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Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro
European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro James A. Root n, Dorren A. Davey, Kerry J. Af Forselles Genitourinary Biology, Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
art ic l e i nf o
a b s t r a c t
Article history: Received 22 October 2015 Received in revised form 12 November 2015 Accepted 18 November 2015
Selective prostaglandin EP1 antagonists have been suggested for the treatment of bladder dysfunction. This study assessed the contractile prostanoid receptor subtypes in human and non-human bladder in vitro. Classical tissue bath studies were conducted using bladder strips exposed to prostanoid agonists and antagonists. Prostaglandin E2 (PGE2) contracted rat, macaque and human bladder smooth muscle strips (pEC50 7.91 70.06 (n ¼7), 6.40 70.13 (n ¼ 7), and 6.077 0.11 (n ¼5), respectively). The EP1 receptor antagonist, PF2907617 (300 nM), caused a rightward shift of the PGE2 concentration–response curve in the rat bladder only (pKB 8.40 70.15, n ¼3). PGE2 responses in rat and macaque bladders, but not human, were antagonised by the EP3 antagonist CJ24979 (1 mM). Sulprostone, a mixed EP1/EP3/FP receptor agonist, induced potent contractions of rat bladder muscle (pEC50 7.94 70.31, n ¼ 6). The FP receptor agonist, prostaglandin F2α (PGF2α), induced bladder contraction in all species tested, but with a lower potency in rat. The selective FP receptor agonist latanoprost caused potent contractions of macaque and human bladder strips only. SQ29548, a selective TP antagonist, and GW848687X, a mixed EP1/TP antagonist caused rightward shifts of the concentration–response curves to the selective TP agonist, U46619 (pKB estimates 8.53 70.07 and 7.567 0.06, n ¼3, respectively). Responses to U46619 were absent in rat preparations. These data suggest significant species differences exist in bladder contractile prostanoid receptor subtypes. We conclude that the EP1 subtype does not represent the best approach to the clinical treatment of bladder disorders targeting inhibition of smooth muscle contraction. & 2015 Published by Elsevier B.V.
Keywords: Prostaglandin Human Rat Macaque Urinary bladder Urothelium
1. Introduction Prostaglandins are synthesized from arachidonic acid and mediate a wide array of biological responses (Coleman et al., 1994), including modulation of bladder smooth muscle tone (Coleman et al., 1994; Palea et al., 1998; Schröder et al., 2004; Su et al., 2008). Prostaglandins are synthesized by the urinary bladder (Jeremy et al., 1987; Zwergel et al., 1991) and their effects on bladder function have been reported in several species (Palea et al., 1998; Larsson, 1980). Reductions in bladder capacity have been attributed to one or more of the contractile prostanoid receptor subtypes EP1, EP3, FP and TP (Schröder et al., 2004; Wang et al., 2008; Su et al., 2008; Palea et al., 1998). Furthermore, inhibition of the EP1 prostanoid receptor has been shown to increase bladder capacity in rats in vivo by Lee et al. (2007). In patients with disorders of the bladder such as overactive n Correspondence to: AstraZeneca R&D, RIA iMED, Pepparedsleden 1, S-431 83 Mölndal, Sweden. E-mail address: [email protected] (J.A. Root).
bladder, urinary PGE2 and PGF2α levels are increased compared with control subjects (Kim et al., 2006). Urinary excretion of PGE2 is also increased in patients with interstitial cystitis (Lynes et al., 1987). In addition, nonsteroidal anti-inflammatory drugs, such tiaprofenic acid, that inhibit prostanoid synthesis are reported to cause cystitis (Ahmed and Davison., 1991). Prostaglandin biology is thus of great interest in bladder disorder research. The characterization of these receptors in various tissues has been hampered by the lack of agonists and antagonists with good selectivity for the different prostanoid receptors. Furthermore, to our knowledge, neither detailed cross-species investigations, nor studies that examine the role of the urothelium in the in vitro smooth muscle response to prostanoids have been reported. Our aim was to characterize the prostanoid receptor subtypes responsible for contraction of bladder smooth muscle in rat and cynomolgus macaque, two species commonly used in preclinical bladder research, and compare with the receptors in the human bladder. To assess the role of the urothelium and sub-mucosal neurones in prostanoid-induced contractions we also conducted studies in intact bladder muscle preparations.
http://dx.doi.org/10.1016/j.ejphar.2015.11.030 0014-2999/& 2015 Published by Elsevier B.V.
Please cite this article as: Root, J.A., et al., Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.11.030i
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2. Methods 2.1. Tissue preparation Urinary bladders from female Sprague Dawley CD rats (225– 300 g, n ¼16) and male cynomolgus macaque monkeys (macaca fascicularis) from placebo-treated unrelated studies (n ¼8) were acquired in accordance with UK Home Office regulations. Human bladder specimens (12 donors; 4 female, 8 male; age range 47–77 years) were acquired with informed consent and in accordance with The Human Tissue Act. After transport to the laboratory in Krebs solution (NaCl 118.2 mM, KCl 4.69 mM, MgSO4 7H2O 0.5 mM, KH2PO4 1.19 mM, glucose 11.1 mM, NaHCO3 25.0 mM), bladder muscle from non-trigone regions was cut into strips approximately 2 mm wide by 5 mm long, and where required the urothelium was removed. Histological confirmation of this method to remove the urothelium was performed in previous (unpublished) studies. Strips were mounted in 5 ml tissue baths containing modified Krebs solution (NaCl 118.2 mM, KCl 4.69 mM, MgSO4 7H2O 0.5 mM, KH2PO4 1.19 mM, glucose 11.1 mM, NaHCO3 25.0 mM, CaCl2 6H2O 2.5 mM, naproxen 0.01 mM) pH 7.4 at 37 °C and gassed with 95% CO2 and 5% O2. Tissues were attached to isometric force-displacement transducers by silk sutures and placed under 1.5 g tension. During a minimum of 60 min, the bladder strips were perfused continuously with modified Krebs solution at 5 ml/min and tension adjusted if required. Perfusion was stopped and tissues left to equilibrate for 10 min, followed by addition of 80 mM KCl to assess viability. Tissues that contracted less than 0.5 g were discounted from the study. After 50 min continuous perfusion with modified Krebs solution and 10 min equilibration without perfusion, antagonists or vehicle were added to each bath 30 min prior to challenging the tissues with cumulatively increasing concentrations of prostanoid agonists. Each concentration of agonist was left in contact for 5 min or until response plateau. Neuronal involvement in the PGE2 response was assessed by a 30 min pre-incubation of tissues with 1 mM tetrodotoxin (TTX) prior to PGE2 challenge. 2.2. Data analysis Measurements were recorded using an amplifier connected to Notocord acquisition software (v4.2). The average tension for a 30 s interval upon plateau of response was calculated as a percentage of the maximal response to 80 mM KCl. Unconstrained concentration response curves were fitted using an in house Microsoft Excel add-in package by non-linear, 4 parameter, logistical regression analysis. From these curves pEC50, Emax and slope values were estimated. pKB values were estimated using the Gaddum-Schild equation (apparent pKB ¼log[concentration ratio-1]log[antagonist]). Statistical analysis was conducted where appropriate using one-way ANOVA, and P o0.05 was considered significant. All values are mean 7S.E.M., n values equate to the number of individual bladders used in a study. 2.3. Chemicals The following compounds were used: SQ29548 (IDS, U.K.), PGE2, PGF2α, latanoprost, naproxen, sulprostone, tetrodotoxin, U-46619 (Sigma Aldrich, U.K.). KCl (Sigma Aldrich, U.K.) was dissolved in distilled water. CJ-24979 (5-bromo-2-methoxy-N-[3(naphthalen-2-yl-methylphenyl)-acryloyl]-benzenesulphonamide, Juteau et al., 2001), GW848687X (6-[2-(5-chloro-2-{[(2,4-difluorophenyl)methyl]oxy}phenyl)-1-cyclopenten-1-yl]-2-pyridinecarboxylic acid, Giblin et al., 2007) and PF-2907617 (Lee et al., 2007) were synthesized in–house (purities 495%). Compounds were dissolved in 100% dimethylsulphoxide (DMSO) and diluted in
modified Krebs solution. Volumes added to the baths for each concentration point were 3.5–10 ml, totalling a maximum DMSO concentration of 0.3% at the highest concentration assessed.
3. Results 3.1. Effect of PGE2 Application of PGE2 to urothelium-free rat, cynomolgus macaque and human bladder detrusor muscle strips caused concentration-dependent contractions, with estimated pEC50 values of 7.91 70.06, 6.40 70.13, and 6.07 70.11, respectively (n ¼7, 7 and 5 respectively; Fig. 1A, Table 1). The nature of the PGE2-induced contractions differed between species; with an increase in spontaneous activity predominant in the rat, but an increase in tone accompanied by a small degree of increasing spontaneous activity in the macaque and human. Relative to the response to 80 mM KCl, maximum responses to PGE2 were smaller in rat bladder strips (18.0 70.8%, n ¼5, Fig. 1A), than those of macaque and human strips (85.8 79.4% and 111.8723.9% respectively, P o0.05, Fig. 1A). 3.2. Effect of agonists In the absence of urothelium, the mixed EP1/EP3/FP agonist, sulprostone 4 300 nM, caused weak contractions in macaque and human bladder strips, however, no response plateau was obtained (Fig. 1C). In rat bladder strips without urothelium, sulprostone induced potent concentration-dependent contractions (pEC50 7.95 70.32, n ¼6, Fig. 1C). PGF2α was more potent in macaque and human bladder strips than rat bladder strips (pEC50 values of 7.63 70.14 (n ¼4), 7.03 70.25 (n ¼5), and 6.29 70.15 (n ¼ 6) respectively (Fig. 1D); with significance (P o0.01) only between macaque and rat). The selective FP agonist, latanoprost, did induce small contractions in rat detrusor strips (Fig. 1E, n ¼4), but only at concentrations above 1 mM). In contrast, latanoprost was a potent agonist in the macaque and human bladder, producing complete concentration response curves with pEC50 values of 8.057 0.12 and 7.50 70.09 respectively (n ¼4, Fig. 1E). The selective TP agonist, U46619, induced substantial contractions in macaque detrusor strips with a mean pEC50 of 7.74 70.13 (Emax 787 9% 80 mM KCl, n ¼3, Fig. 1F). However, similar to latanoprost, U46619 induced small contractions in rat detrusor muscle strips only at high concentrations (10 μM, 11 7 1% 80 mM KCl, Fig. 1F). No differences in any agonist response was noted when data from human strips were analysed by sex. 3.3. Effect of antagonists Pre-incubation of rat bladder strips without urothelium with 300 nM PF-2907617 or 1 mM CJ24979 produced rightward parallel shifts of the PGE2 concentration response curves with apparent pKB values of 8.45 70.07 and 6.3370.08 respectively (n ¼3–4, Fig. 2A). The EP1 antagonist PF-2907617 (300 nM) did not inhibit the PGE2-induced contractions in macaque and human bladder strips (Fig. 2C and D respectively). However, the EP3 antagonist CJ24979 (1 mM) induced rightward shifts of the PGE2 concentration response curves in macaque bladder smooth muscle, yielding an apparent pKB of 5.96 70.22, n ¼4, Fig. 2C). CJ24979 (1 mM) had no effect on the PGE2-induced contractions in human bladder strips (Fig. 2D). Additional studies with U46619 were completed only in macaque bladder due to availability of human samples and the lack of potency in rat. Both the selective TP antagonist SQ29548 (100 nM)
Please cite this article as: Root, J.A., et al., Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.11.030i
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Fig. 1. Effect of prostaglandin agonists on denuded bladder smooth muscle from rat, macaque and human. Concentration–response curves show effect of (A) PGE2, (B) PGE2 on rat bladder strips with or without urothelium, (C) sulprostone, (D) PGF2α, (E) latanoprost, (F) U46619. Data are mean7 S.E.M., n¼ 3–7.
Table 1 Summary of potency values (pEC50) of prostaglandin agonists in rat, macaque and human bladder smooth muscle strips without urothelium (n ¼3–7). Agonist
Rat pEC50 (mean7 S.E.M.)
Macaque pEC50 (mean 7 S.E.M.)
Human pEC50 (mean7 S.E.M.)
PGE2 Sulprostone PGF2α Latanoprost U46619
7.91 70.06 7.94 7 0.31 6.29 7 0.15 ND ND
6.40 7 0.13 ND 7.63 70.14 8.05 7 0.12 7.747 0.13
6.077 0.11 ND 7.03 7 0.25 7.50 7 0.09 NT
Abbreviations: PGE2, prostaglandin E2; PGF2α, prostaglandin F2α; ND, not determined; NT, not tested.
and the EP1/TP antagonist GW848687X (50 nM) induced rightward shifts of the PGE2 concentration response curves in macaque bladder strips with apparent pKB values of 8.53 70.07 (n ¼3) and 7.56 7 0.06 (n ¼ 3) respectively (Fig. 2D).
3.4. Effect of tetrodotoxin In macaque bladder strips pre-incubation with 1 mM TTX did not affect the responses to PGE2 (Fig. 2F), however in rat bladder strips TTX induced a statistically significant depression of the maximal responses (mean 3.2 70.4% vs 18 7 0.8% for control,
Please cite this article as: Root, J.A., et al., Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.11.030i
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Fig. 2. Effect of prostaglandin antagonists on denuded bladder smooth muscle from rat, macaque and human. Concentration–response curves show effect on bladder strips of PF2907617 and CJ24979 on PGE2 responses in (A) rat without urothelium, (B) rat with urothelium (C) monkey, (D) human, (E) Effect of SQ29548 and GW848687X on responses to U46619 in monkey bladder. (F) Effect of TTX on PGE2 responses in rat and macaque bladder strips. Data are mean 7S.E.M., n¼ 3–7.
P o0.01) and a 5-fold decrease in potency (pEC50 7.29 for TTX vs 7.91 for controls; Fig. 2F).
abscence of urothelium (6.37 70.05 vs 6.33 70.08 respectively, n¼ 4, Fig. 2B).
3.5. Effect of urothelium 4. Discussion There were no visual differences in PGE2-induced contractions in macaque or human tissues with or without intact urothelia (data not shown). The magnitude of the PGE2-induced activity in intact rat bladder was decreased compared with denuded, but not significantly (Po 0.10), with no change in potency (pEC50 7.78 70.07 vs 7.917 0.06 for intact vs denuded tissues, Fig. 1B). In the presence of the urothelium, the apparent pKB of PF2907617 (300 nM) for inhibition of PGE2-induced contractions was 7.54 70.18 compared with 8.45 70.07 in urothelium denuded strips, Fig. 2B). In contrast, in the presence of urothelium the antagonist potency of CJ24979 (1 mM) was similar to that in the
This study demonstrates species differences in the prostanoid receptor subtypes responsible for evoking prostaglandin-induced contractions in bladder detrusor muscle of rats, cynomolgus macaques and humans. Compared with the magnitude of the contraction induced by 80 mM KCl, contractions induced by PGE2 were much greater in macaque and human bladder tissues than rat. The contractions also differed in phenotype, with a more phasic-like contraction observed in the rat muscle strips. In contrast, the potency of PGE2 and the magnitude of PGE2-induced responses were not different in macaque compared with human bladder strips.
Please cite this article as: Root, J.A., et al., Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.11.030i
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There was no evidence of modulation of the PGE2-induced response by the urothelium in human or macaque bladder strips, as there was no difference in the magnitude and potency of the PGE2induced responses in bladder strips with intact urothelia compared with strips without urothelium. The response to PGE2 in the presence of urothelium in rat bladder strips was numerically greater than in strips where the urothelium had been removed, however this did not reach significance. A further species difference between rat and macaque was seen in the response to PGE2 in the presence of TTX. TTX significantly suppressed the magnitude of PGE2-induced contractions in rat bladder strips, while the responses evoked by PGE2 in the macaque showed no difference in magnitude in the presence or absence of TTX. This study supports the in vivo findings of a role for the EP1 prostanoid receptor in the rat micturition reflex (Lee et al., 2007) and provides evidence of the mechanism of action of EP1, suggesting a role of urothelial (or sub-urothelial) neurones in the PGE2-induced contractions in rat bladder. The high binding affinity of PGE2 for the human EP1 and EP3 receptors (9.1 and 0.33 nM respectively, Abramovitz et al., 2000) and the low-potency of PGE2 in human bladder strips support the hypothesis that the prostanoid subtype involved in the PGE2-induced contraction in human bladder in vitro is not an EP1 or EP3 receptor. The low-potency contractile effects of PGE2 in monkey and humans are likely mediated through FP and/or TP prostanoid receptor subtypes as PGE2 has weak efficacy at these subtypes (Abramovitz et al., 2000). The potent PGE2-induced contraction in rat bladder and its inhibition by the selective EP1 antagonist, PF-2907617 corroborates the in vivo data that the PGE2-induced response in rat bladder is mediated via the EP1 receptor subtype (Lee at al., 2007). There was also evidence of involvement of the EP3 subtype in the PGE2-induced contraction in rat and macaque bladder muscle as the EP3 antagonist CJ24979 inhibited the PGE2-induced contractions. In contrast, CJ24979 had no effect on the PGE2-induced contraction in human bladder. However, the potency of the CJ24979 was low compared with the published nanomolar binding affinity of this compound (Juteau et al., 2001). This may be due to the majority of the PGE2-induced contraction being mediated via the EP1 subtype with only a small contribution by the EP3 subtype. Sulprostone is a potent agonist of human EP3 receptors (Abramovitz et al., 2000; Qian et al., 1994), however its affinity and efficacy for the rat and macaque EP3 receptors have not been reported. Nevertheless, the potency of contractions elicited by sulprostone in the rat bladder suggests involvement of EP3 receptor subtype, whilst the lower potency of this agonist in the macaque suggests otherwise. The potent and selective FP agonist, latanoprost, has been reported to have efficacy at FP receptor subtypes in rat (Sharif, 2008). However, unlike the potent contractions in response to PGF2α and latanoprost seen in macaque and human bladder smooth muscle, no response was seen at the expected concentration range in rat bladder smooth muscle, suggesting yet a further species difference. The weak PGF2α-mediated contraction seen is likely to have been mediated by other prostanoid receptor subtypes, such as EP1 or EP3. Palea and colleagues clearly demonstrated the role of the TP receptor in human bladder using highly selective compounds (Palea et al., 1998), and the role of the FP receptor suggested by contractions seen to PGF2α in their study are confirmed here with data using the potent and selective FP agonist latanoprost. The TP subtype data displays the same divergence and a clear separation exists between the species studied; primates, unlike rodents, appear to have evolved to utilise the FP and TP subtypes for control of micturition. These differences suggest that the rat is not a suitable species for translational studies on the control of micturition by prostanoids. In vivo studies support the functional role of EP1 receptors in the rat seen in this study. Bladder capacity, micturition interval
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and volume in conscious urodynamic studies in rats are attenuated by the EP1 antagonist PF2907617 (Lee et al., 2007). Other EP1 antagonists have shown efficacy in other rat models of bladder disease (Wada et al., 2013). Such pre-clinical data and the clinical findings of increased urinary prostanoid concentration in humans with bladder disorders (Kim et al., 2006) have lead researchers to propose the therapeutic use of EP1 antagonists in overactive bladder. However the failure of the EP1 antagonist to inhibit the PGE2-induced contraction response in human and macaque bladder, combined with the weak response to sulprostone, indicates that this receptor subtype, in contrast to the rat, plays a minor, if any role, in human or macaque bladder muscle function. Extrapolation of these findings to in vivo responses should be performed with caution however, as these in vitro data have been generated in the presence of an unknown degree of neuronal innervation and modulation. Regional differences in the lamina propria expression of COX isoforms and EP receptor subtype have been reported (Rahnama'I et al., 2010) and, due to the discrete regional areas studied here, may have a bearing on the in vivo relevance of our findings. Nevertheless, the futility of pursuing such a therapeutic approach towards bladder overactive disorders is indicated by the recent failure of an EP1 antagonist in human clinical trials (Chapple et al., 2014). Differences in prostanoid receptor subtype between rodent (guinea pig) and primate species have been previously reported (Poli et al., 1992), which, further demonstrate the wide species differences in prostanoid biology in the bladder of rodents and primates. In conclusion, the major functional prostanoid receptors in vitro in the rat detrusor smooth muscle are the EP1 and EP3 receptors, in contrast to the human and macaque bladders, where no evidence of functional EP1 receptors in smooth muscle contraction was found. The macaque bladder smooth muscle possesses functional EP3 receptors, but there was no evidence in this study for contraction-mediating EP3 receptors in rat or human bladder strips. Evidence for functional FP and TP receptors in bladder smooth muscle was only observed in the two primate species studied here. Contractions mediated by PGE2 in the rat, but not the monkey, are sensitive to TTX, suggesting that they are elicited via the neuronal release of an unknown mediator. Further studies to identify the contractile mediator should consider the tachykinins as potential candidates as they are known have a role in the modulation of micturition (Ishizuka et al., 1995). There is no evidence at the level of the isolated bladder smooth muscle to support the use of an EP1 antagonist in the treatment of overactive bladder in humans. However, the role of the FP and TP receptors in primate bladder biology and potential therapeutic use does merit further study.
References Abramovitz, M., et al., 2000. The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs. Biochim. Biophys. Acta 17 (2), 285–293. Ahmed, M., Davison, O.W., 1991. Severe cystitis associated with tiaprofenic acid. Br. Med. J. 30 (6814), 1376. Chapple, C.R., et al., 2014. Phase II study on the efficacy and safety of the EP1 receptor antagonist ONO-8539 for nonneurogenic overactive bladder syndrome. J. Urol. 191 (1), 253–260. Coleman, R.A., et al., 1994. International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmocol. Rev. 46 (2), 205–229. Giblin, G.M., et al., 2007. The discovery of 6-[2-(5-chloro-2-{[(2,4-difluorophenyl) methyl]oxy}phenyl)-1-cyclopenten-1-yl]-2-pyridinecarboxylic acid, GW848687X, a potent and selective prostaglandin EP1 receptor antagonist for the treatment of inflammatory pain. Bioorg. Med. Chem. Lett. 15 (2), 385–389. Ishizuka, O., et al., 1995. Prostaglandin E2-induced bladder hyperactivity in normal, conscious rats: involvement of tachykinins? J. Urol. 153 (6), 2034–2038. Jeremy, J.Y., et al., 1987. Eicosanoid synthesis by human urinary bladder mucosa: pathological implications. Br. J. Urol. 59 (1), 36–39.
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Juteau, H., et al., 2001. Structure–activity relationship of cinnamic acylsulfonamide analogues on the human EP3 prostanoid receptor. Bioorg. Med. Chem. 9 (8), 1977–1984. Kim, J.C., et al., 2006. Nerve growth factor and prostaglandins in the urine of female patients with overactive bladder. J. Urol. 175 (5), 1773–1776. Larsson, C., 1980. Production and effects of prostaglandins in the detrusor from homo, cat, rabbit, and rat. Adv. Prostaglandin. Thromboxane Res. 8, 1263–1267. Lee, T., et al., 2007. Urodynamic effects of a novel EP(1) receptor antagonist in normal rats and rats with bladder outlet obstruction. J. Urol. 177 (4), 1562–1567. Lynes, W.L., et al., 1987. Mast cell involvement in interstitial cystitis. J. Urol. 138 (4), 746–752. Palea, S., et al., 1998. Pharmacological characterization of thromboxane and prostanoid receptors in human isolated urinary bladder. Br. J. Pharmacol. 124 (5), 865–872. Poli, E., et al., 1992. Actions of two novel prostaglandin analogs, SC-29169 and SC31391, on guinea pig and human isolated urinary bladder. Gen. Pharmacol. 23 (5), 805–809. Qian, Y.M., et al., 1994. Potent contractile actions of prostanoid EP3-receptor
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Please cite this article as: Root, J.A., et al., Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.11.030i
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro James A. Root n, Dorren A. Davey, Kerry J. Af Forselles Genitourinary Biology, Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
art ic l e i nf o
a b s t r a c t
Article history: Received 22 October 2015 Received in revised form 12 November 2015 Accepted 18 November 2015
Selective prostaglandin EP1 antagonists have been suggested for the treatment of bladder dysfunction. This study assessed the contractile prostanoid receptor subtypes in human and non-human bladder in vitro. Classical tissue bath studies were conducted using bladder strips exposed to prostanoid agonists and antagonists. Prostaglandin E2 (PGE2) contracted rat, macaque and human bladder smooth muscle strips (pEC50 7.91 70.06 (n ¼7), 6.40 70.13 (n ¼ 7), and 6.077 0.11 (n ¼5), respectively). The EP1 receptor antagonist, PF2907617 (300 nM), caused a rightward shift of the PGE2 concentration–response curve in the rat bladder only (pKB 8.40 70.15, n ¼3). PGE2 responses in rat and macaque bladders, but not human, were antagonised by the EP3 antagonist CJ24979 (1 mM). Sulprostone, a mixed EP1/EP3/FP receptor agonist, induced potent contractions of rat bladder muscle (pEC50 7.94 70.31, n ¼ 6). The FP receptor agonist, prostaglandin F2α (PGF2α), induced bladder contraction in all species tested, but with a lower potency in rat. The selective FP receptor agonist latanoprost caused potent contractions of macaque and human bladder strips only. SQ29548, a selective TP antagonist, and GW848687X, a mixed EP1/TP antagonist caused rightward shifts of the concentration–response curves to the selective TP agonist, U46619 (pKB estimates 8.53 70.07 and 7.567 0.06, n ¼3, respectively). Responses to U46619 were absent in rat preparations. These data suggest significant species differences exist in bladder contractile prostanoid receptor subtypes. We conclude that the EP1 subtype does not represent the best approach to the clinical treatment of bladder disorders targeting inhibition of smooth muscle contraction. & 2015 Published by Elsevier B.V.
Keywords: Prostaglandin Human Rat Macaque Urinary bladder Urothelium
1. Introduction Prostaglandins are synthesized from arachidonic acid and mediate a wide array of biological responses (Coleman et al., 1994), including modulation of bladder smooth muscle tone (Coleman et al., 1994; Palea et al., 1998; Schröder et al., 2004; Su et al., 2008). Prostaglandins are synthesized by the urinary bladder (Jeremy et al., 1987; Zwergel et al., 1991) and their effects on bladder function have been reported in several species (Palea et al., 1998; Larsson, 1980). Reductions in bladder capacity have been attributed to one or more of the contractile prostanoid receptor subtypes EP1, EP3, FP and TP (Schröder et al., 2004; Wang et al., 2008; Su et al., 2008; Palea et al., 1998). Furthermore, inhibition of the EP1 prostanoid receptor has been shown to increase bladder capacity in rats in vivo by Lee et al. (2007). In patients with disorders of the bladder such as overactive n Correspondence to: AstraZeneca R&D, RIA iMED, Pepparedsleden 1, S-431 83 Mölndal, Sweden. E-mail address: [email protected] (J.A. Root).
bladder, urinary PGE2 and PGF2α levels are increased compared with control subjects (Kim et al., 2006). Urinary excretion of PGE2 is also increased in patients with interstitial cystitis (Lynes et al., 1987). In addition, nonsteroidal anti-inflammatory drugs, such tiaprofenic acid, that inhibit prostanoid synthesis are reported to cause cystitis (Ahmed and Davison., 1991). Prostaglandin biology is thus of great interest in bladder disorder research. The characterization of these receptors in various tissues has been hampered by the lack of agonists and antagonists with good selectivity for the different prostanoid receptors. Furthermore, to our knowledge, neither detailed cross-species investigations, nor studies that examine the role of the urothelium in the in vitro smooth muscle response to prostanoids have been reported. Our aim was to characterize the prostanoid receptor subtypes responsible for contraction of bladder smooth muscle in rat and cynomolgus macaque, two species commonly used in preclinical bladder research, and compare with the receptors in the human bladder. To assess the role of the urothelium and sub-mucosal neurones in prostanoid-induced contractions we also conducted studies in intact bladder muscle preparations.
http://dx.doi.org/10.1016/j.ejphar.2015.11.030 0014-2999/& 2015 Published by Elsevier B.V.
Please cite this article as: Root, J.A., et al., Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.11.030i
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2. Methods 2.1. Tissue preparation Urinary bladders from female Sprague Dawley CD rats (225– 300 g, n ¼16) and male cynomolgus macaque monkeys (macaca fascicularis) from placebo-treated unrelated studies (n ¼8) were acquired in accordance with UK Home Office regulations. Human bladder specimens (12 donors; 4 female, 8 male; age range 47–77 years) were acquired with informed consent and in accordance with The Human Tissue Act. After transport to the laboratory in Krebs solution (NaCl 118.2 mM, KCl 4.69 mM, MgSO4 7H2O 0.5 mM, KH2PO4 1.19 mM, glucose 11.1 mM, NaHCO3 25.0 mM), bladder muscle from non-trigone regions was cut into strips approximately 2 mm wide by 5 mm long, and where required the urothelium was removed. Histological confirmation of this method to remove the urothelium was performed in previous (unpublished) studies. Strips were mounted in 5 ml tissue baths containing modified Krebs solution (NaCl 118.2 mM, KCl 4.69 mM, MgSO4 7H2O 0.5 mM, KH2PO4 1.19 mM, glucose 11.1 mM, NaHCO3 25.0 mM, CaCl2 6H2O 2.5 mM, naproxen 0.01 mM) pH 7.4 at 37 °C and gassed with 95% CO2 and 5% O2. Tissues were attached to isometric force-displacement transducers by silk sutures and placed under 1.5 g tension. During a minimum of 60 min, the bladder strips were perfused continuously with modified Krebs solution at 5 ml/min and tension adjusted if required. Perfusion was stopped and tissues left to equilibrate for 10 min, followed by addition of 80 mM KCl to assess viability. Tissues that contracted less than 0.5 g were discounted from the study. After 50 min continuous perfusion with modified Krebs solution and 10 min equilibration without perfusion, antagonists or vehicle were added to each bath 30 min prior to challenging the tissues with cumulatively increasing concentrations of prostanoid agonists. Each concentration of agonist was left in contact for 5 min or until response plateau. Neuronal involvement in the PGE2 response was assessed by a 30 min pre-incubation of tissues with 1 mM tetrodotoxin (TTX) prior to PGE2 challenge. 2.2. Data analysis Measurements were recorded using an amplifier connected to Notocord acquisition software (v4.2). The average tension for a 30 s interval upon plateau of response was calculated as a percentage of the maximal response to 80 mM KCl. Unconstrained concentration response curves were fitted using an in house Microsoft Excel add-in package by non-linear, 4 parameter, logistical regression analysis. From these curves pEC50, Emax and slope values were estimated. pKB values were estimated using the Gaddum-Schild equation (apparent pKB ¼log[concentration ratio-1]log[antagonist]). Statistical analysis was conducted where appropriate using one-way ANOVA, and P o0.05 was considered significant. All values are mean 7S.E.M., n values equate to the number of individual bladders used in a study. 2.3. Chemicals The following compounds were used: SQ29548 (IDS, U.K.), PGE2, PGF2α, latanoprost, naproxen, sulprostone, tetrodotoxin, U-46619 (Sigma Aldrich, U.K.). KCl (Sigma Aldrich, U.K.) was dissolved in distilled water. CJ-24979 (5-bromo-2-methoxy-N-[3(naphthalen-2-yl-methylphenyl)-acryloyl]-benzenesulphonamide, Juteau et al., 2001), GW848687X (6-[2-(5-chloro-2-{[(2,4-difluorophenyl)methyl]oxy}phenyl)-1-cyclopenten-1-yl]-2-pyridinecarboxylic acid, Giblin et al., 2007) and PF-2907617 (Lee et al., 2007) were synthesized in–house (purities 495%). Compounds were dissolved in 100% dimethylsulphoxide (DMSO) and diluted in
modified Krebs solution. Volumes added to the baths for each concentration point were 3.5–10 ml, totalling a maximum DMSO concentration of 0.3% at the highest concentration assessed.
3. Results 3.1. Effect of PGE2 Application of PGE2 to urothelium-free rat, cynomolgus macaque and human bladder detrusor muscle strips caused concentration-dependent contractions, with estimated pEC50 values of 7.91 70.06, 6.40 70.13, and 6.07 70.11, respectively (n ¼7, 7 and 5 respectively; Fig. 1A, Table 1). The nature of the PGE2-induced contractions differed between species; with an increase in spontaneous activity predominant in the rat, but an increase in tone accompanied by a small degree of increasing spontaneous activity in the macaque and human. Relative to the response to 80 mM KCl, maximum responses to PGE2 were smaller in rat bladder strips (18.0 70.8%, n ¼5, Fig. 1A), than those of macaque and human strips (85.8 79.4% and 111.8723.9% respectively, P o0.05, Fig. 1A). 3.2. Effect of agonists In the absence of urothelium, the mixed EP1/EP3/FP agonist, sulprostone 4 300 nM, caused weak contractions in macaque and human bladder strips, however, no response plateau was obtained (Fig. 1C). In rat bladder strips without urothelium, sulprostone induced potent concentration-dependent contractions (pEC50 7.95 70.32, n ¼6, Fig. 1C). PGF2α was more potent in macaque and human bladder strips than rat bladder strips (pEC50 values of 7.63 70.14 (n ¼4), 7.03 70.25 (n ¼5), and 6.29 70.15 (n ¼ 6) respectively (Fig. 1D); with significance (P o0.01) only between macaque and rat). The selective FP agonist, latanoprost, did induce small contractions in rat detrusor strips (Fig. 1E, n ¼4), but only at concentrations above 1 mM). In contrast, latanoprost was a potent agonist in the macaque and human bladder, producing complete concentration response curves with pEC50 values of 8.057 0.12 and 7.50 70.09 respectively (n ¼4, Fig. 1E). The selective TP agonist, U46619, induced substantial contractions in macaque detrusor strips with a mean pEC50 of 7.74 70.13 (Emax 787 9% 80 mM KCl, n ¼3, Fig. 1F). However, similar to latanoprost, U46619 induced small contractions in rat detrusor muscle strips only at high concentrations (10 μM, 11 7 1% 80 mM KCl, Fig. 1F). No differences in any agonist response was noted when data from human strips were analysed by sex. 3.3. Effect of antagonists Pre-incubation of rat bladder strips without urothelium with 300 nM PF-2907617 or 1 mM CJ24979 produced rightward parallel shifts of the PGE2 concentration response curves with apparent pKB values of 8.45 70.07 and 6.3370.08 respectively (n ¼3–4, Fig. 2A). The EP1 antagonist PF-2907617 (300 nM) did not inhibit the PGE2-induced contractions in macaque and human bladder strips (Fig. 2C and D respectively). However, the EP3 antagonist CJ24979 (1 mM) induced rightward shifts of the PGE2 concentration response curves in macaque bladder smooth muscle, yielding an apparent pKB of 5.96 70.22, n ¼4, Fig. 2C). CJ24979 (1 mM) had no effect on the PGE2-induced contractions in human bladder strips (Fig. 2D). Additional studies with U46619 were completed only in macaque bladder due to availability of human samples and the lack of potency in rat. Both the selective TP antagonist SQ29548 (100 nM)
Please cite this article as: Root, J.A., et al., Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.11.030i
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Fig. 1. Effect of prostaglandin agonists on denuded bladder smooth muscle from rat, macaque and human. Concentration–response curves show effect of (A) PGE2, (B) PGE2 on rat bladder strips with or without urothelium, (C) sulprostone, (D) PGF2α, (E) latanoprost, (F) U46619. Data are mean7 S.E.M., n¼ 3–7.
Table 1 Summary of potency values (pEC50) of prostaglandin agonists in rat, macaque and human bladder smooth muscle strips without urothelium (n ¼3–7). Agonist
Rat pEC50 (mean7 S.E.M.)
Macaque pEC50 (mean 7 S.E.M.)
Human pEC50 (mean7 S.E.M.)
PGE2 Sulprostone PGF2α Latanoprost U46619
7.91 70.06 7.94 7 0.31 6.29 7 0.15 ND ND
6.40 7 0.13 ND 7.63 70.14 8.05 7 0.12 7.747 0.13
6.077 0.11 ND 7.03 7 0.25 7.50 7 0.09 NT
Abbreviations: PGE2, prostaglandin E2; PGF2α, prostaglandin F2α; ND, not determined; NT, not tested.
and the EP1/TP antagonist GW848687X (50 nM) induced rightward shifts of the PGE2 concentration response curves in macaque bladder strips with apparent pKB values of 8.53 70.07 (n ¼3) and 7.56 7 0.06 (n ¼ 3) respectively (Fig. 2D).
3.4. Effect of tetrodotoxin In macaque bladder strips pre-incubation with 1 mM TTX did not affect the responses to PGE2 (Fig. 2F), however in rat bladder strips TTX induced a statistically significant depression of the maximal responses (mean 3.2 70.4% vs 18 7 0.8% for control,
Please cite this article as: Root, J.A., et al., Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.11.030i
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Fig. 2. Effect of prostaglandin antagonists on denuded bladder smooth muscle from rat, macaque and human. Concentration–response curves show effect on bladder strips of PF2907617 and CJ24979 on PGE2 responses in (A) rat without urothelium, (B) rat with urothelium (C) monkey, (D) human, (E) Effect of SQ29548 and GW848687X on responses to U46619 in monkey bladder. (F) Effect of TTX on PGE2 responses in rat and macaque bladder strips. Data are mean 7S.E.M., n¼ 3–7.
P o0.01) and a 5-fold decrease in potency (pEC50 7.29 for TTX vs 7.91 for controls; Fig. 2F).
abscence of urothelium (6.37 70.05 vs 6.33 70.08 respectively, n¼ 4, Fig. 2B).
3.5. Effect of urothelium 4. Discussion There were no visual differences in PGE2-induced contractions in macaque or human tissues with or without intact urothelia (data not shown). The magnitude of the PGE2-induced activity in intact rat bladder was decreased compared with denuded, but not significantly (Po 0.10), with no change in potency (pEC50 7.78 70.07 vs 7.917 0.06 for intact vs denuded tissues, Fig. 1B). In the presence of the urothelium, the apparent pKB of PF2907617 (300 nM) for inhibition of PGE2-induced contractions was 7.54 70.18 compared with 8.45 70.07 in urothelium denuded strips, Fig. 2B). In contrast, in the presence of urothelium the antagonist potency of CJ24979 (1 mM) was similar to that in the
This study demonstrates species differences in the prostanoid receptor subtypes responsible for evoking prostaglandin-induced contractions in bladder detrusor muscle of rats, cynomolgus macaques and humans. Compared with the magnitude of the contraction induced by 80 mM KCl, contractions induced by PGE2 were much greater in macaque and human bladder tissues than rat. The contractions also differed in phenotype, with a more phasic-like contraction observed in the rat muscle strips. In contrast, the potency of PGE2 and the magnitude of PGE2-induced responses were not different in macaque compared with human bladder strips.
Please cite this article as: Root, J.A., et al., Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.11.030i
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There was no evidence of modulation of the PGE2-induced response by the urothelium in human or macaque bladder strips, as there was no difference in the magnitude and potency of the PGE2induced responses in bladder strips with intact urothelia compared with strips without urothelium. The response to PGE2 in the presence of urothelium in rat bladder strips was numerically greater than in strips where the urothelium had been removed, however this did not reach significance. A further species difference between rat and macaque was seen in the response to PGE2 in the presence of TTX. TTX significantly suppressed the magnitude of PGE2-induced contractions in rat bladder strips, while the responses evoked by PGE2 in the macaque showed no difference in magnitude in the presence or absence of TTX. This study supports the in vivo findings of a role for the EP1 prostanoid receptor in the rat micturition reflex (Lee et al., 2007) and provides evidence of the mechanism of action of EP1, suggesting a role of urothelial (or sub-urothelial) neurones in the PGE2-induced contractions in rat bladder. The high binding affinity of PGE2 for the human EP1 and EP3 receptors (9.1 and 0.33 nM respectively, Abramovitz et al., 2000) and the low-potency of PGE2 in human bladder strips support the hypothesis that the prostanoid subtype involved in the PGE2-induced contraction in human bladder in vitro is not an EP1 or EP3 receptor. The low-potency contractile effects of PGE2 in monkey and humans are likely mediated through FP and/or TP prostanoid receptor subtypes as PGE2 has weak efficacy at these subtypes (Abramovitz et al., 2000). The potent PGE2-induced contraction in rat bladder and its inhibition by the selective EP1 antagonist, PF-2907617 corroborates the in vivo data that the PGE2-induced response in rat bladder is mediated via the EP1 receptor subtype (Lee at al., 2007). There was also evidence of involvement of the EP3 subtype in the PGE2-induced contraction in rat and macaque bladder muscle as the EP3 antagonist CJ24979 inhibited the PGE2-induced contractions. In contrast, CJ24979 had no effect on the PGE2-induced contraction in human bladder. However, the potency of the CJ24979 was low compared with the published nanomolar binding affinity of this compound (Juteau et al., 2001). This may be due to the majority of the PGE2-induced contraction being mediated via the EP1 subtype with only a small contribution by the EP3 subtype. Sulprostone is a potent agonist of human EP3 receptors (Abramovitz et al., 2000; Qian et al., 1994), however its affinity and efficacy for the rat and macaque EP3 receptors have not been reported. Nevertheless, the potency of contractions elicited by sulprostone in the rat bladder suggests involvement of EP3 receptor subtype, whilst the lower potency of this agonist in the macaque suggests otherwise. The potent and selective FP agonist, latanoprost, has been reported to have efficacy at FP receptor subtypes in rat (Sharif, 2008). However, unlike the potent contractions in response to PGF2α and latanoprost seen in macaque and human bladder smooth muscle, no response was seen at the expected concentration range in rat bladder smooth muscle, suggesting yet a further species difference. The weak PGF2α-mediated contraction seen is likely to have been mediated by other prostanoid receptor subtypes, such as EP1 or EP3. Palea and colleagues clearly demonstrated the role of the TP receptor in human bladder using highly selective compounds (Palea et al., 1998), and the role of the FP receptor suggested by contractions seen to PGF2α in their study are confirmed here with data using the potent and selective FP agonist latanoprost. The TP subtype data displays the same divergence and a clear separation exists between the species studied; primates, unlike rodents, appear to have evolved to utilise the FP and TP subtypes for control of micturition. These differences suggest that the rat is not a suitable species for translational studies on the control of micturition by prostanoids. In vivo studies support the functional role of EP1 receptors in the rat seen in this study. Bladder capacity, micturition interval
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and volume in conscious urodynamic studies in rats are attenuated by the EP1 antagonist PF2907617 (Lee et al., 2007). Other EP1 antagonists have shown efficacy in other rat models of bladder disease (Wada et al., 2013). Such pre-clinical data and the clinical findings of increased urinary prostanoid concentration in humans with bladder disorders (Kim et al., 2006) have lead researchers to propose the therapeutic use of EP1 antagonists in overactive bladder. However the failure of the EP1 antagonist to inhibit the PGE2-induced contraction response in human and macaque bladder, combined with the weak response to sulprostone, indicates that this receptor subtype, in contrast to the rat, plays a minor, if any role, in human or macaque bladder muscle function. Extrapolation of these findings to in vivo responses should be performed with caution however, as these in vitro data have been generated in the presence of an unknown degree of neuronal innervation and modulation. Regional differences in the lamina propria expression of COX isoforms and EP receptor subtype have been reported (Rahnama'I et al., 2010) and, due to the discrete regional areas studied here, may have a bearing on the in vivo relevance of our findings. Nevertheless, the futility of pursuing such a therapeutic approach towards bladder overactive disorders is indicated by the recent failure of an EP1 antagonist in human clinical trials (Chapple et al., 2014). Differences in prostanoid receptor subtype between rodent (guinea pig) and primate species have been previously reported (Poli et al., 1992), which, further demonstrate the wide species differences in prostanoid biology in the bladder of rodents and primates. In conclusion, the major functional prostanoid receptors in vitro in the rat detrusor smooth muscle are the EP1 and EP3 receptors, in contrast to the human and macaque bladders, where no evidence of functional EP1 receptors in smooth muscle contraction was found. The macaque bladder smooth muscle possesses functional EP3 receptors, but there was no evidence in this study for contraction-mediating EP3 receptors in rat or human bladder strips. Evidence for functional FP and TP receptors in bladder smooth muscle was only observed in the two primate species studied here. Contractions mediated by PGE2 in the rat, but not the monkey, are sensitive to TTX, suggesting that they are elicited via the neuronal release of an unknown mediator. Further studies to identify the contractile mediator should consider the tachykinins as potential candidates as they are known have a role in the modulation of micturition (Ishizuka et al., 1995). There is no evidence at the level of the isolated bladder smooth muscle to support the use of an EP1 antagonist in the treatment of overactive bladder in humans. However, the role of the FP and TP receptors in primate bladder biology and potential therapeutic use does merit further study.
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Please cite this article as: Root, J.A., et al., Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.11.030i