Excitatory effects of bombesin receptors in urinary tract of normal and diabetic rats in vivo

Life Sciences 100 (2014) 35–44

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Excitatory effects of bombesin receptors in urinary tract of normal and diabetic rats in vivo☆ F. Aura Kullmann ⁎, Grace I. Wells, David McKenna, Karl B. Thor Department of Pharmacology, Urogenix, Inc./Astellas Pharma, Durham, NC 27703, USA

a r t i c l e

i n f o

Article history: Received 23 August 2013 Accepted 23 January 2014 Available online 2 February 2014 Keywords: Underactive bladder Urethane anesthesia Streptozotocin Cystometry

a b s t r a c t Aims: Bombesin receptors (BB receptors) and bombesin related peptides are expressed in the lower urinary tract of rodents. Here we investigated whether in vivo activation of BB receptors can contract the urinary bladder and facilitate micturition in sham rats and in a diabetic rat model of voiding dysfunction. Material and methods: In vivo cystometry experiments were performed in adult female Sprague–Dawley rats under urethane anesthesia. Diabetes was induced by streptozotocin (STZ; 65 mg/kg, i.p.) injection. Experiments were performed 9 and 20 weeks post STZ-treatment. Drugs included neuromedin B (NMB; BB1 receptor preferring agonist), and gastrin-releasing peptide (GRP; BB2 receptor preferring agonist). Key findings: NMB and GRP (0.01–100 μg/kg in sham rats; 0.1–300 μg/kg in STZ-treated rats, i.v.) increased micturition frequency, bladder contraction amplitude and area under the curve dose dependently in both sham and STZ-treated rats. In addition, NMB (3, 10 μg/kg i.v.) triggered voiding in N 80% of STZ-treated rats when the bladder was filled to a sub-threshold voiding volume. NMB and GRP increased mean arterial pressure and heart rate at the highest doses, 100 and 300 μg/kg. Significance: Activation of bombesin receptors facilitated neurogenic bladder contractions in vivo. Single applications of agonists enhanced or triggered voiding in sham rats as well as in the STZ-treated rat model of diabetic voiding dysfunction. These results suggest that BB receptors may be targeted for drug development for conditions associated with poor detrusor contraction such as an underactive bladder condition. © 2014 Elsevier Inc. All rights reserved.

Introduction Several patient populations such as the elderly and diabetic experience voiding dysfunction characterized by the inability to completely void urine from the bladder during micturition. This results in elevated post-void residual urine volumes and symptoms of frequency, nocturia, and urinary tract infections. This condition has recently been recognized as an underactive bladder and represents a highly unmet medical need and a major focus area for basic research and drug development (Miyazato et al., 2013). Current therapies, such as bethanechol and distigmine which increase bladder contractions via activation of muscarinic receptors, have limited efficacy and tolerability due to severe side effects associated with the use of parasympathomimetics (Barendrecht et al., 2007). New therapies with agents that contract the bladder with minimal side effects are needed. The bombesin receptors (BB receptors) are G-protein coupled receptors and are divided into three subtypes: bombesin receptor 1, 2, and 3. BB1 receptors and BB2 receptors are preferentially activated by the endogenous peptides neuromedin B (NMB) and gastrin-releasing ☆ Source of support: This work was supported by Astellas Pharma. ⁎ Corresponding author at: Urogenix Inc./Astellas 801 Capitola Dr., Durham, NC, USA. Tel.: +1 919 923 9248. E-mail addresses: [email protected], [email protected] (F.A. Kullmann). 0024-3205/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2014.01.071

peptide (GRP), respectively. No naturally occurring ligand has been identified for BB3 receptor. NMB and GRP along with their receptors are distributed throughout the brain, spinal cord and peripheral tissues and are involved in a variety of functions ranging from control of smooth muscle contractions, gastric acid secretions, glucoregulation, thermoregulation, satiety, pain and itch (review (Jensen et al., 2008)). In the lower urinary tract, BB receptors and/or bombesin related peptides are expressed in the bladder, urethra and pelvic ganglia of rat and/or guinea-pig (Dalsgaard et al., 1983; Ghatei et al., 1985; Keast and Chiam, 1994; Kilgore et al., 1993; Panula, 1986; Radziszewski et al., 1996, 2011; Watts and Cohen, 1991) and in intramural ganglia of the human male urinary bladder (Dixon et al., 1997). Functional studies have shown that activation of these receptors in the bladder (Falconieri Erspamer et al., 1988; Kullmann et al., 2008; Maggi et al., 1992; Rouissi et al., 1991; Watts and Cohen, 1991) and urethra (Radziszewski et al., 2011) produces smooth muscle contraction. It is not known whether the BB receptors are involved in micturition. Patients with diabetes experience bladder dysfunction characterized by increased capacity, poor emptying and overflow incontinence, characteristics of an underactive bladder. In fact, diabetic cystopathy has been recognized as one of the leading causes of underactive bladder condition (Daneshgari et al., 2009; Gomez et al., 2011; Miyazato et al., 2013). The STZ-treated diabetic rat model has been extensively used as a rodent model of the voiding dysfunction

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Table 1 Changes induced by STZ treatment: blood glucose, body and bladder weight, fluid intake and excreted, voiding frequency and average voided volume per void, in awake rats tested in metabolism cages. Parameter

Sham 9 weeks (n = 6)

STZ-treated 9 weeks (n = 6, unless noted otherwise)

Sham 20 weeks (n = 3)

STZ-treated 20 weeks (n = 5, unless noted otherwise)

Blood glucose (mg/dl) a Body weight (g)b Bladder weight (mg)a Total fluid intake (ml)b Total excreted volume (ml)b Voiding frequency (voids/h)b Voided vol/void (ml) b

127.6 ± 6.9

340.29 ± 15.11, (n = 14)

124.6 ± 8.9

306.40 ± 10.13, (n = 15)

322.3 ± 3.9

251.1 ± 17.4 ⁎

375.0 ± 13.3

267.5 ± 7.3

131.5 30.8 17.8 1.00 1.10

283.6 ± 29.8 ⁎ 237.8 ± 12.8 ⁎ 221.9 ± 9.2 ⁎ 2.22 ± 0.12 ⁎ 4.61 ± 0.2 ⁎

128.8 18.2 13.9 0.94 1.18

375.1 ± 13.3 229.3 ± 21.3 201.5 ± 35.4 2.27 ± 0.42 5.17 ± 0.78

± ± ± ± ±

3.4 5.0 2.8 0.06 0.17

± ± ± ± ±

8.8 1.1 3.9 0.43 0.42

n: number of rats. a Blood glucose measured on the day of the terminal experiment. b Parameters measured in awake metabolism cage experiments. ⁎ Indicate statistically significant values (p b 0.05) between sham and STZ-treated rats at 9 weeks, tested with non-parametric unpaired t-test with Welch's correction. For the 20 weeks treatment, although differences are large, no statistics was attempted because of the low number of rats in control group.

encountered in diabetic patients. In this model, there are timedependent morphological and functional changes in myogenic and neurogenic components of the bladder and urethra that affect voiding and storage phases and result in a large capacity bladder and increased residual volume (Daneshgari et al., 2006a, 2006b, 2009; Liu and Daneshgari, 2005, 2006; Malmgren et al., 1989). In the present study we used the STZ-treated diabetic rat as a model of voiding dysfunction to test the effectiveness of BB receptor agonists to improve voiding.

Materials and methods Animals Experiments were conducted in accordance to protocols established within Urogenix, an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited facility, and approved by the Institutional Animal Care and Use Committee which follows NIH Guidelines for the Care and Use of Laboratory Animals. Female Sprague Dawley rats (200–250 g; ~77 days when received) were obtained from Charles Rivers (Raleigh, NC).

Induction of diabetes Rats were injected with streptozotocin (STZ) (N-(Methylnitrosocarbamoyl)-α-D-glucosamine; Sigma, St. Louis, MO) at 65 mg/kg, intraperitoneal (i.p.) dissolved in 0.02 M sodium citrate buffer. Blood glucose (BG) levels were measured on day 7 post-treatment and on the day of the experiment, using a Precision Xtra system (Abbott Laboratories, Alameda, CA). Successful induction of diabetes was determined if the animal's BG levels were above 250 mg/dl. Approximately 90% of animals in the colony met this criterion. Animals were used at 9 weeks (BG: 340.29 ± 15.11 mg/dl; n = 14) and 20 weeks (BG: 306.40 ± 10.13 mg/dl; n = 15) after treatment with STZ. Sham rats injected with the vehicle for STZ were used as controls (n = 21 for the 9 week STZ-treated group and n = 3 for 20 week STZ-treated group). The 9 and 20 week post-treatment time points were chosen based on previous studies (Daneshgari et al., 2006a, 2006b; Liu and Daneshgari, 2005, 2006). These studies have shown that at 9 weeks post STZ treatment, most STZ-induced changes in bladder function and morphology reach a plateau, but the bladder is not decompensated. From 9 to 20 weeks, there are further time dependent changes in bladder function, with the bladder undergoing a transition from a compensated state to a decompensated state, atonic state (Daneshgari et al., 2006b).

Metabolism cages studies In order to verify that the STZ treatment was effective at reproducing changes in bladder function previously observed in other studies (Daneshgari et al., 2006b; Liu and Daneshgari, 2006; Malmgren et al., 1989), a subset of rats used in this study was tested in metabolism cages. Rats at 9 and 20 weeks after STZ or vehicle treatment were placed in metabolic cages for 4 h in day 1, to allow cage accommodation, and for 24 h in day 2, to acquire functional data. The light cycle was from 6:00 AM to 6:00 PM; food and water were provided ad libitum. Voided urine was collected in cups attached to force displacement transducers (Grass Technologies, Warwick, RI) or balances (A/D GF-1200, Fisher Scientific) connected to a computer. Data analysis included voiding frequency and volume per void for 24 h period. Voiding frequency was calculated as the number of voiding events per hour. Volume per void was calculated as an average of the voids occurring during 24 h. These data (Table 1) indicate that STZ-treatment, 9 and 20 weeks, impacted body weight, bladder weight, fluid intake and excreted, voiding frequency and voided volume, consistent with previously reported data on this model (Daneshgari et al., 2006b; Liu and Daneshgari, 2006; Malmgren et al., 1989). Drugs NMB, a BB1 receptor agonist, and GRP, a BB2 receptor agonist, were purchased from Tocris (Bristol, UK) and dissolved in saline. Doses were based on preliminary pilot studies and on existing literature from the bladder (e.g. bombesin 0.3 ng/kg i.v. (Broccardo et al., 1975)) or other systems (GRP 3 μg/kg/min i.v. (Gu and Lee, 2005)). Specificity of NMB for BB1 receptors and of GRP for BB2 receptors in the rat bladder tissue was preliminarily tested in a previous in vitro study (Kullmann et al., 2013a). Cystometry Cystometry was performed in urethane (1.2 g/kg, Sigma, subcutaneous injection) anesthetized rats. The carotid artery and jugular vein were cannulated to allow blood pressure measurements and intravenous (i.v.) drug delivery, respectively. The ureters were ligated and cut distally. The urinary bladder was catheterized through the dome using a flared catheter connected to a pump (Harvard Apparatus, MA) for saline infusion and to a pressure transducer (Argon Medical Devices, Athens, TX) for bladder pressure recording. Voiding responses were elicited by continuously infusing room temperature (~22 ºC) saline at a rate of 0.1–0.3 ml/min. The rate was adjusted for each animal

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(and maintained constant throughout the experiment) to produce a voiding event every ~ 3–5 min, allowing for acquiring sufficient data when using drugs with short duration of action (5–15 min depending on the dose). Control cystometry was performed for 1.5–2 h followed by vehicle (saline, 0.5 ml/kg) and increasing drug doses, given i.v. every ~ 30–40 min. In some experiments, electrodes (MT Giken Co, Tokyo, Japan) were placed into the external urethral sphincter (EUS) and electromyogram (EMG) activity was recorded using a Grass amplifier (P511AC; Astro-Med, West Warwick, RI). EUS-EMG activity was sampled at 3 kHz and filtered at 60 Hz.

Experimental design Three experimental protocols, P1–3, described below were used in this study. Within each protocol each rat was tested with a

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single compound (i.e. no combination of drugs was tested in a single rat). In order to determine whether BB receptor agonists have an effect on bladder function, the BB1 or BB2 receptor preferring agonists, NMB and GRP, respectively, were tested in sham rats by means of protocol P1. Tachyphylaxis was assessed by means of protocol P2. To evaluate whether BB1 and BB2 receptor agonists are effective in a diabetic model of voiding dysfunction, NMB and GRP were tested in STZtreated rats at 9 weeks post-treatment, by means of protocol P1. At this time point previous studies have shown that most STZ-induced changes in bladder function and morphology are stable, but the bladder is not decompensated (Daneshgari et al., 2006a, 2006b; Liu and Daneshgari, 2005, 2006). To further evaluate whether BB receptor agonists are effective at inducing voiding, NMB was tested by means of protocol P3, in STZ-treated rats at 20 weeks post-treatment. This time point was chosen based on previous studies that have shown

Fig. 1. Effects of NMB and GRP on bladder contraction in sham rats. Ai, Bi. Examples of bladder contractions induced by NMB (0.01–100 μg/kg, i.v.; n = 6 rats) (Ai) and GRP (0.01–100 μg/kg, i.v.; n = 8 rats) (Bi). Arrows indicate application of the respective compounds. Dotted gray lines indicate zero intravesical pressure. Aii–v, Bii–v. Summary of the effects of these compounds on parameters measured in cystometry: IMI (Aii, Bii), BC-AUC (Aiii, Biii), BCA (Aiv, Biv) and PVBP (Av, Bv). Data from each parameter were normalized to the vehicle (saline — abbreviated V; white bars), which was set to 100%. (*) indicates values significantly different values from the vehicle (p b 0.05 using one-way ANOVA followed by Bonferroni's Multiple Comparison post-test). Data are from rats at 9 weeks post vehicle treatment.

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Fig. 2. Repeated administration of NMB and GRP in sham rats. Ai, Bi. Examples of repeated applications of NMB (Ai; n = 3 rats) and GRP (Bi; n = 4 rats), 10 μg/kg. Arrows indicate application of the respective compounds. Aii–v, Bii–v. Summary of effects of NMB and GRP on IMI (Aii, Bii), BC-AUC (Aiii, Biii), BCA (Aiv, Biv) and PVBP (Av, Bv). Data from each parameter were normalized to the vehicle (saline, white bars), which was set to 100%. (*) indicates values significantly different values from the vehicle (p b 0.05), ns indicates no significant differences (p N 0.05), tested using one-way ANOVA followed by Bonferroni's Multiple Comparison post-test. Data are from rats at 9 weeks post vehicle treatment.

time dependent changes in bladder function from 9 to 20 weeks, with bladder undergoing a transition from a compensated state to a decompensated state, atonic state (Daneshgari et al., 2006b). The protocols are as following: P1) Dose responses to NMB and GRP using continuous cystometry in sham and STZ-treated rats (Figs. 1, 3; number of rats is included in each figure legend). Control cystometry was performed for a period of 1–1.5 h followed by a vehicle (saline) and increasing doses of drugs given intravenously every ~30–40 min. P2) Investigation of tachyphylaxis using multiple injections (2–5) of a single dose of 10 μg/kg NMB and 10 μg/kg GRP, given every 30– 40 min, in continuous cystometry (Fig. 2). P3) Drug-induced voiding at sub-threshold voiding volume which was chosen as 50% of functional bladder capacity in SZT-treated rats 20 weeks post-treatment (n = 15 rats, out of which 12 completed the experiment) (Fig. 4). To determine

functional bladder capacity, three cystometrograms (CMGs) were performed and the volume necessary to fill the bladder from an empty state to the threshold for triggering voiding was measured. These three volumes were averaged and taken as the functional bladder capacity. This volume was 1.06 ± 0.12 ml, (n = 12 rats 20 weeks STZtreated), which was much higher than that observed in sham rats (0.28 ± 0.04 ml, n = 3 rats 20 weeks STZ-treated). The bladder was then emptied and infused to ~ 50% capacity, the infusion pump was turned off, and the rat dosed with the vehicle. After 1 min, in which the vehicle's effects were monitored, the bladder was emptied and residual volume measured. The infusion pump was restarted and distension induced contractions were observed for 20–30 min to allow the bladder to return to regular voiding. This cycle was repeated 2–3 times per rat with different doses of NMB (0.1, 1, 3, 10 μg/kg) and

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Fig. 3. Effects of NMB and GRP on bladder contraction in STZ-treated rats. Ai–iv, Bi–iv. Summary of the effects of NMB (n = 6 rats) and GRP (n = 8 rats) on parameters measured in cystometry: IMI (Ai and Bi), BC-AUC (Aii and Bii), BCA (Aiii and Biii), and PVBP (Aiv and Biv). Data from each parameter were normalized to vehicle (saline — abbreviated V, white bars), which was set to 100%. (*) indicates values significantly different from the vehicle (p b 0.05 using one-way ANOVA followed by Bonferroni's Multiple Comparison post-test). Rats were at 9 week post STZ treatment.

data from all rats tested with a specific dose pooled. Data from 3 rats were excluded because they did not complete the experiment. Data analysis Parameters measured during continuous cystometry (protocols P1, P2) included: intermicturition interval (IMI), measured between the peaks of two consecutive voiding contractions; post voiding bladder pressure (PVBP), measured immediately after voiding; bladder contraction amplitude (BCA), the difference between bladder pressure at the contraction peak minus PVBP; bladder contraction area under the curve (BC-AUC), defined as the product of contraction duration, measured from the micturition threshold to the point where the pressure returned to baseline after voiding, and contraction amplitude; mean arterial pressure (MAP) and heart rate (HR). Each parameter was analyzed in a 10 min window following dosing. Data are reported as percentage changes relative to the vehicle, which was set to 100%. Parameters monitored for experiments in which voiding was triggered at 50% functional bladder capacity (protocol P3) included: BC-AUC; EUS-EMG activity defined as root-mean-square (RMS) activity which included both tonic and phasic EUS activity; duration of high frequency

oscillations (HFOs) of the EUS, defined as the period of time in which HFOs were present during a void; voided volume (VV), measured using a balance (A&D, Ann Arbor, MI); residual volume (RV), amount of fluid retained in the bladder following a void, and voiding efficiency (VE), calculated as ((VV / (VV + RV)) ∗ 100). All these parameters were measured in a 1 minute window following vehicle/drug dosing. A drug-induced bladder contraction was defined as a ≥3 mm Hg increase in intravesical pressure occurring within 1 min after dosing. Parameters measured during drug-induced voiding are compared to parameters obtained during a voiding induced at full bladder capacity prior to drug application. Data were recorded using LabChart software (version 7; ADInstruments, Australia) and analyzed using Excel (Microsoft, Redmond, WA) and Prism 5 (GraphPad Software Inc., San Diego, CA). Statistics Results are expressed as mean ± SEM and analyzed using nonparametric one-way ANOVA followed by Bonferroni's Multiple Comparison post-test (significance set at p b 0.05), or non-parametric unpaired t-test with Welch's correction (significance set at p b 0.05), using Prism 5.

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Fig. 4. Drug-induced voiding at sub-threshold bladder capacity in STZ rats. A. The protocol for this model involves stopping the infusion pump (^) and emptying the bladder (#). Infusion pump is then started and the bladder filled to 50% capacity (@), pump stopped (^) and compound (saline/NMB) dosed (arrow). Animals were observed for 1 min (&) before emptying the bladder (#) and measuring the residual volume (RV). B shows an example of the drug-induced voiding, using the protocol described in A (for clarity of the figure the signs ^, #, and @ depicted in A are not depicted in B). Inserts C, D show the coordinated bladder contraction and EUS-EMG activity from a normal voiding when bladder was filled to 100% functional capacity and following the NMB (10 μg/kg)-induced voiding when bladder was filled to 50% functional capacity. E–I. Summary of parameters measures during drug-induced voiding at subthreshold bladder capacity: BC-AUC (E), EUS-EMG RMS activity (F), Voided volume (G), Residual volume (H), Voiding efficiency (I). In all figures, gray bars represent parameters measured during the control period when the bladder was completely filled to 100% full, white bars and black bars represents parameters measured after saline or NMB dosing, respectively, when the bladder was filled to 50% functional capacity. Number of rats tested with each drug dose is given in Table 2 column 2. (*) indicates values significantly different from saline (p b 0.05 using one-way ANOVA followed by Bonferroni's Multiple Comparison post-test). For RV, the p value from ANOVA was 0.0426, but posthoc tests did not show significant differences between selective doses and saline. Data are from rats at 20 weeks post STZ treatment.

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Results Cystometry in sham rats Sham rats (9 weeks post-treatment) under urethane anesthesia displayed regular voiding patterns with bladder contraction amplitude (BCA) of 19.16 ± 1.04 mm Hg, bladder contraction area under the curve (BC-AUC) of 131.32 ± 14.51 mm Hg∗s, post voiding bladder pressure (PVBP) 5.24 ± 0.50 mm Hg and an intermicturition interval (IMI) of ~ 3–5 min (Fig. 1, n = 14). NMB (0.01–100 μg/kg, i.v.; Fig. 1A; n = 6), a BB1 receptor preferring agonist, significantly decreased IMI, as much as 50% of control (Fig. 1Aii), and increased BC-AUC, BCA and PVBP (Fig. 1Aiii–v). Although ANOVA across doses was significant for all parameters (IMI: p = 0.0015; BC-AUC: p = 0.0013; BCA: p = 0.035; PVBC: p = 0.0005), posthoc tests for individual doses versus vehicle were significant only for some parameters (Fig. 1Aii–iv). GRP (0.01–100 μg/kg, i.v.; Fig. 1B; n = 8), a BB2 receptor preferring agonist, decreased IMI, as much as 50% of control, and increased BC-AUC, BCA and PVBP (Fig. 1Bii–v). ANOVA across doses was significant for all parameters (IMI: p = 0.0051; BC-AUC: p = 0.0004; BCA: p = 0.0131; PVBC: p b 0.0001) but posthoc tests for individual doses versus vehicle were not. In summary, in sham rats at 9 weeks post-treatment, NMB and GRP had excitatory effects on bladder function, increasing bladder contractility and voiding frequency. Regarding the cardiovascular system, NMB increased mean MAP and HR significantly at 10 μg/kg and 100 μg/kg (Fig. 5). GRP increased MAP and HR reaching significance at the highest dose, 100 μg/kg for MAP, but not for HR (Fig. 5). Repeated administration of 10 μg/kg NMB (n = 3 sham rats at 9 weeks post-treatment) and GRP (n = 4 sham rats at 9 weeks post-treatment) every ~ 30 min, produced consistent effects on all parameters in sham rats (Fig. 2; shows two applications of NMB and GRP) and STZ-treated rats (data not shown). Additional applications up to 5 times yielded similar results (data not shown). These data suggest no tachyphylaxis. Cystometry in STZ rats To determine whether the excitatory effects seen on bladder activity in sham rats are also present in a model of voiding dysfunction, we used STZ-treated diabetic rats at 9 weeks post-treatment, a time point when STZ-induced changes in bladder morphology and physiology become stable (Liu and Daneshgari, 2006). Under urethane anesthesia these rats displayed regular voiding patterns with BCA of 16.79 ± 0.58 mm Hg, BC-AUC of 134.23 ± 13.51 mm Hg∗s, PVBP of 7.65 ± 0.98 mm Hg and IMI of ~ 4–6 min (n = 14 rats at 9 weeks posttreatment). NMB (0.1–300 μg/kg, i.v.; Fig. 3A; n = 6) and GRP (0.1–300 μg/kg, i.v.; Fig. 3B; n = 8) produced dose dependent responses similar to those observed in sham rats: reduced IMI and increased BC-AUC, BCA and PVBP (Fig. 3Ai–Aiv, Bi–Biv; for NMB ANOVA p values are IMI: p = 0.0007; BC-AUC: p = 0.0004; BCA: p = 0.086; PVBC: p = 0.0001; for GRP ANOVA p values are IMI: p = 0.0027; BC-AUC: p = 0.0002; BCA: p = 0.0045; PVBC: p = 0.0005). In summary, in STZ rats at 9 weeks post-treatment, NMB and GRP had excitatory effects on bladder function, increasing bladder contractility and voiding frequency. On the cardiovascular system, NMB had no significant effect on MAP and HR. GRP had no effect on MAP and increased HR significantly at the highest doses 100 and 300 μg/kg (Fig. 5). Drug-induced voiding at sub-threshold voiding volumes BB receptor agonist-induced enhancement of bladder activity during continuous infusion cystometry suggests that BB receptor agonists might be used to pharmacologically trigger urine release. To test this hypothesis, we evaluated the ability of NMB to induce voiding in STZ

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diabetic rats when their bladder was filled to sub-threshold voiding volume, set to approximately 50% of the functional capacity, defined as the volume necessary to induce voiding (Fig. 4). The STZ-treated rats were at 20 weeks post-treatment because previous studies have shown that the bladder becomes decompensated, atonic state (Daneshgari et al., 2006b), thus appropriate to test drug-induced voiding. These rats had large functional bladder capacity (Fig. 4G gray bars; 1.06 ± 0.12 ml; n = 12 rats 20 weeks post STZ, compared to ~ 0.2–0.4 ml in sham rats at 20 weeks post-treatment — n = 3 rats, data not shown), large post residual volumes (Fig. 4H gray bars; 0.70 ± 0.27 ml; n = 12 rats 20 weeks post STZ compared to ~0.1–0.2 in sham rats at 20 weeks post-treatment — n = 3 rats, data not shown) and ~50% voiding efficiency (Fig. 4I gray bars). When the bladder was filled to 50% of functional capacity, administration of saline (0.5 ml i.v.) did not produce urine release, bladder contraction, or changes in the EUS-EMG activity (Fig. 4A, E–I white bars; Table 2). On the other hand, NMB (0.1, 1, 3, 10 μg/kg) dose dependently produced urine release (Table 2; Fig. 4B, G) and bladder contraction (Fig. 4B, E) associated with phasic high frequency oscillation (HFO) activity of EUS-EMG (Fig. 4D, F) in 0%, 28%, 86% and 100% of rats, respectively (Table 2). NMB increased the duration of HFO and the RMS activity, which included both tonic and phasic EUSEMG activity (Table 2, Fig. 4F). Pharmacologically-induced voided volumes when the bladder was filled to ~50% capacity were similar or larger than physiologically-induced voided volumes when the bladder was filled at full capacity, which is reflected in the decreased residual volumes and improvement of voiding efficiency (Fig. 4G–I). Discussion This study demonstrates that the BB receptor agonists, NMB and GRP, enhanced and/or triggered voiding in sham as well as in STZtreated diabetic rats. The effects of NMB and GRP were similar, raising the possibility that more than one subtype of BB receptors (e.g. BB1 receptor, BB2 receptor) may be present in the micturition pathways. No major differences were found between STZ-treated and sham (aged matched) rats, suggesting that diabetes may not alter the effects of BB receptors on bladder contraction. As the peptide agonists used in this study do not readily cross the blood brain barrier, several peripheral sites of action of BB receptor agonists, including bladder smooth muscle, efferent nerves and/or afferent nerves and urethral smooth muscle (Radziszewski et al., 2011) may account for their excitatory effects. The most likely site of action is the bladder smooth muscle and this is supported by agonist-induced increases in BC-AUC and PVBP (Figs. 1–3), increases in baseline intravesical pressure immediately after drug delivery (Figs. 1–3), and previous in vitro studies that demonstrated direct contraction of bladder smooth muscle in response to bombesin, NMB and GRP (Watts and Cohen, 1991) (Kullmann et al., 2013a). Increases in BCAUC could also be explained by increased neurotransmitter release from the efferent parasympathetic nerves projecting to the bladder. Although the expression of BB receptors in the efferent terminals of parasympathetic nerves has not been shown, indirect evidence, such as staining for bombesin related peptides in rodent pelvic ganglia neurons and in human intramural ganglia (Dixon et al., 1997; Keast and Chiam, 1994), together with our preliminary in vitro tissue contractility data (Kullmann et al., 2013a) support this possibility. In other systems, activation of BB receptors increased cholinergic and/or other components of neuronally-induced smooth muscle responses in vitro. For example, GRP (10− 9 M) enhanced nerve-induced cholinergic contractions in human appendix tissue (Ekblad et al., 1989) and bombesin (10−8 M) produced calcium-dependent ACh release in the guinea pig antrum (Kantoh et al., 1985). It would be of high interest to determine whether BB receptor agonists increase transmitter release in the bladder as well as to investigate additional sites of action (e.g. the afferent pathways, urethra) and mechanisms of action. The effects of BB receptor agonists, to increase bladder contraction and voiding frequency in rat cystometry experiments, are qualitatively

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Table 2 NMB-induced voiding when bladder was filled to 50% functional capacity in STZ rats. Data included were taken from the 1 minute time period following saline/drug treatment. n is the total number of rats tested with a specific dose. Results in columns 3, 4 show the number of animals and in parenthesis the percentage of animals that demonstrated occurrence of voiding and increase in bladder pressure, respectively. The last column shows the duration of high frequency oscillations (HFOs) during the normal physiological voiding (100% full bladder) and during drug-induced voiding. HFOs were observed only when voiding occurred. (*) indicates values significantly different from values measured during normal physiological void (p b 0.05 using one-way ANOVA followed by Bonferroni's Multiple Comparison post-test). Data are from rats at 20 weeks post STZ treatment. Treatment

n = (rats)

Voiding

Bladder pressure increase

Duration of HFO (s)

100% full bladder Saline 0.1 μg/kg 1 μg/kg 3 μg/kg 10 μg/kg

12 12 7 7 7 7

12 (100%) 0 (0%) 0 (0%) 2 (28%) 6 (86%) 7 (100%)

12 (100%) 0 (0%) 1 (14%) 6 (86%) 7 (100%) 7 (100%)

4.6 ± 0.5, n = 0.0 ± 0.0, n = 0.0 ± 0.0, n = 4.4 ± 0.3, n = 9.4 ± 4.1, n = 15.0 ± 1.8 ⁎, n

12 0 0 2 6 =7

Fig. 5. Effects of BB receptor agonists on cardiovascular parameters. A–D. Raw average values of mean arterial blood pressure (MAP; mm Hg) in Sham (A; n = 6 rats, C; n = 8 rats) and STZ rats (B; n = 6 rats, D; n = 8 rats) in response to NMB (A,B) and GRP (C,D). E–F. Raw average values of heart rate (HR; beats per min) in the same Sham (E,G) and STZ rats (F, H) in response to NMB (E,F) and GRP (G,H). Data are collected during the control period and after each dose of NMB and GRP in continuous cystometry. (*) indicates values significantly different from vehicle (p b 0.05 using one-way ANOVA followed by Bonferroni's Multiple Comparison post-test). Data are from rats at 9 weeks post-treatment with vehicle or STZ.

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similar to those of the muscarinic agonist, bethanechol or the acetylcholine esterase inhibitor, distigmine (Kullmann et al., 2013b; Nagabukuro et al., 2004). However, NMB increased BCA by ~20% and decreased IMI by ~50% at 10 μg/kg (Fig. 1), a dose that increased mean arterial pressure ~5% (Fig. 5). In contrast, bethanechol increased BCA by ~30% and decreased IMI by ~50% at 1 mg/kg, a dose that decreased mean arterial pressure by ~70% (Kullmann et al., 2013b). Thus, it is expected that BB receptor agonists would improve voiding without greatly affecting the cardiovascular system. To evaluate agonist effects on voiding efficiency, a specific experimental protocol (Fig. 4) was developed, in which the bladder was filled at sub-threshold voiding volume (~ 50% capacity), and drug-induced urine release was tested using a diabetic rat model of voiding dysfunction. The diabetic STZ-treated rat model at 20 weeks post-treatment was chosen because it is a well characterized model (Daneshgari et al., 2006a, 2006b; Liu and Daneshgari, 2005, 2006; Malmgren et al., 1989) that shares symptoms similar to diabetic patients, including impaired bladder sensation, large bladder capacity, large post residual volumes (Gomez et al., 2011), also confirmed by our results (Fig. 4 gray bars). In these experiments a single dose of NMB (3 or 10 μg/kg) triggered very efficient urine release when the bladder was filled to subthreshold micturition volumes (Fig. 4, Table 2). Taking into account that in diabetic overdistended bladders, the sub-threshold volume is likely equal or maybe higher than the threshold voiding volume in a normal bladder, triggering voiding before further overdistension occurs may be beneficial and may prevent overflow incontinence. Drug-induced voiding contractions were associated with phasic, pulsatile activity of the external urethral sphincter (EUS), also known as high frequency oscillations (HFOs), which facilitate voiding in rats (Streng et al., 2004). NMB dose-dependently increased the duration of the HFOs as well as both phasic and tonic activity of the EUS (Table 2, Fig. 4), suggesting that NMB is acting directly or indirectly on urethral afferents to amplify the micturition reflex. Although the relevance of the effects of BB receptor agonists on sphincter HFOs should be interpreted with caution when extrapolating to humans, where voiding reflex is organized somewhat differently than in rats (i.e. HFOs are absent in human), an action on urethral afferents if proven, may be beneficial in enhancing reflex voiding. The duration of action of BB receptor agonists was short lasting (~5–15 min dose dependent) and there was no evidence of tachyphylaxis (Fig. 2). This would suggest that under normal physiological conditions when voiding occurs approximately every 1 h in the rat and every 5 h in the human, BB receptor agonist administration may be sufficient to increase voiding efficiency or trigger voiding while not interfering with the filling phase to produce overactive bladder for example. However, long term administration should be investigated to determine whether overactive bladder develops. In humans, exogenous intravenous bombesin, NMB or GPR, have been shown to affect a variety of functions including reduction of food intake, stimulation of gastric acid secretion, pancreatic secretion, gall bladder contractions, alteration of gastric emptying and intestinal motor activity (review (Jensen et al., 2008)). These clinical trials suggest that bombesin agonists are safe for human administration and moreover the receptors may not undergo tachyphylaxis. For diabetic patients, with large bladders and inability to void efficiently, BB receptor-induced increases in voiding frequency associated with increases in bladder contraction may be beneficial for better emptying of the bladder. Conclusion Systemic administration of bombesin agonists has excitatory effects in the bladders of normal and STZ-treated diabetic rats. It also triggers voiding when the bladder is filled to a sub-threshold voiding volume in STZ-treated diabetic rats. These results suggest that BB receptors may be further investigated for their modulatory role in bladder

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function, especially for the underactive bladder condition, a highly unmet medical need. Conflict of interest statement The authors declare that there is no conflict of interest.

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