- Email: [email protected]
Effects of Tolterodine on Afferent Neurotransmission in Normal and Resiniferatoxin Treated Conscious Rats
Effects of Tolterodine on Afferent Neurotransmission in Normal and Resiniferatoxin Treated Conscious Rats Petter Hedlund, Tomi Streng, Tack Lee and Karl-Erik Andersson* From the Department of Clinical and Experimental Pharmacology, Lund University Hospital (PH, TS, TL, KEA), Lund, Sweden, and Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine (KEA), Winston Salem, North Carolina
Purpose: The beneficial effects of antimuscarinics on detrusor overactivity and overactive bladder syndrome are exerted during bladder filling, when there is no parasympathetic outflow from the spinal cord. We tested the hypothesis that, if tolterodine exerts some of its effects on afferent nerves, the functional elimination of C-fiber afferents should affect the actions of the drug on urodynamic parameters. Materials and Methods: The study was performed in normal female Sprague Dawley rats and rats treated with resiniferatoxin to eliminate vanilloid sensitive afferent nerves. Tolterodine was given intravenously to normal and resiniferatoxin treated animals. To test if tolterodine at the doses used affects efferent neurotransmission the drug was given to normal and resiniferatoxin treated animals in which detrusor activity was induced by apomorphine. Results: In resiniferatoxin treated animals (0.3 mg kg⫺1 subcutaneously) the mean micturition interval and volume, and mean residual volume increased significantly compared to those in controls. Baseline and micturition pressures in control and resiniferatoxin treated animals were similar, whereas threshold pressures were higher in resiniferatoxin treated animals. In controls 10 g kg⫺1 tolterodine administered intravenously increased the mean micturition interval, bladder capacity and micturition volume. In resiniferatoxin treated rats 1 and 10 g kg⫺1 tolterodine increased the mean micturition interval, bladder capacity and micturition volume. Subcutaneous administration of 100 g kg⫺1 apomorphine induced detrusor overactivity in all rats. The AUC of intravesical pressure during the initial 10 minutes from the start of detrusor overactivity showed no difference between normal and resiniferatoxin treated rats with or without tolterodine pretreatment. Conclusions: Tolterodine increased the micturition interval and bladder capacity in controls and in resiniferatoxin treated animals, suggesting that these effects were exerted independently of resiniferatoxin sensitive afferents. Tolterodine did not decrease the contractile effects of apomorphine at the doses used, suggesting that the drug had no effect on efferent neurotransmission during voiding. Key Words: bladder; tolterodine; urodynamics; nerve fibers, unmyelinated; rats, Sprague-Dawley
dowed with muscarinic receptors and participating in afferent signaling. Extraneuronally generated acetylcholine or acetylcholine leaking from nerves during the storage phase may further stimulate the contraction of detrusor myocytes, which in bladders with detrusor overactivity already have increased myogenic activity.8 Enhanced myogenic contractions can generate an enhanced afferent signal, contributing to urgency and/or initiation of the micturition reflex.3 However, effects on suburothelial interstitial cells (myofibroblasts), on which muscarinic receptors can be demonstrated,9 may also contribute. We tested the hypothesis that, if tolterodine exerts some of its effects on afferent nerves as suggested,10 the functional elimination of C-fiber afferents should affect the actions of the drug on urodynamic parameters. Therefore, we treated rats with RTX systemically at a dose that was previously shown to functionally eliminate vanilloid sensitive afferents (C fibers). To establish if tolterodine at the doses used had effects on the efferent side of the micturition reflex, we studied the effects of the drug on apomorphine stimulated detrusor activity. Apomorphine is believed to activate the bladder independently of afferent activation, initiating activity at a central site of action, and increase the parasympathetic outflow from the spinal cord.11
oluntary or involuntary bladder contraction involves stimulation of the muscarinic receptors on detrusor smooth muscle by acetylcholine released from activated cholinergic nerves.1 Antimuscarinic agents at clinically recommended doses have little effect on voiding contractions2 and they act mainly during the bladder storage phase,3 during which there is normally no parasympathetic outflow from the spinal cord.4 Supporting this, antimuscarinic agents were shown to decrease bladder tone during storage and increase cystometric bladder capacity.2 Baseline release of acetylcholine from nonneuronal (urothelial) as well as neuronal sources was demonstrated in isolated human detrusor muscle.5,6 It was suggested that this release, which is increased by muscle stretching and in the aging bladder, contributes to detrusor overactivity and overactive bladder symptoms by eventually increasing bladder afferent activity during storage.7 This may be caused by a direct effect on suburothelial afferents or on other structures en-
V
Submitted for publication September 27, 2006. Study received Lund/Malmö Animal Ethics Committee approval. * Correspondence: Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Medical Center Blvd., Winston Salem, North Carolina 27157 (telephone: 336-713-1195; FAX: 336-713-7290; e-mail: [email protected]).
0022-5347/07/1781-0326/0 THE JOURNAL OF UROLOGY® Copyright © 2007 by AMERICAN UROLOGICAL ASSOCIATION
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Vol. 178, 326-331, July 2007 Printed in U.S.A. DOI:10.1016/j.juro.2007.03.006
EFFECTS OF TOLTERODINE ON AFFERENT NEUROTRANSMISSION METHODS Animals A total of 32 female or male Sprague-Dawley rats weighing approximately 300 gm were used. The animals were kept under clean conditions at the Biomedical Centre animal facility at Lund University Hospital and they had free access to pellets and water during a 12:12-hour dark-light cycle. Experimental procedures were approved by the Lund/ Malmö Animal Ethics Committee.
Surgical Procedures Rats were anesthetized with ketamine (Ketalar®, 75 mg kg⫺1 intraperitoneally) and xylazine (Rompun®, 15 mg kg⫺1 intraperitoneally), and placed on a thermoregulated surgical area. After an abdominal incision was made a polyethylene collar fitted PE-50 catheter (Clay-Adams, Parsippany, New Jersey) was inserted into the bladder dome and held in place with a purse-string suture. The free end of the catheter was sealed. A PE-50 polyethylene catheter was elongated to about 1.5 times its original length at the tip of the inserting side and filled with heparinized saline (100 IU ml⫺1). At the same time that the bladder catheter was implanted the elongated catheter was inserted into the femoral vein. At the same session another PE-50 catheter was filled with heparinized saline and introduced subcutaneously from neck to lower back. All catheters were tunneled subcutaneously, anchored to the skin of the neck and closed.
RTX Treatment To induce C-fiber desensitization RTX (0.3 mg kg⫺1) dissolved in dimethyl sulfoxide was injected subcutaneously into the rats during anesthesia, as described, 3 days before the experiment. Control animals received corresponding volumes per weight of dimethyl sulfoxide given subcutaneously.
Experimental Design Cystometry was performed without anesthesia 3 days after catheter implantation. The conscious rats were placed in metabolic cages without restraint and the bladder catheters were connected via a T tube to a P23 DC pressure transducer (Statham Instruments, Oxnard, California) and a CMA 100 microinjection pump (Carnegie Medicine AB, Solna, Sweden). Micturition volumes were recorded with a fluid collector connected to an FT03 D force displacement transducer (Grass Instrument, Quincy, Massachusetts). Room temperature saline was infused into the bladder continuously at a rate of 10 ml hour–1. Pressures and micturition volumes were recorded continuously with AcqKnowledge 3.8.1 software and an MP100 data acquisition system (Biopac Systems, Santa Barbara, California) connected to a Model 7E polygraph (Grass Instrument). At the beginning of cystometry the bladder was emptied via the bladder catheter. Apomorphine (100 g kg⫺1 subcutaneously) was used to stimulate efferent activity and induce detrusor overactivity. Tolterodine was given intravenously to controls (10 g kg⫺1) and RTX treated (1 and 10 g kg⫺1) animals before and after apomorphine administration. The doses chosen were based on published information.10
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Evaluations and Calculations Urodynamic parameters were investigated, including micturition pressure (maximum bladder pressure during micturition), baseline pressure (the lowest bladder pressure during filling), threshold pressure (bladder pressure immediately before micturition), bladder capacity (residual volume at the most recent previous micturition plus the volume of infused saline at micturition), micturition volume (volume of expelled urine), residual volume (bladder capacity minus micturition volume), the intercontraction interval and the AUC of intravesical pressure. Reproducible micturition cycles, together approximately corresponding to a 30-minute period, were recorded before drug administration. They served as baseline values to be compared with the 3 micturition cycles in the first 30-minute period after the administration of test substances. Comparisons of the means of all groups were performed with 1-way ANOVA (Holm-Sidak). Pairwise comparisons were made by Student’s t test. All statistical calculations were based on the number of individual animals and significant differences were considered at p ⬍0.05. RESULTS Effect of RTX Treatment on Micturition Parameters After RTX treatment (0.3 mg kg⫺1 subcutaneously) in 22 animals 8 showed decompensated bladders with dribbling incontinence. These animals were used only to assess the effects of RTX and tolterodine on apomorphine induced activity. Three rats showed intermittent dribbling incontinence as well as complete voiding cycles. One RTX treated animal and 1 control were excluded due to catheter problems. One RTX treated animal died of postoperative complications. Compared to the 9 controls RTX treatment in 12 rats increased the micturition interval (p ⬍0.001), bladder capacity (p ⬍0.0001), micturition volume (p ⬍0.05), threshold pressure p ⬍0.05) and residual volume (p ⬍0.05, fig. 1). There were no significant effects on mean ⫾ SD baseline pressure, which was 10.2 ⫾ 0.6 and 10.4 ⫾ 0.8 cm H2O in controls and RTX treated rats, respectively. No statistical difference was observed for micturition pressure between controls and RTX treated rats (98.0 ⫾ 8.6 and 72.2 ⫾ 10.0 cm H2O, respectively, p ⬍0.06). Effect of Tolterodine on Micturition Parameters in Control and RTX Treated Rats In controls intravenous administration of 10 g kg⫺1 tolterodine increased the mean micturition interval (p ⬍0.05), bladder capacity (p ⬍0.05), micturition volume (p ⬍0.05) and residual volume (p ⬍0.05), and decreased micturition pressure (p ⬍0.001, fig. 2). Baseline pressure was 9.4 ⫾ 1.0 and 8.4 ⫾ 2.6 cm H2O in 6 rats before and after drug administration, respectively. Threshold pressure was also unaffected by tolterodine in 6 rats (31.2 ⫾ 2.6 and 30.0 ⫾ 1.5 cm H2O, respectively). In 6 RTX treated rats tolterodine (1 g kg⫺1 intravenously) increased the mean micturition interval from 10.6 ⫾ 3.7 to 13.6 ⫾ 5.5 minutes (p ⬍0.05), increased bladder capacity from 1.6 ⫾ 0.3 to 1.9 ⫾ 0.3 ml (p ⬍0.05) and increased micturition volume from 1.5 ⫾ 0.3 to 1.8 ⫾ 0.2 ml (p ⬍0.05, fig. 3). At 10 g/kg given intravenously in 6 rats the micturition interval, bladder capacity and micturition volume
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EFFECTS OF TOLTERODINE ON AFFERENT NEUROTRANSMISSION
FIG. 1. Effects of subcutaneous administration of 0.3 mg kg⫺1 RTX on micturition parameters in 9 controls (filled bars) and 12 RTX treated rats (open bars). Single asterisk indicates unpaired t test p ⬍0.05. Double asterisks indicate unpaired t test p ⬍0.01. Triple asterisk indicate unpaired t test p ⬍0.001.
were also significantly increased (figs. 3 and 4). There were no significant effects on residual volume, baseline, threshold and micturition pressures at either investigated tolterodine dose. After 10 g kg⫺1 tolterodine mean baseline pressure was 8.6 ⫾ 1.0 cm H2O in 6 rats in comparison to 10.2 ⫾ 1.4 cm H2O in 6 without the antimuscarinic agent. Mean threshold pressure was 39.4 ⫾ 5.1 and 37.6 ⫾ 6.4 cm H2O, and micturition pressure was 73.5.4 ⫾ 15.7 and 61.8 ⫾ 18.2 cm H2O in 6 rats each before and after 10 g kg⫺1 tolterodine, respectively.
Effect of Tolterodine on Apomorphine Induced Overactivity in Control and RTX Treated Rats Subcutaneous administration of 100 g kg⫺1 apomorphine induced characteristic detrusor overactivity with dribbling incontinence in controls and in RTX treated rats. Mean maximal pressure and AUC were almost identical in controls and RTX treated animals after apomorphine (100 g kg⫺1) (fig. 4). Tolterodine (1 and 10 g kg⫺1) had no effect on apomorphine induced activity (fig. 5).
FIG. 2. Micturition parameters in 6 controls before (filled bars) and after (open bars) intravenous administration of 10 g kg tolterodine. Single asterisk indicates paired t test p ⬍0.05. Double asterisks indicate paired t test p ⬍0.01.
EFFECTS OF TOLTERODINE ON AFFERENT NEUROTRANSMISSION
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FIG. 3. Original tracings of intravesical pressure and voided volumes during continuous cystometry in 2 RTX treated rats before (A) and after (C) intravenous administration of 1 g kg⫺1 (B) and 10 g kg⫺1 (D) tolterodine.
DISCUSSION The current experiments showed that the RTX treatment had significant effects on bladder function, as reflected by cystometric parameters and the development of dribbling incontinence in some animals. RTX increased the mean micturition interval, bladder capacity and voided volume, suggesting that structures involved in afferent signaling were affected (desensitized). However, functional elimination of RTX sensitive afferents did not affect the actions of tolterodine, suggesting that they were exerted independently of this type of C fibers. However, since TRPV1, the receptor for RTX, is located not only on a subpopulation of afferent nerves,12 but also on urothelium,13 interstitial cells and even on detrusor
myocytes,14 there may be effects not only on afferent nerves. It is well known that RTX desensitizes afferent nerves.15 However, actions on extraneuronal structures important for afferent signaling and sensitive to muscarinic receptor stimulation may not undergo desensitization.16 As shown in the current study, tolterodine at the concentrations used had no effect on apomorphine induced detrusor activity. As reported previously, apomorphine is believed to activate the bladder independently of afferent activation, initiating activity at a central site of action, and increase the parasympathetic outflow from the spinal cord11 with a consequent release of large amounts of acetylcholine at the cholinergic nerve terminals and the direct stimulation of
FIG. 4. Micturition parameters before (black bars) and after (white bars) intravenous administration of 10 g kg tolterodine in 6 rats given 0.3 mg kg RTX subcutaneously. Single asterisk indicates paired t test p ⬍0.05.
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FIG. 5. Apomorphine induced detrusor overactivity in 5 controls and 8 rats treated with 0.3 mg kg RTX subcutaneously before (filled bars) and after (open bars) intravenous administration of 10 g kg⫺1 tolterodine. s., second.
detrusor myocytes, resulting in a voiding contraction. Since the effects of tolterodine and acetylcholine are competitive, the amounts of tolterodine used in the current study may not have been sufficient to inhibit the acetylcholine effect during voiding. The lack of an effect of the antimuscarinic on apomorphine induced activity supports the assumption that efferent neurotransmission was not affected. This is different from what happens during filling, when only small amounts of acetylcholine are available to the receptors. This implies that in this situation the tolterodine concentrations achieved at the current dose are sufficient to inhibit the acetylcholine effects. The finding that the effects of tolterodine were independent of RTX sensitive afferent nerves are in apparent disagreement with the results of Yokoyama et al, who concluded that the actions of tolterodine depended on the suppression of C-fiber bladder afferent activity.10 They found in a rat model of increased detrusor activity caused by occlusion of the middle cerebral artery that bladder capacity was markedly decreased after occlusion in RTX at 0.3 mg kg⫺1, which is the same dose as in the current investigation, and in vehicle treated rats. Low tolterodine doses of 0.2 and 2 nM kg⫺1 (0.1 and 1 g kg⫺1, respectively) significantly increased bladder capacity in vehicle treated rats without increasing residual volume but had no effects in RTX treated rats. They suggested that at low doses tolterodine exerts an inhibitory effect on C-fiber bladder afferent nerves, thereby improving bladder capacity during the storage phase. The reasons for the discrepancy in results between the current study and that of Yokoyama et al are unclear but they may depend on the models used.
Kim et al administered muscarinic receptor agonists and antagonists intravesically to rats and were able to separate the local inhibitory effects of antimuscarinic agents during the storage phase from a decrease in voiding pressure.17 They found that intravesical instillation of antimuscarinics at clinically meaningful concentrations suppressed carbachol induced detrusor overactivity. Kim et al concluded that antimuscarinic agents may be effective for treating overactive bladder, not only by suppression of muscarinic receptor mediated detrusor muscle contractions, but also by blocking muscarinic receptors in bladder-afferent pathways. Since the effects of antimuscarinics are believed to be attributable to a competitive antagonism of acetylcholine at muscarinic receptor sites and the direct involvement of RTX sensitive afferents seems controversial, possible sites of action other than C fibers may be discussed. M2 and M3 receptor immunoreactivity was observed in the urothelium, nerve fibers and detrusor layers in the human bladder.9 A significant increase in suburothelial myofibroblast-like M2 and M3 receptor immunoreactivity was seen in patients with idiopathic detrusor overactivity. This immunoreactivity significantly correlated with the urgency score and M2 receptor immunoreactivity also correlated with the frequency score. The increase in muscarinic receptor immunostaining in myofibroblast-like cells and its correlation with clinical scores suggested to us a potential role in pathophysiological mechanisms and in the therapeutic effect of antimuscarinic agents. There may be differences in muscarinic receptor distribution, including subtypes, between the rat and the human bladder. However, muscarinic receptors can be expected in
EFFECTS OF TOLTERODINE ON AFFERENT NEUROTRANSMISSION the same structures in the rat bladder as in the human bladder. The current results suggest that tolterodine, which blocks M2 and M3 receptors, exerted an effect on afferent signaling from the bladder, leading to decreased voiding frequency, and consequent increases in bladder capacity and voided volumes independent of RTX sensitive afferents. It may be assumed that there is a release of small amounts of acetylcholine from nerves or from extraneuronal sources, eg urothelium, during the bladder storage phase5,6 and the released transmitter stimulates afferent signaling. The low concentrations of released acetylcholine can be effectively counteracted by tolterodine at concentrations that have no effect on detrusor contraction. However, this is not the case when there is a massive release of the transmitter, as can be expected during apomorphine stimulated voiding.
3. 4.
5.
6.
7.
CONCLUSIONS
8.
The current experiments show that tolterodine increases the micturition interval and bladder capacity in controls and in RTX treated animals. Thus, functional elimination of RTX sensitive afferents did not affect these actions of tolterodine, suggesting that they were exerted independently of this population of C fibers. This does not necessarily mean that the effect was not exerted on the afferent side of the micturition reflex since A␦-fiber mediated activity may be less affected by RTX treatment, and urothelium and interstitial cells may not be desensitized. Because tolterodine did not decrease the contractile effects of apomorphine, the drug at the doses used seemed to have no effect on efferent neurotransmission during voiding. Thus, tolterodine at low doses exerted an inhibitory effect on afferent neurotransmission without influencing the effect of efferent stimulation during voiding.
9.
10.
11.
12.
13.
14.
Abbreviations and Acronyms RTX ⫽ resiniferatoxin TRPV1 ⫽ transient receptor potential channel V1 REFERENCES 1.
2.
Andersson KE and Wein AJ: Pharmacology of the lower urinary tract: basis for current and future treatments of urinary incontinence. Pharmacol Rev 2004; 56: 581. Finney SM, Andersson K-E, Gillespie JI and Stewart LH: Antimuscarinic drugs in detrusor overactivity: motor or sensory actions? BJU Int 2006; 98: 503.
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16.
17.
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Andersson KE: Antimuscarinics for treatment of overactive bladder. Lancet Neurol 2004; 3: 46. Morrison J, Birder L, Craggs M, de Groat W, Downie J, Drake M et al: Incontinence. In: 3rd International Consultation on Incontinence. Edited by P Abrams, L Cardozo, S Khoury and A Wein. Plymouth, United Kingdom: Health Publication 2005; pp 363– 422. Yoshida M, Miyamae K, Iwashita H, Otani M and Inadome A: Management of detrusor dysfunction in the elderly: changes in acetylcholine and adenosine triphosphate release during aging. Urology, suppl., 2004; 63: 17. Yoshida M, Inadome A, Maeda Y, Satoji Y, Masunaga K, Sugiyama Y et al: Non-neuronal cholinergic system in human bladder urothelium. Urology 2006; 67: 425. Andersson KE and Yoshida M: Antimuscarinics and the overactive detrusor—which is the main mechanism of action? Eur Urol 2003; 43: 1. Brading AF: A myogenic basis for the overactive bladder. Urology, suppl., 1997; 50: 57. Mukerji G, Yiangou Y, Grogono J, Underwood J, Agarwal SK, Khullar V et al: Localization of M2 and M3 muscarinic receptors in human bladder disorders and their clinical correlations. J Urol 2006; 176: 367. Yokoyama O, Yusup A, Miwa Y, Oyama N, Aoki Y and Akino H: Effects of tolterodine on an overactive bladder depend on suppression of C-fiber bladder afferent activity in rats. J Urol 2005; 174: 2032. Kontani H, Inoue T and Sakai T: Effects of apomorphine on urinary bladder motility in anesthetized rats. Jpn J Pharmacol 1990; 52: 59. Avelino A, Cruz C, Nagy I and Cruz F: Vanilloid receptor 1 expression in the rat urinary tract. Neuroscience 2002; 109: 787. Birder LA, Kanai AJ, de Groat WC, Kiss S, Nealen ML, Burke NE et al: Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells. Proc Natl Acad Sci U S A 2001; 98: 13396. Ost D, Roskams T, Van Der Aa F and De Ridder D: Topography of the vanilloid receptor in the human bladder: more than just the nerve fibers. J Urol 2002; 168: 293. Cortright DN and Szallasi A: Biochemical pharmacology of the vanilloid receptor TRPV1. An update. Eur J Biochem 2004; 271: 1814. Gevaert T, Vandepitte J, Ost D, Nilius B and De Ridder D: Autonomous contractile activity in the isolated rat bladder is modulated by a TRPV1 dependent mechanism. Neurourol Urodyn 2006; Epub. Kim Y, Yoshimura N, Masuda H, de Miguel F and Chancellor MB: Antimuscarinic agents exhibit local inhibitory effects on muscarinic receptors in bladder-afferent pathways. Urology 2005; 65: 238.
Purpose: The beneficial effects of antimuscarinics on detrusor overactivity and overactive bladder syndrome are exerted during bladder filling, when there is no parasympathetic outflow from the spinal cord. We tested the hypothesis that, if tolterodine exerts some of its effects on afferent nerves, the functional elimination of C-fiber afferents should affect the actions of the drug on urodynamic parameters. Materials and Methods: The study was performed in normal female Sprague Dawley rats and rats treated with resiniferatoxin to eliminate vanilloid sensitive afferent nerves. Tolterodine was given intravenously to normal and resiniferatoxin treated animals. To test if tolterodine at the doses used affects efferent neurotransmission the drug was given to normal and resiniferatoxin treated animals in which detrusor activity was induced by apomorphine. Results: In resiniferatoxin treated animals (0.3 mg kg⫺1 subcutaneously) the mean micturition interval and volume, and mean residual volume increased significantly compared to those in controls. Baseline and micturition pressures in control and resiniferatoxin treated animals were similar, whereas threshold pressures were higher in resiniferatoxin treated animals. In controls 10 g kg⫺1 tolterodine administered intravenously increased the mean micturition interval, bladder capacity and micturition volume. In resiniferatoxin treated rats 1 and 10 g kg⫺1 tolterodine increased the mean micturition interval, bladder capacity and micturition volume. Subcutaneous administration of 100 g kg⫺1 apomorphine induced detrusor overactivity in all rats. The AUC of intravesical pressure during the initial 10 minutes from the start of detrusor overactivity showed no difference between normal and resiniferatoxin treated rats with or without tolterodine pretreatment. Conclusions: Tolterodine increased the micturition interval and bladder capacity in controls and in resiniferatoxin treated animals, suggesting that these effects were exerted independently of resiniferatoxin sensitive afferents. Tolterodine did not decrease the contractile effects of apomorphine at the doses used, suggesting that the drug had no effect on efferent neurotransmission during voiding. Key Words: bladder; tolterodine; urodynamics; nerve fibers, unmyelinated; rats, Sprague-Dawley
dowed with muscarinic receptors and participating in afferent signaling. Extraneuronally generated acetylcholine or acetylcholine leaking from nerves during the storage phase may further stimulate the contraction of detrusor myocytes, which in bladders with detrusor overactivity already have increased myogenic activity.8 Enhanced myogenic contractions can generate an enhanced afferent signal, contributing to urgency and/or initiation of the micturition reflex.3 However, effects on suburothelial interstitial cells (myofibroblasts), on which muscarinic receptors can be demonstrated,9 may also contribute. We tested the hypothesis that, if tolterodine exerts some of its effects on afferent nerves as suggested,10 the functional elimination of C-fiber afferents should affect the actions of the drug on urodynamic parameters. Therefore, we treated rats with RTX systemically at a dose that was previously shown to functionally eliminate vanilloid sensitive afferents (C fibers). To establish if tolterodine at the doses used had effects on the efferent side of the micturition reflex, we studied the effects of the drug on apomorphine stimulated detrusor activity. Apomorphine is believed to activate the bladder independently of afferent activation, initiating activity at a central site of action, and increase the parasympathetic outflow from the spinal cord.11
oluntary or involuntary bladder contraction involves stimulation of the muscarinic receptors on detrusor smooth muscle by acetylcholine released from activated cholinergic nerves.1 Antimuscarinic agents at clinically recommended doses have little effect on voiding contractions2 and they act mainly during the bladder storage phase,3 during which there is normally no parasympathetic outflow from the spinal cord.4 Supporting this, antimuscarinic agents were shown to decrease bladder tone during storage and increase cystometric bladder capacity.2 Baseline release of acetylcholine from nonneuronal (urothelial) as well as neuronal sources was demonstrated in isolated human detrusor muscle.5,6 It was suggested that this release, which is increased by muscle stretching and in the aging bladder, contributes to detrusor overactivity and overactive bladder symptoms by eventually increasing bladder afferent activity during storage.7 This may be caused by a direct effect on suburothelial afferents or on other structures en-
V
Submitted for publication September 27, 2006. Study received Lund/Malmö Animal Ethics Committee approval. * Correspondence: Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Medical Center Blvd., Winston Salem, North Carolina 27157 (telephone: 336-713-1195; FAX: 336-713-7290; e-mail: [email protected]).
0022-5347/07/1781-0326/0 THE JOURNAL OF UROLOGY® Copyright © 2007 by AMERICAN UROLOGICAL ASSOCIATION
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Vol. 178, 326-331, July 2007 Printed in U.S.A. DOI:10.1016/j.juro.2007.03.006
EFFECTS OF TOLTERODINE ON AFFERENT NEUROTRANSMISSION METHODS Animals A total of 32 female or male Sprague-Dawley rats weighing approximately 300 gm were used. The animals were kept under clean conditions at the Biomedical Centre animal facility at Lund University Hospital and they had free access to pellets and water during a 12:12-hour dark-light cycle. Experimental procedures were approved by the Lund/ Malmö Animal Ethics Committee.
Surgical Procedures Rats were anesthetized with ketamine (Ketalar®, 75 mg kg⫺1 intraperitoneally) and xylazine (Rompun®, 15 mg kg⫺1 intraperitoneally), and placed on a thermoregulated surgical area. After an abdominal incision was made a polyethylene collar fitted PE-50 catheter (Clay-Adams, Parsippany, New Jersey) was inserted into the bladder dome and held in place with a purse-string suture. The free end of the catheter was sealed. A PE-50 polyethylene catheter was elongated to about 1.5 times its original length at the tip of the inserting side and filled with heparinized saline (100 IU ml⫺1). At the same time that the bladder catheter was implanted the elongated catheter was inserted into the femoral vein. At the same session another PE-50 catheter was filled with heparinized saline and introduced subcutaneously from neck to lower back. All catheters were tunneled subcutaneously, anchored to the skin of the neck and closed.
RTX Treatment To induce C-fiber desensitization RTX (0.3 mg kg⫺1) dissolved in dimethyl sulfoxide was injected subcutaneously into the rats during anesthesia, as described, 3 days before the experiment. Control animals received corresponding volumes per weight of dimethyl sulfoxide given subcutaneously.
Experimental Design Cystometry was performed without anesthesia 3 days after catheter implantation. The conscious rats were placed in metabolic cages without restraint and the bladder catheters were connected via a T tube to a P23 DC pressure transducer (Statham Instruments, Oxnard, California) and a CMA 100 microinjection pump (Carnegie Medicine AB, Solna, Sweden). Micturition volumes were recorded with a fluid collector connected to an FT03 D force displacement transducer (Grass Instrument, Quincy, Massachusetts). Room temperature saline was infused into the bladder continuously at a rate of 10 ml hour–1. Pressures and micturition volumes were recorded continuously with AcqKnowledge 3.8.1 software and an MP100 data acquisition system (Biopac Systems, Santa Barbara, California) connected to a Model 7E polygraph (Grass Instrument). At the beginning of cystometry the bladder was emptied via the bladder catheter. Apomorphine (100 g kg⫺1 subcutaneously) was used to stimulate efferent activity and induce detrusor overactivity. Tolterodine was given intravenously to controls (10 g kg⫺1) and RTX treated (1 and 10 g kg⫺1) animals before and after apomorphine administration. The doses chosen were based on published information.10
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Evaluations and Calculations Urodynamic parameters were investigated, including micturition pressure (maximum bladder pressure during micturition), baseline pressure (the lowest bladder pressure during filling), threshold pressure (bladder pressure immediately before micturition), bladder capacity (residual volume at the most recent previous micturition plus the volume of infused saline at micturition), micturition volume (volume of expelled urine), residual volume (bladder capacity minus micturition volume), the intercontraction interval and the AUC of intravesical pressure. Reproducible micturition cycles, together approximately corresponding to a 30-minute period, were recorded before drug administration. They served as baseline values to be compared with the 3 micturition cycles in the first 30-minute period after the administration of test substances. Comparisons of the means of all groups were performed with 1-way ANOVA (Holm-Sidak). Pairwise comparisons were made by Student’s t test. All statistical calculations were based on the number of individual animals and significant differences were considered at p ⬍0.05. RESULTS Effect of RTX Treatment on Micturition Parameters After RTX treatment (0.3 mg kg⫺1 subcutaneously) in 22 animals 8 showed decompensated bladders with dribbling incontinence. These animals were used only to assess the effects of RTX and tolterodine on apomorphine induced activity. Three rats showed intermittent dribbling incontinence as well as complete voiding cycles. One RTX treated animal and 1 control were excluded due to catheter problems. One RTX treated animal died of postoperative complications. Compared to the 9 controls RTX treatment in 12 rats increased the micturition interval (p ⬍0.001), bladder capacity (p ⬍0.0001), micturition volume (p ⬍0.05), threshold pressure p ⬍0.05) and residual volume (p ⬍0.05, fig. 1). There were no significant effects on mean ⫾ SD baseline pressure, which was 10.2 ⫾ 0.6 and 10.4 ⫾ 0.8 cm H2O in controls and RTX treated rats, respectively. No statistical difference was observed for micturition pressure between controls and RTX treated rats (98.0 ⫾ 8.6 and 72.2 ⫾ 10.0 cm H2O, respectively, p ⬍0.06). Effect of Tolterodine on Micturition Parameters in Control and RTX Treated Rats In controls intravenous administration of 10 g kg⫺1 tolterodine increased the mean micturition interval (p ⬍0.05), bladder capacity (p ⬍0.05), micturition volume (p ⬍0.05) and residual volume (p ⬍0.05), and decreased micturition pressure (p ⬍0.001, fig. 2). Baseline pressure was 9.4 ⫾ 1.0 and 8.4 ⫾ 2.6 cm H2O in 6 rats before and after drug administration, respectively. Threshold pressure was also unaffected by tolterodine in 6 rats (31.2 ⫾ 2.6 and 30.0 ⫾ 1.5 cm H2O, respectively). In 6 RTX treated rats tolterodine (1 g kg⫺1 intravenously) increased the mean micturition interval from 10.6 ⫾ 3.7 to 13.6 ⫾ 5.5 minutes (p ⬍0.05), increased bladder capacity from 1.6 ⫾ 0.3 to 1.9 ⫾ 0.3 ml (p ⬍0.05) and increased micturition volume from 1.5 ⫾ 0.3 to 1.8 ⫾ 0.2 ml (p ⬍0.05, fig. 3). At 10 g/kg given intravenously in 6 rats the micturition interval, bladder capacity and micturition volume
328
EFFECTS OF TOLTERODINE ON AFFERENT NEUROTRANSMISSION
FIG. 1. Effects of subcutaneous administration of 0.3 mg kg⫺1 RTX on micturition parameters in 9 controls (filled bars) and 12 RTX treated rats (open bars). Single asterisk indicates unpaired t test p ⬍0.05. Double asterisks indicate unpaired t test p ⬍0.01. Triple asterisk indicate unpaired t test p ⬍0.001.
were also significantly increased (figs. 3 and 4). There were no significant effects on residual volume, baseline, threshold and micturition pressures at either investigated tolterodine dose. After 10 g kg⫺1 tolterodine mean baseline pressure was 8.6 ⫾ 1.0 cm H2O in 6 rats in comparison to 10.2 ⫾ 1.4 cm H2O in 6 without the antimuscarinic agent. Mean threshold pressure was 39.4 ⫾ 5.1 and 37.6 ⫾ 6.4 cm H2O, and micturition pressure was 73.5.4 ⫾ 15.7 and 61.8 ⫾ 18.2 cm H2O in 6 rats each before and after 10 g kg⫺1 tolterodine, respectively.
Effect of Tolterodine on Apomorphine Induced Overactivity in Control and RTX Treated Rats Subcutaneous administration of 100 g kg⫺1 apomorphine induced characteristic detrusor overactivity with dribbling incontinence in controls and in RTX treated rats. Mean maximal pressure and AUC were almost identical in controls and RTX treated animals after apomorphine (100 g kg⫺1) (fig. 4). Tolterodine (1 and 10 g kg⫺1) had no effect on apomorphine induced activity (fig. 5).
FIG. 2. Micturition parameters in 6 controls before (filled bars) and after (open bars) intravenous administration of 10 g kg tolterodine. Single asterisk indicates paired t test p ⬍0.05. Double asterisks indicate paired t test p ⬍0.01.
EFFECTS OF TOLTERODINE ON AFFERENT NEUROTRANSMISSION
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FIG. 3. Original tracings of intravesical pressure and voided volumes during continuous cystometry in 2 RTX treated rats before (A) and after (C) intravenous administration of 1 g kg⫺1 (B) and 10 g kg⫺1 (D) tolterodine.
DISCUSSION The current experiments showed that the RTX treatment had significant effects on bladder function, as reflected by cystometric parameters and the development of dribbling incontinence in some animals. RTX increased the mean micturition interval, bladder capacity and voided volume, suggesting that structures involved in afferent signaling were affected (desensitized). However, functional elimination of RTX sensitive afferents did not affect the actions of tolterodine, suggesting that they were exerted independently of this type of C fibers. However, since TRPV1, the receptor for RTX, is located not only on a subpopulation of afferent nerves,12 but also on urothelium,13 interstitial cells and even on detrusor
myocytes,14 there may be effects not only on afferent nerves. It is well known that RTX desensitizes afferent nerves.15 However, actions on extraneuronal structures important for afferent signaling and sensitive to muscarinic receptor stimulation may not undergo desensitization.16 As shown in the current study, tolterodine at the concentrations used had no effect on apomorphine induced detrusor activity. As reported previously, apomorphine is believed to activate the bladder independently of afferent activation, initiating activity at a central site of action, and increase the parasympathetic outflow from the spinal cord11 with a consequent release of large amounts of acetylcholine at the cholinergic nerve terminals and the direct stimulation of
FIG. 4. Micturition parameters before (black bars) and after (white bars) intravenous administration of 10 g kg tolterodine in 6 rats given 0.3 mg kg RTX subcutaneously. Single asterisk indicates paired t test p ⬍0.05.
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EFFECTS OF TOLTERODINE ON AFFERENT NEUROTRANSMISSION
FIG. 5. Apomorphine induced detrusor overactivity in 5 controls and 8 rats treated with 0.3 mg kg RTX subcutaneously before (filled bars) and after (open bars) intravenous administration of 10 g kg⫺1 tolterodine. s., second.
detrusor myocytes, resulting in a voiding contraction. Since the effects of tolterodine and acetylcholine are competitive, the amounts of tolterodine used in the current study may not have been sufficient to inhibit the acetylcholine effect during voiding. The lack of an effect of the antimuscarinic on apomorphine induced activity supports the assumption that efferent neurotransmission was not affected. This is different from what happens during filling, when only small amounts of acetylcholine are available to the receptors. This implies that in this situation the tolterodine concentrations achieved at the current dose are sufficient to inhibit the acetylcholine effects. The finding that the effects of tolterodine were independent of RTX sensitive afferent nerves are in apparent disagreement with the results of Yokoyama et al, who concluded that the actions of tolterodine depended on the suppression of C-fiber bladder afferent activity.10 They found in a rat model of increased detrusor activity caused by occlusion of the middle cerebral artery that bladder capacity was markedly decreased after occlusion in RTX at 0.3 mg kg⫺1, which is the same dose as in the current investigation, and in vehicle treated rats. Low tolterodine doses of 0.2 and 2 nM kg⫺1 (0.1 and 1 g kg⫺1, respectively) significantly increased bladder capacity in vehicle treated rats without increasing residual volume but had no effects in RTX treated rats. They suggested that at low doses tolterodine exerts an inhibitory effect on C-fiber bladder afferent nerves, thereby improving bladder capacity during the storage phase. The reasons for the discrepancy in results between the current study and that of Yokoyama et al are unclear but they may depend on the models used.
Kim et al administered muscarinic receptor agonists and antagonists intravesically to rats and were able to separate the local inhibitory effects of antimuscarinic agents during the storage phase from a decrease in voiding pressure.17 They found that intravesical instillation of antimuscarinics at clinically meaningful concentrations suppressed carbachol induced detrusor overactivity. Kim et al concluded that antimuscarinic agents may be effective for treating overactive bladder, not only by suppression of muscarinic receptor mediated detrusor muscle contractions, but also by blocking muscarinic receptors in bladder-afferent pathways. Since the effects of antimuscarinics are believed to be attributable to a competitive antagonism of acetylcholine at muscarinic receptor sites and the direct involvement of RTX sensitive afferents seems controversial, possible sites of action other than C fibers may be discussed. M2 and M3 receptor immunoreactivity was observed in the urothelium, nerve fibers and detrusor layers in the human bladder.9 A significant increase in suburothelial myofibroblast-like M2 and M3 receptor immunoreactivity was seen in patients with idiopathic detrusor overactivity. This immunoreactivity significantly correlated with the urgency score and M2 receptor immunoreactivity also correlated with the frequency score. The increase in muscarinic receptor immunostaining in myofibroblast-like cells and its correlation with clinical scores suggested to us a potential role in pathophysiological mechanisms and in the therapeutic effect of antimuscarinic agents. There may be differences in muscarinic receptor distribution, including subtypes, between the rat and the human bladder. However, muscarinic receptors can be expected in
EFFECTS OF TOLTERODINE ON AFFERENT NEUROTRANSMISSION the same structures in the rat bladder as in the human bladder. The current results suggest that tolterodine, which blocks M2 and M3 receptors, exerted an effect on afferent signaling from the bladder, leading to decreased voiding frequency, and consequent increases in bladder capacity and voided volumes independent of RTX sensitive afferents. It may be assumed that there is a release of small amounts of acetylcholine from nerves or from extraneuronal sources, eg urothelium, during the bladder storage phase5,6 and the released transmitter stimulates afferent signaling. The low concentrations of released acetylcholine can be effectively counteracted by tolterodine at concentrations that have no effect on detrusor contraction. However, this is not the case when there is a massive release of the transmitter, as can be expected during apomorphine stimulated voiding.
3. 4.
5.
6.
7.
CONCLUSIONS
8.
The current experiments show that tolterodine increases the micturition interval and bladder capacity in controls and in RTX treated animals. Thus, functional elimination of RTX sensitive afferents did not affect these actions of tolterodine, suggesting that they were exerted independently of this population of C fibers. This does not necessarily mean that the effect was not exerted on the afferent side of the micturition reflex since A␦-fiber mediated activity may be less affected by RTX treatment, and urothelium and interstitial cells may not be desensitized. Because tolterodine did not decrease the contractile effects of apomorphine, the drug at the doses used seemed to have no effect on efferent neurotransmission during voiding. Thus, tolterodine at low doses exerted an inhibitory effect on afferent neurotransmission without influencing the effect of efferent stimulation during voiding.
9.
10.
11.
12.
13.
14.
Abbreviations and Acronyms RTX ⫽ resiniferatoxin TRPV1 ⫽ transient receptor potential channel V1 REFERENCES 1.
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