ANTIMUSCARINIC AGENTS EXHIBIT LOCAL INHIBITORY EFFECTS ON MUSCARINIC RECEPTORS IN BLADDERAFFERENT PATHWAYS YONGTAE KIM, NAOKI YOSHIMURA, HITOSHI MASUDA, FERNANDO AND MICHAEL B. CHANCELLOR
ABSTRACT Objectives. To investigate the potential of antimuscarinic agents for sensory mechanisms in overactive bladder using intravesical instillation. Methods. Antimuscarinic agents were instilled intravesically in rats using two protocols. In the high-dose protocol, 5 mg atropine, oxybutynin, and dimethindene (M2-selective muscarinic receptor antagonist) were instilled into the bladder, and cystometric parameters, such as bladder capacity, intercontraction interval, pressure threshold, and maximal voiding pressure were monitored. In the low-dose protocol, 0.1 and 0.5 g/mL oxybutynin, trospium, tolterodine, and dimethindene were continuously infused into the bladder. The doses chosen were based on the calculated urine-excreted concentrations of trospium typically achieved from human oral treatment of 40 mg/day. The effect of carbachol with and without the low-dose agents was then assessed. Results. With the high-dose protocol, bladder capacity, intercontraction interval, and pressure threshold were increased when atropine and oxybutynin were instilled, but not when dimethindene was used. The maximal voiding pressure was not affected by any of the agents tested. In the low-dose protocol, none of the cystometric parameters were altered with antimuscarinic agents alone. The intercontraction interval decreased with intravesical carbachol (65% ⫾ 0.1% compared with baseline), but this was prevented with concomitant antimuscarinic agents. Conclusions. We have separated the local inhibitory effects of antimuscarinic agents during the storage phase from a decrease in voiding pressure. Intravesical instillation of antimuscarinic agents at clinically meaningful concentrations also suppressed carbachol-induced bladder overactivity. Antimuscarinic agents may be effective in treating overactive bladder, not only by suppression of muscarinic receptor-mediated detrusor muscle contractions, but also by blocking muscarinic receptors in bladder-afferent pathways. UROLOGY 65: 238–242, 2005. © 2005 Elsevier Inc.
isturbances of normal control of the bladder reflexes can lead to an overactive bladder.1 The most common pharmacotherapy for overactive bladThis study was supported by National Institutes of Health grants NIH HD39768 and DK067226. N. Yoshimura and F. de Miguel are paid consultants to Indevus. M. B. Chancellor is a paid consultant to Indevus, Pfizer, and Ortho McNeill. From the Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and Department of Urology, Chungbuk National University College of Medicine, Cheongju, Chungbuk, Republic of Korea Reprint requests: Michael B. Chancellor, M.D., Department of Urology, University of Pittsburgh School of Medicine, 3471 Fifth Avenue, Suite 700, Pittsburgh, PA 15213. E-mail: [email protected]
msx.upmc.edu Submitted: September 25, 2004, accepted (with revisions): November 15, 2004 © 2005 ELSEVIER INC. 238
ALL RIGHTS RESERVED
der is antimuscarinic agents. Conventional wisdom states that antimuscarinic agents act by blocking muscarinic receptors on the detrusor muscle that are stimulated by acetylcholine released from activated cholinergic (parasympathetic) nerves, thereby decreasing the ability of the bladder to contract. However, antimuscarinic drugs mainly act to increase the bladder capacity and decrease the urge to urinate during the storage phase, when normally the parasympathetic nerves are not active.2 Furthermore, antimuscarinic drugs are usually competitive antagonists, implying that when a massive release of acetylcholine occurs, such as during micturition, the effects of the drugs should be decreased. A paradox, therefore, exists between what can be explained by smooth muscle physiology and what 0090-4295/05/$30.00 doi:10.1016/j.urology.2004.11.021
TABLE I. Cystometric parameters—high-dose protocol Atropine (n ⴝ 6)
Oxybutynin (n ⴝ 6)
Dimethindene (n ⴝ 6)
BC (mL) ICI (s) PT (cm H2O) MVP (cm H2O)
0.33 ⫾ 0.05 405 ⫾ 44 6.1 ⫾ 1.1 28.7 ⫾ 2.1
0.47 ⫾ 0.05* 654 ⫾ 100* 9.4 ⫾ 1.6* 26.6 ⫾ 0.9
0.33 ⫾ 0.02 426 ⫾ 42 4.9 ⫾ 0.6 32.9 ⫾ 3.1
0.44 ⫾ 0.04* 604 ⫾ 69* 6.8 ⫾ 0.8* 30.2 ⫾ 2.9
0.39 ⫾ 0.04 565 ⫾ 82 5.0 ⫾ 0.6 28.1 ⫾ 3.2
0.38 ⫾ 0.08 610 ⫾ 126 6.2 ⫾ 1.5 27.5 ⫾ 3.7
KEY: BC ⫽ bladder capacity; ICI ⫽ intercontraction interval; MVP ⫽ maximal voiding pressure; PT ⫽ pressure threshold. * Statistically significant compared with baseline (P ⬍ 0.05).
is clinically observed regarding where antimuscarinic agents are producing their effects. Recently, Yoshida et al.3 demonstrated the basal release of acetylcholine from the urothelium that increases with age and stretch. Immunohistochemistry4 and reverse transcriptase-polymerase chain reaction5 techniques have recently been used to reveal M2 muscarinic receptors on human urothelium that are closely associated with sensory nerves.6 We investigated the muscarinic bladder sensory mechanism with intravesical instillation of antimuscarinic agents. We also simulated detrusor overactivity with intravesical instillation of carbachol chloride. The antimuscarinic agents were instilled at concentrations achievable in the bladder with typical oral dosages of trospium to determine whether they would have local effects on the muscarinic receptors involved in bladder-afferent pathways. MATERIAL AND METHODS The University of Pittsburgh Institutional Animal Care and Use Committee approved the experimental protocol. Adult female Sprague-Dawley rats (Hilltop Lab Animals, Scottsdale, Pa) weighing 250 to 300 g were used throughout the experiment.
CYSTOMETROGRAPHY Under urethane anesthesia (1.2 g/kg subcutaneous injection), a midline abdominal incision was made. The ureters were ligated and a transvesical catheter with a fire-flared tip (PE-50) was inserted into the dome of the bladder for bladder filling and pressure recording, and the abdomen was closed. A three-way stopcock was connected to the transvesical tube to monitor the bladder pressure during cystometry (continuous infusion of normal saline at rate of 0.04 mL/min). The voided volume, bladder capacity, intercontraction interval (ICI), pressure threshold, and maximal voiding pressure were monitored both before and after drug instillation.
EXPERIMENTAL PROTOCOLS Antimuscarinic agents were instilled intravesically in two protocols. In the high-dose protocol, 5 mg atropine sulfate (3.0 ⫻ 10⫺4 mol/L), 5 mg oxybutynin chloride (4.2 ⫻ 10⫺4 mol/L), and dimethindene maleate (4.1 ⫻ 10⫺4 mol/L) were dissolved in 30 mL saline. The concentrations chosen were based on reported clinical trials of intravesical instillation of these agents,7 except for dimethindene,8 an M2-selective muscarinic receptor antagonist, which was used for experimental purposes only. After baseline cystometry, 0.3 mL of the antimuscarinic agents was slowly instilled through a transvesical tube for 5 minutes and retained UROLOGY 65 (2), 2005
for 30 minutes. The bladder was then rinsed with saline and cystometric analysis was repeated for 1 hour. In the low-dose protocol, dimethindene maleate, oxybutynin chloride, tolterodine tartrate, and trospium chloride were used at concentrations of 0.1 g/mL (2.1 to 2.5 ⫻ 10⫺7 mol/L) and 0.5 g/mL (1.1 to 1.5 ⫻ 10⫺6 mol/L). The drug concentration used was based on the excreted urine concentration (0.1 to 0.5 g/mL) of trospium in humans after a steady-state oral dose of 40 mg/24 hr.9 We performed cystometry with continuous infusion of antimuscarinic agents (0.04 mL/min), followed by 30 M carbachol chloride with or without antimuscarinic agents (0.04 mL/min). We compared the cystometric parameters during continuous infusion of each drug for 1 hour duration. This was done for antimuscarinic agents alone and for carbachol with and without the antimuscarinic agents.
All data are presented as the mean ⫾ standard error of the mean. P ⬍0.05 was considered significant. The overall comparisons among groups were performed using Prism statistical software (GraphPad Software, San Diego, Calif). An unpaired t test was used to compare the cystometric parameters among the groups.
RESULTS With the high-dose protocol, bladder capacity, ICI, and pressure threshold all increased with atropine and oxybutynin instillation, but not with dimethindene. The maximal voiding pressure was not affected by any of the three agents (Table I). This effect appeared to be mediated predominately by the M3 muscarinic receptor, because dimethindene did not increase any of the cystometric parameters tested. In the low-dose experiments, we first demonstrated that the ICI decreased with intravesical carbachol chloride alone (Fig. 1A). By contrast, dimethindene, oxybutynin, tolterodine, and trospium did not affect the ICI (Fig. 1B). Similarly, when carbachol was instilled with any of the antimuscarinic agents, ICI did not differ significantly from the baseline values. Figure 2 illustrates the ratio of ICI after instillation of the four antimuscarinic agents compared with baseline. No statistically significant change in the ICI was observed after antimuscarinic drug infusion versus baseline saline infusion (ie, a drug treatment/baseline value ratio of 1.0; Fig. 2A). 239
FIGURE 1. Typical cystometric findings in low-dose protocol. (A) ICI decreased with intravesical carbachol chloride 30 M at rate of 0.04 mL/min. (B) All cystometric parameters were unchanged with any of four antimuscarinic agents, at either 0.1 or 0.5 g/mL. ICI returned to baseline when carbachol was instilled concurrently with antimuscarinic agents. CMG ⫽ cystometrogram.
These data, therefore, demonstrated that a low urine concentration of antimuscarinic agents had no effect on normal bladder storage and contractile function. The ICI of the bladder decreased with intravesical carbachol instillation by 65% ⫾ 0.1% compared with baseline (Fig. 2B). However, when carbachol was instilled with either 0.1 or 0.5 g/mL of each of the four antimuscarinic agents, the carbachol-induced ICI reduction was prevented (drug treatment/baseline ratio 1.0; Fig. 2B). The pressure threshold and maximal voiding pressure were not affected by instillation of either carbachol or 0.1 and 0.5 g/mL antimuscarinic agents (Table II). COMMENT The muscarinic receptors found in the human detrusor are of the M2 and M3 subtypes. M2 receptors predominate in number over M3 receptors.10,11 M3 receptors are mainly responsible for normal micturition contraction. The role of the M2 receptors in bladder function is less clear. M2 receptors may oppose sympathetically mediated (by way of 240
FIGURE 2. ICI ratio of antimuscarinic agents compared with baseline saline infusion in low-dose protocol. (A) ICI ratio to baseline cystometry unchanged after intravesical instillation of any of four antimuscarinic agents alone. (B) ICI ratio to baseline significantly decreased with intravesical carbachol chloride 30 M (65% ⫾ 0.1% compared with baseline) but returned to baseline with intravesical instillation of either 0.1 or 0.5 g/mL antimuscarinic agents. *P ⬍0.05. Carb ⫽ carbachol chloride; Oxyb1 ⫽ oxybutynin 0.1 g/mL; Oxyb5 ⫽ oxybutynin 0.5 g/mL; Trosp1 ⫽ trospium 0.1 g/mL; Trosp5 ⫽ trospium 0.5 g/mL; Dimet1 ⫽ dimethindene 0.1 g/mL; Dimet5 ⫽ dimethindene 0.5 g/mL; Tolter1 ⫽ tolterodine 0.1 g/ mL; Tolter5 ⫽ tolterodine 0.5 g/mL.
TABLE II. ICI ratio to baseline—low-dose protocol Dose (g/mL) Saline Dimethindene Oxybutynin Tolterodine Trospium
— 0.1 0.5 0.1 0.5 0.1 0.5 0.1 0.5
Carbachol ⴙ Antimuscarinic Agent
— 1.12 ⫾ 0.05 1.06 ⫾ 0.11 0.93 ⫾ 0.09 0.89 ⫾ 0.14 1.15 ⫾ 0.12 1.19 ⫾ 0.08 1.07 ⫾ 0.13 1.04 ⫾ 0.08
0.65 ⫾ 0.02 1.15 ⫾ 0.11* 0.93 ⫾ 0.13* 0.95 ⫾ 0.10* 1.00 ⫾ 0.13* 0.85 ⫾ 0.08* 0.96 ⫾ 0.14* 0.84 ⫾ 0.08* 0.99 ⫾ 0.09*
All experiments used 6 animals. KEY: ICI⫽intercontraction interval. * Statistically significant compared with baseline (P ⬍ 0.05).
beta-adrenoceptors) detrusor relaxation, because activation of M2 receptors results in inhibition of adenylyl cyclase.12 Muscarinic receptors are also found on bladder urothelial cells at high density.6 The role of the urothelium in bladder activation has attracted considerable interest.13 Yoshida et al.3 UROLOGY 65 (2), 2005
used microdialysis analysis of acetylcholine to show that basal acetylcholine release occurs in the human bladder. This release was resistant to tetrodotoxin and was considerably reduced when the urothelium was removed, suggesting that the released acetylcholine was non-neuronal in origin and at least partly generated by the urothelium. In our study, we instilled three nonspecific antimuscarinic drugs and one specific muscarinic antagonist into the bladder. Our aim was to tease out the cystometric characteristics that would indicate an afferent nerve (ICI) versus smooth muscle (maximal detrusor contraction pressure) effect. We have shown that atropine and oxybutynin, nonspecific muscarinic receptor antagonists, increased the bladder capacity and ICI without decreasing the detrusor contractility when applied intravesically at the same concentrations used in clinical trials (ie, high-dose protocol). The results also support a direct, local, afferent nerve action of antimuscarinic drugs. Because the drugs were instilled intravesically and their effects investigated for only a short duration immediately after instillation, a direct effect on the detrusor smooth muscle is unlikely. It is more probable that the agents affected the muscarinic receptors in the urothelial or suburothelial tissues. Muscarinic receptors have recently been identified in the urothelium and may be involved in the afferent pathways.6 Although oxybutynin has a local anesthetic effect in the bladder, de Wachter and Wyndaele14 reported that bladder C-fiber afferents responded significantly less to intravesical pressure and volume after oxybutynin instillation. Thus, our results support the hypothesis that oxybutynin desensitizes C-fiber afferents by acting on muscarinic receptors in the urothelium or on afferent nerves. We recognize that our hypothesis is hinged on the carbachol-induced bladder contractions and that it may be controversial. Alternative opinions include that all the drugs reaching the urothelium are from the systemic circulation and not by urine uptake. In this study, carbachol-induced detrusor overactivity was suppressed with all four antimuscarinic agents tested, including dimethindene. Thus, antimuscarinic agents could be effective in treating detrusor overactivity by local inhibitory effects by acting on the urothelium or suburothelial afferent nerve terminals or a local anesthetic effect. Trospium is a quaternary amine and has not been shown to cross the blood-brain barrier in humans. When taken orally, up to 60% of metabolically active, absorbed trospium is excreted in the urine, allowing a potential local intravesical effect.9 The doses of the drugs used in the low-dose protocol study were thus based on the urine-excreted concentration of trospium at a human steady-state oral dose of 40 mg/24 hr. The physioUROLOGY 65 (2), 2005
logic effect seen in this study could possibly represent clinical physiologic concentrations and effects. One potential weakness of this study was that only high and low doses of intravesical antimuscarinic agents were used rather than a dose-response curve. However, the doses chosen were based on what is used in clinical practice for intravesical drug instillation (high dose) or the urine concentration achieved with orally prescribed antimuscarinic agents (low dose). Because dimethindene did not affect bladder capacity and ICI in normal voiding even at a nonphysiologic high dose, but was able to suppress carbachol-induced detrusor overactivity at the low concentration, M2 receptors may not be involved in the afferent pathways during normal voiding, but could be involved in these pathways in a situation with high acetylcholine release. CONCLUSIONS Antimuscarinic drugs may be effective in treating detrusor overactivity, not only by suppression of muscarinic receptor-mediated detrusor muscle contraction, but also by local inhibitory effects on muscarinic receptors in bladder-afferent pathways. Antimuscarinic agents that are excreted into the urine may have an effect on the afferent nerves located near the urothelium. REFERENCES 1. Abrams P, Cardozo L, Fall M, et al: The standardisation of terminology of lower urinary tract function: report from the Standardisation Sub-Committee of the International Continence Society. Neurourol Urodyn 21: 167–178, 2002. 2. de Groat WC, Booth AM, and Yoshimura N: Neurophysiology of micturition and its modifications in animal models of human disease, in Maggi CA (Ed): The Autonomic Nervous System: Nervous Control of the Urogenital System. London, Harwood, 1993, vol 6, pp 227–289. 3. Yoshida M, Miyamae K, Iwashita H, et al: Management of detrusor dysfunction in the elderly: changes in acetylcholine and adenosine triphosphate release during aging. Urology 63: 17–23, 2004. 4. Haberberger R, Scholz R, Kummer W, et al: M2-receptor subtype does not mediate muscarine-induced increases in [Ca(2⫹)](i) in nociceptive neurons of rat dorsal root ganglia. J Neurophysiol 84: 1934 –1941, 2000. 5. de Miguel F, Nagatomi J, Torimoto K, et al: Differential expression of muscarinic receptor subtype mRNA in two rat models of neurogenic bladder. Scottsdale, Arizona, Society for Urodynamics and Female Urology, 2004. 6. Hawthorn MH, Chapple CR, Cock M, et al: Urothelium-derived inhibitory factor(s) influences on detrusor muscle contractility in vitro. Br J Pharmacol 129: 416 – 419, 2000. 7. Deaney C, Glickman S, Gluck T, et al: Intravesical atropine suppression of detrusor hyperreflexia in multiple sclerosis. J Neurol Neurosurg Psychiatry 65: 957–958, 1998. 8. Bohme TM, Keim C, Kreutzmann K, et al: Structureactivity relationships of dimethindene derivatives as new M2selective muscarinic receptor antagonists. J Med Chem 46: 856 – 867, 2003. 9. Guay DR: Clinical pharmacokinetics of drugs used to treat urge incontinence. Clin Pharmacokinet 42: 1243–1285, 2003. 241
10. Fetscher C, Fleichman M, Schmidt M, et al: M(3) muscarinic receptors mediate contraction of human urinary bladder. Br J Pharmacol 136: 641– 643, 2002. 11. Chess-Williams R, Chapple CR, Yamanishi T, et al: The minor population of M3-receptors mediate contraction of human detrusor muscle in vitro. J Auton Pharmacol 21: 243–248, 2001. 12. Yamanishi T, Chapple CR, Yasuda K, et al: The role of M2 muscarinic receptor subtypes in mediating contraction of
the pig bladder base after cyclic adenosine monophosphate elevation and/or selective M3 inactivation. J Urol 167: 397– 401, 2002. 13. Andersson KE: Bladder activation: afferent mechanisms. Urology 59: 43–50, 2002. 14. de Wachter S, and Wyndaele JJ: Intravesical oxybutynin: a local anesthetic effect on bladder C afferents. J Urol 169: 1892–1895, 2003.
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