Effects of the β3-Adrenergic Receptor Agonist Disodium 5-[(2R)-2-[[(2R)-2-(3-Chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL-316243) on Bladder Micturition Reflex in Spontaneously Hypertensive Rats

The overactive bladder (OAB) is defined by the symptom of urgency (Abrams et al., 2002), which is the complaint of a sudden compelling desire to pass urine. The evaluation of urgency as a sensation is not easy in humans; however, several scales are available to measure the intensity of urgency. In animals, “urgency” cannot be used as a measurement, and only a few parameters of physiological endpoints, which are unique to humans as a consequence of urgency, can be measured by urodynamic studies. For example, spontaneously hypertensive rats (SHRs) exhibit such an animal model that mimics the pathophysiology of OAB patients with increased voiding frequency and lower bladder capacity (Persson et al., 1998; Spitsbergen et al., 1998). The mechanism behind the detrusor overactivity (DO) in SHRs and how the model simulates urgency in humans is not known. The alternations of both the afferent pathway and the efferent pathway in SHRs compared with their normotensive controls [Wistar-Kyoto (WKY)] have been proposed. The evidence for an enhanced afferent limb of the micturition reflex pathway in SHRs was based on an increased production of nerve growth factor by bladder smooth muscle (Spitsbergen et al., 1998; Clemow et al., 1999). The consequence of an increased nerve growth factor level includes enlargement of bladder sensory neuron size (Clemow et al., 1997) and sensitization of the bladder afferent nerve activity (Dmitrieva and McMahon, 1996). However, direct evidence of enhanced afferent activity in SHRs is still lacking.

The function of adrenoceptors (ARs) in the efferent limb (efferent outflow/bladder function) may also underlie the DO in SHRs. Blockade of spinal α1-AR removed the abnormal bladder contractions, and activation of peripherally postjunctional α1-AR produced a stronger contraction of smooth muscle in SHRs (Persson et al., 1998). Differential activation of inhibitory prejunctional α2-AR produced a stronger relaxation of smooth muscle in WKY rats (Persson et al., 1998). In addition, β-AR dysfunction has been proposed to contribute to the DO in SHRs (Frazier et al., 2006).

It is well accepted that β-AR activation results in relaxation of bladder smooth muscle during normal urine storage phase (Andersson and Wein, 2004). In the urinary bladder of SHRs, there is actually increased sympathetic innervation (Tong et al., 1996; Spitsbergen et al., 1998). Following this evidence, greater adrenergic innervation might cause more relaxation of the bladder smooth muscle or greater sensitivity to β-AR activation. Thus, the pharmacology of β-AR activation of bladder detrusor in SHRs is interesting to study.

Several subtypes of β-AR have been identified, namely, β1-, β2-, and β3-ARs (Bylund et al., 1994). In in vitro studies, bladder relaxation evoked by β-AR agonists is mediated mainly via β2- and β3-ARs in rats (Yamazaki et al., 1998). There is compelling in vivo evidence to support a role for β3-AR in increasing bladder capacity in anesthetized rats as well as in an animal model of pathological bladder instability and hyperreflexia (Woods et al., 2001; Kaidoh et al., 2002). There have been no reports to date profiling the in vivo efficacy of a β3-AR agonist in SHRs. Thus, the objectives of the present study were to compare bladder afferent and smooth muscle functions between SHRs and WKY rats and to evaluate β3-AR activation by CL-316243 on bladder function in SHRs. To study afferent pathway changes in SHRs, we used the visceromotor reflex (VMR) and cardiovascular (pressor) responses to noxious urinary bladder distension (UBD), a model that has been previously demonstrated as a reliable measure of bladder afferent signal transmission (Su et al., 2008a,b). Bladder smooth muscle function along with its modulations was tested in the isovolumetric bladder rhythmic contraction (RC) model in vivo.

Materials and Methods

Eighty-nine female SHRs (weighing 150–250 g) and 56 female WKY rats (weighing 150–250 g) were used in this study. Core body temperature of the rats was maintained at 36°C through use of a circulating hot water pad placed under the rat with a feedback-controlled system. Rats were euthanized upon completion of experimental procedures by an intravenous overdose of sodium pentobarbital (120 mg/kg; Vortech Pharmaceuticals, Dearborn, MI). The experimental protocol was approved by the Institutional Animal Care and Use Committee of GlaxoSmithKline Pharmaceuticals (King of Prussia, PA).

Gene Expression by TaqMan Study. Rats were anesthetized initially with 3% isoflurane, and then they were euthanized by exsanguination. The dorsal root ganglions (DRGs) at levels L6/S1 and T13/L1 and bladders were removed immediately, and they were kept at –80°C. Tissues were homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA), and, after phase separation with chloroform, total RNA was extracted using the RNeasy Mini kit (QIAGEN, Valencia, CA) following the manufacturer's instructions. Any genomic DNA contamination was removed using DNase I (Ambion, Austin, TX). RNA samples were judged to be free of genomic DNA by no amplification in a standard TaqMan assay using 10 ng of RNA and ACTB primer/probe oligonucleotides. The RNA was quantified using Ribogreen RNA quantitation reagent (Invitrogen) and converted to cDNA by reverse transcription using the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). The equivalent of 10 ng of mRNA per well was arrayed into 384-well plates using a Biomek FX robot (Beckman Coulter, Fullerton, CA), and quantitative reverse transcription-PCR was carried out using a 7900HT Sequence Detector System (Applied Biosystems) in a 5-μl reaction volume. TaqMan Universal PCR Master Mix 2X (Applied Biosystems) and universal PCR conditions recommended by the manufacturer were followed. All primers and probes (5-carboxyfluorescein and 5-carboxytetramethyl rhodamine) are listed in Table 1. To normalize the data, samples were scaled relative to each other by the geometric mean of the set of valid housekeeper data points for that sample. Each data point was then expressed as the ratio of the housekeeper abundance in the sample to the average of that housekeeper in all samples, and it was marked invalid if it had statistically inconsistent behavior with the other housekeepers in those samples with similar tissue types. The housekeeping genes used were β-actin (ACTB), GAPDH, and cyclophilin A (PPIA), and the data were floored at a relative abundance of 30.

In Vivo VMR and Pressor Responses to Urinary Bladder Distension. Rats were anesthetized initially with 3% isoflurane. For blood pressure measurement, the right carotid artery was catheterized with polyethylene 50 tubing. The arterial catheter was linked with a low-volume transducer and signal was amplified through a DC amplifier (Neurolog, NL108; Digitimer Ltd., Hertfordshire, UK). One jugular vein was cannulated with polyethylene tubing for intravenous administration of drug or vehicle (saline). Polyethylene 90 tubing was inserted into the urinary bladder via the urethra and secured by a tight ligature around the distal urethral orifice. The bladder catheter was also linked to a bladder distension control device (B482C-1; Department of Biomedical Engineering, University of Iowa, Iowa City, IA) (Su et al., 2008b). The bladder was distended with saline by regulating air inflow into a Marriott bottle from a valve interface distension control device. Two needle electrodes were sutured into the oblique abdominal musculature just above the inguinal ligament. Abdominal contractions were quantified by action potentials of electromyographic activity. Action potentials were initially amplified through a low-noise AC differential amplifier (Neurolog NL104; Digitimer Ltd.), processed with a filter system (NL125; Digitimer Ltd.), and collected using the CED power 1401 data acquisition program (CED, Cambridge, UK). Raw action potentials of myoelectric activities, bladder pressure, and blood pressure were displayed online continuously. All data were analyzed off-line using the spike2 program (CED). Following completion of the surgical preparation, isoflurane anesthesia was reduced until flexion reflex response could be evoked by pinch of the foot without spontaneous escape behaviors (approximately 1% isoflurane).

For UBD, all rats received phasic bladder distensions for 30 s at 3-min intervals. Stimulus-response functions (SRFs) were generated by applying UBDs at 5, 10, 20, 30, 40, and 60 mm Hg. Following SRFs, a series of at least six phasic UBDs at 60 mm Hg were applied to evaluate response stability to repeated UBD. Drug or saline was administered only after four consistent responses were elicited.

The electromyographic activity was integrated and calculated as the area under the curve. The VMR response to the stimulus was defined as the increase in electromyographic activity during UBD from the baseline activity before each response. Pressor response was quantified as the peak change in mean arterial pressure during UBD compared with the average level during a baseline period immediately before UBD. SRFs to graded UBD were plotted for each experiment, and a least-squares regression line was obtained from the linear part of the SRF. The regression line was then extrapolated to the ordinate (representing distension pressure) to estimate the response threshold (Su et al., 2008b). After drug administration, response was modified to the percentile of mean control response: the average of four UBD responses before drug treatment.

In Vivo Bladder Rhythmic Contraction. All surgical procedures were performed under 3% isoflurane anesthesia. In each animal, one jugular vein was cannulated with polyethylene-50 tubing for intravenous administration of urethane anesthesia, drug, or saline (vehicle), and a tracheostomy tube was inserted to facilitate respiration. A cannula (polyethylene-50) was placed into the bladder via the urethra, and the urethra was ligated to ensure an isovolumetric bladder. Upon completion of surgical procedures, slow intravenous infusion of urethane (1.2 g/kg ethyl carbonate; Sigma-Aldrich, St. Louis, MO) was given over 15 min. Isoflurane anesthesia was terminated. Saline bladder infusion procedures were begun 30 min after final dose of urethane.

The urethral cannula was connected with a T connector and linked with a low-volume pressure transducer (MLT0380D; ADInstruments, Colorado Springs, CO), and the signal was amplified through a DC amplifier (ML119; ADInstruments). The other end of the T connector was linked to a 20-ml syringe with a perfusion pump. To evaluate the bladder compliance, the bladder was filled with saline at a rate of 250 μl/min. The infusion was terminated once the bladder pressure reached approximately 30 mm Hg. Infusion volume and intravesical pressure response functions were generated, and the response threshold was the volume that induced a rapid rising of intravesical pressure.

For the pharmacological study of β3-AR agonist on bladder RC, the saline infusion into bladder was slowed to a rate of 50 μl/min to induce the micturition reflex (here defined as bladder contraction with amplitude >10 mm Hg). The infusion rate was then lowered to 10 μl per minute until three to five rhythmic bladder contractions were established, and infusion was terminated. The vehicle or drug was administered intravenously after a 15-min control period. Following drug administration, the bladder RC was recorded for 20 min. Two parameters of bladder RC were evaluated: frequency and amplitude of the contraction. The mean controls were calculated by the averages of readouts during the last 5-min interval of the control period. The inhibition of CL-316243 in saline was calculated by the mean response beginning 10 min after injection, where the maximal effects were observed.

Pharmacokinetic Studies. As in in vivo bladder contraction studies, rats were studied under urethane anesthesia (1.2 g/kg) and dosed with CL-316243 (1 mg/kg i.v.). Arterial blood (0.4 ml) was drawn every 5 min after dose into lithium heparin-containing tubes. Plasma was separated by centrifugation, and it was stored at –20°C before analysis. Analysis of plasma samples was performed using liquid chromatography/tandem mass spectrometric detection. Rat plasma samples were thawed; plasma proteins were precipitated with 200 μl of 95:5 acetonitrile/10 mM aqueous ammonium formate, pH 3.0, containing an appropriate mass spectral internal standard; and the resulting mixture was vortex-mixed for 2 min followed by centrifugation for 30 min at >2000g. Using a sensitive and selective liquid chromatography/tandem mass spectrometric method on an HTS PAL autosampler (CTC Analytics, Zwingen, Switzerland) coupled to a Sciex API5000 triple-quadruple mass spectrometer (Applied Biosystems), samples were analyzed for quantitative concentrations of CL-316243. Analytical standards for CL-316243 (2–5000 ng/ml) were prepared in both SHR and WKY rat plasma to ensure accurate calibration of the mass spectrometer for each biological matrix.

Data Analysis. All data are expressed as mean ± S.E.M. Results were analyzed with Student's t test or analysis of variance (ANOVA) with repeated measures using Bonferroni post-test by Prism 4 (GraphPad Software Inc., San Diego, CA). A value of p < 0.05 was considered statistically significant.

Drugs. CL-316243 (mol. wt. 465.79) was purchased from Sigma-Aldrich, and it was dissolved in saline.

Results

Gene Expression by Quantitative PCR

The three housekeeping genes, β-actin (ACTB), GAPDH, and, PPIA, were expressed in a stable manner across all samples, allowing for proper normalization. The localization of AR mRNA in rat bladder and DRGs normalized to the housekeeping genes is shown in Fig. 1. The expression of all ARs was greatly enriched in detrusor muscle compared with DRGs. β1 and β2 were only detectable at a low level in DRGs, whereas the amount of β3 mRNA fell below the limit of detection.

Similar expression profiles were obtained in both SHRs and WKY rats (Fig. 1). Both β1 and β2 were more abundant in detrusor muscle in SHRs (p < 0.05). However, β3 expression levels in detrusor were no different in both SHRs and WKY rats (p > 0.05).

In Vivo Visceromoter Reflex and Pressor Responses to Urinary Bladder Distension

Stimulus-Response Functions. VMR and pressor responses to graded UBD exhibited a similar SRF as described previously in SD rats (Su et al., 2008a,b); both VMR and pressor responses increased to graded, phasic UBD (p < 0.001). Figure 2, A and B, illustrates the SRFs of VMR and pressor responses to graded UBD in SHRs and WKY rats. The VMR was not different between SHRs and WKY rats; however, the pressor response was significantly higher than that in WKY rats (p < 0.05) (Fig. 2; Table 2). Reflex responses were reproducible, with repeated noxious UBDs as illustrated in Fig. 2, C and D.

Effects of CL-316243. Intravenous administration of saline or CL-316243 (3 mg/kg i.v.) did not produce significant changes in VMR or pressor responses to UBD in either SHRs or WKY rats (Fig. 3).

CL-316243 at 3 mg/kg significantly reduced the mean blood pressure in SHRs (p < 0.05, unpaired t test; n = 8) after 10 min, and it was sustainable through the length of the assay. No effect was seen in WKY rats (Fig. 4A). CL-316243 (3 mg/kg i.v.) did not alter the heart rate in either strain (Fig. 4B).

In Vivo Bladder Rhythmic Contraction

Bladder compliance was first evaluated in both SHRs and WKY rats by fast intravesical saline infusion (250 μl/min). Figure 5 is the summary of volume-intravesical pressure response in two strains. As the volume of saline infusion into bladder increases to the threshold level, the intravesical pressure rises. The threshold volume in SHRs was significantly lower than that in WKY rats (0.51 ± 0.09 and 1.06 ± 0.13 ml, respectively; p < 0.001). Intravesical pressure increased as a function of infusion volume in SHRs significantly greater than in WKY rats (p < 0.001, two-way ANOVA). Thus, pressures produced by same volumes of bladder distension in SHRs were higher than those in WKY rats.

To evaluate the pharmacology of CL-316243 in bladder functions in both SHRs and WKY rats, isovolumetric bladder RC was evoked by slow intravesical saline infusion (50 and 10 μl/min as described under Materials and Methods). Figure 6 shows an example of a raw trace recording in an SHR before and after i.v. injection of CL-316243 (0.1 mg/kg). The data are summarized in Fig. 7. The administration of CL-316243 dose-dependently inhibited or decreased the frequency of the RCs in SHRs, and it elicited relatively weak inhibition on the amplitude of bladder contractions at 0.1 and 1 mg/kg. An unpaired t test showed a stronger inhibition of CL-316243 at 1 mg/kg on the frequency than that on the amplitude of contraction (p < 0.001). CL-316243 did not have a significant effect in WKY rats.

Pharmacokinetic Studies

The plasma levels of CL-316243 following a single 1-mg/kg i.v. dose in SHRs and WKY rats are shown in Fig. 8. C_max values of CL-316243 were determined 5 min after dosing (3.94 ± 0.77 μg/ml in SHRs and 3.21 ± 1.35 μg/ml in WKY rats). Following administration of CL-316243, the corresponding exposures (area under the curve_0-t) in each strain were similar (44.6 ± 8.0 μg min/ml in SHRs and 38.5 ± 13.4 μg min/ml in WKY rats).

Discussion

The present study evaluated the role of β3-AR on bladder afferent and smooth muscle functions in SHRs and WKY rats. The β3-AR agonist CL-316243 inhibited bladder RCs in SHRs, suppressing both frequency and, to a lesser extent, amplitude of contractions. However, CL-316243 failed to attenuate RC in WKYs. Because CL-316243 failed to attenuate VMR and pressor responses to UBD, the mechanism of inhibition on bladder functions by CL-316243 was directly on the detrusor; this is also supported by the abundant β3-mRNA expression in detrusor. Reduced compliance was detected in the SHR detrusor accompanied by a reduced RC threshold compared with WKY rats. In contrast, using the VMR responses to noxious UBD as a measure of bladder afferent signal transmission, sensitized bladder afferent activity in SHRs was not detected. Thus, alternation of efferent/detrusor function seems to be important in the mechanism of the DO phenotype in SHRs, whereas afferent hypersensitivity seems to have less of a role. Moreover, the SHR detrusor seems to be more sensitive to β3-AR activation.

Mechanisms of DO Phenotype in SHRs. This study found that the detrusor of SHRs behaves significantly different from WKY rats, with increased detrusor tones and reduced bladder compliance. In contrast, the afferent signals as measured by VMR response were not enhanced in SHRs. The increased pressor response in SHRs, a strain with high sympathetic impact genetically (Hendley et al., 1988), may provide less reliable information than the VMR response because the latter is unlikely to be affected by autonomic nerve system outflow. Based on afferent mechanisms, urgency and nociception seem to have different pathways (Pandita et al., 1997). However, they do have many features in common (Steers, 2002; Andersson and Wein, 2004). In fact, because OAB is a phenomenon of “hypersensitivity” (Yamaguchi et al., 2007), it is reasonable to use VMR and pressor responses to bladder distension as a tool to study pure afferent mechanisms in bladder hypersensitivity (Su et al., 2008b).

The decreased compliance of the detrusor in SHRs likely leads to their DO phenotype. The volume-pressure relationship was assessed during fast bladder filling as an index of compliance. The curves were significantly left-shifted in SHRs. Meanwhile, a decreased threshold to saline infusion in SHRs was also detected.

All β-AR subtypes exist in the rat urinary bladder body (Longhurst and Levendusky, 1999), and they mediate rat detrusor relaxation (Yamazaki et al., 1998). Sustained stimulation of β-AR did not modify β3-AR subsequent functional effects, but it diminished β1- and β2-AR responses (Rozec and Gauthier, 2006). Thus, enhanced sympathetic outflow in the SHR detrusor would likely desensitize the β1- and β2-ARs, leading to DO. Functional studies of β2- and/or β1-AR in SHRs should be conducted to establish a better understanding of their contribution to DO. It should be noted that many other mechanisms of the efferent pathway and smooth muscle function also seem important to DO in SHRs. For example, activation of α1-AR at central and peripheral levels with impaired prejunctionally inhibitory α2-AR may contribute to DO in SHRs (Persson et al., 1998). In addition, overexpression of RhoA in SHR smooth muscle may also be involved in the high bladder smooth muscle contraction tone in SHRs (Rajasekaran et al., 2005).

Effects of β3-AR Agonist on DO in SHRs. In the present study, the β3-AR agonist CL-316243 attenuated RC selectively in SHRs, largely reducing contraction frequency and moderately suppressing contraction amplitude. The effects of CL-316243 observed here on bladder function may be related to its direct effect on smooth muscle since previous in vitro studies have shown CL-316243 to inhibit bladder strip contractions (Longhurst and Levendusky, 1999; Takeda et al., 1999). Activation of β3-AR seems not to suppress the bladder afferent pathway. In our study, mRNA of β3-AR was highly expressed in detrusor with little detected in the nervous system. Consistently, CL-316243 did not attenuate VMR responses to UBD in either strain, implicating a limited role on afferent activity arising from the bladder. As predicted, the alteration of bladder compliance by CL-316243 did not change the readout of VMR and pressor responses since a bladder distension device was used to control bladder pressure.

The beneficial effects of β3-AR agonist in bladder function of SHRs concur with previous studies of OAB associated with bladder outlet obstruction and intravesical instillation of acetic acid (Woods et al., 2001), cerebral infarction (Kaidoh et al., 2002), and intravesical instillation of prostaglandin E₂ (Takeda et al., 2002). The minimal effective dose for CL-316243 in this preparation was ∼1 μg/kg, which is consistent with doses used in the above-mentioned studies involving functional measurements. Interestingly, CL-316243 produced less suppression on the contraction amplitude in SHRs. Likewise, CL-316243 suppressed mechanically or chemically induced bladder overactivity and improved urine storage function without affecting voiding contraction. The decreased relaxation of β3-AR activation during voiding has been proposed to be due to the compensation of adenylyl cyclase activity through activation of postjunctional muscarinic M2 receptors by endogenous acetylcholine release during voiding (Woods et al., 2001; Kaidoh et al., 2002; Takeda et al., 2002; Ehlert et al., 2007; Yamaguchi and Chapple, 2007). This character distinguishes β3-AR agonists from antimuscarinic agents, which produce urinary retention by decreasing the intensity of detrusor contraction.

It was found that β-AR activation produced more relaxation in SHRs than in WKY rats. However, β-AR activation produced less relaxation in KCl-precontracted bladder strips in vitro from SHRs (Frazier et al., 2006). This discrepancy is considered attributable to different experimental conditions, in vitro versus in vivo. Similar inconsistency can be found in other incidences. For example, increased inhibition of a Rho-kinase inhibitor was observed in SHR detrusor in vivo (Rajasekaran et al., 2005) but not in vitro (Schneider et al., 2005). CL-316243 selectivity on the SHR detrusor can be interpreted several ways. 1) Exposure may vary in the two strains. To explore this possibility, pharmacokinetic profiling was performed following i.v. injection of CL-316243. The corresponding exposures of CL-316243 in each strain were similar, which does not explain the increased efficacy in SHRs observed. 2) An altered β3-AR expression could contribute to the selective inhibition of bladder rhythmic contraction in SHRs. However, expression of the mRNA of β3-AR showed no difference in either strain. It should be noted that it has not been established whether alterations of β3-AR mRNA are predictive for those of functional receptor protein. Unfortunately, β3-AR expression cannot be assessed with any certainty at the moment because no validated methods of protein quantification exist. Thus, functional mRNA expression may be a suitable alternative (Vrydag and Michel, 2007; Yamaguchi and Chapple, 2007). 3) The β-AR signal transduction pathway depends on whether the detrusor is prestimulated (Yamaguchi and Chapple, 2007). In vitro studies have shown favorite relaxations for bladder strips were in the order of passive tension > KCl- (Frazier et al., 2005) > carbachol-induced contraction of the rat bladder (Longhurst and Levendusky, 1999). If a high basal tone of smooth muscle contraction is required for efficacy of β3-AR activation in vivo, then decreased compliance of detrusor in SHRs provides a precontracted condition. Lastly, β3-AR could be activated where catecholamine tone is high (Rozec and Gauthier, 2006). The enhanced sympathetic outflow in SHRs contributes to additional activation on preserved β3-AR by CL-316243 to relax the SHR detrusor. Thus, we believe that the selective inhibition by CL-316243 on bladder RC in SHRs is due to the precondition in the SHR detrusor with enhanced endogenous catecholamine level and increased detrusor tone.

CL-316243 had weak cardiovascular side effects as assessed by changes in heart rate and blood pressure (Yamaguchi and Chapple, 2007). Although CL-316243 did not change heart rate at the high dose (3 mg/kg), it did have a depressor effect in SHRs. The depressor effect may be unavoidable due to the genetic link and functional coupling between DO and hypertension in SHRs (Clemow et al., 1999). CL-316243 also reduced blood pressure in anesthetized Sprague-Dawley rats; however, it has the weakest cardiovascular side effects among nonselective β-AR agonists (Takeda et al., 2000, 2002; Kaidoh et al., 2002).

The DO phenotype in SHR rats acts as an animal model that reflects abnormal efferent/detrusor function without direct mechanosensory afferent hypersensitivity. The DO leads to the stimulation of the afferent pathway and subsequent sensations of urgency. β3-AR activation to relieve symptoms of OAB might be most effective under conditions of high cholinergic tone or a precontracted state. The beneficial effects of activation of β3-AR directly on the detrusor muscle in SHRs in the current study along with other DO models highlight the potential therapeutic significance of β3-AR in the treatment of OAB.