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EFFICACY OF THE β3-ADRENERGIC RECEPTOR AGONIST CL-316243 ON EXPERIMENTAL BLADDER HYPERREFLEXIA AND DETRUSOR INSTABILITY IN THE RAT
0022-5347/01/1663-1142/0 THE JOURNAL OF UROLOGY® Copyright © 2001 by AMERICAN UROLOGICAL ASSOCIATION, INC.®
Vol. 166, 1142–1147, September 2001 Printed in U.S.A.
EFFICACY OF THE 3-ADRENERGIC RECEPTOR AGONIST CL-316243 ON EXPERIMENTAL BLADDER HYPERREFLEXIA AND DETRUSOR INSTABILITY IN THE RAT MORGAN WOODS, NANCYLEIGH CARSON, N. WESLEY NORTON, JEFFREY H. SHELDON THOMAS M. ARGENTIERI
AND
From the Urologic Disorders, Wyeth-Ayerst Women’s Health Discovery Research, Radnor, Pennsylvania
ABSTRACT
Purpose: Recent evidence indicates that in a number of species detrusor relaxation is mediated through activation of the 3-adrenergic receptor. We determined whether activation of the 3-adrenergic receptor would be a useful therapeutic approach for bladder instability. We profiled in vitro activity of the 3-adrenergic receptor agonist CL-616243 and the efficacy of this compound in experimental models of detrusor instability and bladder hyperreflexia. Materials and Methods: Isolated rat detrusor strips were contracted by depolarizing the preparations with 20 mM. KCl. CL-316423 was added to the tissue bath in increasing concentrations and contraction inhibition was assessed. Efficacy against bladder instability was evaluated using the obstructed hypertrophied bladder model in the rat. The acetic acid bladder cystometry model was used to assess the efficacy of CL-316423 in bladder hyperreflexia. Isovolumetric contractions were evoked by electrical stimulation using a silver bipolar electrode. Data are expressed as the mean plus or minus standard error of mean. Results: CL-316243 inhibited spontaneously contracting, isolated rat detrusor strips in a concentration dependent manner with a mean concentration inhibiting 50% of maximal response of 2.65 ⫾ 0.36 nM. Intrinsic activity relative to forskolin was 1. In vivo CL-316243 administered intravenously or orally significantly increased the voiding interval and bladder compliance. In addition, there was a decrease in the number of spontaneous contractions during the filling phase in a model of neurogenic and obstruction induced bladder instability. The amplitude of electrically evoked isovolumetric contractions was significantly smaller after CL-316243 exposure. Conclusions: These data suggest that activating the 3-adrenergic receptor in rat bladder using CL-316243 may directly inhibit smooth muscle contractility, experimental hyperreflexia and detrusor instability, and be useful for urge urinary incontinence. KEY WORDS: bladder; rats, Sprague-Dawley; adrenergic agonists
Urinary incontinence is rapidly becoming recognized as a significant unmet therapeutic need. In the United States more than 10 million individuals have some form of urinary incontinence. Urge urinary incontinence, which includes hyperreflexive and hyperactive bladder syndromes, represents the underlying etiology in 50% to 70% of all cases of urinary incontinence.1 Contraction in the rat bladder is accomplished mainly through activating cholinergic parasympathetic neurons, while relaxation is mediated mostly through activating adrenergic sympathetic nerves.2 Because antimuscarinics inhibit parasympathetic stimulation, they have been the pharmacological mainstay for treating urge urinary incontinence for the last 25 years. However, the clinical efficacy and usefulness of these compounds has been limited by their antimuscarinic side effects, resulting in an overall patient compliance of only approximately 30%. Recently a growing number of newly marketed compounds have attempted to improve the dry mouth symptom, which is the most significant side effect associated with antimuscarinic therapy. While some improvements in the side effect profile have been realized, the mechanism of action is the same (antimuscarinic) and improved side effects may have been achieved at the expense of overall efficacy. To our knowledge no new mechanisms of action have been exploited to date for treating detrusor instability.
The role of the -adrenergic receptor in detrusor relaxation has been well documented.3– 6 Recently the 3-adrenergic receptor subtype was identified in a number of different species as an important receptor responsible for transduction of the autonomic relaxation of the bladder detrusor.7–10 Data now indicate that the 3 receptor is present and responsible for relaxation of isolated smooth muscle preparations in human tissues11–14 and increased bladder capacity in anesthetized rats.15, 16 However, to our knowledge there have been no reports to date profiling the in vivo efficacy of a 3-adrenergic agonist in an animal model of pathological bladder instability and hyperreflexia. We confirmed the in vitro findings of others of the selective 3-agonist CL-316243 (fig. 1) and demonstrated its efficacy for inhibiting voiding hyperreflexia and the spontaneous contractions associated with detrusor instability.
Accepted for publication March 9, 2000.
FIG. 1. Structure of CL-316243 1142
EFFICACY OF -ADRENERGIC RECEPTOR AGONIST IN RAT BLADDER MATERIALS AND METHODS
Isolated rat bladder strip. Male Sprague-Dawley rats weighing 200 to 400 gm. were euthanized by CO2 inhalation and exsanguination. The bladders were rapidly removed and placed in 37C physiological salt solution containing 118.4 mM. NaCl, 5 mM. KCl, 2.5 mM. CaCl2 1.2 mM. MgSO4, 1.2 mM. KH2PO4, 24.9 mM. NaHCO3 and 11.1 mM. D-glucose gassed with 95% O2/5% CO2 to achieve a pH of 7.4. The bladder dome was isolated from the trigone region and cut into 4 to 5 ⫻ 10 mm. strips. One end was secured to the bottom of a water jacket tissue bath and the other was secured to an isometric force transducer (Grass Instruments, Quincy, Massachusetts). Tissues were pre-tensioned at 0.25 to 0.5 gm. After 30 minutes of equilibration they were contracted with an additional 15 mM. KCl (total 20 mM.) and again allowed to equilibrate until the preparations were contracting steadily. Compounds were administered directly into the tissue baths as sequential concentrations. Transducer signals were digitized at 12-bit resolution and analyzed on line using a 586 base computer and custom software. The area under the contraction curve was used as a measure of contractility since spontaneous bladder contractions were irregular in amplitude and frequency. A 5-minute area under the curve value was obtained 30 minutes after administering each concentration to the tissue bath. Isovolumetric contractions. Female Sprague-Dawley rats weighing 190 to 210 gm. were anesthetized with urethane at a dose of 1 gm./kg./10 ml. intraperitoneal and 1 gm./kg./10 ml. subcutaneous. The bladders were exposed through a short midline incision and the bladder dome was cannulated with an 24 gauge polytetrafluoroethylene angiocatheter that was heat flared at the end, inserted into the dome of the bladder and secured with 4-zero silk. The cannula was connected with a T connector to a Model P23Db pressure transducer (Statham Instruments, Inc., Oxnard, California) and to a 5 cc syringe. The bladders were inflated with saline to a pressure of approximately 20 cm. water. One jugular vein was cannulated with polyethylene tubing for intravenous administration of compound or vehicle. Sutures were attached to the skin flaps to form a pouch around the abdominal opening. The abdominal cavity was then filled with mineral oil. Bladder contractions were evoked by electrical stimulation at 10 to 50 Hz. and 10 to 50 V. with a 0.5 ms. pulse duration delivered by a silver bipolar electrode. The stimulus was maintained for 5 to 7 seconds or until a maximum contraction was achieved. Bladders rested 5 minutes between stimulations. Hyperreflexic bladders. Voiding frequency was enhanced by stimulating sensory afferents with a dilute acetic acid solution in the cystometry infusate, as previously described by Birder and de Groat.17 Briefly, female Sprague-Dawley rats weighing 190 to 210 gm. were anesthetized with urethane at a dose of 1 gm./kg./10 ml. intraperitoneal and 1 gm./kg./10 ml. subcutaneous. The trachea was cannulated with a polyethylene tube to ensure a patent airway. The external jugular was cannulated with polyethylene tubing to administer the compound. The bladder was exposed through a midline incision and a 24 gauge polytetrafluoroethylene angiocatheter was heat flared at the end, inserted into the bladder dome and secured with 4-zero silk. The bladder was flushed with normal saline and allowed to equilibrate for 1 hour before cystometry was performed. Using a T connector the bladder catheter was connected to a pressure transducer and to an infusion pump (Harvard Apparatus, Millis, Massachusetts). Cystometry recordings were monitored on a polygraph, while infusing the bladder with saline containing 0.25% acetic acid at a rate of 2.4 ml. per hour for 1 hour. Compound was then administered intravenously and cystometry was monitored for an additional 2 hours. Cystometry parameters recorded were voiding interval, amplitude and
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threshold pressure, bladder capacity and compliance, and the number of spontaneous bladder contractions during the filling phase. The control period was considered the 30-minute time of acetic acid saline perfusion before dose administration. The compound effect was analyzed 15 minutes after the dose for 105 minutes. Hypertrophied bladders. The method for producing hypertrophied unstable bladders was modified from that reported by Malmgren18 and Wojdan19 et al. Briefly, female SpragueDawley rats weighing 190 to 210 gm. were used. Animals were anesthetized with isoflurane. After anesthetization the bladder and urethra were exposed through a midline incision and a 4-zero silk ligature was tied around the proximal urethra in the presence of a stainless steel rod 1 mm. in diameter. When the rod was removed, calibrated partial urethral occlusion resulted. The abdominal muscle was closed using 3-zero silk and the skin was closed with surgical staples. Each rat received 150,000 units penicillin G benzathine intramuscularly. During the next 6 to 9 weeks bladder hypertrophy and instability resulted from partial outlet obstruction. Catheter Implantation: After approximately 6 to 9 weeks the animals were re-anesthetized with isoflurane. The ligature was removed and a flared polyethylene catheter was placed in the bladder dome and secured with a suture. The catheter was exteriorized under the skin through an opening in the back of the neck. The abdominal incision was sutured and the free end of the catheter was sealed. After surgery each animal was given another dose of 150,000 units penicillin intramuscularly. Cystometry Evaluation: Two days after catheter implantation animals were placed in a metabolic cage and the bladder catheter was attached using a T connector to a pressure transducer and infusion pump. Urine volume was monitored with a plastic beaker attached to an FTO3 force displacement transducer (Grass Instruments). Cystometry evaluation of bladder function was started by infusing the bladder with saline at a rate of 10 to 20 ml. per hour depending on the degree of hypertrophy. Cystometry parameters recorded were number of spontaneous bladder contractions during the filling phase, and voiding amplitude and volume. Cystometry recordings were made on a polygraph and included at least 2 voiding intervals or 20 minutes. The rats rested for 2 hours and then underwent oral gavage with the test compound. Cystometry was repeated approximately 60 minutes after administering the test compound. Another group of vehicle (saline) treated animals with hypertrophied bladders served as time/vehicle controls. Data analysis. Data are presented as the mean plus or minus standard error of mean. Appropriate statistical tests are indicated. Differences were considered statistically significant at p ⬍0.05. RESULTS
Detrusor strip contraction. Isolated detrusor strips exposed to 20 mM. KCl contract spontaneously with irregular amplitude and frequency. Exposure to 0.1 to 30 nM. CL-316243 produced a concentration dependent inhibition of contraction, as measured by the decrease in contraction area under the curve. Exposure to 200 nM. carbachol at the end of the experiment produced a robust contractile response (fig. 2, A). Concentration response data were expressed as the percent of the maximal contraction above zero with a pre-tension weight of 0.25 to 0.5 gm. The average concentration inhibiting 50% of maximal response was 2.65 ⫾ 0.36 nM. In 6 animals intrinsic activity relative to forskolin was 1 (fig. 2, B). Isovolumetric contractions. Animals were separated into 2 groups. Five contractions per animal were elicited and averaged in each group, followed by intravenous administration
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EFFICACY OF -ADRENERGIC RECEPTOR AGONIST IN RAT BLADDER
FIG. 2A. Tracing of rat bladder strip contraction. After contractions stabilized CL-316243 was added to tissue bath in increasing concentrations. Area under curve was monitored for 5-minute interval 30 minutes after each addition. Addition of 200 nM. carbachol at experiment end produced robust contraction. Total time of trace was 240 minutes. g, gm. B, concentration response curve for inhibition of rat bladder strip contraction by CL-316243. Line is best fit Michaelis-Menten logistic through data points. Concentration inhibiting 50% of response was 2.6 nM.
of 10 mg./kg. CL-316243 in 5 and saline in 4. At 15 minutes another 5 contractions were elicited and averaged. Mean pretreatment contraction amplitude was 47.6 ⫾ 4.2 and 46.3 ⫾ 7.2 cm. water in the drug and vehicle treated groups, respectively (p ⬎0.05). After dosing there was a significant mean decrease in contraction amplitude in the CL-316243 treated group to 34.5 ⫾ 3.3 cm. water (p ⬍0.05). There was no significant mean change in the vehicle treated group (46.1 ⫾ 7.2 cm. water). In addition to a decrease in contraction amplitude, mean baseline pressure in the CL-316243 treated group decreased 56.8% ⫾ 7.4% (p ⬍0.05). In the vehicle treated group mean baseline pressure decreased 3.3% ⫾ 19.2% (p ⬎0.05). Hyperreflexia. The average voiding interval in animals in which the bladder was perfused with saline containing 0.25% acetic acid was 47% shorter than that interval in animals that received saline perfusion alone (p ⬍0.05). Spontaneous contractions during the filling phase with acetic acid were also typically observed in these animals during cystometry. Administering CL-316243 0.01 to 30 mg./kg. CL-316243 intravenously in these animals significantly increased the voiding interval in a dose dependent manner (p ⬍0.05, fig. 3). Despite the continued exposure to acetic acid at doses above 0.1 mg./kg. the voiding interval was equivalent to the cystometry interval with saline alone. There was also a dose dependent decrease in voiding amplitude, an increase in bladder capacity and compliance, and a decrease in the void-
FIG. 3. Increase in voiding interval produced by increasing doses of CL-316243 in acetic acid induced bladder hyperreflexia model. Asterisk indicates ANOVA Student-Newman-Keuls test p ⬍0.05 versus vehicle.
ing pressure threshold (table 1). Furthermore, CL-316243 treatment produced a decrease in the frequency of spontaneous contractions during the filling phase. For example, immediately after intravenous dosing no evidence of spontaneous bladder contractions was observed at any dose tested (fig. 4). Saline dosing had no effect on the voiding interval or number of spontaneous bladder contractions. Hypertrophied bladder. Hypertrophied bladder weight was 0.8 to 1.1 gm. Cystometrography of hypertrophied bladders is characterized by small spontaneous bladder contractions during most of the filling phase. Exposure to CL-316243 produced a dose dependent decrease in the observed number of spontaneous bladder contractions (table 2). A maximum inhibition of spontaneous bladder contractions of ⫺54.9% was observed at the 3 mg./kg. dose and above. The dose that produced half of this maximum effect was 0.44 mg./kg. (fig. 5). While there was a trend toward a dose dependent decrease in voiding amplitude, voiding volume was unaffected. Figure 6 shows representative cystometry recordings before and after CL-316243 administration. DISCUSSION
Urge urinary incontinence and its associated bladder instability is often reported as the most prevalent form of incontinence.1 To date the most accepted pharmacological therapy for bladder instability is antimuscarinic agents to inhibit the action of acetylcholine on detrusor smooth muscle. Agents such as oxybutynin have been shown to have reasonably good efficacy,20 although anticholinergic side effects, including dry mouth,21 often limits usefulness. While recent reports of sustained formulations have indicated an improvement in the dry mouth profile,22 there have been no new successful pharmacological approaches for more than 25 years. The -adrenergic receptor has been known to mediate relaxation in several species, including humans.4, 5, 23 Li et al first described 1 and 2-adrenergic receptor subtypes in the guinea pig.2 Recently the 3-adrenergic receptor has been described in a number of animal species7–10 and in humans.11–13 Selective activation of the 3-adrenergic receptor has been shown to modulate bladder function and urine storage in the rat.13, 15, 16 Disruption of -adrenergic responsiveness may partially underlie bladder instability associated with aging.24 CL-316243 is a potent and selective agonist of the 3adrenergic receptor. The compound has been shown to bind to the rat 3 receptor with an inhibition constant of 1 M. and stimulate cyclic adenosine monophosphate production with an activation constant of 0.7 nM. CL-316243 did not
EFFICACY OF -ADRENERGIC RECEPTOR AGONIST IN RAT BLADDER
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TABLE 1. Effect of CL-316243 on micturition parameters in rat hyperreflexive bladders Mean Voiding Dose No. Interval ⫾ SEM (mg./kg.) Subjects (mins.)
Mean Voiding Amplitude ⫾ SEM (cm. water)
Mean Voiding Threshold ⫾ SEM (cm. water)
Mean Capacity ⫾ SEM (l.)
Mean Compliance ⫾ SEM (l./cm. water⫺1)
Vehicle 12 2.6 ⫾ 0.3 26.6 ⫾ 2.1 5.4 ⫾ 0.5 137 ⫾ 18 39 ⫾ 7 0.01 6 3.0 ⫾ 0.4* 28.1 ⫾ 2.5 NT NT NT 0.1 9 4.3 ⫾ 0.4* 21.1 ⫾ 2.8 NT NT NT 1.0 12 4.8 ⫾ 0.5* 20.0 ⫾ 1.1* NT NT NT 10 6–8 4.5 ⫾ 0.6* 20.2 ⫾ 1.2* 3.5 ⫾ 0.5* 227 ⫾ 37* 70 ⫾ 12* 30 7 5.0 ⫾ 0.7* 20.7 ⫾ 1.6 NT NT NT * Versus respective parameter for vehicle dosed (acetic acid/time control) ANOVA Student-Newman-Keuls test p ⬍0.05.
FIG. 4. Cystometry recordings using saline containing 0.25% acetic acid. Vehicle had no effect. There were spontaneous contractions during bladder filling. Arrow indicates administration of 1 ml./kg. saline intravenously (i.v.). Before CL-316243 delivery there were contractions but after CL-316243 (arrow) there was immediate inhibition of spontaneous contractions.
demonstrate significant binding to the 1 and 2-adrenergic receptors at concentrations up to 100 M.25 These CL-316243 findings are similar to those described by Takeda et al,15 suggesting that CL-316243 is a full agonist of the rat 3-adrenergic receptor and a potent direct inhibitor of bladder smooth muscle contractility. The tissues retained their ability to respond to muscarinic stimulation at the end of the experiment, suggesting that 3-adrenergic receptor stimulation may result in less impairment of normal contractility than antimuscarinic therapy. The effects of CL-316243 on in situ isovolumetric bladder contractions were consistent with the data generated from isolated strips. CL-316243 significantly inhibited contractions, suggesting that the compound has the direct ability to inhibit bladder smooth muscle contractility when administered systemically. Recently Birder and de Groat described a model of neurogenic bladder instability.17 Hyperreflexia was produced by adding acetic acid to the saline used for bladder cystometry. Data indicate that acetic acid stimulates sensory neurons that form the afferent arm of the micturition reflex. The result is a shortening of the voiding interval and the appearance of spontaneous contractions during the filling phase. Animals given CL-316243 responded with an increase in the voiding interval back to control values, a decrease in voiding threshold pressure, an increase in bladder capacity and compliance, and elimination of spontaneous bladder contractions. Spontaneous bladder contractions appeared to be more sensitive to compound than the voiding interval. At 0.01 mg./kg. there was no significant effect on the voiding interval and yet spontaneous bladder contractions were virtually eliminated. Similar findings were reported by Takeda et al.15 In their study of normal rats they noted a significant increase in the voiding interval and decrease in amplitude with 10 and 100 g./kg. CL-316243 intravenously. It was not clear in this study whether the effect of CL-316243 was through phosphorolation of phospholamban or some other mechanisms. The in vitro and isovolumetric contraction results suggest that activating the 3-adrenergic receptor results in a decrease in smooth muscle tone and excitability. Recent evidence also suggests that activating -adrenergic receptors in the blad-
Mean % Change in Contractions ⫾ SEM 4.5 ⫾ 24.2 ⫺97.8 ⫾ 1.8* ⫺94.9 ⫾ 2.0* ⫺85.5 ⫾ 4.6* ⫺92.7 ⫾ 2.6* ⫺86.0 ⫾ 8.3*
der facilitates conductance through the large conductance calcium dependent K channel via a protein kinase-A pathway.26 Activating this channel would lead to hyperpolarization of bladder smooth muscle and increased stability, and would significantly contribute to the underlying mechanism of action of 3-adrenergic receptor agonists. Oral administration of CL-316243 produced a dose dependent inhibition of spontaneous bladder contractions during cystometry filling of the hypertrophied bladders. While no statistically significant effects were observed, there was a trend toward a dose dependent decrease in voiding amplitude. Despite this finding no effect on voided volume was noted. Bladder instability due to outlet obstruction may arise from a myogenic and a neurogenic mechanism.27, 28 The simplest explanation for the observations using CL-316243 would be that the compound increases bladder compliance and capacity, resulting in an increased voiding interval by the direct inhibition of bladder smooth tone. The decrease in isovolumetric contraction amplitude and spontaneous bladder contractions would support this conclusion. Nevertheless, it cannot be ruled out that CL-316243 may have some additional peripheral or central neuronal effects. Furthermore, while it is well accepted that sympathetic stimulation of the bladder produces relaxation, others have shown that adrenergic innervation is elevated in spontaneously hypertensive rats and yet spontaneously hypertensive rat bladders are hyperactive relative to Wistar-Kyoto rat bladders.29, 30 If activation of adrenergic nerves relaxes bladder smooth muscle, it is difficult to reconcile why the bladders of spontaneously hypertensive rats are hyperactive despite greater adrenergic innervation. Clemow et al demonstrated that with an increase in adrenergic efferent innervation there is an increase in sensory afferent innervation in spontaneously hypertensive bladders.31 Their data suggest that the increase in sensory afferent nerve density may underly an enhanced voiding reflex. In addition, while there is an increase in adrenergic innervation and norepinephrine content, it is possible that post-synaptic -adrenergic receptor density is diminished, making smooth muscle less responsive to norepinephrine. CONCLUSIONS
Our study confirms the findings of others that the activation of 3-adrenergic receptors has an important role in normal rat bladder relaxation. Our data also show that a 3adrenergic receptor agonist may increase bladder capacity and inhibit the bladder instability associated with hyperreflexia and detrusor hypertrophy. Takeda et al reported that administering CL-316243 to anesthetized rats had only a slight influence on blood pressure and heart rate, whereas other non selective -adrenergic receptor agonists and cyclic adenosine monophosphate phosphodiesterase inhibitors often had profound cardiovascular sequelae.15 This finding suggest that 3-adrenergic receptor agonist may be useful for treating urge urinary incontinence without the side effects associated with antimuscarinic therapy or the potential cardiovascular liability associated with potassium channel openers.32–34
EFFICACY OF -ADRENERGIC RECEPTOR AGONIST IN RAT BLADDER
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TABLE 2. Effect of CL-316243 on micturition parameters in rat hypertrophied bladder Treatment (mg./kg.)
No. Subjects
Mean No. Contractions During Filling ⫾ SEM Pretreatment
Saline 6 0.1 7 0.3 8 1.0 9 3.0 6 10.0 7 * Versus pretreatment paired
Posttreatment
19.6 ⫾ 3.7 27.9 ⫾ 3.7 24.4 ⫾ 2.5 26.1 ⫾ 1.9 19.3 ⫾ 1.7 24.0 ⫾ 3.6 Student’s t test p
19.6 ⫾ 5.7 27.6 ⫾ 3.3 21.4 ⫾ 2.0 11.8 ⫾ 2.7* 10.2 ⫾ 4.1* 9.4 ⫾ 2.7* ⬍0.05.
Mean Cm. Water Voiding Amplitude ⫾ SEM Pretreatment
Posttreatment
Pretreatment
Posttreatment
Mean Bladder Wt. ⫾ SEM (gm.)
38.5 ⫾ 8.8 31.5 ⫾ 7.9 30.1 ⫾ 7.2 30.8 ⫾ 4.1 24.6 ⫾ 8.0 40.3 ⫾ 6.6
39.0 ⫾ 14.7 26.6 ⫾ 6.7 20.3 ⫾ 2.1 24.0 ⫾ 2.7 20.2 ⫾ 4.1 25.9 ⫾ 5.4
1.4 ⫾ 0.2 3.3 ⫾ 0.8 3.2 ⫾ 0.7 2.3 ⫾ 0.2 3.9 ⫾ 0.3 1.7 ⫾ 0.2
1.6 ⫾ 0.2 3.1 ⫾ 0.5 3.2 ⫾ 0.5 2.3 ⫾ 0.3 3.7 ⫾ 0.4 2.0 ⫾ 0.2
1.1 ⫾ 0.2 1.0 ⫾ 0.1 1.0 ⫾ 0.1 0.9 ⫾ 0.1 1.0 ⫾ 0.1 0.8 ⫾ 0.2
Mean Ml. Voided Vol. ⫾ SEM
activating 1 and 2 receptors results in a positive inotropic response or in heart failure cases when the relative ratios of -adrenergic receptor subtypes may be altered. Despite these mentioned precautions selective 3-adrenergic receptor agonists may represent a new useful class of agents for treating bladder instability. REFERENCES
FIG. 5. Dose response curve of inhibition of spontaneous bladder contractions (SBC) in rat hypertrophied bladder by CL-316243. Line is best fit Michaelis-Menten logistic through data points. Concentration inhibiting 50% of response was 0.44 mg./kg. Asterisk indicates ANOVA Student-Newman-Keuls test p ⬍0.05 versus vehicle.
FIG. 6. Cystometry recordings of bladder pressure and urine output volume in animal with bladder hypertrophy induced instability before treatment as control and 60 minutes after dosing with 1 mg./kg. CL-316243 orally (p.o.). Significant inhibition of spontaneous contractions occurred with slight but statistically insignificant decrease in voiding amplitude and no effect on voided volume.
In addition, it should be noted that, while the 3adrenergic receptor may have a minor role in the cardiovascular system of the rat, the contribution of the 3-adrenergic receptor to cardiovascular function may be species dependent.35 Specifically Gauthier et al suggested that selective activation of 3-adrenergic receptors results in a negative inotropic response in isolated human myocardium.36, 37 However, the net effect of 3-adrenergic receptor activation is still not clear in the setting of normal adrenergic tone, in which
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uation of bladder instability in rats with infravesical outflow obstruction. J Urol, 137: 1291, 1987 Wojdan, A., Freeden, C., Woods, M. et al: Comparison of the potassium channel openers, WAY-133537, ZD6169, and celikalim on isolated bladder tissue and in vivo bladder instability in rat. J Pharmacol Exp Ther, 289: 1410, 1999 Holmes, D. M., Montz, F. J. and Stanton, S. L.: Oxybutinin versus propantheline in the management of detrusor instability. A patient-regulated variable dose trial. Br J Obstet Gynaecol, 96: 607, 1989 Yarker, Y. E., Goa, K. L. and Fitton, A.: Oxybutynin. A review of its pharmacodynamic and pharmacokinetic properties, and its therapeutic use in detrusor instability. Drugs Aging, 6: 243, 1995 Versi, E., Appell, R., Mobley, D. et al: Dry mouth with conventional and controlled-release oxybutynin in urinary incontinence. Ditropan XL Study Group. Obstet Gynecol, 95: 718, 2000 Larsen, J. J.: Alpha and beta-adrenoceptors in the detrusor muscle and bladder base of the pig and beta-adrenoceptors in the detrusor muscle of man. Br J Pharmacol, 65: 215, 1979 Derweesh, I. H., Wheeler, M. A. and Weiss, R. M.: Alterations in G-proteins and -adrenergic responsive adenylyl cyclase in rat urinary bladder during aging. J Pharmacol Exp Ther, 294: 969, 2000 Dolan, J. A., Muenkel, H. A., Burns, M. G. et al: Beta-3 adrenoceptor selectivity of the dioxolane dicarboxylate phenethanolamines. J Pharmacol Exp Ther, 269: 1000, 1994 Kobayashi, H., Adachi-Akahane, S. and Nagano, T.: Involvement of BKCa channels in the relaxation of detrusor muscle via -adrenoceptors. Eur J Pharmacol, 404: 231, 2000 Steers, W. D., Ciambotti, J., Etzel, B. et al: Alterations in afferent pathways from the urinary bladder of the rat in response to partial urethral obstruction. J Comparative Neurol, 310: 401, 1991
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28. Yoshimura, N.: Bladder afferent pathway and spinal cord injury: possible mechanisms inducing hyperreflexia of the urinary bladder. Prog Neurobiol, 57: 583, 1999 29. Tong, Y. C., Hung, Y. C., Lin, S. N. et al: Comparisons of neurotransmitter concentrations in the synaptosomal preparation of the normotensive and hypertensive rat urinary bladder. Neurosci Lett, 204: 141, 1996 30. Spitsbergen, J. M., Clemow, D. B., McCarty, R. et al: Neurally mediated hyperactive voiding in spontaneously hypertensive rats. Brain Res, 790: 151, 1998 31. Clemow, D. B., McCarty, R., Steers, W. D. et al: Efferent and afferent neuronal hypertrophy associated with micturition pathways in spontaneously hypertensive rats. Neurourol Urodyn, 16: 293, 1997 32. Nurse, D. E., Restorick, J. M. and Mundy, A. R.: The effect of cromakalim on the normal and hyper-reflexic human detrusor muscle. Br J Urol, 68: 27, 1991 33. Hedlund, H., Mattiasson, A. and Andersson, K.-E.: Effects of pinacidil on detrusor instability in men with bladder outlet obstruction. J Urol, 146: 1345, 1991 34. Komersova, K., Rogerson, J. W., Conway, E. L. et al: The effect of levcromakalim (BRL 38227) on bladder function in patients with high spinal cord lesions. Br J Clin Pharmacol, 39: 207, 1995 35. Gauthier, C., Tavernier, G., Trochu, J. N. et al: Interspecies differences in the cardiac negative inotropic effects of beta(3)adrenoceptor agonists. J Pharmacol Exp Ther, 290: 687, 1999 36. Gauthier, C., Tavernier, G., Charpentier, F. et al: Functional beta3-adrenoceptor in the human heart. J Clin Invest, 98: 556, 1996 37. Gauthier, C., Leblais, V., Kobzik, L. et al: The negative inotropic effect of beta3-adrenoceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle. J Clin Invest, 102: 1377, 1998
Vol. 166, 1142–1147, September 2001 Printed in U.S.A.
EFFICACY OF THE 3-ADRENERGIC RECEPTOR AGONIST CL-316243 ON EXPERIMENTAL BLADDER HYPERREFLEXIA AND DETRUSOR INSTABILITY IN THE RAT MORGAN WOODS, NANCYLEIGH CARSON, N. WESLEY NORTON, JEFFREY H. SHELDON THOMAS M. ARGENTIERI
AND
From the Urologic Disorders, Wyeth-Ayerst Women’s Health Discovery Research, Radnor, Pennsylvania
ABSTRACT
Purpose: Recent evidence indicates that in a number of species detrusor relaxation is mediated through activation of the 3-adrenergic receptor. We determined whether activation of the 3-adrenergic receptor would be a useful therapeutic approach for bladder instability. We profiled in vitro activity of the 3-adrenergic receptor agonist CL-616243 and the efficacy of this compound in experimental models of detrusor instability and bladder hyperreflexia. Materials and Methods: Isolated rat detrusor strips were contracted by depolarizing the preparations with 20 mM. KCl. CL-316423 was added to the tissue bath in increasing concentrations and contraction inhibition was assessed. Efficacy against bladder instability was evaluated using the obstructed hypertrophied bladder model in the rat. The acetic acid bladder cystometry model was used to assess the efficacy of CL-316423 in bladder hyperreflexia. Isovolumetric contractions were evoked by electrical stimulation using a silver bipolar electrode. Data are expressed as the mean plus or minus standard error of mean. Results: CL-316243 inhibited spontaneously contracting, isolated rat detrusor strips in a concentration dependent manner with a mean concentration inhibiting 50% of maximal response of 2.65 ⫾ 0.36 nM. Intrinsic activity relative to forskolin was 1. In vivo CL-316243 administered intravenously or orally significantly increased the voiding interval and bladder compliance. In addition, there was a decrease in the number of spontaneous contractions during the filling phase in a model of neurogenic and obstruction induced bladder instability. The amplitude of electrically evoked isovolumetric contractions was significantly smaller after CL-316243 exposure. Conclusions: These data suggest that activating the 3-adrenergic receptor in rat bladder using CL-316243 may directly inhibit smooth muscle contractility, experimental hyperreflexia and detrusor instability, and be useful for urge urinary incontinence. KEY WORDS: bladder; rats, Sprague-Dawley; adrenergic agonists
Urinary incontinence is rapidly becoming recognized as a significant unmet therapeutic need. In the United States more than 10 million individuals have some form of urinary incontinence. Urge urinary incontinence, which includes hyperreflexive and hyperactive bladder syndromes, represents the underlying etiology in 50% to 70% of all cases of urinary incontinence.1 Contraction in the rat bladder is accomplished mainly through activating cholinergic parasympathetic neurons, while relaxation is mediated mostly through activating adrenergic sympathetic nerves.2 Because antimuscarinics inhibit parasympathetic stimulation, they have been the pharmacological mainstay for treating urge urinary incontinence for the last 25 years. However, the clinical efficacy and usefulness of these compounds has been limited by their antimuscarinic side effects, resulting in an overall patient compliance of only approximately 30%. Recently a growing number of newly marketed compounds have attempted to improve the dry mouth symptom, which is the most significant side effect associated with antimuscarinic therapy. While some improvements in the side effect profile have been realized, the mechanism of action is the same (antimuscarinic) and improved side effects may have been achieved at the expense of overall efficacy. To our knowledge no new mechanisms of action have been exploited to date for treating detrusor instability.
The role of the -adrenergic receptor in detrusor relaxation has been well documented.3– 6 Recently the 3-adrenergic receptor subtype was identified in a number of different species as an important receptor responsible for transduction of the autonomic relaxation of the bladder detrusor.7–10 Data now indicate that the 3 receptor is present and responsible for relaxation of isolated smooth muscle preparations in human tissues11–14 and increased bladder capacity in anesthetized rats.15, 16 However, to our knowledge there have been no reports to date profiling the in vivo efficacy of a 3-adrenergic agonist in an animal model of pathological bladder instability and hyperreflexia. We confirmed the in vitro findings of others of the selective 3-agonist CL-316243 (fig. 1) and demonstrated its efficacy for inhibiting voiding hyperreflexia and the spontaneous contractions associated with detrusor instability.
Accepted for publication March 9, 2000.
FIG. 1. Structure of CL-316243 1142
EFFICACY OF -ADRENERGIC RECEPTOR AGONIST IN RAT BLADDER MATERIALS AND METHODS
Isolated rat bladder strip. Male Sprague-Dawley rats weighing 200 to 400 gm. were euthanized by CO2 inhalation and exsanguination. The bladders were rapidly removed and placed in 37C physiological salt solution containing 118.4 mM. NaCl, 5 mM. KCl, 2.5 mM. CaCl2 1.2 mM. MgSO4, 1.2 mM. KH2PO4, 24.9 mM. NaHCO3 and 11.1 mM. D-glucose gassed with 95% O2/5% CO2 to achieve a pH of 7.4. The bladder dome was isolated from the trigone region and cut into 4 to 5 ⫻ 10 mm. strips. One end was secured to the bottom of a water jacket tissue bath and the other was secured to an isometric force transducer (Grass Instruments, Quincy, Massachusetts). Tissues were pre-tensioned at 0.25 to 0.5 gm. After 30 minutes of equilibration they were contracted with an additional 15 mM. KCl (total 20 mM.) and again allowed to equilibrate until the preparations were contracting steadily. Compounds were administered directly into the tissue baths as sequential concentrations. Transducer signals were digitized at 12-bit resolution and analyzed on line using a 586 base computer and custom software. The area under the contraction curve was used as a measure of contractility since spontaneous bladder contractions were irregular in amplitude and frequency. A 5-minute area under the curve value was obtained 30 minutes after administering each concentration to the tissue bath. Isovolumetric contractions. Female Sprague-Dawley rats weighing 190 to 210 gm. were anesthetized with urethane at a dose of 1 gm./kg./10 ml. intraperitoneal and 1 gm./kg./10 ml. subcutaneous. The bladders were exposed through a short midline incision and the bladder dome was cannulated with an 24 gauge polytetrafluoroethylene angiocatheter that was heat flared at the end, inserted into the dome of the bladder and secured with 4-zero silk. The cannula was connected with a T connector to a Model P23Db pressure transducer (Statham Instruments, Inc., Oxnard, California) and to a 5 cc syringe. The bladders were inflated with saline to a pressure of approximately 20 cm. water. One jugular vein was cannulated with polyethylene tubing for intravenous administration of compound or vehicle. Sutures were attached to the skin flaps to form a pouch around the abdominal opening. The abdominal cavity was then filled with mineral oil. Bladder contractions were evoked by electrical stimulation at 10 to 50 Hz. and 10 to 50 V. with a 0.5 ms. pulse duration delivered by a silver bipolar electrode. The stimulus was maintained for 5 to 7 seconds or until a maximum contraction was achieved. Bladders rested 5 minutes between stimulations. Hyperreflexic bladders. Voiding frequency was enhanced by stimulating sensory afferents with a dilute acetic acid solution in the cystometry infusate, as previously described by Birder and de Groat.17 Briefly, female Sprague-Dawley rats weighing 190 to 210 gm. were anesthetized with urethane at a dose of 1 gm./kg./10 ml. intraperitoneal and 1 gm./kg./10 ml. subcutaneous. The trachea was cannulated with a polyethylene tube to ensure a patent airway. The external jugular was cannulated with polyethylene tubing to administer the compound. The bladder was exposed through a midline incision and a 24 gauge polytetrafluoroethylene angiocatheter was heat flared at the end, inserted into the bladder dome and secured with 4-zero silk. The bladder was flushed with normal saline and allowed to equilibrate for 1 hour before cystometry was performed. Using a T connector the bladder catheter was connected to a pressure transducer and to an infusion pump (Harvard Apparatus, Millis, Massachusetts). Cystometry recordings were monitored on a polygraph, while infusing the bladder with saline containing 0.25% acetic acid at a rate of 2.4 ml. per hour for 1 hour. Compound was then administered intravenously and cystometry was monitored for an additional 2 hours. Cystometry parameters recorded were voiding interval, amplitude and
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threshold pressure, bladder capacity and compliance, and the number of spontaneous bladder contractions during the filling phase. The control period was considered the 30-minute time of acetic acid saline perfusion before dose administration. The compound effect was analyzed 15 minutes after the dose for 105 minutes. Hypertrophied bladders. The method for producing hypertrophied unstable bladders was modified from that reported by Malmgren18 and Wojdan19 et al. Briefly, female SpragueDawley rats weighing 190 to 210 gm. were used. Animals were anesthetized with isoflurane. After anesthetization the bladder and urethra were exposed through a midline incision and a 4-zero silk ligature was tied around the proximal urethra in the presence of a stainless steel rod 1 mm. in diameter. When the rod was removed, calibrated partial urethral occlusion resulted. The abdominal muscle was closed using 3-zero silk and the skin was closed with surgical staples. Each rat received 150,000 units penicillin G benzathine intramuscularly. During the next 6 to 9 weeks bladder hypertrophy and instability resulted from partial outlet obstruction. Catheter Implantation: After approximately 6 to 9 weeks the animals were re-anesthetized with isoflurane. The ligature was removed and a flared polyethylene catheter was placed in the bladder dome and secured with a suture. The catheter was exteriorized under the skin through an opening in the back of the neck. The abdominal incision was sutured and the free end of the catheter was sealed. After surgery each animal was given another dose of 150,000 units penicillin intramuscularly. Cystometry Evaluation: Two days after catheter implantation animals were placed in a metabolic cage and the bladder catheter was attached using a T connector to a pressure transducer and infusion pump. Urine volume was monitored with a plastic beaker attached to an FTO3 force displacement transducer (Grass Instruments). Cystometry evaluation of bladder function was started by infusing the bladder with saline at a rate of 10 to 20 ml. per hour depending on the degree of hypertrophy. Cystometry parameters recorded were number of spontaneous bladder contractions during the filling phase, and voiding amplitude and volume. Cystometry recordings were made on a polygraph and included at least 2 voiding intervals or 20 minutes. The rats rested for 2 hours and then underwent oral gavage with the test compound. Cystometry was repeated approximately 60 minutes after administering the test compound. Another group of vehicle (saline) treated animals with hypertrophied bladders served as time/vehicle controls. Data analysis. Data are presented as the mean plus or minus standard error of mean. Appropriate statistical tests are indicated. Differences were considered statistically significant at p ⬍0.05. RESULTS
Detrusor strip contraction. Isolated detrusor strips exposed to 20 mM. KCl contract spontaneously with irregular amplitude and frequency. Exposure to 0.1 to 30 nM. CL-316243 produced a concentration dependent inhibition of contraction, as measured by the decrease in contraction area under the curve. Exposure to 200 nM. carbachol at the end of the experiment produced a robust contractile response (fig. 2, A). Concentration response data were expressed as the percent of the maximal contraction above zero with a pre-tension weight of 0.25 to 0.5 gm. The average concentration inhibiting 50% of maximal response was 2.65 ⫾ 0.36 nM. In 6 animals intrinsic activity relative to forskolin was 1 (fig. 2, B). Isovolumetric contractions. Animals were separated into 2 groups. Five contractions per animal were elicited and averaged in each group, followed by intravenous administration
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EFFICACY OF -ADRENERGIC RECEPTOR AGONIST IN RAT BLADDER
FIG. 2A. Tracing of rat bladder strip contraction. After contractions stabilized CL-316243 was added to tissue bath in increasing concentrations. Area under curve was monitored for 5-minute interval 30 minutes after each addition. Addition of 200 nM. carbachol at experiment end produced robust contraction. Total time of trace was 240 minutes. g, gm. B, concentration response curve for inhibition of rat bladder strip contraction by CL-316243. Line is best fit Michaelis-Menten logistic through data points. Concentration inhibiting 50% of response was 2.6 nM.
of 10 mg./kg. CL-316243 in 5 and saline in 4. At 15 minutes another 5 contractions were elicited and averaged. Mean pretreatment contraction amplitude was 47.6 ⫾ 4.2 and 46.3 ⫾ 7.2 cm. water in the drug and vehicle treated groups, respectively (p ⬎0.05). After dosing there was a significant mean decrease in contraction amplitude in the CL-316243 treated group to 34.5 ⫾ 3.3 cm. water (p ⬍0.05). There was no significant mean change in the vehicle treated group (46.1 ⫾ 7.2 cm. water). In addition to a decrease in contraction amplitude, mean baseline pressure in the CL-316243 treated group decreased 56.8% ⫾ 7.4% (p ⬍0.05). In the vehicle treated group mean baseline pressure decreased 3.3% ⫾ 19.2% (p ⬎0.05). Hyperreflexia. The average voiding interval in animals in which the bladder was perfused with saline containing 0.25% acetic acid was 47% shorter than that interval in animals that received saline perfusion alone (p ⬍0.05). Spontaneous contractions during the filling phase with acetic acid were also typically observed in these animals during cystometry. Administering CL-316243 0.01 to 30 mg./kg. CL-316243 intravenously in these animals significantly increased the voiding interval in a dose dependent manner (p ⬍0.05, fig. 3). Despite the continued exposure to acetic acid at doses above 0.1 mg./kg. the voiding interval was equivalent to the cystometry interval with saline alone. There was also a dose dependent decrease in voiding amplitude, an increase in bladder capacity and compliance, and a decrease in the void-
FIG. 3. Increase in voiding interval produced by increasing doses of CL-316243 in acetic acid induced bladder hyperreflexia model. Asterisk indicates ANOVA Student-Newman-Keuls test p ⬍0.05 versus vehicle.
ing pressure threshold (table 1). Furthermore, CL-316243 treatment produced a decrease in the frequency of spontaneous contractions during the filling phase. For example, immediately after intravenous dosing no evidence of spontaneous bladder contractions was observed at any dose tested (fig. 4). Saline dosing had no effect on the voiding interval or number of spontaneous bladder contractions. Hypertrophied bladder. Hypertrophied bladder weight was 0.8 to 1.1 gm. Cystometrography of hypertrophied bladders is characterized by small spontaneous bladder contractions during most of the filling phase. Exposure to CL-316243 produced a dose dependent decrease in the observed number of spontaneous bladder contractions (table 2). A maximum inhibition of spontaneous bladder contractions of ⫺54.9% was observed at the 3 mg./kg. dose and above. The dose that produced half of this maximum effect was 0.44 mg./kg. (fig. 5). While there was a trend toward a dose dependent decrease in voiding amplitude, voiding volume was unaffected. Figure 6 shows representative cystometry recordings before and after CL-316243 administration. DISCUSSION
Urge urinary incontinence and its associated bladder instability is often reported as the most prevalent form of incontinence.1 To date the most accepted pharmacological therapy for bladder instability is antimuscarinic agents to inhibit the action of acetylcholine on detrusor smooth muscle. Agents such as oxybutynin have been shown to have reasonably good efficacy,20 although anticholinergic side effects, including dry mouth,21 often limits usefulness. While recent reports of sustained formulations have indicated an improvement in the dry mouth profile,22 there have been no new successful pharmacological approaches for more than 25 years. The -adrenergic receptor has been known to mediate relaxation in several species, including humans.4, 5, 23 Li et al first described 1 and 2-adrenergic receptor subtypes in the guinea pig.2 Recently the 3-adrenergic receptor has been described in a number of animal species7–10 and in humans.11–13 Selective activation of the 3-adrenergic receptor has been shown to modulate bladder function and urine storage in the rat.13, 15, 16 Disruption of -adrenergic responsiveness may partially underlie bladder instability associated with aging.24 CL-316243 is a potent and selective agonist of the 3adrenergic receptor. The compound has been shown to bind to the rat 3 receptor with an inhibition constant of 1 M. and stimulate cyclic adenosine monophosphate production with an activation constant of 0.7 nM. CL-316243 did not
EFFICACY OF -ADRENERGIC RECEPTOR AGONIST IN RAT BLADDER
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TABLE 1. Effect of CL-316243 on micturition parameters in rat hyperreflexive bladders Mean Voiding Dose No. Interval ⫾ SEM (mg./kg.) Subjects (mins.)
Mean Voiding Amplitude ⫾ SEM (cm. water)
Mean Voiding Threshold ⫾ SEM (cm. water)
Mean Capacity ⫾ SEM (l.)
Mean Compliance ⫾ SEM (l./cm. water⫺1)
Vehicle 12 2.6 ⫾ 0.3 26.6 ⫾ 2.1 5.4 ⫾ 0.5 137 ⫾ 18 39 ⫾ 7 0.01 6 3.0 ⫾ 0.4* 28.1 ⫾ 2.5 NT NT NT 0.1 9 4.3 ⫾ 0.4* 21.1 ⫾ 2.8 NT NT NT 1.0 12 4.8 ⫾ 0.5* 20.0 ⫾ 1.1* NT NT NT 10 6–8 4.5 ⫾ 0.6* 20.2 ⫾ 1.2* 3.5 ⫾ 0.5* 227 ⫾ 37* 70 ⫾ 12* 30 7 5.0 ⫾ 0.7* 20.7 ⫾ 1.6 NT NT NT * Versus respective parameter for vehicle dosed (acetic acid/time control) ANOVA Student-Newman-Keuls test p ⬍0.05.
FIG. 4. Cystometry recordings using saline containing 0.25% acetic acid. Vehicle had no effect. There were spontaneous contractions during bladder filling. Arrow indicates administration of 1 ml./kg. saline intravenously (i.v.). Before CL-316243 delivery there were contractions but after CL-316243 (arrow) there was immediate inhibition of spontaneous contractions.
demonstrate significant binding to the 1 and 2-adrenergic receptors at concentrations up to 100 M.25 These CL-316243 findings are similar to those described by Takeda et al,15 suggesting that CL-316243 is a full agonist of the rat 3-adrenergic receptor and a potent direct inhibitor of bladder smooth muscle contractility. The tissues retained their ability to respond to muscarinic stimulation at the end of the experiment, suggesting that 3-adrenergic receptor stimulation may result in less impairment of normal contractility than antimuscarinic therapy. The effects of CL-316243 on in situ isovolumetric bladder contractions were consistent with the data generated from isolated strips. CL-316243 significantly inhibited contractions, suggesting that the compound has the direct ability to inhibit bladder smooth muscle contractility when administered systemically. Recently Birder and de Groat described a model of neurogenic bladder instability.17 Hyperreflexia was produced by adding acetic acid to the saline used for bladder cystometry. Data indicate that acetic acid stimulates sensory neurons that form the afferent arm of the micturition reflex. The result is a shortening of the voiding interval and the appearance of spontaneous contractions during the filling phase. Animals given CL-316243 responded with an increase in the voiding interval back to control values, a decrease in voiding threshold pressure, an increase in bladder capacity and compliance, and elimination of spontaneous bladder contractions. Spontaneous bladder contractions appeared to be more sensitive to compound than the voiding interval. At 0.01 mg./kg. there was no significant effect on the voiding interval and yet spontaneous bladder contractions were virtually eliminated. Similar findings were reported by Takeda et al.15 In their study of normal rats they noted a significant increase in the voiding interval and decrease in amplitude with 10 and 100 g./kg. CL-316243 intravenously. It was not clear in this study whether the effect of CL-316243 was through phosphorolation of phospholamban or some other mechanisms. The in vitro and isovolumetric contraction results suggest that activating the 3-adrenergic receptor results in a decrease in smooth muscle tone and excitability. Recent evidence also suggests that activating -adrenergic receptors in the blad-
Mean % Change in Contractions ⫾ SEM 4.5 ⫾ 24.2 ⫺97.8 ⫾ 1.8* ⫺94.9 ⫾ 2.0* ⫺85.5 ⫾ 4.6* ⫺92.7 ⫾ 2.6* ⫺86.0 ⫾ 8.3*
der facilitates conductance through the large conductance calcium dependent K channel via a protein kinase-A pathway.26 Activating this channel would lead to hyperpolarization of bladder smooth muscle and increased stability, and would significantly contribute to the underlying mechanism of action of 3-adrenergic receptor agonists. Oral administration of CL-316243 produced a dose dependent inhibition of spontaneous bladder contractions during cystometry filling of the hypertrophied bladders. While no statistically significant effects were observed, there was a trend toward a dose dependent decrease in voiding amplitude. Despite this finding no effect on voided volume was noted. Bladder instability due to outlet obstruction may arise from a myogenic and a neurogenic mechanism.27, 28 The simplest explanation for the observations using CL-316243 would be that the compound increases bladder compliance and capacity, resulting in an increased voiding interval by the direct inhibition of bladder smooth tone. The decrease in isovolumetric contraction amplitude and spontaneous bladder contractions would support this conclusion. Nevertheless, it cannot be ruled out that CL-316243 may have some additional peripheral or central neuronal effects. Furthermore, while it is well accepted that sympathetic stimulation of the bladder produces relaxation, others have shown that adrenergic innervation is elevated in spontaneously hypertensive rats and yet spontaneously hypertensive rat bladders are hyperactive relative to Wistar-Kyoto rat bladders.29, 30 If activation of adrenergic nerves relaxes bladder smooth muscle, it is difficult to reconcile why the bladders of spontaneously hypertensive rats are hyperactive despite greater adrenergic innervation. Clemow et al demonstrated that with an increase in adrenergic efferent innervation there is an increase in sensory afferent innervation in spontaneously hypertensive bladders.31 Their data suggest that the increase in sensory afferent nerve density may underly an enhanced voiding reflex. In addition, while there is an increase in adrenergic innervation and norepinephrine content, it is possible that post-synaptic -adrenergic receptor density is diminished, making smooth muscle less responsive to norepinephrine. CONCLUSIONS
Our study confirms the findings of others that the activation of 3-adrenergic receptors has an important role in normal rat bladder relaxation. Our data also show that a 3adrenergic receptor agonist may increase bladder capacity and inhibit the bladder instability associated with hyperreflexia and detrusor hypertrophy. Takeda et al reported that administering CL-316243 to anesthetized rats had only a slight influence on blood pressure and heart rate, whereas other non selective -adrenergic receptor agonists and cyclic adenosine monophosphate phosphodiesterase inhibitors often had profound cardiovascular sequelae.15 This finding suggest that 3-adrenergic receptor agonist may be useful for treating urge urinary incontinence without the side effects associated with antimuscarinic therapy or the potential cardiovascular liability associated with potassium channel openers.32–34
EFFICACY OF -ADRENERGIC RECEPTOR AGONIST IN RAT BLADDER
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TABLE 2. Effect of CL-316243 on micturition parameters in rat hypertrophied bladder Treatment (mg./kg.)
No. Subjects
Mean No. Contractions During Filling ⫾ SEM Pretreatment
Saline 6 0.1 7 0.3 8 1.0 9 3.0 6 10.0 7 * Versus pretreatment paired
Posttreatment
19.6 ⫾ 3.7 27.9 ⫾ 3.7 24.4 ⫾ 2.5 26.1 ⫾ 1.9 19.3 ⫾ 1.7 24.0 ⫾ 3.6 Student’s t test p
19.6 ⫾ 5.7 27.6 ⫾ 3.3 21.4 ⫾ 2.0 11.8 ⫾ 2.7* 10.2 ⫾ 4.1* 9.4 ⫾ 2.7* ⬍0.05.
Mean Cm. Water Voiding Amplitude ⫾ SEM Pretreatment
Posttreatment
Pretreatment
Posttreatment
Mean Bladder Wt. ⫾ SEM (gm.)
38.5 ⫾ 8.8 31.5 ⫾ 7.9 30.1 ⫾ 7.2 30.8 ⫾ 4.1 24.6 ⫾ 8.0 40.3 ⫾ 6.6
39.0 ⫾ 14.7 26.6 ⫾ 6.7 20.3 ⫾ 2.1 24.0 ⫾ 2.7 20.2 ⫾ 4.1 25.9 ⫾ 5.4
1.4 ⫾ 0.2 3.3 ⫾ 0.8 3.2 ⫾ 0.7 2.3 ⫾ 0.2 3.9 ⫾ 0.3 1.7 ⫾ 0.2
1.6 ⫾ 0.2 3.1 ⫾ 0.5 3.2 ⫾ 0.5 2.3 ⫾ 0.3 3.7 ⫾ 0.4 2.0 ⫾ 0.2
1.1 ⫾ 0.2 1.0 ⫾ 0.1 1.0 ⫾ 0.1 0.9 ⫾ 0.1 1.0 ⫾ 0.1 0.8 ⫾ 0.2
Mean Ml. Voided Vol. ⫾ SEM
activating 1 and 2 receptors results in a positive inotropic response or in heart failure cases when the relative ratios of -adrenergic receptor subtypes may be altered. Despite these mentioned precautions selective 3-adrenergic receptor agonists may represent a new useful class of agents for treating bladder instability. REFERENCES
FIG. 5. Dose response curve of inhibition of spontaneous bladder contractions (SBC) in rat hypertrophied bladder by CL-316243. Line is best fit Michaelis-Menten logistic through data points. Concentration inhibiting 50% of response was 0.44 mg./kg. Asterisk indicates ANOVA Student-Newman-Keuls test p ⬍0.05 versus vehicle.
FIG. 6. Cystometry recordings of bladder pressure and urine output volume in animal with bladder hypertrophy induced instability before treatment as control and 60 minutes after dosing with 1 mg./kg. CL-316243 orally (p.o.). Significant inhibition of spontaneous contractions occurred with slight but statistically insignificant decrease in voiding amplitude and no effect on voided volume.
In addition, it should be noted that, while the 3adrenergic receptor may have a minor role in the cardiovascular system of the rat, the contribution of the 3-adrenergic receptor to cardiovascular function may be species dependent.35 Specifically Gauthier et al suggested that selective activation of 3-adrenergic receptors results in a negative inotropic response in isolated human myocardium.36, 37 However, the net effect of 3-adrenergic receptor activation is still not clear in the setting of normal adrenergic tone, in which
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