European Journal of Pharmacology 641 (2010) 160–167

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European Journal of Pharmacology

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Cardiovascular Pharmacology

Activation of prostanoid EP receptors by prostacyclin analogues in rabbit iliac artery:Implications for anti-restenotic potential

Christopher McCormick a,⁎, Robert L. Jones a, Simon Kennedy b, Roger M. Wadsworth a

a Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, John Arbuthnot Building, 27 Taylor Street, Glasgow, G4 0NR, United Kingdomb Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, United Kingdom

⁎ Corresponding author. Bioengineering Department, UnBuilding, Rottenrow, Glasgow, G4 0NW, United Kingdom.

E-mail addresses: christopher.mccormick@strath.ac.robert.l.jones@strath.ac.uk (R.L. Jones), S.Kennedy@bio.gr.m.wadsworth@strath.ac.uk (R.M. Wadsworth).

0014-2999/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.ejphar.2010.04.035

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 October 2009Received in revised form 31 March 2010Accepted 23 April 2010Available online 5 May 2010

Keywords:Prostanoid receptorProstacyclin analogueSmooth muscle cell proliferationRestenosisDrug-eluting stentRO-1138452SC-51322L-798106

Prostacyclin analogues have the potential to be effective agents in a new generation of drug-eluting stents byvirtue of prostanoid IP receptor mediated anti-proliferative effects on smooth muscle cells. However,prostanoid IP receptor mediated vessel relaxation is reduced at elevated analogue concentrations. Themechanisms underlying this loss of activity are unclear, and its influence on the anti-proliferative potential ofthese compounds remains to be determined. A classical organ bath approach was used to examine thefunctional response of the rabbit iliac artery to the prostacyclin analogues, AFP-07 and cicaprost. Selectivereceptor antagonists for prostanoid IP (RO-1138452), EP1 (SC-51322) and EP3 (L-798106) receptors wereused to characterise the receptors involved. The effects of these agents on proliferation ([3H]-thymidineincorporation) of rabbit iliac artery smooth muscle cells stimulated by foetal calf serum were then studied.AFP-07 gave a bell-shaped log concentration–response curve consisting of prostanoid IP receptor mediatedrelaxation followed by reversal at higher concentrations. SC-51322 and L-798106 potentiated this relaxation,although only L-798106 completely removed the contractile element. The prostanoid EP3 receptor agonist,sulprostone, produced constriction, which was attenuated by L-798106. RO-1138452 blocked the inhibitoryaction of AFP-07 and cicaprost on proliferation, implicating an involvement of prostanoid IP receptors. L-798106 had no effect on the anti-proliferative effect of cicaprost, but reduced the effect of AFP-07. Non-selective activation of prostanoid EP3 receptors (and possibly prostanoid EP1 receptors) compromises therelaxant activity of prostacyclin analogues, although it does not reduce the anti-proliferative capacity ofthese compounds in the model studied.

iversity of Strathclyde, WolfsonTel.: +44 0141 548 3030.uk (C. McCormick),la.ac.uk (S. Kennedy),

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Although drug-eluting stents were a significant improvement onbaremetal stents, concerns around delayed healing (Joner et al., 2006)and high restenosis rates in certain patient groups (Ishio et al., 2007;Colombo et al., 2004), mean that intense effort is now focussed on thedevelopment of a next generation drug-eluting stent. Restenosis ischaracterised by thrombus formation, inflammation, and excessivesmooth muscle cell proliferation and migration (Babapulle andEisenberg, 2002). Prostacyclin inhibits platelet activation and smoothmuscle cell proliferation, making it an ideal candidate for inclusion ona drug-eluting stent (McCormick et al., 2007). Alt et al. (2000)developed a drug-eluting stent containing the prostacyclin analogue,iloprost, and the thrombin inhibitor, hirudin, which reduced in-stentrestenosis in pigs.

It is assumed that prostacyclin analogues exert their beneficialeffects via activation of the prostanoid IP receptor, leading toincreased intracellular cAMP (Hata and Breyer, 2004). cAMP elevationcan be reversed at elevated analogue concentrations, although themechanisms are unclear (Accomazzo et al., 2002). Structuraldifferences between prostacyclin analogues are believed to result invariations in activity at prostanoid I- and E-type (EP1,2,3,4) receptors.The prostanoid EP3 receptor has splice variants, which can couple toGi, Gq, and Gs-type G proteins (Pierce and Regan, 1998), with Gi

coupling producing inhibitory effects on cAMP. Therefore, the loss ofactivity observed by Accomazzo et al. (2002) may be due to non-selective activity at prostanoid EP3 receptors.

There is good evidence for the presence of prostanoid IP receptors indiseased human coronary artery (Buerke et al., 1996; Voisard et al.,2005), while there is little information on prostanoid EP receptors.However, COX-2 is present in most vascular tissues, including diseasedhuman coronary artery (Baker et al., 1999). Taken together with theincrease in PGE2 production that accompanies COX-2 upregulation inmost vascular cells (Norel, 2007), the presence of prostanoid E-typereceptors in this artery is not unlikely. Evidence of a prostanoid EP3receptor mediated stimulatory effect on proliferation is emerging

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(Chuang et al., 2006), although there is a paucity of data in smoothmuscle cells. Data on human smooth muscle cells has revealed a role forthe prostanoid EP3 receptor in stimulatingmigration (Blindt et al., 2002).Taken together with the potential vasoconstriction produced byprostanoid EP1 and EP3 receptor activation (Hata and Breyer, 2004), itis clear that prostanoid EP receptor activation may produce deleteriouseffects at the elevated prostacyclin analogue concentrations likely to beobserved following stenting (Hwang et al., 2001).

The rabbit iliac artery develops neointima in response to stenting(Rogers et al., 1996), and is a leading pre-clinical model of stenttherapy (Schwartz et al., 2002), although little is known of theprostanoid receptors within this model. The present study addressedthis issue, while also examining the properties of two prostacyclinanalogues on the myogenic tone of rabbit iliac artery and theproliferation of its smooth muscle cells. AFP-07 (Chang et al., 1997)is the most potent prostacyclin analogue yet reported, while cicaprosthas the highest selectivity for the IP receptor (Dong et al., 1986;Lawrence et al., 1992).

2. Materials and methods

2.1. Artery function studies

Male New Zealand white rabbits, weighing 2.5–3.5 kg, weresacrificed by terminal anaesthesia induced by sodium pentobarbi-tone. The right and left iliac arteries were then excised andimmersed in Krebs' solution ((mM): NaCl 118.4; NaHCO3 25.0;KCl 4.7; MgSO4 1.2; CaCl2 2.5; glucose 11.0). After pinning out on aSylgard coated block and removal of surrounding fat and connectivetissue, rings (4 mm long) were cut. The rings were set up in con-ventional 10 ml organ baths under a resting tension of 1 g andallowed to stabilise for 1 h (Coats et al., 2008). Isometric tensionwas recorded with Grass FT03 transducers linked to an AD Instru-ments PowerLab pre-amplifier-digitizer/desk-top computer. Con-tractile tone was established by addition of an EC70 concentration(0.3 μM) of phenylephrine, followed by exposure to cumulativeconcentrations of the prostacyclin analogues in the absence orpresence of various prostanoid receptor antagonists (Table 2).Analogue concentrations examined were selected to mimic the highlocal drug concentrations that are found with stent-based drugrelease (Hwang et al., 2001). The presence of the prostanoid EP1,EP2, EP3 and EP4 receptors was assessed by cumulative addition of aselective receptor agonist for each receptor. In experimentsinvolving prostanoid EP1,3 receptor agonists, an EC50 concentrationof phenylephrine (0.1 μM) was used to precontract the artery rings;an EC70 concentration of phenylephrine (0.3 μM) was applied inthose experiments using prostanoid EP2,4 receptor agonists. Thesedata were expressed as a percentage of the phenylephrine inducedprecontractile tone.

In separate experiments, the prostanoid receptor agonist, andreceptor antagonist where available, were added in the absence ofphenylephrine induced tone; data were expressed as a percentage ofthe maximum contraction achieved to the second addition of 80 mMKCl performed prior to commencement of the protocol.

In all experiments, each prostanoid receptor antagonist was added15 min prior to the addition of the prostanoid receptor agonist. Inendothelium denuded artery rings, denudation was performed bygentle scraping of endothelium using a rough wire, and confirmed bythe absence of relaxation to a single dose of carbachol (1 μM) added atthe end of each experiment. Indomethacin (1 μM) was present for allprotocols, and the prostanoid TP receptor antagonist, BMS-180291([1S-(exo,exo)]-2-[[3-[4-[(pentylamino)carbonyl]-2-oxazolyl]-7-oxa-bicyclo[2.2.1]hept-2-yl]methyl]-benzenepropanoic acid) (300 nM)was present in all experiments involving prostanoid EP receptoragonists.

2.2. Guinea-pig isolated preparations

Thoracic trachea and proximal vas deferens were excised frommale Dunkin-Hartley guinea-pigs (450–500 g) killed by exposure to100% CO2. Organ bath and tension recording conditionswere the sameas above; resting tension was set at 1.0 and 0.5 g respectively. Inaddition, twitch responses of the vas deferens were elicited by shorttrains of electrical field stimulation (60 V, 0.5 ms, 10 Hz for 1 s) every40 s using platinum ring electrodes; the EP3 agonist response wasmeasured as the % inhibition of the control twitch tension.

2.3. Smooth muscle cell isolation and culture

Rabbit iliac artery smooth muscle cells were isolated and culturedby an explant method. Briefly, the left and right iliac arteries wereexcised from freshly sacrificed male New Zealand White rabbits, andtransferred immediately to a sterile culture dish containing 5 mlgrowth medium (F12-Ham:Weymouth 50:50, 1% (v/v) penicillin–streptomycin). Artery rings of 3 mm length were prepared, andtransferred to separate cell culture flasks containing 5 ml growthmedium supplemented with 10% (v/v) foetal calf serum. Smoothmuscle cells explant from the tissue over 10 days, and these cells weregrown in culture at 37 °C in a humidified atmosphere containing 5%CO2:95% air. Growth medium was replaced every two days, and cellswere passaged to fresh culture plates at regular intervals to preventcells reaching complete confluence.

2.4. Smooth muscle cell proliferation

Cell proliferation was measured by [3H]-thymidine incorporationas described previously (Work et al., 2001). Briefly, cells of passage 3–6 were grown to 70% confluence in 24-well culture plates. Quiescencewas induced by incubation in serum free media for 24 h. Cells werethen stimulated by varying concentrations of foetal calf serum for afurther 24 h. [3H]-Thymidine (975 Bq/ml)was added for the last 5 h ofthe stimulation period. At the end of the incubation, cells were rinsedin ice cold phosphate buffered saline, followed by 10% trichloroaceticacid. Lauryl sulphate (10%) plus sodium hydroxide (0.2 M) was thenadded and radioactivity wasmeasured by liquid scintillation counting.All drugs were added immediately following serum addition.

2.5. Materials

The following compounds were gifts: AFP-07 (7,7-difluoro-16S,20-dimethyl-18,19-didehydro PGI2, Asahi Glass Co, Japan),cicaprost (Schering, AG, Germany), L-798106 (sodium [(2E)-N-[(5-bromo-2-methoxyphenyl)-sulphonyl]-3-[2-(2-naphthylmethyl)phe-nyl]acrylamide, synthesized at GlaxoSmithKline, UK), ONO-AE1-259(16R-9β-chloro-19,20-didehydro-9,15-dideoxy-16-hydroxy-17,17-trimethylene PGE2, ONO Pharmaceuticals, Japan) and tetrazolo PGE1analogue-19a (8-aza-1-decarboxy-11-deoxy-16-phenyl-ω-tetranor-2-(5-tetrazolo) PGE1, synthesized at Allergan Inc., CA, USA). Thefollowing compounds were purchased: RO-1138452 (4,5-dihydro-N-[[4-(1-methylethoxy)phenyl)]methyl]phenyl]-1H-imidazol-2-amine,CAY-10441) (Cayman Chemical, MI, USA); phenylephrine hydrochlo-ride and carbachol (carbamylcholine chloride) (Sigma-Aldrich, Poole,UK); SC-51322 (8-chlorodibenz[b,f][1,4]oxazepine-10(11H)-carbox-ylic acid 2-[3-2-(furanylmethyl)thio]-1-oxopropyl]hydrazide (BiomolInternational, Exeter, UK); BMS-180291 ([1S-(exo,exo)]-2-[[3-[4-[(pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]-benzenepropanoic acid, Bristol-Myers Squibb, USA) and GR-32191 ((E)-7-[(1R,2R,3S,5S)-3-hydroxy-5-[(4-phenylphenyl)meth-oxy]-2-(1-piperidyl)cyclopentyl]hept-4-enoic acid, GlaxoSmithKline,UK). Growthmedia reagents were purchased as follows: Weymouth'sMB722/1 1×+L-Glutamine, and F-12 Nutrient Mixture (Ham) 1×+L-Glutamine, and TryplE solution (Invitrogen, Glasgow, UK); foetal calf

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serum (European source) and Penicillin–Streptomycin (Sigma-Aldrich, Poole, UK).

2.6. Data analysis and statistical procedures

All data are presented as the mean±one standard error of themean, where n represents the number of animals. In cellular studies,one observation is the average of four replicates. In organ bath studies,sigmoidal curves were fitted to log concentration–response data usinga non-linear regression equation (GraphPad Prism software); the low-concentration asymptote was constrained to the resting tension or theinitial phenylephrine tone level as appropriate. Log concentration–response data were further analyzed by repeated-measures two-wayANOVA, with comparison of pairs of curves by a main-effects contrast(SuperANOVA software). In cellular proliferation studies, a one-wayANOVA was applied and Dunnett's post test was performed asappropriate. In all cases, differences were considered significant whenthe P value was less than 0.05 (Pb0.05).

3. Results

3.1. Artery function

Fig. 1 shows effects of the prostacyclin analogues cicaprost andAFP-07 on established tone induced by phenylephrine in rabbit iliacartery. Cicaprost induced complete relaxation while AFP-07 showed abell-shaped concentration–response relationship consisting of relax-ation to a maximum value of 25.7±8.8% of established tone at 30 nM,followed by contraction back to a final value of 69.6±13.5% at 1 μM.Endothelium removal did not significantly affect the concentration–response curve for either agonist. Endothelium-intact vessels wereused in all subsequent experiments.

In order to understand the profile of AFP-07, the experiments wererepeated in the presence of the prostanoid IP receptor antagonist, RO-

Fig. 1. Concentration–response curves for (A) cicaprost and (B) AFP-07 in endotheliumintact and denuded rabbit iliac arteries (n=6). Artery rings were precontracted with anEC70 concentration of phenylephrine. BMS-180291 (300 nM) was present for all tests.NS — no significant difference between curves (ANOVA/main-effects contrast).

1138452 (1 µM), the prostanoid EP1 receptor antagonist, SC-51322(1 µM), or the prostanoid EP3 receptor antagonist, L-798106 (1 µM)(Fig. 2). In the presence of RO-1138452, AFP-07 produced aconcentration dependent contraction of the rabbit iliac artery to avalue of 130.7±0.4% of phenylephrine induced tone. In contrast, boththe prostanoid EP1 and EP3 receptor antagonists significantlypotentiated the relaxation element of the artery response to AFP-07,producing final values of 26.4±10.6% and 1.1±2.6% respectively.Only L-798106 completely removed the contractile element of theresponse at the highest concentrations of AFP-07. In the presence ofeither L-798106, or SC-51322, the cicaprost concentration–responsecurve was shifted slightly to the left, although this effect was lesspronounced when compared with AFP-07 (data not shown). None ofthe prostanoid receptor antagonists produced an appreciable changein established tone during the 15 min incubation period prior toaddition of the prostanoid receptor agonist.

In artery rings precontracted with an EC50 concentration ofphenylephrine, 17-phenyl PGE2 (which shows moderate prostanoidEP1/EP3 receptor agonist selectivity) produced further contraction to amaximum level of 7.0±1.0 g (Fig. 3A). Sulprostone (which showsmoderate prostanoid EP3/EP1 receptor agonist selectivity) behavedsimilarly (maximum value of 7.23±1.11 g) and was some 5–10 timesmore potent than 17-phenyl PGE2 (Fig. 3A). The selective prostanoidEP2 receptor agonist, ONO-AE1-259, had no effect on phenylephrineinduced tone up to 300 nM, and produced a weak contraction at 1 µM(Fig. 3B). The selective prostanoid EP4 receptor agonist, tetrazolo PGE1analogue-19a, produced weak relaxation to 4.33±1.01 g, followingcontraction with an EC70 concentration of phenylephrine (Fig. 3B)from a starting tone of 5.24±0.94 g.

17-Phenyl PGE2 and sulprostone added to artery rings at restingtone (no pre-contraction) induced contraction to levels of 37.5±7.5%and 53.9±10.7% of the maximal tone induced by 80 mM KCl (Fig. 4).The contraction to sulprostone was significantly attenuated in thepresence of L-798106 (1 µM), while the response to 17-phenyl PGE2was unchanged in the presence of SC-51322 (1 µM).

3.2. Activity on established prostanoid EP1 and EP3 receptor preparations

The prostanoid EP1 receptor agonist activity of AFP-07 wascompared with 17-phenyl PGE2 on guinea-pig trachea in the presenceof the prostanoid TP receptor antagonist GR-32191 (1 µM, n=5)(Table 1). AFP-07 was a full agonist and had a potency 64 times lessthan 17-phenyl PGE2. Similarly, the prostanoid EP3 receptor agonistactivity of AFP-07 was 11-fold less than PGE2 on guinea-pig vasdeferens (n=4); again full agonism was observed.

Fig. 2. Concentration–response curves for AFP-07 in endothelium intact rabbit iliacartery in the presence of SC-51322 (1 μM, n=6), L-798106 (1 μM, n=6) or RO-1138452 (1 μM, n=2–3). Artery rings were precontracted with an EC70 concentrationof phenylephrine (n=6). BMS-180291 (300 nM) was present for all tests. *Pb0.05compared to vehicle (ANOVA/main-effects contrast).

Fig. 3. Concentration–response curves for (A) the prostanoid EP1 and EP3 receptoragonists, 17-phenyl PGE2, and sulprostone, and (B) the prostanoid EP2 and EP4 receptoragonists ONO-AE1-259 and tetrazolo PGE1 analogue-19a, respectively, in endotheliumintact rabbit iliac artery. Unfilled symbols represent the starting tone induced byphenylephrine (A 0.1 µM, B 0.3 µM) prior to addition of the prostanoid agonist (n=6).BMS-180291 (300 nM) was present for all tests.

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3.3. Smooth muscle cell proliferation

Proliferation increased 23-fold over baseline in smooth musclecells stimulated with 10% foetal calf serum, compared to a 9-foldincrease in 2% stimulated cells (data not shown). AFP-07 and cicaprost

Fig. 4. Concentration–response curves for (A) 17-phenyl PGE2 in the presence of theprostanoid EP1 receptor antagonist, SC-51322 (1 µM), and for (B) sulprostone in thepresence of the prostanoid EP3 receptor antagonist, L-798106 (1 µM) in the rabbit iliacartery (n=6). Responses are expressed as a percentage of the maximum response to80 mM KCl obtained at the start of the experiment. *Pb0.05 compared to vehicle(ANOVA/main-effects contrast). BMS-180291 (300 nM) was present for all tests.

weakly inhibited cell proliferation induced by 10% foetal calf serum(Fig. 5A and B). In contrast, more pronounced inhibition was seenunder stimulation by 2% foetal calf serum, (Fig. 5C and D), with thehighest concentration (1 µM) of AFP-07 and cicaprost reducingproliferation to 39.8±9.5% and 39.7±10.8% of control respectively.AFP-07 was about 5 times more potent than cicaprost.

In order to determine the effect of non-selective activation ofprostanoid EP3 receptors on the anti-proliferative action of AFP-07, theexperimentwas repeated in the presence of the prostanoid EP3 receptorantagonist L-798106 (1 μM). A concentration of 10 nM for theprostacyclin analogue was selected since this would allow anyadditional inhibition as a consequence of the use of L-798106 to beobserved. It can be seen fromFig. 6A that L-798106 significantly reducedthe anti-proliferative effect of AFP-07 at 10 nM, and had no effect oncicaprostmediated inhibition of proliferation at the same concentration.In order to confirm that the dominant anti-proliferative effect wasmediated by the prostanoid IP receptor, the experimentwas repeated inthe presence of the prostanoid IP receptor antagonist, RO-1138452. Thesignificant anti-proliferative effect of each analogue, which waspreviously demonstrated at 100 nM (Fig. 5B), was attenuated whenthe experiment was repeated in the presence of RO-1138452 (1 μM)(Fig. 6B).

4. Discussion

AFP-07was reported to be a highly selective and potent prostanoidIP receptor agonist in ligand binding studies (Chang et al., 1997). Thishigh potency was confirmed in isolated preparations of piglet carotidartery, rabbit mesenteric artery and guinea-pig aorta, where AFP-07induced complete relaxation and was about 9, 7 and 6 times morepotent than cicaprost respectively (Jones and Chan, 2001). In thecurrent study on rabbit iliac artery, AFP-07 induced endothelium-independent relaxation and was 2–3 times more potent thancicaprost. Activation of prostanoid IP receptors is likely to be thesole mechanism based firstly on suppression of relaxation by theselective prostanoid IP receptor antagonist RO-1138452 (Bley et al.,2006; Jones et al., 2006). The pA2 value of RO-1138452 on rabbitmesenteric artery is 8.12 (Jones et al., 2006) (Table 2); the predicteddose-ratio for 1 µMon the iliac artery is therefore about 130. Secondly,there was no/minimal relaxation response to either the prostanoidEP2 receptor agonist ONO-AE1-259 (Suzawa et al., 2000) or theprostanoid EP4 receptor agonist tetrazolo PGE1 analogue-19a (Billotet al., 2003).

The bell-shaped log concentration–response curve for AFP-07 onrabbit iliac arterywas of paramount interest to us.We have observed asimilar profile for AFP-07 in the pig coronary artery (unpublished), inagreement with previous reports of its activity on rat femoral artery(Hung et al, 2006) and for carbacyclin on piglet carotid artery (Jonesand Chan, 2001). Accomazzo et al. (2002) has also reported reversal ofthe cAMP-elevating action of prostacyclin and iloprost in rabbitmesenteric artery smooth muscle cells with increasing agonistconcentration. The reversal was blocked by pertussis toxin treatment,implicating a Gi protein-driven event. However, it was not clearwhether the response was due to a switch in prostanoid IP receptorcoupling from Gs to Gi proteins, or to activation of a second distinctreceptor. The absence of a major contractile element in the rabbit iliacartery response to cicaprost in the present study is consistent withhigh prostanoid IP receptor agonist selectivity, as originally proposedby Dong et al. (1986) and in agreement with binding studies(Abramovitz et al., 2000), and suggests that the bell-shaped responseto AFP-07 may be due to activation of two (or more) receptors, asopposed to a switch in the coupling of a single receptor. It is knownthat the prostanoid EP3 receptor preferentially couples to the Gi

protein in a number of cell types, leading to inhibition of cAMP (Hataand Breyer, 2004).

Table 1Activities/affinities of prostacyclin analogues at prostanoid EP1 and EP3 receptors.

Prostanoidreceptor

Assay system Activity (EC50) or affinity (Kd) (nM) Reference

Standard agonist/radioliganda

Carbacyclin Iloprost AFP-07 Cicaprost

EP1 Human rc-EP1 receptor: reporter gene 4.0 – 20 – – Durocher et al. (2000)Human rc-EP1 receptor: radioligand binding 9.1 23 11 – N1340 Abramovitz et al. (2000)Guinea-pig trachea: muscle contraction 2.2b, 3.0b,e 30 27c 193e N3000 Dong et al. (1986);

ethis studyMouse rc-EP1 receptor: radioligand bindingd 37 – 40 1500 – Chang et al. (1997)Mouse rc-EP1 receptor: radioligand binding 20 N10,000 21 – N10,000 Kiriyama et al. (1997)

EP3 Human rc-EP3 receptor: radioligand binding 0.33 14 56 – 255 Abramovitz et al. (2000)Guinea-pig vas deferens: Inhibition of EFS twitch 1.7, 2.7e 110 160 30e N1000 Lawrence et al. (1992);

ethis studyMouse rc-EP3 receptor: radioligand bindingd 2.0 – 53 280 – Chang et al. (1997)Mouse rc-EP3 receptor: radioligand binding 0.85 31 22 – 170 Kiriyama et al. (1997)

aPGE2 or [3H]-PGE2 unless otherwise specified; b17-phenyl PGE2; cpartial agonist ∼75% of maximum response; dIC50 values taken from the published competition curves.EFS = electrical field stimulation (EP3 receptor is located on sympathetic varicosities), rc = recombinant receptor present in carrier cell; eindicate that numbers were derived fromthe present study.

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The selective prostanoid EP3 receptor antagonist, L-798106 (Belleyet al., 2006; Gallant et al., 2002) (Table 2) suppressed the contractileresponse to sulprostone (EP3NEP1), demonstrating for the first time thatfunctional prostanoid EP3 receptors are present within the rabbit iliacartery. Clarke et al. (2004) reported pA2 values of 7.48 and 7.82 for L-798106 on the prostanoid EP3 receptor systems in guinea-pig vasdeferens and trachea respectively, while Hung et al. (2006) reportedvalues of 7.43–8.03 on rat femoral artery (Table 2). The right-shift (dose-ratio ∼40) seen with 1 µM L-798106 on rabbit iliac artery (Fig. 4B) isconsistent with this level of affinity. Under resting tone, sulprostone at1 nM elicited minimal contraction, whereas the same concentrationelicited a net response≥E50 in the presence of phenylephrine inducedtone. This profile corresponds to marked synergism between the α1-adrenoceptor and the prostanoid EP3 receptor systems in the iliac artery,and is similar to that seen in rat femoral artery (Hung et al., 2006).Crucially, any prostanoid EP3 receptor agonist activity of AFP-07 on rabbit

Fig. 5. Effect of AFP-07 and cicaprost on DNA synthesis in rabbit iliac artery smooth muscleveh = vehicle. *Pb0.05 compared to vehicle (n=6, ANOVA/Dunnett's post test).

iliac artery in the presence of phenylephrine tone will be subject to thesame synergism, thereby accounting for the bell-shaped response curve.Indeed, Fig. 2 shows that the prostanoid EP3 receptor contractile potencyof AFP-07 in the presence of IP receptor blockade is quite high (EC50∼10 nM). The difference between cicaprost and AFP-07 on rabbit iliacartery (and the differences between cicaprost and carbacyclin/iloprost inassays listed in Table 1) reflects the greater IP/EP1,3 agonist selectivity ofthe former. It is likely that the linear 13,14-triple bond in cicaprostrestricts the locus of the 15-hydroxyl group much more than the transdouble bond in AFP-07, carbacyclin and iloprost (Wise and Jones, 2000).

The presence of a functional prostanoid EP1 receptor populationwithin the rabbit iliac artery is suggested by the modest potentiationof the relaxation response to AFP-07 in the presence of the prostanoidEP1 receptor antagonist SC-51322. However, the contribution ofprostanoid EP1 receptors is likely to be small in view of the inability ofSC-51322 to affect the concentration–response curve to 17-phenyl

cells stimulated by 10% foetal calf serum (A and B), or 2% foetal calf serum (C and D);

Fig. 6. DNA synthesis in rabbit iliac artery cells stimulated by 2% foetal calf serum. (A)The prostanoid EP3 receptor antagonist L-798106 reduced the anti-proliferative effectof AFP-07 and had no effect in cicaprost-treated cells. (B) Anti-proliferative effect ofAFP-07 and cicaprost were attenuated by the prostanoid IP receptor antagonist, RO-1138452 (1 µM). Agonist concentrations were 10 nM and 100 nM in panels A and Brespectively. *Pb0.05, compared to control (n=4, ANOVA/Dunnett's post test).

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PGE2. Indeed, contraction induced by 17-phenyl PGE2 could beentirely due to activation of prostanoid EP3 receptors, on the basisof previous estimates of its prostanoid EP3 receptor agonism(Lawrence et al., 1992). The shift of the AFP-07 curve by SC-51322may be due to weak block of prostanoid EP3 receptors by SC-51322coupled with the finely balanced nature of the functional antagonism.Further studies with improved prostanoid EP1 receptor antagonistse.g. GW848687X (Giblin et al., 2007) are required to confirm thepresence of the prostanoid EP1 receptor subtype within this artery.

In addition to vasodilation, prostacyclin analogues can exert anti-proliferative effects on smooth muscle cells, although significantvariation exists between studies, and negative results have also been

Table 2Selectivities of prostanoid receptor antagonists used in the current study.

Prostanoidantagonist

Assay system pKi or pA2 for prostan

IP EP1

RO-1138452 (IP) Human rc-prostanoid receptor 8.70 b5.0Guinea-pig tracheaGuinea-pig aorta 8.39Rabbit mesenteric artery 8.12

SC-51322 (EP1) Human rc-prostanoid receptor b4.0 7.86Human rc-prostanoid receptor 8.80Guinea-pig trachea 8.45Rat femoral artery

L-798106 (EP3) Human rc-prostanoid receptor b6.3 4.41Guinea-pig vas deferensRat femoral artery

Recombinant (rc) receptor assays involve radioligand binding, except for Durocher et al. (200muscle preparations.

reported (Clapp et al., 2002;Voisard et al., 2005;Willis et al., 1986). Giventhat the anti-restenotic therapeutic potential of prostacyclin analoguesdepends, at least in part, on their anti-proliferative capacity, it isnecessary to understand the source of such variation. We sought toexamine the effect that the degree of growth stimulation has on the anti-proliferative activity of these analogues, and todetermine if non-selectiveactivation of E-type prostanoid receptors would alter this activity.

The foetal calf serum concentration used to stimulate the smoothmuscle cells had a strong bearing on the anti-proliferative potential ofeach analogue, with both analogues displaying weak anti-prolifera-tive effects in maximally stimulated cells. It is possible that highconcentrations of foetal calf serum activate growth pathways that areindependent of the anti-proliferative effects of the prostacyclinanalogues, which are believed to act via cAMP (Kothapalli et al.,2003; Narumiya et al., 1999), or that the proliferative stimulusoverwhelms the inhibitory capacity of the analogues. Further studieson their anti-proliferative effect against a variety of individual,pathway specific, stimuli are required to confirm this explanation.

Within this study, it has been shown that AFP-07 and cicaprostexert distinct effects in functional organ bath studies, and that thesedifferences are due, at least in part, to differences in selectivity forprostanoid EP3 receptors. The extent to which such differences affect,or are a predictor of, the anti-proliferative potential of each compoundhas not been previously explored. The present study is the first to usea prostanoid IP receptor antagonist, RO-1138452, to demonstrate thatthe anti-proliferative effects of these analogues are mediated, at leastin part, by activation of the IP receptor. Given the affinity of RO-1138452 for prostanoid IP receptors (Table 2), an antagonistconcentration of 1 µM ought to have been sufficient to expose aprostanoid EP3 receptor mediated proliferative effect of AFP-07 inrabbit iliac artery cells, if present. However, this was not the case andthe RO-1138452 only partially removed the anti-proliferative effect ofthe AFP-07. There may be a number of explanations for thisobservation. Prostanoid EP2 and EP4 receptor activation has beendemonstrated to inhibit smooth muscle cell proliferation in smoothmuscle cells (Fujino et al., 2002; Yau and Zahradka, 2003). Therefore,non-selective activation of these receptors by the analogues mayaccount for this observation. However, this seems unlikely given thelimited functional effects observed in the present study to theprostanoid EP2 and EP4 receptor agonists. This suggests an absenceof functional prostanoid EP2 and EP4 receptors in this artery type,although expression studies would be required to confirm this.

We found that L-798106 significantly reduced the anti-prolifera-tive effect of AFP-07, which is consistent with studies in murinetumour cells, where activation of splice variants of the EP3 receptorwas shown to inhibit proliferation (Macias-Perez et al., 2008). Re-interpretation of our data on RO-1138452, where prostanoid IP

oid receptor Reference

EP2 EP3 EP4

b5.0 b5.0 Bley et al. (2006)b6.0 Jones et al. (2006)

b6.0b4.0 6.16 4.85 Abramovitz et al. (2000)

Durocher et al. (2000)Hung et al. (2006)

≤6.0b4.3 9.22 6.05 Belley et al. (2006)

7.48 Clarke et al. (2004)7.35–8.10 Hung et al. (2006)

0), which is a reporter gene assay; other assays are functional assays on isolated smooth

166 C. McCormick et al. / European Journal of Pharmacology 641 (2010) 160–167

receptor blockade, did not completely restore proliferation to controllevels, may also suggest that activation of prostanoid EP3 receptorsenhances the anti-proliferative effect. Evidence of the ability of splicevariants of this receptor to positively couple to adenylate cyclase mayprovide an explanation for this observation (Narumiya et al., 1999). Itmay have been expected that L-798106 would increase the anti-proliferative effect of AFP-07, given the enhanced relaxation weobserved in the presence of the blocker. It is possible that cell cultureconditions induce a change in prostanoid EP3 receptor expression; thepresence of serum may lead to a down regulation of cAMP inhibitoryGI-coupled prostanoid EP3 receptor splice variants (Loffler et al.,2008). Prostanoid EP3 receptor isoforms can undergo agonist-inducedinternalisation, which may also account for the apparent discrepancybetween functional studies and cell culture studies reported here(Bilson et al., 2004).

5. Conclusions

It has been demonstrated that the rabbit iliac artery has functionalpopulations of prostanoid IP and EP3 receptors, and that one of themost advanced prostacyclin analogues developed to date has non-selective activity at these receptors in this artery. The functionalsignificance of such activity is prostanoid IP receptor mediatedrelaxation of smooth muscle followed by prostanoid EP3 receptormediated contraction at increased concentrations. However, theabsence of any deleterious effects of non-selective E-type receptoractivation on anti-proliferative potential confirms potential utility in anext generation drug-eluting stent.

Acknowledgements

Dr C. McCormick was funded by an Engineering and PhysicalSciences Research Council doctoral training award.

We acknowledge the following generous gifts: AFP-07 (Asahi GlassCo, Japan), cicaprost (Schering, AG, Germany), L-798106 (GlaxoS-mithKline, UK), ONO-AE1-259 (ONO Pharmaceuticals, Japan) andtetrazolo PGE1 analogue-19a (DF Woodward, Allergan Inc., CA, USA).

All protocols were approved by the relevant Ethics Committee atthe University of Strathclyde.

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