit solution was supplemented with prazosin(0.3 mM) or atropine (1 mM) for experiments using carbachol andnoradrenaline respectively.

2.3.3. Concentrations of inhibitors usedThe concentration of inhibitors used in these experiments were

selected from the literature to ensure inhibition of: Rho-kinase:Y-27632 (1, 10, 30 μM) 10, 100 and 300 fold higher than the Ki

(0.1 μM) respectively (Braverman et al., 2006); protein kinase C:GF109203X (1 and 10 μM) approximately 100 and 1000 fold higherthan the Ki (10 nM) respectively (Toullec et al., 1991); phospholi-pase C: U 73122 (3 μM and 10 μM), where 10 μM U 73122completely inhibits carbachol mediated inositol phosphate accu-mulation in the rat urinary bladder (Schneider et al., 2004b);L-type Ca2þ channels: nifedipine (1 μM) approximately 30 foldhigher than IC50 (�30 nM) for carbachol mediated contraction(Boess et al., 1990) and 10 fold higher (100 nM) than the concen-tration required for 90% inhibition of carbachol mediated bladdercontraction (Schneider et al., 2004b).

2.3.4. Data analysisThe peak force (g) of electrical field stimulation or agonist mediated

contraction was measured at each frequency or concentration.

Mean frequency and concentration response curves were cal-culated from pooled data from n experiments, where n is equal tothe number of mice used. Results are expressed as the mean7S.E.M.Differences between the initial and subsequent drug exposed con-centration–response curve(s) were analysed by GraphPad Prism ver-sion 5.00 using a two way repeated measure analysis of variance(ANOVA), or by analysis of the non-linear regression best fit curvesusing a F-test. The p-values stated were used to evaluate the statisticalsignificance of any difference between the two curves, or non-linearregression best fit curves, and represent the probability of the drugtreatment causing a significant change. po0.05 was consideredsignificant.

2.4. Immunohistochemistry

Following dissection, tissues were either embedded in optimumcutting temperature compound (Tissuetek, Sakura Fineteck) andsnap frozen on melting methyl butane cooled with liquid nitrogenor fixed with 4% (v/v) formaldehyde then snap-frozen followingfixation. 4% (v/v) formaldehyde fixed tissues were placed in PBScontaining 4% (v/v) formaldehyde for 2 h at room temperature thenwashed four times for 10 min with 7% (w/v) sucrose in PBScontaining 0.01% (w/v) sodium azide and stored in this solutionfor 48 h at 4 1C before being snap frozen as above. 10 μm sections ofembedded frozen tissues were then cut at �20 1C using a cryostat(Leica CM 1850) and thawed onto Superfrosts Plus (Menzel-Glaser) glass slides for immunohistochemical staining.

2.4.1. ROCK2 localisationFollowing cutting, slide-mounted 10 μm sections of 4% formal-

dehyde fixed tissue were left to air dry for 1 h at room tempera-ture. Sections were then washed for 10 min with PBS and blockedfor 30 min at room temperature with 10% (v/v) donkey serum and1% (w/v) BSA in PBS containing 0.1% (w/v) lysine, 0.1% (w/v)sodium azide and 0.2% (v/v) Tween-20. After blocking, slide-mounted sections were incubated overnight at 4 1C with rabbitpolyclonal anti-ROCK2 primary antibodies (1:1000, Abcam,ab71598) in PBS containing 0.01% (w/v) BSA, 5% (v/v) donkeyserum, 0.1% (w/v) lysine, 0.1% (w/v) sodium azide and 0.1% (v/v)Tween-20. For negative control sections, rabbit polyclonal anti-ROCK2 primary antibodies were omitted. Sections were thenwashed four times for 10 min with PBS and incubated withfluorescein isothiocyanate (FITC)-conjugated AffiniPure donkeyanti-rabbit antibodies (HþL) (1:200 in PBS containing 0.1% (v/v)Tween-20, Jackson ImmunoResearch) for 1 h at room temperaturein the dark. Sections were then washed four times for 10 min eachtime with PBS at room temperature in the dark and mounted inVectashields (Vector Laboratories) mounting medium and cover-slipped for imaging.

2.4.2. RhoA localisationFollowing cutting, 10 μm fresh-frozen slide-mounted tissue

sections were left to air dry for 30 min at room temperature.Sections were then fixed with ice cold 4% (v/v) formaldehyde inPBS for 5 min followed by three 10 min washes with PBS. Sectionswere then blocked for 30 min at room temperature with 10% (v/v)donkey serum and 1% (w/v) BSA in PBS containing 0.1% (w/v)lysine, 0.1% (w/v) sodium azide and 0.1% (v/v) Triton X-100. Afterblocking, slide-mounted sections were incubated overnight at 4 1Cwith primary rabbit polyclonal anti-RhoA antibodies (1:500,Abcam, ab68826) in PBS containing: 5% (v/v) donkey serum,0.01% (w/v) BSA, 0.1% (w/v) lysine, 0.1% (w/v) sodium azide and0.1% (v/v) Triton X-100. For negative control sections, primaryrabbit polyclonal anti-RhoA antibodies were omitted. Sectionswere then washed four times for 10 min with PBS and incubated

C.W. White et al. / European Journal of Pharmacology 721 (2013) 313–321 315

with fluorescein isothiocyanate (FITC)-conjugated AffiniPure don-key anti-rabbit antibodies (HþL) (1:200; Jackson ImmunoRe-search) for 1 h at room temperature in the dark. Sections wereprotected from light and washed four times for 10 min with PBS atroom temperature and mounted in Vectashields mounting med-ium and cover-slipped for imaging

2.4.3. ImagingSections were viewed using an Olympus BX60 fluorescence

microscope fitted with an Olympus mercury burner light sourceattachment and Olympus UPlanSApo 4� , 10� , 20� and 40�objectives. Fluorescence was viewed using a U-MNIBA Olympusfilter cube consisting of a DM505 dichroic mirror, BP470-490exciter filter and BA515-550 barrier filter. To acquire fluorescentphotomicrographs a SPOT RT slider digital camera and SPOT RTsoftware (v. 3.5, Diagnostic Instruments) run on a personalcomputer were used.

2.5. Reagents used

Atropine sulphate, carbamylcholine chloride (carbachol), don-key serum, L-(-)-norepinephrine bitartrate salt monohydrate(noradrenaline) sodium azide, nifedipine and prazosin hydro-chloride were purchased from Sigma-Aldrich (St. Louis, MO,USA). 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), Trans-4-[(1R)-1-Ami-noethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride

Fig. 2. (a and b) Mean log concentration responses curves to carbachol in the isolated wild type mouse prostate in the absence (circles) or presence (squares) of U73122(3 μM; a and 10 mM; b). (c and d) Comparison of mean contractile responses to electrical field stimulation (0.5 ms, 60 V, 20 Hz, 10 s trains of pulses) in prostates taken from,(c) wild-type or (d) α1A-adrenoceptor knockout (α1AKO) mice in the absence (open bars) or presence (black bars) of U73122 (10 μM). Points or bars represent the meanforce7S.E.M., where n¼6. p-values were determined using a F-test or two-way repeated-measures of ANOVA and represent the probability of the drug treatment causing asignificant change difference in the non-linear regression best fit curves or in the contractile response for (a and b) and (c and d) respectively.

Table 1Effect of inhibitors on the mean potency (� log10[EC50]) and efficacy (maximumcontractile response) of carbachol and noradrenaline in the mouse prostate gland.

Agonist� log10[EC50](mean7S.E.M.)

Maximum response% of control

Carbachol Control 5.9470.08 100%U73122(3 μM)

5.8770.18 8973.9a

U73122(10 μM)

5.5670.32 7774.7c

GF109203X(1 μM)

5.6670.46 9677.4

GF109203X(10 μM)

5.5870.18 10275.0

Nifedipine(1 μM)

5.8870.33 8176.4a

Y-27632(10 μM)

5.1770.22a 8871.5b

Y-27632(30 μM)

5.1470.25a 6473.2b

Noradrenaline Control 5.7970.10 100%Y-27632(10 μM)

5.3170.14a 9874.5

Y-27632(30 μM)

5.4170.12a 8673.2a

p-Values represent a significant difference from control value. Calculated using anF-test (� log10[EC50] values) or by a paired one-tailed t-test (maximum responses).

a po0.05.b po0.01.c po0.001.

C.W. White et al. / European Journal of Pharmacology 721 (2013) 313–321316

(Y-27632) and 2-[1-(3-Dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF203109X) were purchased from Tocris (Bristol,UK). Atropine, carbachol, prazosin, Y-27632 were dissolved indistilled water, nifedipine in ethanol and GF203109X and U-73122 in dimethyl sulfoxide. All further dilutions were made indistilled water. Noradrenaline was dissolved and diluted in cate-cholamine diluent (NaCl 154 mM, NaH2PO4 1.2 mM and ascorbicacid 0.2 mM, in distilled water).

3. Results

3.1. Isolated organ bath studies

In the wild type mouse prostate gland, the phospholipase Cinhibitor U73122 reduced contractile responses to the muscarinicreceptor agonist carbachol in a concentration dependent manner,with the maximum response reduced to 8973.9% and 7774.7%of the control response with 3 μM (po0.05; Fig. 2a) and 10 μM(po0.001; Fig. 2b) U73122 respectively. Only at 10 μM did U73122produce a decrease (po0.05; Table 1) of the potency of carbachol,whereas 3 μM U73122 (p¼0.16; Table 1) did not. FurthermoreU73122 (10 μM) did not inhibit the nerve mediated contractileresponse in prostates taken from wild-type mice (Fig. 2c; p¼0.07)but caused a reduction in the nerve mediated contractile responseat 2 and 5 Hz in prostates taken from α1A-adrenoceptor knock-out mice (Fig. 2; po0.05). In control experiments the diluted

dissolving solvents dimethyl sulfoxide and ethanol did not haveany observable effect on carbachol or electrical field stimulationmediated contractile responses.

In isolated prostates taken from wild type mice, the carbacholmediated contractile response was unaffected by the proteinkinase C inhibitor GF109203X (10 μM; p¼0.27, Fig. 3a, Table 1),however contractile responses mediated by electrical field stimu-lation were inhibited by GF109203X (10 μM; Fig. 3b, po0.001).

In the presence of the L-type calcium channel blocker nifedi-pine (1 μM), the maximum contractile response to carbachol inprostates taken from wild type mice was reduced to 8176.4% ofthe control maximum contractile response to carbachol (po0.05;

Fig. 3. (a) Mean log concentration responses curves to carbachol in the isolatedwild type mouse prostate in the absence (circles) or presence (squares) ofGF109203X (10 μM). (b) Comparison of mean contractile responses to electricalfield stimulation (0.5 ms, 60 V, 20 Hz, 10 s trains of pulses) in the wild type mouseprostate absence (open bars) or presence (black bars) of GF109203X (10 μM). Pointsor bars represent the mean force7S.E.M., where n¼6. p-Values were determinedusing a F-test or two-way repeated-measures of ANOVA and represent theprobability of the drug treatment causing a significant change difference in thenon-linear regression best fit curves or in the contractile response for (a) and(b) respectively.

Fig. 4. (a) Mean log concentration responses curves to carbachol in the isolatedwild type mouse prostate in the absence (circles) or presence (squares) ofnifedipine (1 μM). (b and c) Comparison of mean contractile responses to electricalfield stimulation (0.5 ms, 60 V, 20 Hz, 10 s trains of pulses) in prostates taken from,(b) wild-type or (c) α1A-adrenoceptor knockout (α1AKO) mice in the absence (openbars) or presence (black bars) of nifedipine (1 μM). Points or bars represent themean force7S.E.M., where n¼6. p-values were determined using a F-test or two-way repeated-measures of ANOVA and represent the probability of the drugtreatment causing a significant change difference in the non-linear regression bestfit curves or in the contractile response for (a) and (b and c) respectively.

C.W. White et al. / European Journal of Pharmacology 721 (2013) 313–321 317

Fig. 4a). However, nifedipine (1 μM) had no effect on the potencyof carbachol (Table 1). Nifedipine (1 μM) inhibited contractileresponses elicited by electrical field stimulation in prostates takenfrom wild-type (Fig. 4b; po0.001) and α1A-adrenoceptor knockout(Fig. 4c; po0.001) mice.

The Rho kinase inhibitor Y-27632 (10 and 30 μM) reducedcontractile responses of the isolated wild type mouse prostate toboth carbachol and noradrenaline in a concentration dependentmanner (Fig. 5). The maximum contractile response to carbacholwas reduced to 8871.5% and 6473.2% of the control response inthe presence of 10 μM (po0.01; Fig. 5a) and 30 μM (po0.01;Fig. 5b) Y-27632, respectively. The maximum contractile responseto noradrenaline, on the other hand, was reduced to 86%73.2% ofthe control response by 30 μM Y-27632 (po0.05; Fig. 5d) but wasunaffected by 10 μM Y-27632 (Fig. 5c). A decrease in the potency ofcarbachol and noradrenaline was observed at both concentrationsof Y-27632 (Table 1) in the wild type mouse prostate. Furthermore,Y-27632 (10 μM) reduced the nerve mediated contractileresponses in prostates taken from wild-type (Fig. 6a; po0.001),α1A-adrenoceptor knockout (Fig. 6b; po0.001) as well as M3

muscarinic receptor knockout (Fig. 6c, po0.001) mice.

3.2. Immunolocalisation of RhoA and ROCK2

RhoA (Fig. 7a) and ROCK2 (Fig. 7b) polyclonal antibodiespositively immunostained the mouse prostatic stroma, containingsmooth muscle, in slide mounted sections of wild type mouseprostate. No staining was observed in control sections where theRhoA and ROCK2 antibodies were omitted from the incubationmedium (Fig. 7c and d).

4. Discussion

The prototypical signalling pathway for M3 muscarinic recep-tors is via activation of PLC, resulting in the formation of thesecond messengers DAG and IP3 (Caulfield, 1993) leading to anincrease in cytosolic Ca2þ as well as PKC activation (Caulfield andBirdsall, 1998). In smooth muscle preparations, the resultantincrease in cytosolic Ca2þ leads to a contractile response. It istherefore not surprising that a concentration-dependent inhibitionof the carbachol mediated contractile response in the mouseprostate was observed with the PLC selective inhibitor U 73122.As PLC results in the production of IP3, this suggests that Ca2þ

release form intracellular stores is involved in the carbacholmediated contractile response (Fig. 1). However, no observableeffect on the nerve mediated contractile response was seen inprostates taken from wild-type mice at the same concentration ofU 73122 (10 μM) that markedly inhibited carbachol mediatedcontractions and has previously been shown to inhibit IP3 accu-mulation (Schneider et al., 2004b). It should be noted though, thatthe nerve mediated contractions of the prostate are elicited, inaddition to M3 muscarinic receptors, by α1A-adrenoceptors that arethought to largely mediate contraction via PKC mediated openingof L-type Ca2þ channels (Eckert et al., 1995; Guh et al., 1995;Preston and Haynes, 2003), as well as by activation of the Rhokinase pathway (Rees et al., 2003; Takahashi et al., 2007). This mayindicate that activation of the α1A-adrenoceptor masks any reduc-tion in nerve mediated contraction caused by inhibition of PLCsignalling normally activated by M3 muscarinic receptors. Inagreement with this, U 73122 inhibited nerve mediated contrac-tion in prostates taken from α1A-adrenoceptor knockout mice.

Fig. 5. Mean log concentration responses curves to carbachol (a and b) or noradrenaline (c and d) in the isolated wild type mouse prostate in the absence (circles) orpresence (squares) of Y-27632 (10 μM; a and c) or Y-27632 (30 μM; b and d). Points represent the mean force7S.E.M., where n¼6. p-Values were determined by comparisonof the non-linear regression best fit curves using a F-test and represent the probability of the test drug causing a significant difference in the non-linear regression best fitcurves.

C.W. White et al. / European Journal of Pharmacology 721 (2013) 313–321318

The influx of Ca2þ from the extracellular environment isknown to be important in prostatic smooth muscle contractionin the human (Drescher et al., 1994; Guh et al., 1995; Preston andHaynes, 2003), rabbit (Seki et al., 1988), guinea-pig (Haynes andHill, 1997) and rat (Maruyama et al., 1998). In these studies, thevoltage gated L-type Ca2þ channel blocker nifedipine or Ca2þ freephysiological bathing solution was used to inhibit prostatic con-tractions elicited by electrical field stimulation or exogenousapplication of α1-adrenoceptor agonists. In the present study,nifedipine inhibited carbachol and nerve mediated contractileresponses in prostates taken from wild-type and α1A-adrenoceptor

knockout mice, indicating a clear role for extracellular Ca2þ incholinergically mediated contractions of the mouse prostate.

Smooth muscle contraction mediated by α1-adrenoceptors inthe prostate has previously implicated PKC in the activation of L-type Ca2þ channels (Haynes et al., 2002; Preston and Haynes,2003). This results in an increase in cytosolic Ca2þ and ultimatelycontraction. As both the carbachol and nerve mediated contractileresponses of the mouse prostate were reduced by nifedipine, theinhibitor GF109203X was used to investigate the role of PKC incontraction of the mouse prostate. Inhibition of PKC in the mouseprostate reduced the nerve mediated contractile response, but hadno observable effect on contractions mediated by carbachol. Thereduction in the nerve mediated response is most likely due toinhibition of the α1A-adrenoceptor mediated component of pros-tate contraction. However, in addition to its post-junctionalactions, activation of PKC is involved in neurotransmitter releasein various tissues (Somogyi et al., 1996; Takata et al., 1991), andthis could also influence the current results.

In addition to Gq, α1A-adrenoceptors in the prostates of humans(Rees et al., 2003; Takahashi et al., 2007) and rats (Saito et al.,2011) also couple to the G-protein RhoA which activates Rho-kinase. Rho-kinase catalyses the phosphorylation of the regulatorysubunit of MLCP to prolong contraction (Christ and Andersson,2007) (Fig. 1). Contractions of the rat prostate mediated bycarbachol are also sensitive to inhibition of Rho-kinase (Saitoet al., 2011). In agreement with studies on the prostates of humans(Rees et al., 2003; Takahashi et al., 2007) and rats (Saito et al.,2011), this study shows both Rho-kinase (ROCK2) and its activatorRhoA to be localised at the protein level in the mouse prostaticsmooth muscle layer. Furthermore, Rho-kinase inhibition mark-edly reduced the contractile responses to both carbachol andelectrical field stimulation of prostates taken from α1A-adrenocep-tor knockout mice. This indicates that in the mouse prostate,activation of the Rho-kinase pathway is an important mediator forcontractions elicited by M3 muscarinic receptor stimulation. Nervemediated contractile responses in prostates taken from M3 mus-carinic receptor knockout mice were similarly reduced in thepresence of the Rho-kinase inhibitor Y-27632. The increased effectof Rho-kinase inhibition seen in the prostates of mice compared toprevious rat studies is most likely due to the cholinergic contrac-tile component being much greater in the prostate of the mouse(Gray and Ventura, 2005; White et al., 2010) compared to the rat(Najbar-Kaszkiel et al., 1997).

RhoA/Rho-kinase activation represents a common signallingpathway for noradrenaline and acetylcholine in the mouse pros-tate. If the same applies in the human prostate, it may explain theadreno-muscarinic receptor synergy previously observed (Roosenet al., 2009). In addition, the Rho-kinase pathway is involved inproliferation of the human prostate and has therefore beensuggested as a suitable additional target for a more effectivetreatment for benign prostatic hyperplasia targeting both contrac-tility and proliferation (Ventura et al., 2011).

It is possible that different signalling pathways are involved inthe contractile mechanisms of different prostate lobes since it hasbeen shown that the amplitude of agonist induced contraction islarger in ventral and dorsal rings compared to lateral and anteriorpreparations of the mouse prostate (Kitazawa, 2013). This phe-nomenon is unlikely to affect our findings as we exclusively usedonly preparations of ventral prostate. Nevertheless, the intracel-lular prostatic smooth muscle signalling pathways activated arelikely to be the same for each receptor subtype, and any regionalvariation in prostatic contractility is probably associated withchanges in smooth muscle content and/or receptor populations.

The results presented here show that the contractile responsemediated by M3 muscarinic receptors in the mouse prostateinvolves the prototypical PLC signalling pathway, suggesting a role

Fig. 6. Comparison of mean contractile responses to electrical field stimulation(0.5 ms, 60 V, 1–20 Hz, 10 s trains of pulses) in prostates taken from; (a) wild-type,(b) α1A-adrenoceptor knockout (α1AKO) or (c) M3 muscarinic receptor knockout(M3KO) mice in the absence (open bars) or presence (black bars) of Y-27632(10 μM). Bars represent mean force7S.E.M., n¼5–6. ANOVA p-values were deter-mined by a two-way repeated-measures of ANOVA and represent the probability ofthe drug treatment or solvent causing a significant change in the contractileresponse.

C.W. White et al. / European Journal of Pharmacology 721 (2013) 313–321 319

for IP3-mediated Ca2þ release from intracellular stores as well asCa2þ influx from the extracellular environment via voltage-gatedL-type Ca2þ channels. Furthermore, the M3 muscarinic receptorand α1A-adrenoceptor components of smooth muscle contractionin the mouse prostate involve Ca2þ sensitisation via the activationof the Rho-kinase pathway, suggesting a common pathway thatmay be targeted for more effective treatments of lower urinarytract symptoms associated with benign prostatic hyperplasia.

Acknowledgements

This work was supported by Grants from the ANZ Trustees [09/3164] and the National Health & Medical Research Council(Australia) [334136] to Sabatino Ventura. Carl W. White is arecipient of a Monash University Postgraduate Publications Award.The authors would like to acknowledge Minoru Matsui, Depart-ment of Clinical Research and General Medicine, Tokyo-NishiTokushukai Hospital, Tokyo, Japan, for the approval for the use ofthe M3 muscarinic receptor knockout mice.

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